U.S. patent application number 10/666291 was filed with the patent office on 2007-11-22 for antibody complexes and methods for immunolabeling.
Invention is credited to Joseph Beechem, David Hagen, Iain Johnson.
Application Number | 20070269902 10/666291 |
Document ID | / |
Family ID | 27382120 |
Filed Date | 2007-11-22 |
United States Patent
Application |
20070269902 |
Kind Code |
A1 |
Beechem; Joseph ; et
al. |
November 22, 2007 |
Antibody complexes and methods for immunolabeling
Abstract
The present invention provides labeling reagents and methods for
labeling primary antibodies and for detecting a target in a sample
using an immuno-labeled complex that comprises a target-binding
antibody and one or more labeling reagents. The labeling reagents
comprise monovalent antibody fragments or non-antibody monomeric
proteins whereby the labeling reagents have affinity for a specific
region of the target-binding antibody and are covalently attached
to a label. Typically, the labeling reagent is an anti-Fc Fab or
Fab' fragment that was generated by immunizing a goat or rabbit
with the Fc fragment of an antibody. The present invention provides
for discrete subsets of labeling reagent and immuno-labeled
complexes that facilitate the simultaneous detection of multiple
targets in a sample wherein the immuno-labeled complexes are
distinguished by i) a ratio of label to labeling reagent, or ii) a
physical property of said label, or iii) a ratio of labeling
reagent to said target-binding antibody, or iv) by said
target-binding antibody. This is particularly useful for
fluorophore labels that can be attached to labeling reagents and
subsequently immuno-labeled complexes in ratios for the detection
of multiple targets.
Inventors: |
Beechem; Joseph; (Eugene,
OR) ; Hagen; David; (Eugene, OR) ; Johnson;
Iain; (Eugene, OR) |
Correspondence
Address: |
INVITROGEN CORPORATION;C/O INTELLEVATE
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Family ID: |
27382120 |
Appl. No.: |
10/666291 |
Filed: |
September 17, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10467550 |
Oct 12, 2004 |
|
|
|
PCT/US02/31416 |
Oct 2, 2002 |
|
|
|
10666291 |
Sep 17, 2003 |
|
|
|
10118204 |
Apr 5, 2002 |
|
|
|
10467550 |
Oct 12, 2004 |
|
|
|
60329068 |
Oct 12, 2001 |
|
|
|
60369418 |
Apr 1, 2002 |
|
|
|
Current U.S.
Class: |
436/501 ; 435/5;
435/6.19; 435/7.4 |
Current CPC
Class: |
B82Y 10/00 20130101;
G01N 33/53 20130101; B82Y 30/00 20130101; G01N 33/6857 20130101;
B82Y 5/00 20130101; G01N 33/58 20130101 |
Class at
Publication: |
436/501 ;
435/005; 435/006; 435/007.4 |
International
Class: |
G01N 33/00 20060101
G01N033/00; C12Q 1/68 20060101 C12Q001/68 |
Claims
1. A ligand-detection reagent; wherein said reagent comprises a
ligand-binding antibody and a ligand analog and a labeling reagent
non-covalently bonded to said antibody to form a ternary complex
wherein said ligand analog is covalently bonded to a reporter
molecule and said labeling reagent comprises a monovalent antibody
fragment or a non-antibody protein and a covalently bonded label
moiety.
2. The reagent according to claim 1, wherein said reporter molecule
is selected from the group consisting of a borapolyazaindacene, a
coumarin, a xanthene, a cyanine, a fluorescent protein and a
phosphorescent dye.
3. The reagent according to claim 2, wherein said reporter molecule
is selected from the group consisting of BODIPY, OREGON GREEN or
fluorinated coumarin dyes.
4. The reagent according to claim 2, wherein said xanthene dye
moiety is fluorinated.
5. The reagent according to claim 2, wherein said ligand analog is
selected from the group consisting of an amino acid, an enzyme, a
kinase substrate, a peptide, a protein, a polysaccharide, a
phosphatase substrate, a nucleoside, a nucleotide, an
oligonucleotide, a nucleic acid, a hapten, a cell surface receptor,
a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer,
a polymeric microparticle, a biological cell and a virus.
6. The reagent according to claim 5, wherein said ligand analog is
phosphotyramide, phosphotyrosinamide, phosphoserine,
phosphoethanolamine, phosphorylated kinase peptide substrate,
phosphatase substrate, phosphorylated peptide or digoxigenin.
7. The reagent according to claim 6, wherein said ligand analog is
phosphotyramide, phosphotyrosinamide, phosphoserine or digoxigenin
and said reporter molecule is a xanthene, coumarin or
borapolyazaindacene moiety.
8. The reagent according to claim 7, wherein ligand analog-reporter
molecule is selected from the group consisting of Compounds 2,
4-19, 22-29, 31-43, ##STR26## and salts thereof.
9. The reagent according to claim 5, wherein said monovalent
antibody fragment is a Fab or Fab' fragment and is selected from
the group consisting of anti-Fc antibody fragment, anti-Fab
antibody fragment, anti-kappa light chain antibody fragment,
anti-lambda light chain antibody fragment, and a single chain
variable protein fragment and wherein said non-antibody protein is
selected from the group consisting of a protein G, a protein A, a
protein L, a lectin, and a protein G bound to albumin, wherein said
albumin is covalently linked to one or more label moieties and
albumin is selected from the group consisting of human albumin,
bovine serum albumin, and ovalbumin.
10. The reagent according to claim 9, wherein said label moiety is
selected from a group consisting of a chromophore, a fluorophore, a
quenching moiety, a fluorescent protein and a phosphorescent
dye.
11. The reagent according to claim 10, wherein said label moiety is
a fluorophore or a quenching moiety.
12. The reagent according to claim 11, wherein said fluorophore and
quenching moiety are individually selected from the group
consisting of cyanine and xanthene moieties.
13. The reagent according to claim 12, wherein said monovalent
antibody fragment is an anti-Fc Fab fragment.
14. The reagent according to claim 13, wherein said labeling
reagent comprises an anti-Fc monovalent antibody fragment and a
xanthene moiety.
15. The reagent according to claim 12, wherein said reporter
molecule is an energy donor molecule capable of transferring energy
to said label moiety that is an energy acceptor molecule wherein an
energy transfer pair is selected from the group consisting of
Oregon Green 488-Alexa Fluor 555 dye pair, BODIPY-FL-Alexa Fluor
555 dye pair and BODIPY-FL-QSY 9 dye pair.
16. The reagent according to claim 5, wherein said reagent
comprises a ligand antibody, a ligand analog and a labeling reagent
to form a ternary complex wherein said ligand analog is selected
from the group consisting of phosphotyramide, phosphoserine,
phosphotyrosinamide, phosphoethanolamine, phosphorylated kinase
peptide substrate, phosphatase substrate and a phosphorylated
peptide and said analog is covalently bonded to a xanthene reporter
molecule and said labeling reagent is an anti-Fc monovalent
antibody fragment covalently bonded to a xanthene labeling moiety
or non-fluorescent quenching moiety.
17. A method for determining the presence of a target ligand in a
sample, in which is employed a ligand-detection reagent comprising
a ligand-binding antibody, a ligand analog and a labeling reagent
non-covalently bonded to said antibody to form a ternary complex
wherein said ligand analog is covalently bonded to a reporter
molecule and said labeling reagent comprises a monovalent antibody
fragment or a non-antibody protein and a covalently bonded label
moiety whereby the amount of generated detectable signal from said
reporter molecule is dependent on the presence of said ligand, said
method comprising: a. generating ligand-detection reagent according
to any one of claims 1-16, wherein said ligand-binding antibody,
said ligand analog and said labeling reagent are incubated together
for a sufficient amount of time to form said complex; b. incubating
said reagent with said sample for a sufficient amount of time for
said target ligand to displace said ligand analog from binding
groove of said ligand-binding antibody; c. illuminating said sample
with an appropriate wavelength wherein said reporter molecule
generates a change in detectable signal in the presence of said
target ligand whereby said target ligand is detected.
18. The method according to claim 17, wherein said target ligand is
selected from the group consisting of a phosphorylated biomolecule,
kinase substrate, phosphatase substrate, digoxigenin, small
molecule drugs, dinitrophenyl, cell surface proteins, intracellular
proteins, extracellular proteins, antibodies, immunogenic peptides,
allergens, histamine and cytokines.
19. The method according to claim 18, wherein said ligand is in
solution or immobilized on a solid or semi-solid matrix.
20. The method according to claim 19 wherein said solid or
semi-solid matrix is selected from the group consisting of a
membrane, polymeric gel, polymeric microparticle and an array.
21. The method according to claim 20, wherein presence of said
ligand is determined by a shift in color of said detectable
signal.
22. The method according to claim 20, wherein presence of said
ligand is determined by an increase in intensity of said detectable
signal.
23. The method according to claim 20, wherein presence of said
ligand is determined by a decrease in intensity of said detectable
signal.
24. A method for determining the presence of a phosphorylated
target ligand in a sample, in which is employed a ligand-detection
reagent comprising a ligand-binding antibody that is capable of
binding a phosphotyrosine, phosphoserine or phosphothreonine
moiety, a ligand analog that is selected from the group consisting
of phosphotyramide, phosphotyrosinamide, phosphoserine,
phosphoethanolamine, phosphorylated kinase peptide substrate,
phosphatase substrate and phosphorylated peptide and a labeling
reagent non-covalently bonded to said antibody to form a ternary
complex wherein said ligand analog is covalently bonded to a
reporter molecule and said labeling reagent comprises a monovalent
antibody fragment or a non-antibody protein and a covalently bonded
label moiety whereby the amount of generated detectable signal from
said reporter molecule is dependent on the presence of said ligand,
said method comprising: a. generating a ligand-detection reagent
according to any one of claims 1-16, wherein said ligand-binding
antibody, said ligand analog and said labeling reagent are
incubated together for a sufficient amount of time to form said
complex; b. incubating said ligand-detection reagent with said
sample for a sufficient amount of time for said phosphorylated
molecule to displace said ligand analog from binding groove of said
ligand-binding antibody; c. illuminating said sample with an
appropriate wavelength wherein said reporter molecule generates a
change in detectable signal in the presence of said phosphorylated
molecule whereby the presence of said phosphorylated molecule is
determined.
25. The method according to claim 24, wherein said phosphorylated
target molecule is selected from the group consisting of proteins,
peptides, amino acids, nucleotides, phosphatase substrates, and
kinase substrates.
26. The method according to claim 25, wherein said phosphorylated
target molecules are immobilized on a solid or semi-solid matrix or
are in solution.
27. The method according to claim 26, wherein said solid or
semi-solid matrix is a polymeric gel, a membrane, a polymeric
particle, a polymeric microparticle or an array.
28. The method according to claim 27, wherein presence of said
ligand is determined by a shift in color of said detectable
signal.
29. The method according to claim 27, wherein presence of said
ligand is determined by an increase in intensity of said detectable
signal.
30. The method according to claim 27, wherein presence of said
ligand is determined by a decrease in intensity of said detectable
signal.
31. A ligand-detection solution comprising: a. a ligand-detection
reagent; wherein said reagent comprises a ligand-binding antibody,
a ligand analog and a labeling reagent non-covalently bonded to
said antibody to form a ternary complex wherein said ligand analog
is covalently bonded to a reporter molecule and said labeling
reagent comprises a monovalent antibody fragment or a non-antibody
protein and a covalently bonded label moiety; and, b. a buffer.
32. The solution according to claim 31, wherein said reporter
molecule is selected from the group consisting of a
borapolyazaindacene, a coumarin, a xanthene, a cyanine, a
fluorescent protein and a phosphorescent dye.
33. The solution according to claim 32, wherein said ligand analog
is selected from the group consisting of an amino acid, an enzyme,
a kinase substrate, a peptide, a protein, a polysaccharide, a
phosphatase substrate, a nucleoside, a nucleotide, an
oligonucleotide, a nucleic acid, a hapten, a cell surface receptor,
a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer,
a polymeric microparticle, a biological cell and a virus.
34. The solution according to claim 33, wherein said ligand analog
is phosphotyramide, phosphoethanolamine, phosphoserine,
phosphotyrosinamide, phosphorylated kinase peptide substrate,
phosphatase substrate, phosphorylated peptide or digoxigenin.
35. The solution according to claim 34, wherein said ligand analog
is phosphotyramide, phosphotyrosinamide, phosphoserine,
phosphoethanolamine or digoxigenin and said reporter molecule is a
xanthene, coumarin or borapolyazaindacene moiety.
36. The solution according to claim 35, wherein said ligand analog
is selected from the group consisting of Compound 2, 4-19, 22-29,
31-38, ##STR27## and salts thereof.
37. The solution according to claim 36, wherein said ligand analog
is Compound 15 or Compound 23.
38. The solution according to claim 37, wherein said buffer has
less that 5 mM phosphate.
39. The solution according to claim 33, wherein said monovalent
antibody fragment is a Fab or Fab' fragment and is selected from
the group consisting of anti-Fc antibody fragment, anti-kappa light
chain antibody fragment, anti-lambda light chain antibody fragment,
and a single chain variable protein fragment and wherein said
non-antibody protein is selected from the group consisting of a
protein G, a protein A, a protein L, a lectin, and a protein G
bound to albumin, wherein said albumin is covalently linked to one
or more label moieties and albumin is selected from the group
consisting of human albumin, bovine serum albumin, and
ovalbumin.
40. The solution according to claim 39, wherein said label moiety
is selected from a group consisting of a chromophore, a
fluorophore, a hapten, an enzyme, a quenching moiety, a fluorescent
protein and a phosphorescent dye.
41. The solution according to claim 40, wherein said label moiety
is a fluorophore or a quenching moiety.
42. The solution according to claim 41, wherein said fluorophore
and quenching moiety are individually selected from the group
consisting of cyanine and xanthene moieties.
43. The solution according to claim 42, wherein said monovalent
antibody fragment is an anti-Fc Fab fragment.
44. The solution according to claim 43, wherein said labeling
reagent comprises an anti-Fc Fab antibody fragment and a xanthene
moiety.
45. The solution according to claim 40, wherein said reporter
molecule is an energy donor molecule capable of transferring energy
to said labeling moiety that is an energy acceptor molecule wherein
an energy transfer pair is selected from the group consisting of
Oregon Green 488-Alexa Fluor 555 dye pair, BODIPY-FL-Alexa Fluor
555 dye pair and BODIPY-FL-QSY 9 dye pair.
46. The solution according to claim 36, wherein said reagent
comprises a ligand antibody, a ligand analog and a labeling reagent
to form a ternary complex wherein said ligand analog is selected
from the group consisting of phosphotyramide, phosphoethanolamine,
phosphoserine, phosphotyrosinamide, phosphorylated kinase peptide
substrate, phosphatase substrate and phosphorylated peptide and
said ligand analog is covalently bonded to a xanthene or
borapolyazaindacene reporter molecule and said labeling reagent is
an anti-Fc monovalent antibody fragment covalently bonded to a
xanthene label moiety or non-fluorescent quenching moiety.
47. A kit for the detection of a target ligand, wherein said kit
comprises a ligand analog, a labeling reagent that comprises a
monovalent antibody fragment or a non-antibody protein and a
covalently bonded label moiety and optionally a ligand-binding
antibody.
48. The kit according to claim 47, wherein said ligand analog is
selected from the group consisting of an amino acid, an enzyme, a
kinase substrate, a peptide, a protein, a polysaccharide, a
phosphatase substrate, a nucleoside, a nucleotide, an
oligonucleotide, a nucleic acid, a hapten, a cell surface receptor,
a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer,
a polymeric microparticle, a biological cell and a virus.
49. The kit according to claim 48 wherein said ligand analog is
selected from the group consisting of is a phosphotyramide, a
phosphoserine, a phosphotyrosinamide, a phosphoethanolamine, a
phosphorylated kinase peptide substrate, a phosphatase substrate, a
phosphorylated peptide or a digoxigenin.
50. The kit according to claim 49, wherein said ligand-binding
antibody has affinity for a phosphorylated biomolecule.
51. The kit according to claim 48, wherein said wherein said
monovalent antibody fragment is a Fab or Fab' fragment and is
selected from the group consisting of anti-Fc antibody fragment,
anti-Fab antibody fragment, anti-kappa light chain antibody
fragment, anti-lambda light chain antibody fragment, and a single
chain variable protein fragment and wherein said non-antibody
protein is selected from the group consisting of a protein G, a
protein A, a protein L, a lectin, and a protein G bound to albumin,
wherein said albumin is covalently linked to one or more label
moieties and albumin is selected from the group consisting of human
albumin, bovine serum albumin, and ovalbumin.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Ser. No.
60/329,068, filed Oct. 12, 2001; U.S. Ser. No. 60/369,418 filed
Apr. 1, 2002, U.S. Ser. No. 10/118,204 filed Apr. 5, 2002, and
PCT/US02/31416 filed Oct. 2, 2002, which disclosures are herein
incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to immuno-labeled complexes
and methods for use in the detection and measurement of one or more
targets in a biological sample. The invention has applications in
the fields of molecular biology, cell biology,
immunohistochemistry, diagnostics, and therapeutics.
BACKGROUND OF THE INVENTION
[0003] Immunolabeling is a method for qualitative or quantitative
determination of the presence of a target in a sample, wherein
antibodies are utilized for their specific binding capacity. The
antibodies form a complex with the target (antigen), wherein a
detectable label is present on the antibody or on a secondary
antibody. The detectable label is a key feature of immunolabeling,
which can be detected directly or indirectly. The label provides a
measurable signal by which the binding reaction is monitored
providing a qualitative and/or quantitative measure of the degree
of binding. The relative quantity and location of signal generated
by the labeled antibodies can serve to indicate the location and/or
concentration of the target. The label can also be used to select
and isolate labeled targets, such as by flow sorting or using
magnetic separation media. Examples of labels include but are not
limited to radioactive nucleotides (.sup.125I, .sup.3H, .sup.14C,
.sup.32P), chemiluminescent, fluorescent, or phosphorescent
compounds (e.g., dioxetanes, xanthene, or carbocyanine dyes,
lanthanide chelates), particles (e.g., gold clusters, colloidal
gold, microspheres, quantum dots), and enzymes (e.g., peroxidases,
glycosidases, phosphatases, kinases). Ideally, the label is
attached to the antibody in a manner that does not perturb the
antibody's binding characteristics but enables the label to be
measured by an appropriate detection technology. The choice of
labels is influenced by factors such as ease and sensitivity of
detection, equipment availability, background in the sample
(including other labels) and the degree to which such labels are
readily attached to the particular antibody. Both direct and
indirect labeling of antibodies is utilized for immunolabeling.
Direct labeling utilizes only a primary antibody, i.e. the antibody
specific for the target, bound to the label. In contrast, indirect
labeling utilizes a secondary antibody bound to the label, which is
specific for the primary antibody, e.g. a goat anti-rabbit
antibody. The principal differences in immunolabeling methods and
materials reside in the way that the label is attached to the
antibody-antigen complex, the type of label that is used, and the
means by which the antibody-antigen complex is detected.
[0004] Limitations for direct labeling primary antibodies include
the need for buffers free of primary amines, or carrier proteins
such as bovine serum albumin (BSA), and other compounds such as
tris-(hydroxymethyl)aminomethane (TRIS), glycine, and ammonium
ions. These materials are, however, common components in antibody
buffers and purification methods, and it may not be possible or
feasible to remove them prior to the coupling reaction. In
particular, many monoclonal antibodies are available only as
ascites fluid or in hybridoma culture supernatants, or diluted with
carrier proteins, such as albumins. Thus, direct labeling of
antibodies in ascites fluid or other medias containing interfering
compounds is not attainable.
[0005] The indirect immunolabeling method typically involves a
multi-step process in which an unlabeled first antibody (typically
a primary antibody) is directly added to the sample to form a
complex with the antigen in the sample. Subsequently, a labeled
secondary antibody, specific for the primary antibody, is added to
the sample, where it attaches noncovalently to the primary
antibody-antigen complex. Alternatively, a detectable label is
covalently attached to an immunoglobulin-binding protein such as
protein A and protein G to detect the antibody-antigen complex that
has previously been formed with the target in the sample. Using
ligands, such as streptavidin, that are meant to amplify the
detectable signal also expands this cascade binding.
[0006] Indirect immunolabeling often results in false positives and
high background. This is due to the fact that secondary antibodies,
even when purified by adsorption against related species,
nevertheless can exhibit significant residual cross-reactivity when
used in the same sample. For example, when mouse tissue is probed
with a mouse monoclonal antibody, the secondary antibody must
necessarily be a labeled anti-mouse antibody. This anti-mouse
antibody will detect the antibody of interest but will inevitably
and additionally detect irrelevant, endogenous mouse
immunoglobulins inherent in mouse tissue. This causes a significant
background problem, especially in diseased tissues, which reduces
the usefulness and sensitivity of the assay. Thus, the simultaneous
detection of more than one primary antibody in a sample without
this significant background interference depends on the
availability of secondary antibodies that 1) do not cross-react
with proteins intrinsic to the sample being examined, 2) recognize
only one of the primary antibodies, and 3) do not recognize each
other (Brelje, et al., METHODS IN CELL BIOLOGY 38, 97-181,
especially 111-118 (1993)).
[0007] To address the background problem in indirect labeling, a
number of strategies have been developed to block access of the
anti-mouse secondary antibodies to the endogenous mouse
immunoglobulins. One such strategy for blocking involves complexing
the primary antibody with a selected biotinylated secondary
antibody to produce a complex of the primary and secondary
antibodies, which is then mixed with diluted normal murine serum
(Trojanowski et al., U.S. Pat. No. 5,281,521 (1994)). This method
is limited by the necessity to utilize an appropriate ratio of
primary-secondary complex. Too low a ratio of primary-secondary
complex will cause a decrease in specific staining and increased
background levels due to the uncomplexed secondary anti-mouse
antibody binding to endogenous mouse antibodies. However, the
ability of a whole IgG antibody (as was used in the referenced
method) to simultaneously bind and cross-link two antigens results
in too high a ratio, causing the complex to precipitate or form
complexes that are too large to penetrate into the cell or
tissue.
[0008] Another strategy for blocking access to endogenous
immunoglobulins in the sample involves pre-incubating the sample
with a monovalent antibody, such as Fab' fragments, from an
irrelevant species that recognize endogenous immunoglobulins. This
approach requires large quantities of expensive Fab' fragments and
gives mixed results and adds at least two steps (block and wash) to
the overall staining procedure. The addition of a cross-linking
reagent has resulted in improved reduction of background levels
(Tsao, et al., U.S. Pat. No. 5,869,274 (1997)) but this is
problematic when used with fluorophore-labeled antibodies. The
cross-linking causes an increase in the levels of autofluorescence
and thus the background (J. Neurosci. Meth. 83, 97 (1998); Mosiman
et al., Methods 77, 191 (1997); Commun. Clin. Cytometry 30, 151
(1997); Beisker et al., Cytometry 8, 235 (1987)). In addition,
pre-incubation with a cross-linking reagent often masks or prevents
the antibody from binding to its antigen (J. Histochem. Cytochem.
45, 327 (1997); J. Histochem. Cytochem. 39, 741 (1991); J.
Histochem. Cytochem. 43, 193 (1995); Appl. Immunohistochem.
Molecul. Morphol. 9, 176 (2001)).
[0009] In a variation of this blocking strategy, a multi-step
sequential-labeling procedure is used to overcome the problems of
cross-reactivity. The sample is incubated with a first antibody to
form a complex with the first antigen, followed by incubation of
the sample with a fluorophore-labeled goat Fab anti-mouse IgG to
label the first antibody and block it from subsequently complexing
when the second antibody is added. In the third step, a second
mouse antibody forms a complex with the second antigen. Because the
second antibody is blocked from cross-reacting with the first
antibody, the second mouse antibody is detected with a standard
indirect-labeling method using a goat anti-mouse antibody
conjugated to a different fluorescent dye (J. Histochem. Cytochem.
34, 703 (1986)). This process requires multiple incubation steps
and washing steps and it still cannot be used with mouse antibodies
to probe mouse tissue.
[0010] Another blocking method is disclosed in the animal research
kit (ARK) developed by DAKO. In this kit, a primary antibody is
complexed with biotin-labeled goat Fab anti-mouse IgG and excess
free Fab is blocked with normal mouse serum. However, since the Fab
used in this process is generated from the intact IgG (rather than
a selected region) there is a potential for the formation of
anti-paratope or anti-idiotype antibodies that will block the
antigen-binding site and prevent immunolabeling. The biotinylated
antibody also requires subsequent addition of a labeled avidin or
streptavidin conjugate for its subsequent visualization.
[0011] The present invention is advantageous over previously
described methods and compositions in that it provides the benefits
of indirect labeling with the easy and flexibility of direct
labeling for determination of a desired target in a biological
sample. The present invention provides labeled monovalent proteins
specific for a target-binding antibody, which are complexed prior
to addition with a biological sample. Because these monovalent
proteins are not bivalent antibodies, precipitation and
cross-linking are not a problem. Therefore the compositions of the
present invention can be used with immunologically similar
monoclonal or polyclonal antibodies of either an identical isotype
or different isotypes. The monovalent labeling reagents are
specific for the Fc region of target-binding antibodies, these
reagents will not interfere with the binding region of the primary
antibody. In addition, the monovalent labeling reagents are not
negatively affected by the presence of primary amines like BSA,
gelatin, hybridoma culture supernatants or ascites fluid, thus
primary antibodies present in these media can be effectively
labeled with the labeling reagents of the present invention. Thus,
the present invention provides numerous advantages over the
conventional methods of immunolabeling.
SUMMARY OF THE INVENTION
[0012] The present invention provides labeling reagents and methods
for labeling primary antibodies and for detecting a target in a
sample using an immuno-labeled complex that comprises a
target-binding antibody and one or more labeling reagents. The
labeling reagents comprise monovalent antibody fragments or
non-antibody monomeric proteins whereby the labeling reagents have
affinity for a specific region of the target-binding antibody and
are covalently attached to a label. Typically, the labeling reagent
is an anti-Fc Fab or Fab' fragment that was generated by immunizing
a goat or rabbit with the Fc fragment of an antibody.
[0013] The methods for labeling a target-binding antibody with a
labeling reagent comprise a) contacting a solution of
target-binding antibodies with a labeling reagent, b) incubating
said target-binding antibodies and said labeling reagent wherein a
region of said target binding antibody is selectively bound by
labeling reagent, and c) optionally removing unbound labeling
reagent by adding a capture reagent comprising immunoglobulin
proteins or fragments thereof that are optionally immobilized on a
matrix. The labeling of the target-binding antibody can be
performed irrespective of the solution that the antibody is present
in and includes proteins that are normally present in serum or
ascites. This feature of the labeling process of the target-binding
antibody eliminates the need to purify and concentrate the
target-binding antibody. The time required for the labeling reagent
to selectively bind to the target-binding antibody is typically
very short, often less than 10 minutes. Often the labeling reagent
binds the target-binding antibody in the amount of time it takes to
add and mix the labeling reagent with the target-binding antibody.
This formation of an immuno-labeled complex--a target-binding
antibody and a labeling reagent--results in the formation of an
target detection solution that is used to detect a target in a
sample.
[0014] The labeling steps of the target-binding antibody are
optionally repeated to form a panel of subsets, these
immuno-labeled complex subsets may be used individually or pooled
wherein each subset is distinguished from another subset by i) the
target-binding antibody, or ii) a ratio of label to labeling
reagent, or iii) a ratio of labeling reagent to the target-binding
antibody or iv) by a physical property of the label. Thus, it is
appreciated that a wide range of subsets can be formed wherein the
subsets can be used individually to detect a target in a sample or
pooled to simultaneously detect multiple targets in a sample. The
simultaneous detection of multiple targets in a sample is
especially useful in methods that utilize flow cytometry or methods
that immobilize a population of cells or tissue on a surface.
[0015] The methods for determining a target in a sample using
immuno-labeled subsets comprises forming a subset of immuno-labeled
complexes, as described above, contacting a sample with said
immuno-labeled complexes, incubating the sample for a time
sufficient to allow the immuno-labeled complex to selectively bind
to a desired target, and illuminating the immuno-labeled complex
whereby the target is detected. The sample is any material that may
contain a target and typically comprises a population of cells,
cellular extract, subcellular component, proteins, peptides, tissue
culture, tissue, a bodily fluid, or a portion or combination
thereof. When multiple targets are detected a pooled subset of
immuno-labeled complexes are formed and incubated with the sample
or individual subsets are add sequentially to a sample. For methods
using flow cytometry the population of cells is illuminated when
they pass through an optical examination zone and the data
collected about the label determines the identity and quantity of
the targets.
[0016] In addition the labeling reagents are used to determine the
presence of a target ligand in a sample employing a target-binding
antibody and a ligand analog to form a ligand-detection reagent.
The ligand-detection reagent comprises a ligand-binding antibody, a
ligand analog to form an antibody-ligand analog complex wherein the
ligand analog is covalently bonded to a reporter molecule and a
labeling reagent non-covalently bonded to a region of the antibody
to form a ternary complex. The labeling reagent comprises a
monovalent antibody fragment or a non-antibody protein that is
covalently bonded to a label moiety. The reporter molecule, when
forming part of the ligand-detection reagent is quenched by the
label moiety of the labeling reagent, wherein the amount of
quenching is directly related to the amount of ligand present in
the sample. Thus, the target ligand displaces the ligand analog
relieving quenching of the reporter molecule. Alternatively, the
ligand analog is fluorogenic wherein the ligand analog is
essentially non-fluorescent in solution but when bound by the
ligand-binding antibody the detectable signal increases. In this
instance a decrease in signal, as opposed to the relieving of
quenching, is measured for the presence of a target ligand.
[0017] The ligand analog is covalently attached to a reporter
molecule selected from the group consisting of a
borapolyazaindacene, a coumarin, a xanthene, a cyanine, a
fluorescent protein and a phosphorescent dye. Through careful
selection of the reporter molecule, ligand analog, ligand-binding
antibody and labeling reagent we have demonstrated that the
reporter molecule can be substantially quenched or masked when
bound by the ligand-binding antibody. In this instance, the
labeling reagent is covalently attached to a label wherein the
label is capable of absorbing energy from the reporter molecule to
form an energy transfer pair when complexed with the ligand-binding
antibody. The emitted energy from the reporter molecule is either
absorbed by the label and re-emitted at a longer wavelength than
energy emitted by the reporter molecule or is absorbed with little
or no energy being re-emitted at a longer wavelength. In this way
the label is either considered a quencher or a fluorophore moiety,
both of which are capable of absorbing energy.
[0018] Therefore, a method is provided for determining the presence
of a ligand in a sample employing the ligand-detection reagent. In
carrying out the present methods the ligand analog-reporter
molecule and labeling reagent is complexed with the ligand-binding
antibody wherein the reporter molecule is quenched or masked. The
ligand-detection reagent is incubated with the sample for a
sufficient amount of time to allow for the target ligand present in
the sample to displace the ligand-analog. In this way the unmasking
of the reporter molecule provides either the presence of a
detectable signal or a shift in color compared to when the
ligand-binding antibody bound the ligand analog.
[0019] The present invention provides novel labeling reagents,
ligand-detection reagents, ligand-analogs and a competitive
immunoassay for the determination of the presence of a target
ligand in a sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1: Shows a schematic representation of the formation of
the immuno-labeled complex (target-binding antibody and labeling
reagent).
[0021] FIG. 2: Shows species specificity of goat Fab anti-(mouse
Fc), as observed using a microplate coated with IgG of various
species. The various species were blocked with BSA, reacted with
biotinylated goat Fab anti-(mouse Fc), washed, and then treated
with streptavidin-horseradish peroxidase (HRP), followed by
hydrogen peroxide (H.sub.2O.sub.2) and the Amplex Red peroxidase
detection reagent.
[0022] FIG. 3: Shows a preferred molar ratio of a goat Fab
anti-(mouse Fc) labeling reagent. Varying amounts of an Alexa Fluor
488 dye-labeled Fab fragment of goat anti-(mouse Fc) were added to
a constant amount of anti-biotin monoclonal antibody (mAb). This
mixture was equilibrated for 20 minutes, and then added to
biotinylated-BSA in a microplate well. After allowing time to bind,
the plates were washed and the remaining fluorescence was
quantitated. The analysis was performed in triplicate (circles).
Control experiments were performed, as described above, but without
adding the primary anti-biotin antibody (solid squares).
[0023] FIG. 4: Shows a comparison of the fluorescence intensity
(Example 6) for labeling reagent prepared in homogeneous solution
(Example 4) and labeling reagent prepared on a column (Example
5).
[0024] FIG. 5: Shows detection of multiple targets on T cells using
a labeling reagent attached to a R-phycoerythrin (R-PE) (FIG. 5A)
to detect CD3-positive T cells, a labeling reagent attached to
Alexa Fluor 647 dye (FIG. 5B) to detect CD4-positive T cells and a
labeling reagent attached to Alexa Fluor 488 dye (FIG. 5B) to
detect CD8-positive T cells (Example 18). The CD-3 detected T cells
are shown in the upper left (UL) and upper right (UR) quadrants.
The relative percentages of total lymphocytes that are CD3-positive
cells are 83.3% (UL+UR). The relative percentage of CD8-positive
Alexa Fluor 488 dye-stained lymphocytes and CD3-positive R-PE
dye-stained lymphocytes is 35.1% (UR quadrant). The lower left
quadrant (LL, 20.4%) shows CD3-negative lymphocytes (i.e. non-T
cells) comprised of NK cells, B cells and some monocytes. In the
lower right (LR, 2.7%) region are non-T cells, which are
nonspecifically stained. FIG. 5B further shows CD3-positive T-cells
subdivided into Alexa Fluor 647 dye CD4-positive and Alexa Fluor
488 dye CD8-positive. CD4-positive cells represent 50.9% of total
lymphocytes (UL quadrant) and CD8-positive cells represent 24.5% of
the total lymphocytes (LR quadrant). The 23.1% of cells in the LL
quadrant are non-T cells, while the 1.5% of cells in UR quadrant
are likely nonspecifically stained lymphocytes.
[0025] FIG. 6: Shows high-performance size-exclusion
chromatographic analysis of Alexa Fluor 488 dye-labeled goat Fab
anti-(mouse Fc) labeling reagent binding to a mouse IgG.sub.1
target-binding antibody. The labeling reagent, alone, appears as a
peak at 38 minutes; the target-binding antibody, alone, appears as
a peak at 33 minutes. When labeling reagent and target-binding
antibody are mixed together at a molar ratio of .about.5:1
(labeling reagent:target-binding antibody), the resulting
immunolabeling complex appears as a peak at 29 minutes (Example
10).
[0026] FIG. 7: Shows the production of labeling reagent wherein the
label is attached to the labeling reagent when immobilized on a
column.
[0027] FIG. 8: Shows a schematic representation of the
ligand-detection reagent comprising a ligand analog, ligand-binding
antibody and labeling reagent.
[0028] FIG. 9: Shows the amount of fluorescence quenching by
BODIPY-FL Digoxigenin ligand analog when bound to the
ligand-binding antibody/Fab fragment of goat anti-mouse kappa chain
conjugated to QSY-9 complex. As the amount of anti Digoxigenin/Fab
fragment increases, the fluorescence of the BODIPY-FL Digoxigenin
decreases. When target ligand (Digoxigenin) is added the
fluorescence quenching is partially relieved.
[0029] FIG. 10: Shows the use of a fluorogenic ligand analog
(Compound 4) that upon interaction with the ligand-binding antibody
exhibits fluorescence enhancement.
[0030] FIG. 11: Shows the quenching of the ligand analog (Compound
15) when bound by the ligand-binding antibody and the subsequent
relief of quenching when target ligand, phosphotyrosine peptides,
are added and the ligand analog displaced from the binding groove
of the phosphotyrosine ligand-binding antibody.
[0031] FIG. 12: Shows the off rate of the ligand analog (Compound
15) when target ligand is added to the ligand-detection
reagent.
[0032] FIG. 13: Shows the selectivity of the ligand-binding
antibody for the target ligand and ligand analog (Compound 15)
[0033] FIG. 14: Shows that the use of ATP in a kinase assay does
not compete for binding of the phosphotyrosine ligand-binding
antibody.
[0034] FIG. 15: Shows the detection of Abl kinase activity using
Compound 15 as the ligand analog, phosphotyrosine ligand-binding
antibody as the ligand-binding antibody and MPIJ-5 as the kinase
substrate and subsequent target ligand.
[0035] FIG. 16: Shows the ability of the ligand-detection reagent
to detect the presence of an inhibitor of kinase activity
(staurosporine).
[0036] FIG. 17: Shows the screening of multiple phosphotyrosine
ligand-binding antibodies to optimize the affinity of the ligand
analog (Compound 16) for the ligand-binding antibody.
[0037] FIG. 18: Shows a method for selecting ligands for use in
displacement assays. FIG. 14A shows the measure of fluorescence
polarization as a function of antibody concentration, FIG. 14B
shows the measure of fluorescence intensity as a function of
antibody concentration, FIG. 14C shows a measure of fluorescence
enhancement in the presence of target ligand (phosphopeptide) and
FIG. 14D shows the confirmation of fluorescence enhancement by
depolarization of the ligand analog.
[0038] FIG. 19: Shows the ability of the labeling reagent to quench
the reporter molecule when present as a ligand detection reagent
ternary complex.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0039] Before describing the present invention in detail, it is to
be understood that this invention is not limited to specific
compositions or process steps, as such may vary. It should be noted
that, as used in this specification and the appended claims, the
singular form "a", "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a protein labeling complex" includes a plurality of
complexes and reference to "a target-binding protein" includes a
plurality of proteins and the like.
[0040] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention is related. The
following terms are defined for purposes of the invention as
described herein.
[0041] The term "affinity" as used herein refers to the strength of
the binding interaction of two molecules, such as an antibody and
an antigen or a positively charged moiety and a negatively charged
moiety. For bivalent molecules such as antibodies, affinity is
typically defined as the binding strength of one binding domain for
the antigen, e.g. one Fab fragment for the antigen. The binding
strength of both binding domains together for the antigen is
referred to as "avidity". As used herein "High affinity" refers to
a ligand that binds to an antibody having an affinity constant
(K.sub.a) greater than 10.sup.4 M.sup.-1, typically
10.sup.5-10.sup.11 M.sup.-1; as determined by inhibition ELISA or
an equivalent affinity determined by comparable techniques such as,
for example, Scatchard plots or using K.sub.d/dissociation
constant, which is the reciprocal of the K.sub.a, etc.
[0042] The term "antibody" as used herein refers to a protein of
the immunoglobulin (Ig) superfamily that binds noncovalently to
certain substances (e.g. antigens and immunogens) to form an
antibody-antigen complex. Antibodies can be endogenous, or
polyclonal wherein an animal is immunized to elicit a polyclonal
antibody response or by recombinant methods resulting in monoclonal
antibodies produced from hybridoma cells or other cell lines. It is
understood that the term "antibody" as used herein includes within
its scope any of the various classes or sub-classes of
immunoglobulin derived from any of the animals conventionally
used.
[0043] The term "antibody fragments" as used herein refers to
fragments of antibodies that retain the principal selective binding
characteristics of the whole antibody. Particular fragments are
well-known in the art, for example, Fab, Fab', and F(ab').sub.2,
which are obtained by digestion with various proteases, pepsin or
papain, and which lack the Fc fragment of an intact antibody or the
so-called "half-molecule" fragments obtained by reductive cleavage
of the disulfide bonds connecting the heavy chain components in the
intact antibody. Such fragments also include isolated fragments
consisting of the light-chain-variable region, "Fv" fragments
consisting of the variable regions of the heavy and light chains,
and recombinant single chain polypeptide molecules in which light
and heavy variable regions are connected by a peptide linker. Other
examples of binding fragments include (i) the Fd fragment,
consisting of the VH and CH1 domains; (ii) the dAb fragment (Ward,
et al., Nature 341, 544 (1989)), which consists of a VH domain;
(iii) isolated CDR regions; and (iv) single-chain Fv molecules
(scFv) described above. In addition, arbitrary fragments can be
made using recombinant technology that retains antigen-recognition
characteristics.
[0044] The term "antigen" as used herein refers to a molecule that
induces, or is capable of inducing, the formation of an antibody or
to which an antibody binds selectively, including but not limited
to a biological material. Antigen also refers to "immunogen". The
target-binding antibodies selectively bind an antigen, as such the
term can be used herein interchangeably with the term "target".
[0045] The term "anti-region antibody" as used herein refers to an
antibody that was produced by immunizing an animal with a select
region that is a fragment of a foreign antibody wherein only the
fragment is used as the immunogen. Anti-region antibodies include
monoclonal and polyclonal antibodies. The term "anti-region
fragment" as used herein refers to a monovalent fragment that was
generated from an anti-region antibody of the present invention by
enzymatic cleavage.
[0046] The term "biotin" as used herein refers to any biotin
derivative, including without limitation, substituted and
unsubstituted biotin, and analogs and derivatives thereof, as well
as substituted and unsubstituted derivatives of caproylamidobiotin,
biocytin, desthiobiotin, desthiobiocytin, iminobiotin, and biotin
sulfone.
[0047] The term "biotin-binding protein" as used herein refers to
any protein that binds selectively and with high affinity to
biotin, including without limitation, substituted or unsubstituted
avidin, and analogs and derivatives thereof, as well as substituted
and unsubstituted derivatives of streptavidin, ferritin avidin,
nitroavidin, nitrostreptavidin, and Neutravidin.TM. avidin (a
de-glycosylated modified avidin having an isoelectric point near
neutral).
[0048] The term "buffer" as used herein refers to a system that
acts to minimize the change in acidity or basicity of the solution
against addition or depletion of chemical substances.
[0049] The term "capture reagent" refers to a non-specific
immunoglobulin that is used to remove excess labeling reagent after
the formation of the immuno-labeled complex. The capture reagent is
optionally attached a matrix to facilitate removal of the excess
labeling regent. A matrix typically includes a microsphere, an
agarose bead or any solid surface that the excess labeling reagent
can be passed by.
[0050] The term "chromophore" as used herein refers to a label that
emits light in the visible spectra that can be observed without the
aid of instrumentation.
[0051] The term "complex" as used herein refers to the association
of two or more molecules, usually by non-covalent bonding, e.g.,
the association between an antibody and an antigen or the labeling
reagent and the target-binding antibody.
[0052] The term "detectable response" as used herein refers to an
occurrence of, or a change in, a signal that is directly or
indirectly detectable either by observation or by instrumentation.
Typically, the detectable response is an occurrence of a signal
wherein the fluorophore is inherently fluorescent and does not
produce a change in signal upon binding to a metal ion or
biological compound. Alternatively, the detectable response is an
optical response resulting in a change in the wavelength
distribution patterns or intensity of absorbance or fluorescence or
a change in light scatter, fluorescence lifetime, fluorescence
polarization, or a combination of the above parameters. Other
detectable responses include, for example, chemiluminescence,
phosphorescence, radiation from radioisotopes, magnetic attraction,
and electron density.
[0053] The term "detectably distinct" as used herein refers to a
signal that is distinguishable or separable by a physical property
either by observation or by instrumentation. For example, a
fluorophore is readily distinguishable either by spectral
characteristics or by fluorescence intensity, lifetime,
polarization or photo-bleaching rate from another fluorophore in
the sample, as well as from additional materials that are
optionally present.
[0054] The term "directly detectable" as used herein refers to the
presence of a material or the signal generated from the material is
immediately detectable by observation, instrumentation, or film
without requiring chemical modifications or additional
substances.
[0055] The term "energy transfer" as used herein refers to the
process by which the excited state energy of an excited group, e.g.
fluorescent reporter dye, is conveyed through space or through
bonds to another group, e.g. a quencher moiety or fluorescer, which
may attenuate (quench) or otherwise dissipate or transfer the
energy to another reporter group or emit the energy at a longer
wavelength. Energy transfer typically occurs through fluorescence
resonance energy transfer (FRET).
[0056] The term "energy transfer pair" as used herein refers to any
two moieties that participate in energy transfer. Typically, one of
the moieties acts as a fluorescent reporter, i.e. donor, and the
other acts as an acceptor, which may be a quenching compound or a
compound that absorbs and re-emits energy in the form of a
fluorescent signal ("Fluorescence resonance energy transfer."
Selvin P. (1995) Methods Enzymol 246:300-334; dos Remedios C. G.
(1995) J. Struct. Biol. 115:175-185; "Resonance energy transfer:
methods and applications." Wu P. and Brand L. (1994) Anal Biochem
218:1-13). Fluorescence resonance energy transfer (FRET) is a
distance-dependent interaction between two moieties in which
excitation energy, i.e. light, is transferred from a donor to an
acceptor without emission of a photon. The acceptor may be
fluorescent and emit the transferred energy at a longer wavelength,
or it may be non-fluorescent and serve to diminish the detectable
fluorescence of the reporter molecule (quenching). FRET may be
either an intermolecular or intramolecular event, and is dependent
on the inverse sixth power of the separation of the donor and
acceptor, making it useful over distances comparable with the
dimensions of biological macromolecules. When an energy transfer
pair is part of the present ligand-detection reagent the energy
transfer is an intramolecular event. Thus, the spectral properties
of the energy transfer pair as a whole change in some measurable
way if the distance between the moieties is altered by some
critical amount. Self-quenching probes incorporating fluorescent
donor-non-fluorescent acceptor combinations have been developed
primarily for detection of proteolysis (Matayoshi, (1990) Science
247:954-958) and nucleic acid hybridization ("Detection of Energy
Transfer and Fluorescence Quenching" Morrison, L., in Nonisotopic
DNA Probe Techniques, L. Kricka, Ed., Academic Press, San Diego,
(1992) pp. 311-352; Tyagi S. (1998) Nat. Biotechnol. 16:49-53;
Tyagi S. (1996) Nat. Biotechnol 14:303-308). In most applications,
the donor and acceptor dyes are different, in which case FRET can
be detected by the appearance of sensitized fluorescence of the
acceptor or by quenching of donor fluorescence.
[0057] The term "examination zone" as used herein refers to an
optical zone of a flow cytometer, or a similar instrument, wherein
cells are passed through essentially one at a time in a thin stream
whereby the bound immuno-labeled complex is illuminated and the
intensity and emission spectra of the fluorophore is detected and
recorded. This includes instruments wherein the examination zone
moves and the sample is held in place.
[0058] The term "fluorophore" as used herein refers to a
composition that is inherently fluorescent or demonstrates a change
in fluorescence upon binding to a biological compound or metal ion,
i.e., fluorogenic. Fluorophores may contain substitutents that
alter the solubility, spectral properties or physical properties of
the fluorophore. Numerous fluorophores are known to those skilled
in the art and include, but are not limited to coumarin, cyanine,
benzofuran, a quinoline, a quinazolinone, an indole, a benzazole, a
borapolyazaindacene and xanthenes including fluoroscein, rhodamine
and rhodol as well as other fluorophores described in RICHARD P.
HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND
RESEARCH CHEMICALS (9.sup.th edition, CD-ROM, September 2002).
[0059] The term "immuno-labeled complex" refers to the complex of
target-binding antibody that is non-covalently attached to a
labeling reagent.
[0060] The term "immuno-labeled complex subset" as used herein
refers to a discrete set of immuno-labeled complexes that are
homogenous and can be distinguished from another subset of
immuno-labeled complex by the physical properties of the label, or
the ratio of the label to labeling reagent, or the ratio of
labeling reagent to target-binding antibody, or the target-binding
antibody. Typically an immuno-labeled complex subset is present in
a buffer to provide a "target detection solution".
[0061] The term "kit" as used herein refers to a packaged set of
related components, typically one or more compounds or
compositions.
[0062] The term "label" as used herein refers to a chemical moiety
or protein that retains it's native properties (e.g. spectral
properties, conformation and activity) when attached to a labeling
reagent and used in the present methods. The label can be directly
detectable (fluorophore), indirectly detectable (hapten or enzyme)
or act as a quencher for the reporter molecule of the ligand
analog. Such labels include, but are not limited to, radiolabels
that can be measured with radiation-counting devices; pigments,
dyes or other chromogens that can be visually observed or measured
with a spectrophotometer; quenching moiety that functions to absorb
and not re-emit the energy from the dye moiety of the ligand analog
that is within close proximity; spin labels that can be measured
with a spin label analyzer; and fluorescent labels (fluorophores),
where the output signal is generated by the excitation of a
suitable molecular adduct and that can be visualized by excitation
with light that is absorbed by the dye or can be measured with
standard fluorometers or imaging systems, for example. The label
can be a chemiluminescent substance, where the output signal is
generated by chemical modification of the signal compound; a
metal-containing substance; or an enzyme, where there occurs an
enzyme-dependent secondary generation of signal, such as the
formation of a colored product from a colorless substrate. The term
label can also refer to a "tag" or hapten that can bind selectively
to a conjugated molecule such that the conjugated molecule, when
added subsequently along with a substrate, is used to generate a
detectable signal. For example, one can use biotin as a tag and
then use an avidin or streptavidin conjugate of horseradish
peroxidate (HRP) to bind to the tag, and then use a colorimetric
substrate (e.g., tetramethylbenzidine (TMB)) or a fluorogenic
substrate such as Amplex Red reagent (Molecular Probes, Inc.) to
detect the presence of HRP. Numerous labels are know by those of
skill in the art and include, but are not limited to, particles,
fluorophores, haptens, enzymes and their calorimetric, fluorogenic
and chemiluminescent substrates and other labels that are described
in RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT
PROBES AND RESEARCH PRODUCTS (9.sup.th edition, CD-ROM, September
2002), supra.
[0063] The term "labeling reagent" as used herein refers to a
monovalent antibody fragment or a non-antibody monomeric protein
provided that the labeling reagent has affinity for a selected
region of the target-binding antibody and is covalently attached to
a label.
[0064] The term "labeling reagent subset" as used herein refers to
a discrete set of labeling reagents that are homogenous and can be
distinguished from another subset of labeling reagent either by the
physical properties of the label or the ratio of the label to
labeling reagent.
[0065] The term "labeling solution" as used herein refers to a
solution that is used to form an immuno-labeled complex wherein the
solution comprises labeling reagents and a buffer.
[0066] The term "ligand" as used herein refers to a moiety that
contains an antibody binding epitope. The ligand may contain amino
acids to form peptides or proteins or the ligand may be essentially
free if amino acids. The term "ligand" and "target" as used herein
are used interchangeably.
[0067] The term "ligand analog" as used herein refers to ligand
that has been modified to alter the affinity of the ligand analog
for the ligand-binding antibody compared to an appropriate ligand.
The affinity modification includes, but is not limited to, the
addition of a dye moiety, addition of alkyl groups to the binding
epitope, change of amino acid sequence of the epitope or spacing of
the dye moiety from the epitope. Thus, the ligand analog has the
same spatial and polar organization as the ligand to define one or
more determinant or epitopic sites capable of competing with the
ligand for the binding sites of a receptor, and differs from the
ligand in the absence or presence of an atom or functional group at
the site of binding to another molecule or in having a linking
group which has been introduced in place of one or more atoms
originally present in the ligand.
[0068] The term "ligand-binding antibody" as used herein refers to
an antibody that has affinity for a discrete epitope, antigen or
ligand that can be used with the methods of the present invention.
Typically the discrete epitope is the target but the epitope can be
a marker for the target such as CD3 on T cells. Ligand-binding
antibody can be used interchangeably with the term "primary
antibody" when describing methods that use an antibody that binds
directly to the antigen as opposed to a "secondary antibody" that
binds to a region of the primary antibody. As used herein the term
"ligand-binding antibody" is used interchangeably with
"target-binding antibody".
[0069] The term "ligand-detection reagent" as used herein refers to
an immuno-complex that is used to determine the presence of a
target ligand in a sample. The complex comprises a ligand-binding
antibody, a ligand analog and a labeling reagent wherein the
covalently bonded label is a fluorophore or a quenching moiety. In
this instance, the ligand analog is displaced by the target ligand
resulting in a change in signal intensity or a shift in color
change of the detectable signal whereby the presence of a target
ligand is determined.
[0070] The term "matrix" as used herein refers to a solid or
semi-solid surface that a biological molecule can be attached to,
such as a sample of the present invention or a capture reagent.
Examples include, but are not limited to, agarose, polyacrylamide
gel, polymers, microspheres, glass surface, plastic surface,
membrane, margnetic surface, and an array.
[0071] The term "monovalent antibody fragment" as used herein
refers to an antibody fragment that has only one antigen-binding
site. Examples of monovalent antibody fragments include, but are
not limited to, Fab fragments (no hinge region), Fab' fragments
(monovalent fragments that contain a heavy chain hinge region), and
single-chain fragment variable (ScFv) proteins.
[0072] The term "non-antibody monomeric protein" as used herein
refers to a protein that binds selectively and non-covalently to a
member of the Ig superfamily of proteins, including but not limited
to proteins A, G, and L, hybrids thereof (A/G), recombinant
versions and cloned versions thereof, fusions of these proteins
with detectable protein labels, and lectins but the protein itself
is not an antibody or an antibody fragment.
[0073] The terms "protein" and "polypeptide" are used herein in a
generic sense to include polymers of amino acid residues of any
length. The term "peptide" is used herein to refer to polypeptides
having less than 100 amino acid residues, typically less than 10
amino acid residues. The terms apply to amino acid polymers in
which one or more amino acid residues are an artificial chemical
analogue of a corresponding naturally occurring amino acid, as well
as to naturally occurring amino acid polymers.
[0074] The term "purified" as used herein refers to a preparation
of a target-binding antibody that is essentially free from
contaminating proteins that normally would be present in
association with the antibody, e.g., in a cellular mixture or
milieu in which the protein or complex is found endogenously such
as serum proteins or hybridoma supernatant.
[0075] The term "quenching moiety" or "quencher" as used herein
refers to a compound that is capable of absorbing energy from an
energy donor that is not re-emitted (non-fluorescent) or re-emitted
at a detectably different wavelength from the energy emitted by the
donor molecule. In this respect, quenchers may be essentially
non-fluorescent or fluorescent. Numerous quenching moieties are
well known in the art including xanthene and cyanine compounds and
other compounds disclosed in RICHARD P. HAUGLAND, MOLECULAR PROBES
HANDBOOK OF FLUORESCENT PROBES AND RESEARCH PRODUCTS (9.sup.th
edition, CD-ROM, September 2002), supra.
[0076] The term "reporter molecule" as used herein refers to any
luminescent molecule that is capable of functioning as a member of
an energy transfer pair wherein the reporter molecule retains it's
native properties (e.g. spectral properties, conformation and
activity) when attached to a ligand analog and used in the present
methods. Typically, luminescent molecules, as used herein include
dyes, fluorescent proteins, phosphorescent dyes, chromophores and
chemiluminescent compounds that are capable of producing a
detectable signal upon appropriate activation. The term "dye"
refers to a compound that emits light to produce an observable
detectable signal. "Dye" includes fluorescent and nonfluorescent
compounds that include without limitations pigments, fluorophores,
chemiluminescent compounds, luminescent compounds and chromophores.
The term "chromophore" as used herein refers to a label that emits
light in the visible spectra that can be observed without the aid
of instrumentation. The term "fluorophore" as used herein refers to
a composition that is inherently fluorescent or demonstrates a
change in fluorescence upon binding to a biological compound, i.e.
can be fluorogenic or the intensity can be diminished by quenching.
Fluorophores may contain substitutents that alter the solubility,
spectral properties or physical properties of the fluorophore.
Numerous fluorophores are known to those skilled in the art and
include, but are not limited to coumarin, cyanine, benzofuran, a
quinoline, a quinazolinone, an indole, a benzazole, a
borapolyazaindacene and xanthenes including fluoroscein, rhodamine
and rhodol as well as other fluorophores described in RICHARD P.
HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND
RESEARCH CHEMICALS (9.sup.th edition, CD-ROM, September 2002).
[0077] The term "sample" as used herein refers to any material that
may contain a target, as defined below. Typically, the sample
comprises a population of cells, cellular extract, subcellular
components, tissue culture, a bodily fluid, and tissue. The sample
may be in an aqueous solution, a viable cell culture or immobilized
on a solid or semi solid surface such as a gel, a membrane, a glass
surface, a microparticle or on a microarray.
[0078] The term "target" as used herein refers to any entity that a
target-binding antibody has affinity for such as an epitope or
antigen. This target includes not only the discrete epitope that
the target-binding antibody has affinity for but also includes any
subsequently bound molecules or structures. In this way an epitope
serves as a marker for the intended target. For example, a cell is
a target wherein the target-binding antibody binds a cell surface
protein such as CD3 on a T cell wherein the target marker is CD3
and the target is the T cell.
[0079] The term "target-binding antibody" as used herein refers to
an antibody that has affinity for a discrete epitope or antigen
that can be used with the methods of the present invention.
Typically the discrete epitope is the target but the epitope can be
a marker for the target such as CD3 on T cells. The term can be
used interchangeably with the term "primary antibody" when
describing methods that use an antibody that binds directly to the
antigen as opposed to a "secondary antibody" that binds to a region
of the primary antibody.
[0080] The term "ternary complex" as used herein refers to a
composition that simultaneously comprises a ligand-binding
antibody, a ligand analog of the present invention and labeling
reagent wherein the ligand analog is non-covalently bound in the
binding groove of the ligand-binding antibody and the labeling
reagent is non-covalently bonded to a region, e.g. Fc, of the
ligand-binding antibody.
II. Compositions and Methods of Use
[0081] In accordance with the present invention, labeling reagents,
methods for labeling target-binding antibodies and methods for
using the labeled antibodies to detect a target in a sample
including a target ligand in a competitive immunoassay are
provided. The labeling reagents comprise monovalent antibody
fragments or non-antibody monomeric proteins that are covalently
attached to a label of the present invention. The label covalently
attached to a labeling reagent is directly detectable such as a
fluorophore, a quenching moiety or functions as an indirect label
that requires an additional component such as a calorimetric enzyme
substrate or an enzyme conjugate. The labeling reagents have
affinity for a specific region of the target-binding antibody. The
target-binding antibodies are defined as any antibody known to one
skilled in the art that has an affinity for a target in a sample.
The target-binding antibodies are labeled with the labeling reagent
in a labeling method to form immuno-labeled complexes and then
added to a sample to detect a target.
[0082] The labeling reagent and the methods of the present
invention provide for detection of one or multiple targets in a
sample. Multiple targets are detected when either pooled subsets of
immuno-labeled complexes or a panel of subsets that are
sequentially added to a sample. The subset of immuno-labeled
complexes begins with labeling reagent subsets wherein a labeling
reagent subset is distinguished by the ratio of label to labeling
reagent or by the physical characteristics of the label. The
discrete labeling reagents subsets are added to the target-binding
antibodies wherein the affinity of the antibody and ratio of
labeling reagent to target-binding antibody determines the subsets
of immuno-labeled complexes. This results in an infinite number of
immuno-labeled complex subsets that are distinguished by i) the
target-binding antibody, or ii) a ratio of label to labeling
reagent, or iii) a ratio of labeling reagent to the target-binding
antibody or iv) by a physical property of the label. These subsets
can be used individually in a method of the present invention to
detect a single or multiple targets in a sample or pooled and used
to simultaneously detect multiple targets in a sample. These pooled
subsets allow for not only detection but also identification and
quantitation of the targets.
[0083] In one aspect of the invention, reagent and methods are
provided for the detection of a target ligand employing a
competitive immunoassay wherein a ligand analog is displaced by a
target ligand resulting in a change in detectable signal that
indicates the presence of target ligand. Thus, the present
invention provides ligand-detection reagents, ligand analogs and
methods of employing the reagents for the detection of a target
ligand. The ligand-detection reagents comprise a ligand-binding
antibody, a ligand analog that is covalently attached to a reporter
molecule and a labeling reagent that is covalently attached to a
label. The ligand-detection reagent is a complex wherein the ligand
analog is non-covalently bound by the binding groove of the
antibody and the labeling reagent is non-covalently bound to a
region of the antibody. The methods employ the ligand-detection
reagents wherein in one aspect of the invention energy transfer is
utilized in a competitive immunoassay format to determine the
presence of a ligand in a sample.
A. Ligand Analog
[0084] The ligand analog comprises at least one epitope site for a
desired ligand-binding antibody, a reporter molecule and a linker.
Thus, the ligand analog may be monovalent or polyvalent. Typically
the ligand analog is monovalent or divalent. In one aspect of the
invention the monovalent ligand analog is quenched when bound by
the ligand-binding antibody. Thus, monovalent ligand analogs are
preferred for applications wherein it is desired that the ligand
analog be quenched when bound by the ligand-binding antibody. In
another aspect, the divalent ligand analog is fluorogenic wherein
the ligand analog is essentially non-fluorescent when unbound from
the ligand-binding antibody but when bound by the antibody in such
a way that the each binding groove is bound to the bivalent ligand
analog and the reporter molecule is held in between the two Fab
fragments of the antibody the fluorogenic ligand analog becomes
fluorescent, See Example 32 and Compounds 4 and 5. Thus, in this
aspect a divalent ligand analog is preferred for applications
wherein a fluorogenic ligand analog is employed.
[0085] The ligand analog typically has an altered affinity for the
ligand-binding antibody compared to the target ligand. The altered
affinity may be greater or less than the target ligand, typically
the affinity is less or equal to the affinity of the target ligand.
The affinity of the ligand analog is determined empirically along
with the selection of the ligand-binding antibody, and optionally
the labeling protein to optimize the displacement of the ligand
analog by the target ligand in each assay system. The altered
affinity of the ligand analog can be accomplished by a number of
modifications to the target ligand to make a ligand analog or
alternatively a synthetic chemical strategy can be employed to
design and synthesize a ligand analog with the appropriate
affinity, fluorescence response, and ability to be quenched.
Modification to a ligand to form a ligand analog can include a
change of a single, or multiple, amino acids, either in the epitope
or the surrounding sequence, a change in the post-translational
modification of a protein or peptide such as the addition or
removal of a sugar group or phosphate, the addition of a linker or
simply by the addition of a reporter molecule. Alternatively, the
epitope can be synthesized with an appropriate linker and reporter
molecule, such as was done for the phosphotyramide,
phosphotyrosinamide, phosphoethanoamine and phosphoserine ligand
analogs, See Examples 22-29.
[0086] Synthesis of the epitope, linker and reporter group provide
for the most flexibility for designing a ligand analog with the
appropriate affinity for the ligand-binding antibody and spectral
properties of the reporter group. However, this method is typically
not preferred wherein a ligand-binding antibody was raised against
a ligand that requires a conformational epitope. In this instance,
the sequence, spacing, and folding or conformation of the antigen
is necessary for adequate binding by the ligand-binding antibody.
Thus, for these ligand analogs, the reporter molecule is typically
conjugated to the target ligand to form the analog resulting in
linker that is typically less than 10 atoms in length.
[0087] The ligand analog for the most part will be haptenic, rather
than antigenic, and generally be less than about 10,000 molecular
weight, more usually less than about 6,000 molecular weight, and
frequently in the range of about 125 to 1,000 molecular weight,
excluding the linking group employed for linking to the reporter
molecule.
[0088] Regardless of the method employed to derive a ligand analog,
the reporter molecule is typically conjugated to the ligand analog.
Thus, the reporter molecule and the ligand analog each need to
contain an appropriate reactive or functional group that result in
a covalent bond. The reactive group and functional group are
typically an electrophile and a nucleophile that can generate a
covalent linkage. Alternatively, the reactive group is a
photoactivatable group, and becomes chemically reactive only after
illumination with light of an appropriate wavelength. Typically,
the conjugation reaction between the reactive group of the reporter
molecule and the reactive group of the ligand analog results in one
or more atoms of the reactive group to be incorporated into a new
linkage attaching the reporter molecule to the ligand analog.
Selected examples of functional groups and linkages are shown in
Table 1, where the reaction of an electrophilic group and a
nucleophilic group yields a covalent linkage. TABLE-US-00001 TABLE
1 Examples of some routes to useful covalent linkages Electrophilic
Group Nucleophilic Group Resulting Covalent Linkage activated
esters* amines/anilines carboxamides acrylamides thiols thioethers
acyl azides** amines/anilines carboxamides acyl halides
amines/anilines carboxamides acyl halides alcohols/phenols esters
acyl nitriles alcohols/phenols esters acyl nitriles amines/anilines
carboxamides aldehydes amines/anilines imines aldehydes or ketones
hydrazines hydrazones aldehydes or ketones hydroxylamines oximes
alkyl halides amines/anilines alkyl amines alkyl halides carboxylic
acids esters alkyl halides thiols thioethers alkyl halides
alcohols/phenols ethers alkyl sulfonates thiols thioethers alkyl
sulfonates carboxylic acids esters alkyl sulfonates
alcohols/phenols ethers anhydrides alcohols/phenols esters
anhydrides amines/anilines carboxamides aryl halides thiols
thiophenols aryl halides amines aryl amines aziridines thiols
thioethers boronates glycols boronate esters carbodiimides
carboxylic acids N-acylureas or anhydrides diazoalkanes carboxylic
acids esters epoxides thiols thioethers haloacetamides thiols
thioethers haloplatinate amino platinum complex haloplatinate
heterocycle platinum complex haloplatinate thiol platinum complex
halotriazines amines/anilines aminotriazines halotriazines
alcohols/phenols triazinyl ethers halotriazines thiols triazinyl
thioethers imido esters amines/anilines amidines isocyanates
amines/anilines ureas isocyanates alcohols/phenols urethanes
isothiocyanates amines/anilines thioureas maleimides thiols
thioethers phosphoramidites alcohols phosphite esters silyl halides
alcohols silyl ethers sulfonate esters amines/anilines alkyl amines
sulfonate esters thiols thioethers sulfonate esters carboxylic
acids esters sulfonate esters alcohols ethers sulfonyl halides
amines/anilines sulfonamides sulfonyl halides phenols/alcohols
sulfonate esters *Activated esters, as understood in the art,
generally have the formula --CO.OMEGA., where .OMEGA. is a good
leaving group (e.g., succinimidyloxy (--OC.sub.4H.sub.4O.sub.2)
sulfosuccinimidyloxy (--OC.sub.4H.sub.3O.sub.2--SO.sub.3H),
-1-oxybenzotriazolyl (--OC.sub.6H.sub.4N.sub.3); or an aryloxy
group or aryloxy substituted one or more times by electron
withdrawing substituents such as nitro, fluoro, chloro, cyano, or
trifluoromethyl, or combinations thereof, #used to form activated
aryl esters; or a carboxylic acid activated by a carbodiimide to
form an anhydride or mixed anhydride --OCOR.sup.a or
--OCNR.sup.aNHR.sup.b, where R.sup.a and R.sup.b, which may be the
same or different, are C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
perfluoroalkyl, or C.sub.1-C.sub.6 alkoxy; or cyclohexyl,
3-dimethylaminopropyl, or N-morpholinoethyl). **Acyl azides can
also rearrange to isocyanates
[0089] Choice of the reactive group used to attach the reporter
molecule to the ligand analog typically depends on the reactive or
functional group on the ligand analog and the type or length of
covalent linkage desired. The types of functional groups typically
present on biomolecules include, but are not limited to, amines,
amides, thiols, alcohols, phenols, aldehydes, ketones, phosphates,
imidazoles, hydrazines, hydroxylamines, disubstituted amines,
halides, epoxides, silyl halides, carboxylate esters, sulfonate
esters, purines, pyrimidines, carboxylic acids, olefinic bonds, or
a combination of these groups. A single type of reactive site may
be available on the substance (typical for polysaccharides or
silica), or a variety of sites may occur (e.g., amines, thiols,
alcohols, phenols), as is typical for proteins.
[0090] A linker may be synthesized on the ligand analog or on the
reporter molecule wherein after conjugation the linker is
incorporated into the ligand analog. The linker typically
incorporates 1-30 nonhydrogen atoms selected from the group
consisting of C, N, O, S and P. The linker is optionally a
substituted alkyl, amine or a substituted cycloalkyl. Alternately,
the reporter group may be directly attached (where linker is a
single bond) to the ligand analog or the alkyl may contain a
benzene ring. When the linker is not a single covalent bond, the
linker may be any combination of stable chemical bonds, optionally
including, single, double, triple or aromatic carbon-carbon bonds,
as well as carbon-nitrogen bonds, nitrogen-nitrogen bonds,
carbon-oxygen bonds, sulfur-sulfur bonds, carbon-sulfur bonds,
phosphorus-oxygen bonds, phosphorus-nitrogen bonds, and
nitrogen-platinum bonds. Typically the linker incorporates less
than 20 nonhydrogen atoms and are composed of any combination of
ether, thioether, thiourea, amine, ester, carboxamide, sulfonamide,
hydrazide bonds and aromatic or heteroaromatic bonds. Most
preferred are linkers that contain less than 10 non-hydrogen atoms.
Typically the linker is a combination of single carbon-carbon bonds
and carboxamide, sulfonamide or thioether bonds. The bonds of the
linker typically result in the following moieties that can be found
in the linker: ether, thioether, carboxamide, thiourea,
sulfonamide, urea, urethane, hydrazine, alkyl, aryl, heteroaryl,
alkoky, cycloalkyl and amine moieties.
[0091] Any combination of linkers may be used to attach the
reporter molecule to the ligand analog. For monovalent ligand
analogs the analog typically contains one linker and for divalent
ligand analogs the analogs typically incorporate two linkers, which
may be the same or different. The linker may also be substituted to
alter the physical properties of the ligand analog, such as binding
affinity for the ligand-binding antibody and spectral properties of
the fluorophore.
[0092] We have unexpectedly discovered that the site of attachment
of the linker on the reporter group alters the ability of the
reporter molecule to be quenched when bound to the ligand-binding
antibody and the binding affinity of the analog for the
ligand-binding antibody. This is particularly true when the
reporter molecule is a xanthene dye. By way of example Compound 8
and 9 are isomers but demonstrate different binding affinity for
the same antibody and quenching by the same antibody, See, Example
43 and FIG. 19. In this instance, the 6-isomer (linker is attached
to the 6 position of the pendent phenyl ring; Compound 9)
demonstrates increased binding affinity and increased quenching
compared to the 5-isomer of the same ligand analog, Compound 8.
Therefore, the position of attachment of the linker on the reporter
molecule is important for defining binding affinity of the ligand
analog for the ligand-binding antibody and for the ability of the
reporter molecule to be masked or quenched when bound by the
ligand-binding antibody. This quenching property is particularly
relevant when a monovalent ligand analog is employed.
[0093] The length of the linker is another important aspect for
optimizing the amount of quenching conferred on the reporter
molecule. We have found that a shorter linker results in an
increased quenching of the reporter molecule by the ligand-binding
antibody. Without wishing to be bound by a theory, it appears that
quenching is increased when the reporter molecule is "pulled" into
the binding groove of the antibody, which is facilitated by a short
linker; a short linker preferably containing 10 or less
non-hydrogen atoms. In addition, the linker can be substituted by
substitutents that alter the physical properties of the ligand
analog, such as binding affinity and spectral properties of the
reporter molecule. We have unexpectedly found that substituting the
linker to form a phosphotyrosinamide instead of a phosphotyramide
ligand analog alters the binding affinity and the ability of the
reporter molecule to be quenched when bound by the ligand-binding
antibody. See, Compounds 34-38 and 41-42 and Example 44.
[0094] Therefore, the linker of the ligand analog is important for
attaching the reporter molecule to the ligand analog, for altering
the binding affinity of the analog and for altering the spectral
properties of the reporter group. The lengths of the linker, site
of attachment on the reporter group and linker substituents all are
parameters that can be altered to maximize the binding affinity of
the analog for the antibody and the ability of the reporter
molecule to be quenched when bound by the ligand-binding
antibody.
[0095] The reporter molecules of the present invention include any
detectable label known by one skilled in the art that can be
covalently attached to the ligand analog of the present invention.
When part of the ligand analog, the reporter molecule is typically
capable of transferring energy to another moiety to be absorbed and
optionally re-emitted at a longer wavelength. Alternatively, the
reporter molecule is fluorogenic such that when the ligand analog
is bound to the ligand-binding antibody the reporter group is
fluorescent but when unbound is essentially non-fluorescent.
Reporter molecules include, without limitation, a chromophore, a
fluorophore, a fluorescent protein, and a phosphorescent dye.
Typically, substituents on the fluorophore or ligand analog alter
the spectral properties to form a fluorogenic ligand analog.
Preferred reporter molecules include fluorophores and fluorescent
proteins.
[0096] A fluorophore of the present invention is any chemical
moiety that exhibits an absorption maximum beyond 280 nm, and when
covalently attached to a ligand analog retains its spectral
properties. Fluorophores of the present invention include, without
limitation; a pyrene (including any of the corresponding derivative
compounds disclosed in U.S. Pat. No. 5,132,432), an anthracene, a
naphthalene, an acridine, a stilbene, an indole or benzindole, an
oxazole or benzoxazole, a thiazole or benzothiazole, a
4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a cyanine (including
any corresponding compounds in U.S. Ser. Nos. 09/968,401 and
09/969,853), a carbocyanine (including any corresponding compounds
in U.S. Ser. Nos. 09/557,275; 09/969,853 and 09/968,401; U.S. Pat.
Nos. 4,981,977; 5,268,486; 5,569,587; 5,569,766; 5,486,616;
5,627,027; 5,808,044; 5,877,310; 6,002,003; 6,004,536; 6,008,373;
6,043,025; 6,127,134; 6,130,094; 6,133,445; and publications WO
02/26891, WO 97/40104, WO 99/51702, WO 01/21624; EP 1 065 250 A1),
a carbostyryl, a porphyrin, a salicylate, an anthranilate, an
azulene, a perylene, a pyridine, a quinoline, a borapolyazaindacene
(including any corresponding compounds disclosed in U.S. Pat. Nos.
4,774,339; 5,187,288; 5,248,782; 5,274,113; and 5,433,896), a
xanthene (including any corresponding compounds disclosed in U.S.
Pat. Nos. 6,162,931; 6,130,101; 6,229,055; 6,339,392; 5,451,343 and
U.S. Ser. No. 09/922,333), an oxazine (including any corresponding
compounds disclosed in U.S. Pat. No. 4,714,763) or a benzoxazine, a
carbazine (including any corresponding compounds disclosed in U.S.
Pat. No. 4,810,636), a phenalenone, a coumarin (including an
corresponding compounds disclosed in U.S. Pat. Nos. 5,696,157;
5,459,276; 5,501,980 and 5,830,912), a benzofuran (including an
corresponding compounds disclosed in U.S. Pat. Nos. 4,603,209 and
4,849,362) and benzphenalenone (including any corresponding
compounds disclosed in U.S. Pat. No. 4,812,409) and derivatives
thereof. As used herein, oxazines include resorufins (including any
corresponding compounds disclosed in U.S. Pat. No. 5,242,805),
aminooxazinones, diaminooxazines, and their benzo-substituted
analogs.
[0097] When the fluorophore is a xanthene, the fluorophore is
optionally a fluorescein, a rhodol (including any corresponding
compounds disclosed in U.S. Pat. Nos. 5,227,487 and 5,442,045), or
a rhodamine (including any corresponding compounds in U.S. Pat.
Nos. 5,798,276; 5,846,737; U.S. Ser. No. 09/129,015). As used
herein, fluorescein includes benzo- or dibenzofluoresceins,
seminaphthofluoresceins, or naphthofluoresceins. Similarly, as used
herein rhodol includes seminaphthorhodafluors (including any
corresponding compounds disclosed in U.S. Pat. No. 4,945,171).
Alternatively, the fluorophore is a xanthene that is bound via a
linkage that is a single covalent bond at the 9-position of the
xanthene. Preferred xanthenes include derivatives of
3H-xanthen-6-ol-3-one attached at the 9-position, derivatives of
6-amino-3H-xanthen-3-one attached at the 9-position, or derivatives
of 6-amino-3H-xanthen-3-imine attached at the 9-position.
[0098] Preferred fluorophores of the invention include xanthene
(rhodol, rhodamine, fluorescein and derivatives thereof) coumarin,
cyanine, pyrene, oxazine and borapolyazaindacene. Most preferred
are fluorinated xanthenes, fluorinated coumarins and cyanines. When
conjugated to a ligand analog it is preferred that the fluorophore
not be substituted by a polar group such as SO.sub.3.sup.- due to
poor binding affinity conferred to the ligand analog for the
ligand-binding antibody. The choice of the fluorophore attached to
the ligand analog will determine the absorption and fluorescence
emission properties of the ligand analog, the ligand-detection
reagent and ultimately the assay solution in the presence of a
ligand. Physical properties of a fluorophore label include spectral
characteristics (absorption, emission and stokes shift),
fluorescence intensity, lifetime, polarization and photo-bleaching
rate all of which can be used to distinguish one fluorophore from
another.
[0099] Typically the fluorophore contains one or more aromatic or
heteroaromatic rings, that are optionally substituted one or more
times by a variety of substituents, including without limitation,
halogen, nitro, cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl,
alkynyl, cycloalkyl, arylalkyl, acyl, aryl or heteroaryl ring
system, benzo, or other substituents typically present on
fluorophores known in the art.
[0100] In one aspect of the invention, the fluorophore has an
absorption maximum beyond 480 nm. In a particularly useful
embodiment, the fluorophore absorbs at or near 488 nm to 514 nm
(particularly suitable for excitation by the output of the
argon-ion laser excitation source) or near 546 nm (particularly
suitable for excitation by a mercury arc lamp).
[0101] Many of fluorophores can also function as chromophores and
thus the described fluorophores are also preferred chromophores of
the present invention.
[0102] In one aspect of the invention the ligand analogs are
phospho-tyrosine, -threonine or -serine ligand analogs that are
employed for the detection of phosphorylated biomolecules including
proteins and peptides or for the detection of kinase or phosphatase
enzyme activity. Preferred phospho-ligand analogs typically
comprise a phosphotyramide moiety or a phosphoethanolamide moiety
and include phosphotyramide, phosphotyrosinamide and
phosphoethanolamide ligand analogs.
[0103] Typically a ligand analog comprising a phosphophenol moiety
has the following formula ##STR1## wherein R is a reporter molecule
and linker is a single covalent bond or comprises 1-20 non-hydrogen
atoms to covalently attach the reporter molecule to the
phosphophenol moiety. Preferred reporter molecules include
borapolyazaindacene, coumarin, xanthene, cyanine, fluorescent
protein and phosphorescent dye. Most preferred reporter molecules
are xanthene, borapolyazaindacene and coumarin, typically these
reporter molecules are not substituted by polar groups. The linker
typically contains alkyl and amine groups.
[0104] In one embodiment, ligand analogs comprising a
phosphotyramide moiety are selected from the group consisting of:
##STR2##
[0105] Exemplified Compounds according to Formula II include
Compounds 2, 7-17, 20-21 and 39, exemplified Compounds according to
Formula III include Compounds 22-33 and 40, and exemplified
compounds according to Formula IV include compounds 34-38 and
41-42. Formula IV, as used herein is typically referred to as a
phosphotyrosinamide ligand analog.
[0106] Alternatively, the phosphophenol moiety forms part of a
fluorogenic ligand analog according to formula: ##STR3## wherein R
is a reporter molecule and L is a linker that is a single covalent
bond or comprises 1-20 non-hydrogen atoms to covalently attached
said reporter molecule to phosphophenol moiety. Again, preferred
reporter molecules are selected from the group consisting of
borapolyazaindacene, coumarin, xanthene, cyanine, fluorescent
protein and phosphorescent dye. Most preferred is a
borapolyazaindacene reporter molecule, See Compounds 4 and 5. The
linker typically comprises alkyl and amino groups.
[0107] Thus, in a preferred embodiment a fluorogenic ligand analog
comprising a phosphotyramide moiety is according to formula:
##STR4##
[0108] The fluorogenic ligand analogs are not limited to
phosphotyramide moieties; it is appreciated that any epitope that
has affinity for a ligand-binding antibody can replace the
phosphotyramide moiety of formula VI to form a fluorogenic ligand
analog. Preferred epitopes are single amino acids or portions
thereof including theronine and serine.
[0109] In another aspect of the invention, the ligand analog has
affinity for a phosphothreonine or phosphoserine ligand-binding
antibody. In this instance, the ligand analogs are typically
phosphoethanolamines according to formula
R-Linker-NHCH.sub.2CH.sub.2--O--PO.sub.3 Formula VII wherein R is a
reporter molecule and linker is a single covalent bond or comprises
1-20 non-hydrogen atoms to covalently attached said reporter
molecule to phosphoethanolamine moiety. Typically the linker is a
single covalent bond, See Compound 2.
[0110] In addition, it is also contemplated that serine and
threonine residues conjugated to a reporter molecule also form part
of the invention, See Compound 43 Example 29.
[0111] Theses non-fluorogenic ligand analogs that comprise
phosphoethanolamine, serine, threonine and phosphophenol
(phosphotyramide) moieties are all capable of being quenched when
bound by a ligand-binding antibody. Preferred ligand analogs that
demonstrate a high degree of quenching typically comprise a
xanthene reporter molecule wherein the linker is attached at the
6-position of the pendent phenyl ring including Compounds 7, 9, 12,
15, 19, 23, 25, 27, 33, 34 and 38. Most preferred ligand analogs
for their ability to be quenched when bound by a phosphotyrosine
ligand-binding antibody are Compounds 9, 15, 23 and 34.
B. Labeling Reagents
[0112] In addition to forming immune-complexes for the detection of
a target in a sample, the labeling reagents also form part the
ligand-detection reagent wherein the labeling reagent comprises a
monovalent antibody fragment or a non-antibody protein and a
covalently bound label. Labels that are conjugated to a ligand
analog are selected from the group consisting of a chromophore, a
fluorophore, a quenching moiety, a fluorescent protein and a
phosphorescent dye. Typically the label is a fluorophore or a
quenching moiety that is capable of absorbing energy from the
reporter molecule of the ligand analog when bound by the
ligand-binding antibody. The absorbed energy is either quenched
(not re-emitted) or re-emitted at a longer wavelength resulting is
a color shift of the detectable signal.
[0113] The labeling reagents of the present invention are
monovalent antibody fragments or non-antibody monomeric proteins
that have affinity for a region of a target-binding antibody. The
regions of the target-binding antibody that can be bound by a
labeling reagent include the Fc region, Fab region, the kappa or
lambda light chain region or a heavy chain region. When the
labeling reagent is derived from an antibody the monovalent
fragment can be, anti-Fc, anti-Fab, an anti-Fc isotype, anti-kappa
light chain, anti-lambda light chain, or a single-chain fragment
variable protein. Labeling reagents that are a non-antibody peptide
or protein, are for example but not limited to, soluble Fc
receptor, protein G, protein A, protein L, lectins, or a fragment
thereof. The labeling reagents typically have affinity for the Fc
region of the target-binding antibody but any region, except the
binding domain, may be used as a binding site for the labeling
reagent. The Fc region is preferable because it is the farthest
from the binding domain of the target-binding antibody and is
unlikely to cause steric hinderance, when bound by a labeling
reagent, of the binding domain for the target.
[0114] Antibody is a term of the art denoting the soluble substance
or molecule secreted or produced by an animal in response to an
antigen, and which has the particular property of combining
specifically with the antigen that induced its formation.
Antibodies themselves also serve are antigens or immunogens because
they are glycoproteins and therefore are used to generate
anti-species antibodies. Antibodies, also known as immunoglobulins,
are classified into five distinct classes--IgG, IgA, IgM, IgD, and
IgE. The basic IgG immunoglobulin structure consists of two
identical light polypeptide chains and two identical heavy
polypeptide chains (linked together by disulfide bonds). When IgG
is treated with the enzyme papain, a monovalent antigen-binding
fragment can be isolated, referred herein to as a Fab fragment.
When IgG is treated with pepsin (another proteolytic enzyme), a
larger fragment is produced, F(ab').sub.2. This fragment can be
split in half by treating with a mild reducing buffer that results
in the monovalent Fab' fragment. The Fab' fragment is slightly
larger than the Fab and contains one or more free sulfhydryls from
the hinge region (which are not found in the smaller Fab fragment).
The term "antibody fragment" is used herein to define both the Fab'
and Fab portions of the antibody. It is well known in the art to
treat antibody molecules with pepsin and papain in order to produce
antibody fragments (Gorevic et al., Methods of Enzyol., 116:3
(1985)).
[0115] The monovalent Fab fragments of the present invention are
produced from either murine monoclonal antibodies or polyclonal
antibodies generated in a variety of animals that have been
immunized with a foreign antibody or fragment thereof, U.S. Pat.
No. 4,196,265 discloses a method of producing monoclonal
antibodies. Typically, labeling reagents are derived from a
polyclonal antibody that has been produced in a rabbit or goat but
any animal known to one skilled in the art to produce polyclonal
antibodies can be used to generate anti-species antibodies.
However, monoclonal antibodies are equal, and in some cases,
preferred over polyclonal antibodies provided that the
target-binding antibody is compatible with the monoclonal
antibodies that are typically produced from murine hybridoma cell
lines using methods well known to one skilled in the art. Example 1
describes production of polyclonal antibodies raised in animals
immunized with the Fc region of a foreign antibody. It is a
preferred embodiment of the present invention that the labeling
reagents be generated against only the Fc region of a foreign
antibody. Essentially, the animal is immunized with only the Fc
region fragment of a foreign antibody, such as murine. The
polyclonal antibodies are collected from subsequent bleeds,
digested with an enzyme, pepsin or papain, to produce monovalent
fragments. The fragments are then affinity purified on a column
comprising whole immunoglobulin protein that the animal was
immunized against or just the Fc fragments. As described in detail
below, the labeling reagents are also covalently labeled with
fluorophore labels when bound to the affinity column to eliminate
incorporating label into the binding domain of the monovalent
fragment. One of skill in the art will appreciate that this method
can be used to generate monovalent fragments against any region of
a target-binding protein and that selected peptide fragments of the
target-binding antibody could also be used to generate
fragments.
[0116] Alternatively, a non-antibody protein or peptide such as
protein G, or other suitable proteins, can be used alone or coupled
with albumin wherein albumin is attached with a label of the
present invention. Preferred albumins of the invention include
human and bovine serum albumins or ovalbumin. Protein A, G and L
are defined to include those proteins know to one skilled in the
art or derivatives thereof that comprise at least one binding
domain for IgG, i.e. proteins that have affinity for IgG. These
proteins can be modified but do not need to be and are labeled in
the same manner as the monovalent Fab fragments of the
invention.
[0117] The labels of the present invention include any directly or
indirectly detectable label known by one skilled in the art that
can be covalently attached to the labeling reagent of the present
invention. Labels include, without limitation, a chromophore, a
fluorophore, a fluorescent protein, a phosphorescent dye, a tandem
dye, a particle, a hapten, an enzyme and a radioisotope. Preferred
labels include fluorophores, fluorescent proteins, haptens, and
enzymes.
[0118] For labels (labeling reagent) that are to be used with a
ligand-binding antibody and a ligand analog, the labels, by
definition, are capable of absorbing energy from the reporter
molecule when the ligand analog is bound by the ligand-binding
antibody. The labels included a chromophore, a fluorophore, a
quenching moiety, a fluorescent protein and a phosphorescent dye.
Typically, these labels include fluorophores and quenching
moieties, which include both fluorescent and essentially
non-fluorescent compounds. The fluorophores (and chromophores) can
be any of the compounds disclosed above for use as a reporter
molecule including compounds substituted with polar groups.
[0119] Numerous quenching compounds are known to one of skill in
the art including, but not limited to, compounds disclosed in U.S.
Pat. No. 6,541,618 and U.S. Ser. No. 09/942,342 and cyanine
compounds disclosed in U.S. Pat. Nos. 6,348,596; 6,080,868 and U.S.
Ser. No. 60/491,783, xanthene compounds U.S. Pat. No.
6,399,392.
[0120] In addition to fluorophores, enzymes also find use as labels
for the labeling reagents. Enzymes are desirable labels because
amplification of the detectable signal can be obtained resulting in
increased assay sensitivity. The enzyme itself does not produce a
detectable response but functions to break down a substrate when it
is contacted by an appropriate substrate such that the converted
substrate produces a fluorescent, colorimetric or luminescent
signal. Enzymes amplify the detectable signal because one enzyme on
a labeling reagent can result in multiple substrates being
converted to a detectable signal. This is advantageous where there
is a low quantity of target present in the sample or a fluorophore
does not exist that will give comparable or stronger signal than
the enzyme. However, fluorophores are most preferred because they
do not require additional assay steps and thus reduce the overall
time required to complete an assay. The enzyme substrate is
selected to yield the preferred measurable product, e.g.
calorimetric, fluorescent or chemiluminescence. Such substrates are
extensively used in the art, many of which are described in the
MOLECULAR PROBES HANDBOOK, supra.
[0121] A preferred calorimetric or fluorogenic substrate and enzyme
combination uses oxidoreductases such as horseradish peroxidase and
a substrate such as 3,3'-diaminobenzidine (DAB) and
3-amino-9-ethylcarbazole (AEC), which yield a distinguishing color
(brown and red, respectively). Other colorimetric oxidoreductase
substrates that yield detectable products include, but are not
limited to: 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
(ABTS), o-phenylenediamine (OPD), 3,3',5,5'-tetramethylbenzidine
(TMB), o-dianisidine, 5-aminosalicylic acid, 4-chloro-1-naphthol.
Fluorogenic substrates include, but are not limited to,
homovanillic acid or 4-hydroxy-3-methoxyphenylacetic acid, reduced
phenoxazines and reduced benzothiazines, including Amplex.RTM. Red
reagent and its variants (U.S. Pat. No. 4,384,042) and reduced
dihydroxanthenes, including dihydrofluoresceins (U.S. Pat. No.
6,162,931) and dihydrorhodamines including dihydrorhodamine 123.
Peroxidase substrates that are tyramides (U.S. Pat. Nos. 5,196,306;
5,583,001 and 5,731,158) represent a unique class of peroxidase
substrates in that they can be intrinsically detectable before
action of the enzyme but are "fixed in place" by the action of a
peroxidase in the process described as tyramide signal
amplification (TSA). These substrates are extensively utilized to
label targets in samples that are cells, tissues or arrays for
their subsequent detection by microscopy, flow cytometry, optical
scanning and fluorometry.
[0122] Another preferred colorimetric (and in some cases
fluorogenic) substrate and enzyme combination uses a phosphatase
enzyme such as an acid phosphatase, an alkaline phosphatase or a
recombinant version of such a phosphatase in combination with a
colorimetric substrate such as 5-bromo-6-chloro-3-indolyl phosphate
(BCIP), 6-chloro-3-indolyl phosphate, 5-bromo-6-chloro-3-indolyl
phosphate, p-nitrophenyl phosphate, or o-nitrophenyl phosphate or
with a fluorogenic substrate such as 4-methylumbelliferyl
phosphate, 6,8-difluoro-7-hydroxy-4-methylcoumarinyl phosphate
(DiFMUP, U.S. Pat. No. 5,830,912) fluorescein diphosphate,
3-O-methylfluorescein phosphate, resorufin phosphate,
9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)phosphate (DDAO
phosphate), or ELF 97, ELF 39 or related phosphates (U.S. Pat. Nos.
5,316,906 and 5,443,986).
[0123] Glycosidases, in particular beta-galactosidase,
beta-glucuronidase and beta-glucosidase, are additional suitable
enzymes. Appropriate colorimetric substrates include, but are not
limited to, 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside
(X-gal) and similar indolyl galactosides, glucosides, and
glucuronides, o-nitrophenyl beta-D-galactopyranoside (ONPG) and
p-nitrophenyl beta-D-galactopyranoside. Preferred fluorogenic
substrates include resorufin beta-D-galactopyranoside, fluorescein
digalactoside (FDG), fluorescein diglucuronide and their structural
variants (U.S. Pat. Nos. 5,208,148; 5,242,805; 5,362,628; 5,576,424
and 5,773,236), 4-methylumbelliferyl beta-D-galactopyranoside,
carboxyumbelliferyl beta-D-galactopyranoside and fluorinated
coumarin beta-D-galactopyranosides (U.S. Pat. No. 5,830,912).
[0124] Additional enzymes include, but are not limited to,
hydrolases such as cholinesterases and peptidases, oxidases such as
glucose oxidase and cytochrome oxidases, and reductases for which
suitable substrates are known.
[0125] Enzymes and their appropriate substrates that produce
chemiluminescence are preferred for some assays. These include, but
are not limited to, natural and recombinant forms of luciferases
and aequorins. Chemiluminescence-producing substrates for
phosphatases, glycosidases and oxidases such as those containing
stable dioxetanes, luminol, isoluminol and acridinium esters are
additionally useful.
[0126] In addition to enzymes, haptens such as biotin are also
preferred labels. Biotin is useful because it can function in an
enzyme system to further amplify the detectable signal, and it can
function as a tag to be used in affinity chromatography for
isolation purposes. For detection purposes, an enzyme conjugate
that has affinity for biotin is used, such as avidin-HRP.
Subsequently a peroxidase substrate is added to produce a
detectable signal.
[0127] Haptens also include hormones, naturally occurring and
synthetic drugs, pollutants, allergens, affector molecules, growth
factors, chemokines, cytokines, lymphokines, amino acids, peptides,
chemical intermediates, nucleotides and the like.
[0128] Fluorescent proteins also find use as labels for the
labeling reagents of the present invention. Examples of fluorescent
proteins include green fluorescent protein (GFP) and the
phycobiliproteins and the derivatives thereof. The fluorescent
proteins, especially phycobiliprotein, are particularly useful for
creating tandem dye labeled labeling reagents. These tandem dyes
comprise a fluorescent protein and a fluorophore for the purposes
of obtaining a larger stokes shift wherein the emission spectra is
farther shifted from the wavelength of the fluorescent protein's
absorption spectra. This is particularly advantageous for detecting
a low quantity of a target in a sample wherein the emitted
fluorescent light is maximally optimized, in other words little to
none of the emitted light is reabsorbed by the fluorescent protein.
For this to work, the fluorescent protein and fluorophore function
as an energy transfer pair wherein the fluorescent protein emits at
the wavelength that the fluorophore absorbs at and the fluorphore
then emits at a wavelength farther from the fluorescent proteins
than could have been obtained with only the fluorescent protein. A
particularly useful combination is the phycobiliproteins disclosed
in U.S. Pat. Nos. 4,520,110; 4,859,582; 5,055,556 and the
sulforhodamine fluorophores disclosed in U.S. Pat. No. 5,798,276,
or the sulfonated cyanine fluorophores disclosed in U.S. Ser. Nos.
09/968,401 and 09/969,853; or the sulfonated xanthene derivatives
disclosed in U.S. Pat. No. 6,130,101 and those combinations
disclosed in U.S. Pat. No. 4,542,104. Alternatively, the
fluorophore functions as the energy donor and the fluorescent
protein is the energy acceptor.
[0129] The labeling reagents can be independently attached to one
or more labels of the present invention by a number of methods
known to one skilled in the art and modification of such methods.
Methods include, labeling in a solution or on an affinity column.
For labeling in solution the labeling reagent is optionally
modified to contain a reactive group and the label is modified to
contain a reactive group or is synthesized to contain a reactive
group, as is typically the case with fluorophore labels wherein the
reactive group facilitates covalent attachment. The modification of
the labeling reagent to contain a reactive group includes (1)
chemical addition of such a reactive group or (2) alternatively
takes advantage of the disulfide bonds of the F(ab').sub.2 fragment
wherein the fragment is reduced to break the bond and expose the
thiol group that readily reacts with a reactive group on a label,
as disclosed in U.S. Pat. No. 5,360,895. Typically, covalent
attachment of the label to the fragment is the result of a chemical
reaction between an electrophilic group and a nucleophilic group,
See Table 1 for list of useful electrophile and nucleophile
reactive groups. However, when a label is used that is
photoactivated the covalent attachment results when the labeling
solution is illuminated.
[0130] A method for covalently attaching a label, particularly an
enzyme, a fluorescent protein or a particle, comprises the
following steps: [0131] a) cleaving an intact anti-region antibody
with an enzyme resulting in a F(ab').sub.2 fragment; [0132] b)
contacting said F(ab').sub.2 fragment with a reducing agent to
produce Fab' fragments containing a thiol group; [0133] c)
contacting said Fab' fragments with a solution comprising a label
that contains a reactive group; and, [0134] d) isolating Fab'
fragments of step d) that are covalently attached to a label by
size exclusion or affinity chromatography.
[0135] The whole anti-region antibody is cleaved with pepsin to
generate a bivalent F(ab)'.sub.2 fragment. This fragment is
typically affinity purified on a column comprising immunoglobulin
proteins such as IgG that is immobilized on agarose. The fragment
is then reduced to break the disulfide bond of the hinge region
that connects the two Fab fragments resulting in a Fab' fragment
with an exposed thiol group. This is typically accomplished by
adding a mild reducing buffer to the affinity purified F(ab').sub.2
fragments such as a buffer comprising 0.01 M EDTA and 0.01M
cysteine in phosphate buffer saline (PBS). The resulting thiol
group readily reacts with a reactive group on a label to covalently
attach the label to the fragment. Thus, a solution containing a
label that has been chemically modified to contain a reactive
group, using methods well known to one skilled in the art, is added
to the solution of reduced Fab' fragments. This method is
particularly useful for covalently attaching enzyme and other
protein labels due to their size and the lack of exposed amine
groups on the Fab fragments. One of skill in the art will
appreciate that this method requires the use of Fab' fragments as
apposed to Fab fragments due to the disulfide bonds of the Fab'
fragment and that the use of the enzyme papain or the like results
in such a fragment.
[0136] An alternative labeling of monovalent antibody fragments and
the monomeric non-antibody proteins is also accomplished in a
solution. The method comprises the steps: [0137] a) contacting a
Fab fragment or non-antibody monomeric protein with a solution
comprising a label that contains a reactive group; and, [0138] b)
isolating labeled anti-region Fab fragment or non-antibody
monomeric protein by size exclusion or affinity chromatography.
[0139] When a Fab fragment is to be labeled the whole antibody is
cleaved with an enzyme, such as papain, to generate Fab monovalent
fragments and the fragments are typically purified on an affinity
column prior to addition of the label. The Fab fragment or
non-antibody monomeric proteins are optionally chemically modified
to contain a reactive group. However, for covalently attaching
reactive fluorophore labels it has been found that this
modification of the fragment of non-antibody protein is not
necessary. The reactive label, typically a fluorophore or hapten,
are added to a solution of Fab fragments or non-antibody proteins
and the labeling reagent is separated from excess label by size
exclusion or affinity chromatography. The labeling reagents are
then stored in an appropriate buffer.
[0140] Labeling in solution can have some drawbacks, especially
when labeling of Fab fragments or non-antibody proteins with
fluorophores. Thus, Fab fragments and non-antibody proteins of the
present invention are preferably covalently attached to a
fluorophore label when immobilized on an affinity column. The
fragments and non-antibody proteins are immobilized on an affinity
column that comprises a protein that the fragment has affinity for,
typically IgG, and after immobilization a reactive fluorophore is
added to the column wherein the fragments are labeled and unreacted
fluorophores pass through the column.
[0141] The use of this affinity chromatography method avoids the
incorporation of label into the binding domain of the Fab fragment
or non-antibody protein. When Fab fragments are labeled with
fluorophores using this method unexpected advantages were obtained
wherein the fluorescent signal form fragments labeled on a column
are brighter than fragments labeled in solution when the
fluorophore and ratio of fluorophore to labeling reagent are held
constant. Without wishing to be bound by a theory it is possible
that the decreased brightness observed from the fragments labeled
in solution is due to quenching of fluorphores that are bound in or
near the binding domain by the high concentration of amine groups
in the binding domain. Thus, a preferred embodiment of the
invention for covalently attaching fluorphore labels to Fab
fragments comprises the following steps: [0142] a) cleaving an
intact anti region antibody with an enzyme that generates Fab
fragments; [0143] b) isolating the anti-region Fab fragments of
step a); [0144] c) contacting a matrix comprising intact
immunoglobulin proteins or fragments thereof that specifically bind
anti-region Fab fragments with a solution comprising said
anti-region fragments of step b) wherein said Fab fragments are
immobilized; [0145] d) contacting said matrix of step c) with a
solution comprising a fluorophore label that contains a reactive
group; [0146] e) washing said matrix to remove unbound label, and;
[0147] f) eluting said labeling reagent from said matrix whereby
said labeling reagent is manufactured comprising a label and being
isolated from other proteins and fragments thereof.
[0148] The matrix is typically an agarose column that comprises
either the selected region, such as the Fc region, or the entire
antibody provided that the antibody or fragment thereof is the same
species and isotype that was used to produce the antibodies that
the labeling reagent was generated from. However any matrix known
to one skilled in the art can be used that allows for
immobilization of labeling reagent and removal following attachment
of the fluorophore label. Fab and Fab' fragments can both be
labeled in this manner. However a free thiol group is not necessary
and therefore Fab fragments are typically labeled using this
method.
[0149] Due to the unique properties of the labeling reagent and the
attached labels it is a preferred embodiment of the present
invention that enzyme or other protein labels are covalently
attached to Fab' fragments in solution utilizing the free thiol
group of the Fab' fragment. It is another preferred embodiment that
fluorophore labels be covalently attached to the labeling reagent
when the reagent is immobilized on a affinity column wherein the
labeling reagent is typically an Fab fragment or a non-antibody
monomeric protein.
[0150] The attachment of the label to the fragments or the
non-antibody proteins results in multiple subsets that are
distinguished by the ratio of the label to the labeling reagent and
the physical properties of the label. A labeling reagent subset as
used herein refers to a discrete set of labeling reagents that are
homogenous and can be distinguished from another subset of labeling
reagent either by the physical properties of the label or the ratio
of the label to labeling reagent. The physical properties include
differences within a group of labels, such as emission spectra of
fluorphores, or across groups of labels, such as the difference
between an enzyme and a fluorophore. For fluorphore labels, the
physical properties typically relates to the emission spectra, this
includes modification of the same label, e.g. a cyanine with
different substitutions that shifts the emission wavelength, or
different fluorophores, e.g. a cyanine and a coumarin on the same
labeling reagent. The difference in physical properties also
includes the use of tandem dyes, which is specifically defined to
include an energy transfer pair wherein one is a protein and the
other is a fluorophore or both are fluorophores, or the pairing of
other labels that are not necessarily energy transfer pairs. A few
examples of labeling reagent subsets includes, but are not limited
to, a first subset comprising a single fluorophore at a known
ration attached to a anti-Fc Fab fragment; a second subset
comprises the same fluorophore on the Fab fragment at a different
known ration from the first subset, a third subset comprises the
same fluorophore but that has a shifted wavelength due to a
substitution on the fluorophore. Thus, the attachment of labels to
the labeling reagents results in an extensive selection of subsets
that when complexed with a target-binding antibody results in a
unique method to detect one or multiple targets in a sample whereby
the target is identified and quantitated.
C. Immuno-Labeled Complex
[0151] The subsets of labeling reagent are complexed with
target-binding antibodies to produce subsets of immuno-labeled
complex that for the target detection solution. The methods for
forming the immuno-labeled complex comprises the following steps:
[0152] a) contacting a solution of target-binding antibodies with a
labeling reagent subset, wherein said labeling reagent subsets are
distinguished by i) ratio of label to labeling reagent or ii) a
physical properties of said label; [0153] b) incubating said
target-binding antibodies and said labeling reagent for a time
period sufficient for one or more labeling reagents to form an
immuno-labeled complex with a target-binding antibody wherein a
region of said target binding antibody is selectively bound by
labeling reagent; [0154] c) optionally removing unbound labeling
reagent by adding a capture reagent comprising immunoglobulin
proteins or fragments thereof; and, [0155] d) optionally repeating
said steps a), b), and c) to form individual or pooled subsets of
immuno-labeling complexes wherein each subset is distinguished from
another subset by i) a ratio of label to labeling reagent, or ii) a
physical property of said label, or iii) a ratio of labeling
reagent to said target-binding antibody, or iv) by said
target-binding antibody.
[0156] A particular advantage for the use of labeling reagent of
the present invention to label target-binding antibodies is that
the process is relatively insensitive to the solution the
antibodies are in. Due to the physical nature of the labeling
reagents, small monovalent fragments, the reagents do not
cross-link and fall out of solution in the presence of high
concentration of proteins. For this reason, target-binding
antibodies can be complexed when present in ascites fluid, tissue
culture supernatant, serum or other solutions where there is a high
concentration of proteins. This eliminates the need to purify
target-binding proteins prior to labeling.
[0157] When preparing the immuno-labeled complex using purified
target-binding antibody, stock solutions of both the labeling
reagent and the target-binding antibody are typically near 1 mg/mL
in an appropriate buffer, although more or less concentrated
solutions are also suitable. Generally, the labeling reagent is
mixed in a molar ratio of at least one to 50 moles of labeling
reagent to one mole of the target-binding antibody to be complexed.
More commonly a ratio of at least one to as many as 10 moles of
labeling reagent per mole of target-binding antibody is combined.
With an anti-Fc region Fab to a target-binding antibody, a molar
ratio of approximately 2 to 10 is typical, more typically 3 to 5
(particularly for complexes in which the labeling reagent has been
labeled while immobilized on an affinity matrix). The ease of
formation of the complex permits rapid optimization of the complex
and assessment of the effect of variation in experimental
parameters. A particularly unique advantage of the invention is
that the stoichiometry of the complex is easily adjusted to provide
complexes with different ratios of labeling reagent to
target-binding antibody, and thus there is control over the
ultimate detectability of the target in the sample. Complexes that
have been labeled with the same dye but at different molar ratios
can be separately detected by the differences in their
intensities.
[0158] Complex formation appears to occur almost within the mixing
time of the solutions (<1 minute) but the reaction typically is
allowed to proceed for at least 5 minutes and can be longer before
combining the immuno-labeled complex with the sample. Although
complex formation can be reversed by addition of an unlabeled
antibody that contains the same binding region, reversibility is
very slow; furthermore, following binding of the immuno-labeled
complex to a target in a sample, the sample can be "fixed" using
aldehyde-based fixatives by methods that are commonly practiced by
those skilled in the art of immunolabeling.
[0159] The labeling process optionally further comprises the
addition of a capture component to remove excess labeling reagent.
For applications in which immunolabeling complexes of multiple
primary antibodies from the same species (e.g. mouse monoclonal
antibodies) or cross-reacting species (e.g. mouse and human
antibodies) are to be used simultaneously or sequentially, it is
necessary to quench or otherwise remove any excess labeling reagent
by use of a capture component or by other means to avoid
inappropriate labeling of the sample. The most effective capturing
components to capture excess labeling reagent are those that
contain the binding site of the labeling reagent but are themselves
not labeled, preferably an antibody or antibody fragment. Capture
components may be free in solution or immobilized on a matrix, such
as agarose, cellulose, or a natural or synthetic polymer, to
facilitate separation of the excess capture component from the
immuno-labeled complex. The capture component is optionally
attached to a microsphere or magnetic particle. However, separation
of excess labeling reagent is not essential for successful
utilization of the invention, particularly when using a single
target-binding antibody.
[0160] The steps of the labeling process for the target-binding
antibodies can be repeated to form discrete immuno-labeled complex
subsets that can be used individually or pooled in an assay to
detect individual or multiple targets. As used herein the term
immuno-labeled complex subsets refers to subsets that are
distinguished from each other i) a ratio of label to labeling
reagent, or ii) a physical property of the label, or iii) a ratio
of labeling reagent to the target-binding antibody, or iv) by the
target-binding antibody, or a combination thereof. For example a
panel of subsets may comprise a target-binding antibody that is
bound by a labeling reagent comprising a subset of different ratios
of the same label on the labeling reagent resulting in a discrete
subset of immuno-labeled complexes. This subset of immuno-labeled
complexes can be used individually wherein a target is identified
by the intensity of the detectable label or used in combination
with another subset of immunocomplexes that differ in the
target-binding antibody to identify multiple targets.
C. Ligand-Detection Reagent
[0161] The immuno-labeled complexes can be modified by the addition
of a ligand analog of the present invention to form a
ligand-detection reagent. The present ligand-detection reagents
comprise a ligand-binding antibody, a ligand analog and a labeling
reagent, See FIG. 8. This ligand-detection reagent is formed by
incubating the ligand-binding antibody, the ligand analog and the
labeling reagent for sufficient amount of time to allow for a
complex to form. The formation of the complex happens fairly
rapidly, typically less than 30 minutes, preferably less than 15
minutes and most preferred the complex forms in 5 minutes or
less.
[0162] The reporter molecules that are covalently attached to the
ligand analog are preferably selected from the group consisting of
a borapolyazaindacene, a coumarin, a xanthene, a cyanine, a
fluorescent protein and a phosphorescent dye. Most preferred are
borapolyazaindacene, fluorinated xanthene, fluorinated coumarin,
including dyes sold under the trade name OREGON GREEN, BODIPY,
PACIFIC BLUE and MARINA BLUE (Trade marks owned by Molecular
probes, Inc.) including any dyes disclosed in U.S. Pat. Nos.
6,162,931; 5,830,912; 4,774,339; 5,187,288; 5,248,782; and
5,433,896.
[0163] Ligand analogs containing these preferred reporter molecules
were screened with ligand-binding antibodies specific for
phosphotyrosine. Thus, preferred ligand-detection reagents for this
application include a phosphotyrosine-binding antibody and the
phosphotyramide and phosphotyrosinamide ligand analogs. Preferred
ligand analogs include Compounds 6-38. Most preferred are Compounds
7, 9, 12, 15, 19, 23, 25, 27, 33, 34 and 38. When the
ligand-detection reagent comprise Compound 9 or 15 the respective
reporter group is quenched by 80% or more.
[0164] Thus, a preferred embodiment of the present invention
includes a ligand-detection reagent that comprises a ligand-binding
antibody, a ligand analog and a labeling reagent to form an
antibody-ligand analog-labeling reagent complex wherein said ligand
analog is selected from the group consisting of phosphotyramide,
phosphotyrosinamide, phosphoethanolamine, phosphorylated kinase
peptide substrate, phosphatase substrate and phosphorylated peptide
and said analog is covalently bonded to a xanthene or
borapolyazaindacene reporter molecule and the reporter molecule of
ligand analog is capable of being quenched when bound by said
ligand-binding antibody. Preferably, the reporter molecule is
quenched by about 80% or more. The labeling reagent comprises a
label that is a fluorophore or a quenching moiety and a monovalent
antibody fragment or a non-antibody protein wherein the label
functions as an energy acceptor molecule. The labeling reagent is
incubated with the ligand-binding antibody and the ligand analog
for a sufficient amount of time to form a ligand-detection
reagent.
[0165] In another aspect of the invention, fluorogenic ligand
analogs are used resulting in fluorogenic ligand-detection
reagents. In this instance, the ligand-detection reagent is
fluorescent but when the target ligand displaces the ligand analog
the fluorescent signal intensity decreases. Thus, the presence of a
target ligand is determined by a decrease in fluorescence signal.
Preferred fluorogenic ligand analogs include Compounds 4 and 5.
[0166] When preparing the ligand-detection reagent with a labeling
reagent, the complex is formed as disclosed above for the
immuno-complexes. The ligand analog is added before, with or after
the addition of the labeling reagent to the ligand-binding
antibody. The entire complex forms very rapidly, typically less
than 5 minutes.
[0167] Appropriate matching of the reporter group and label are
necessary to maximize the FRET between the reporter molecule and
label for either optimal quenching or emission of energy at a
longer wavelength. Many energy transfer dye pairs are known to one
of skill in the art. Table 2 lists representative energy transfer
pair dyes wherein the acceptor functions as a quenching moiety.
This list is not intended to be limiting. TABLE-US-00002 TABLE 2
Donor Dye Acceptor Compounds Alexa Fluor 350 Alexa Fluor 488; QSY
36; dabcyl Alexa Fluor 488 Alexa Fluor 546; Alexa Fluor 555; Alexa
Fluor 568; Alexa Fluor 594; Alexa Fluor 647; QSY 35; Dabcyl; QSY 7;
QSY 9 Alexa Fluor 546 Alexa Fluor 568; Alexa Fluor 594; Alexa Fluor
647; QSY 35; Dabcyl; QSY 7; QSY 9 Alexa Fluor 555 Alexa Fluor 594;
Alexa Fluor 647; QSY 7; QSY 9 Alexa Fluor 568 QSY 7; QSY 9; QSY 21
Alexa Fluor 594 Alexa Fluor 647; QSY 21 Alexa Fluor 647 QSY 21
Fluorescein Tetramethylrhodamine; QSY 7; QSY 9 IAEDANS Fluorescein
BODIPY FL Alexa Fluor 555; QSY 9
[0168] Preferred energy transfer dye pairs are selected from the
group consisting of Oregon Green 488-Alexa Fluor 555 dye pair,
BODIPY-FL-Alexa Fluor 555 dye pair and BODIPY-FL-QSY 9 dye
pair.
[0169] Therefore, a preferred embodiment of the present invention
includes a ligand-detection reagent that comprises a ligand
antibody, a ligand analog and a labeling reagent to form a ternary
complex wherein said ligand analog is selected from the group
consisting of phosphotyramide, phosphoethanolamine, phosphorylated
kinase peptide substrate, phosphatase substrate and phosphorylated
peptide and said analog is covalently bonded to a xanthene reporter
molecule and said labeling reagent is an anti-Fc monovalent
antibody fragment covalently bonded to a xanthene label moiety or
non-fluorescent quenching moiety.
[0170] For the detection of phosphorylated molecules and enzymes
that modify the degree of phosphorylation the phosphotyramide,
phosphotyrosinamide, phosphoserine and phosphoethanolamine ligand
analogs are preferred, including Compounds 2 and 4-43. In addition,
proteins or peptides that have been modified to be a ligand analog
are also preferred. Table 3 contains a select list of some peptides
that are specific for phosphotyrosine-binding antibodies that when
conjugated to a reporter molecule of the present invention forms a
ligand analog. TABLE-US-00003 TABLE 3 phosphotyrosine ligands
Peptide Sequence pY-1 ENDpYINASL pY-2 DADEpYLIPQQG EGF Receptor
DADEpYL M-2170 IpYGEF M-2165 IYGEF M-2035 TEPEpYQPGE N-1480 DpYVPML
H-1546 Biotin-EPQpYEEIPIYL H-5458 Biotin-EGPWLEEEEEAYGWMSF pp60
TSTEPQpYQPGENL abl peptide EAIYAAPFAKKK DSIP WAGGDASGE pDSIP
WAGGDApSGE pY pY
[0171] It is appreciated that the ligand-detection reagents can be
designed to detect an unlimited number of target ligands utilizing
a ligand analog and an appropriately matched ligand-binding
antibody and that these reagents are in no way limited to the
detection of phosphorylated biomolecules. Thus, ligand analogs and
or target ligands are preferably selected from the group consisting
of an amino acid, an enzyme, a kinase substrate, a peptide, a
protein, a polysaccharide, a phosphatase substrate, a nucleoside, a
nucleotide, an oligonucleotide, a nucleic acid, a hapten,
digoxigenin, a cell surface receptor, a drug, a hormone, a lipid, a
lipid assembly, a synthetic polymer, a polymeric microparticle, a
biological cell and a virus wherein the ligand analog further
comprise a reporter molecule. Preferred ligand analogs for the
detection of phosphorylated biomolecules are selected from the
group consisting of phosphotyramide, phosphotyrosinamide,
phosphoethanolamine, phosphorylated kinase peptide substrate,
phosphatase substrate and phosphorylated peptide. These ligand
analogs are preferably conjugated to xanthene, borapolyazaindacene
or coumarin reporter molecules.
C. Methods of Use
[0172] The labeling reagents, target-binding antibodies and
resulting immuno-labeled complex that forms the target detection
solution can be used in a wide range of immunoassays, essentially
in any assay a traditional secondary antibody is used including
some assays that secondary antibodies are not used because of their
size and ability to cross-link. Examples of such assays used to
detect a target in a sample include immunoblots, direct detection
in a gel, flow cytometry, immunohistochemistry, confocal
microscopy, fluorometry, ELISA and other modified immunoassays.
Furthermore, the immuno-labeled complex can be modified by the
addition of a ligand analog to form a ligand-detection reagent
complex. In this instance, the ligand-detection reagent is employed
in a competitive immunoassay wherein a target ligand displaces the
ligand analog resulting in a change in detectable signal that
indicates the presence of a target ligand in a sample.
[0173] A method of the present invention for detecting a single
target in a sample comprises the following steps: [0174] a)
contacting a solution of target-binding antibodies with a labeling
reagent subset, wherein said labeling reagent subsets are
distinguished by i) ratio of label to labeling reagent or ii) a
physical properties of said label; [0175] b) incubating said
target-binding antibodies and said labeling reagent subset for a
time period sufficient for one or more labeling reagents to form an
immuno-labeled complex with a target-binding antibody wherein a
region of said target binding antibody is selectively bound by
labeling reagent; [0176] c) contacting said sample with said
immuno-labeled complex of step b); [0177] d) incubating said sample
of step c) for a time sufficient to allow said immuno-labeled
complex to selectively bind to said target; and, [0178] e)
illuminating said immuno-labeled complex whereby said target is
detected.
[0179] A sample is incubated with a preformed immuno-labeled
complex that comprises a labeling reagent and a target-binding
antibody. While this method describes the identification of a
single target, subsets of labeling reagents bound to the same
target-binding antibody can be used to identify and provide
additional information about such targets. For example, subsets of
labeling reagent can be prepared wherein two discrete subsets are
generate each with a distinct fluorophore label that is
distinguished by their emission spectra, e.g. one that emits in the
green spectra and one that emits in the red spectra. The labeling
reagent subsets are then added to a solution of target-binding
antibody in a controlled ratio, e.g. two parts one labeling reagent
(green emission) and one part the other labeling reagent (red
emission) per target binding antibody. In this way the
immuno-labeled complexes can be used to detect a target. If another
immuno-labeled complex were added to the sample the original target
could be distinguished from the subsequently detected target.
[0180] The methods of the present invention also provide for the
detection of multiple targets in a sample. Multiple targets include
the discrete epitope that the target-binding antibody has affinity
for as well as molecules or structures that the epitope is bound
to. Thus, multiple target identification includes phenotyping of
cells based on the concentration of the same cell surface marker on
different cells. In this way multiple target identification is not
limited to the discrete epitope that the target binding antibody
binds, although this is clearly a way that multiple targets can be
identified, i.e. based on the affinity of the target-binding
antibody.
[0181] Therefore, a method for detecting multiple targets in a
sample comprises the following steps: [0182] a) contacting a
solution of target-binding antibodies with a labeling reagent
subset, wherein said labeling reagent subsets are distinguished by
i) ratio of label to labeling reagent or ii) a physical properties
of said label; [0183] b) incubating said target-binding antibodies
and said labeling reagent subset for a time period sufficient for
one or more labeling reagents to form an immuno-labeled complex
with a target-binding antibody wherein a region of said
target-binding antibody is selectively bound by labeling reagent,
wherein steps a) and b) are repeated to form discrete
immuno-labeling complex subsets; [0184] c) contacting said sample
with a solution comprising A) a pooled subset of immuno-labeled
complexes, wherein each subset is distinguished from another subset
by i) a ratio of label to labeling reagent, or ii) a physical
property of said label, or iii) a ratio of labeling reagent to said
target-binding antibody, or iv) by said target-binding antibody or
B) an individual subset wherein step c) with a solution comprising
an individual subset is repeated; [0185] d) incubating said sample
of step c) for a time sufficient to allow said immuno-labeled
complex to selectively bind to said target; and, [0186] e)
illuminating said immuno-labeled complex whereby said target is
detected.
[0187] A selected target-binding antibody and a subset of labeling
reagent are incubated to form an immuno-labeled complex subset.
This procedure is repeated to form a panel of immuno-labeled
complex subsets that may be pooled and added to a sample.
Alternatively each immuno-labeled complex subset is added stepwise
to a sample. The immuno-labeled complex subsets are distinguished
by four characteristics resulting in an infinite number of
immuno-labeled complex subsets. First (i) the subsets can be
distinguished by the target-binding antibody that is determined by
the end user for the information that is desired from a sample.
This means that each subset is distinguished based on the affinity
of the target-binding antibody. The target-binding antibody
typically distinguishes immuno-labeled complexes when multiple
targets are identified, however this is normally combined with
another characteristic to gain information form a sample or
increase the number of targets that can be detected at one time.
The second (ii) distinguishing feature used is the ratio of label
to labeling reagent, as discussed in detail above. A subset based
on this feature would have for example a ratio of two fluorophore
per each labeling reagent. The third (iii) distinguishing feature
is the ratio of labeling reagent to target-binding antibody. This
is accomplished using a controlled concentration of target-binding
antibody mixed with a controlled concentration of a labeling
reagent subset and the subset would comprise a target-binding
antibody that is bound by a discrete number of labeling reagents.
The fourth (iv) feature is the physical feature of the label.
Typically this refers to the physical properties of the fluorophore
labels wherein a subset of this group is distinguished by the label
itself such as a green emitting fluorophore compared to a red
emitting fluorophore. One of skill in the art will appreciate that
while immuno-labeling complex subsets can be distinguished based on
one feature the subsets are typically, and most useful, when
discretely identified based on a combination of the distinguishing
characteristics.
[0188] Another example of detection of multiple targets utilizes
the following immuno-labeled subsets, all of which comprise a
different target-binding antibody but differ in the label and ratio
of label. The first subset comprises a fluorophore label that emits
red-fluorescent light, a second subset comprises a fluorophore
label that emits green fluorescent light, a third subset comprises
a ratio of 1:1 red to green fluorophore label; a fourth subset
comprises a ratio of 2:1 red to green fluorophore label and a fifth
subset comprises a ratio of 1:2 red to green fluorophore label.
These subsets allow for the simultaneous detection of five targets
in a sample. This aspect of the present invention is particularly
important due to the limited range of fluorophores available
wherein the labeling reagents can be utilized to increase the
number of targets that can be detected at one time. One of skill in
the art can appreciate that these subsets could be expanded by
altering the ratio of label to labeling reagent instead of just the
ratio of labeling reagent to target-binding antibody. This same
methodology can also be applied to a single fluorophore label
wherein the ratios are altered and a target is detected based on
the intensity of the signal instead of the color and the ratio of
the color to another color.
[0189] Following the formation of the immuno-labeled complex
subsets the subsets can be pooled and added to a sample or added
stepwise to a sample, either of which is determined by the end user
and the particular assay format. This method of the present
invention provides for maximum flexibility and ease of determining
multiple targets in a sample.
[0190] Another method of the present invention provides for the
determination of multiple targets in a sample specifically using
the flow cytometry assay format. Traditionally targets identified
using flow cytometry used either directly labeled primary antibody
or labeled microspheres that were covalently attached to a primary
antibody wherein the microsphere is the label. Examples include the
fluorescent encapsulated microsphere beads sold by Luminex. The
labeling reagents and the present invention overcome both the need
for directly labeled primary antibody and the need for expensive
microspheres.
[0191] Thus, a method of the present invention for determining
identity and quantity of targets in a sample by detecting multiple
targets comprises the following steps: [0192] a) contacting a
solution of target-binding antibodies with a labeling reagent
subset, wherein said labeling reagent subsets are distinguished by
i) ratio of label to labeling reagent or ii) a physical properties
of said label; [0193] b) incubating said target-binding antibodies
and said labeling reagent for a time period sufficient for one or
more labeling reagents to form an immuno-labeled complex with a
target-binding antibody wherein a region of said target binding
antibody is selectively bound by labeling reagent, wherein steps a)
and b) are repeated to form a pooled subset of immuno-labeling
complexes; [0194] c) contacting a population of cells in a sample
with a solution comprising a pooled subset of immuno-labeled
complexes, wherein each subset is distinguished from another subset
by i) a ratio of label to labeling reagent, or ii) a physical
property of said label, or iii) a ratio of labeling reagent to said
target-binding antibody, or iv) by said target-binding antibody;
[0195] d) incubating said cells for a time period sufficient to
allow said immuno-labeled complex to bind said targets; [0196] e)
passing said incubated population of cells through an examination
zone; and, [0197] f) collecting data from said cells that were
passed through said examination zone wherein said multiple targets
are detected whereby the identity and quantity of said targets is
determined.
[0198] In one aspect, a target-binding antibody is pre-complexed to
the target-binding antibody to form a subset and that subset or a
panel of subsets are added to a sample, that are typically
distinguished by the target binding antibody. This method then
avoids the need for a directly labeled primary. Secondly, when the
panel of subsets is distinguished, for example, by the ratio of
label to labeling reagent or the ratio of labeling reagent to
target-binding antibody the immuno-labeled complex can function
similar to the microsphere beads of Luminex. For example, this is
accomplished wherein three immuno-labeled complex subsets are
distinguished by the target binding antibody and the fluorophore
attached to the labeling reagent and within one of the subsets is
another set of subsets that are distinguished based on the ratio of
label to labeling reagent. In this way three different epitopes are
detected and one of the epitopes is further distinguished and a
phenotype distinction made based on the intensity of the signal
generated from the labeled-immuno complex subsets based on the
ratio of fluorophore to labeling reagent. This determination of
targets is facilitated when a population of cells or cellular
organelles is passed through the examination zone of a flow
cytometer wherein the fluorescent signal and intensity is recorded
for each cell resulting in a histogram of the cell population or
cellular organelles based on the detected epitopes.
[0199] In another aspect of the invention, additional detection
reagents are combined with the sample concurrently with or
following the addition of immuno-labeled complex subsets. Such
additional detection reagents include, but are not limited to
reagents that selectively detect cells or subcellular components,
ions, or indicate the cell viability, life cycle, or proliferation
state. For example, the additional detection reagent is a labeled
target-binding antibody that is directly or indirectly detectable
and another additional detection reagent is a stain for nucleic
acids, for F-actin, or for a cellular organelle.
[0200] In another aspect of the invention the immuno-labeled
complexes are modified to contain a ligand analog wherein the
resulting ternary complex is utilized as a ligand-detection reagent
in a competitive immunoassay. The ligand-detection reagents of the
present invention can be used without limitation for the detection,
analysis and monitoring of target ligands. These ligand-detection
reagents are typically present in a ligand-detection solution,
wherein the solution comprises a ligand antibody, a ligand analog
and a labeling reagent to form a ternary ligand-detection reagent
complex and a buffer. Appropriate buffers include the family of
Good's buffers or any buffer known to one of skill in the art that
is typically used with antibodies.
[0201] Therefore, in one aspect of the invention a method for
determining the presence of a target ligand in a sample comprises
[0202] a) generating a ligand-detection reagent, wherein the
ligand-binding antibody, the ligand analog and the labeling reagent
are incubated together for a sufficient amount of time to form the
ligand-detection reagent; [0203] b) incubating the reagent with
said sample for a sufficient amount of time for said ligand to
displace said ligand analog from binding groove of said
ligand-binding antibody; [0204] c) illuminating said sample with an
appropriate wavelength wherein said reporter molecule generates a
detectable signal in the presence of said ligand whereby said
ligand is detected.
[0205] The ligand-detection reagent is generated as described
above. The reagent is incubated with the sample for a sufficient
amount of time for the target ligand to displace the ligand analog.
Typically this occurs very rapidly, usually within 5 minutes or
less, preferably the displacement occurs within seconds of adding
the ligand-detection reagent to the sample, See, Example 34.
Illumination of the reporter molecule, as described below, depends
on the reporter molecule of the ligand analog.
[0206] It is envisioned that any target ligand, wherein an
appropriate ligand-binding antibody exists, can be detected using
this method of the present invention. This includes the use of
either a fluorogenic or non-fluorogenic ligand analog and a
labeling reagent wherein the labeling reagent functions to
quenching the reporter molecule when bound by the ligand-binding
antibody or to further shift the detectable signal from the
reporter molecule signal.
[0207] In one aspect of the invention, the method for determining
the presence of a target ligand in a sample is used to detect
phosphorylated biomolecules. In this instance the ligand-binding
solution typically comprises a ligand-binding antibody that is
capable of binding a phosphotyrosine, phosphoserine or
phosphothreonine moiety, an appropriately matched ligand analog
that is selected from the group consisting of phosphotyramide,
phosphoserine phosphotyrosinamide, phosphoethanolamine,
phosphorylated kinase peptide substrate, phosphatase substrate and
phosphorylated peptide, and optionally a labeling reagent whereby
the amount of generated detectable signal from said reporter
molecule is dependent on the presence of the phosphorylated target
ligand. Preferably, the ligand analog is covalently bonded to a
xanthene, coumarin, or borapolyazaindacene reporter molecule and
said labeling reagent is an anti-Fc monovalent antibody fragment
covalently bonded to a xanthene labeling moiety or non-fluorescent
quenching moiety.
[0208] Incubating phospho-tyrosine, -threonine or -serine binding
antibodies with an appropriately matched ligand analog and labeling
reagent, generates the ligand-detection reagent. Preferably the
ligand analog comprises a phosphophenol moiety including both
phosphotyramide and phosphotyrosinamide ligand analogs. The
labeling reagent may be added prior to the addition of the ligand
analog, after the addition of the ligand analog or all three
components may be added simultaneously to form a ligand-detection
reagent.
[0209] Following formation of the ligand-detection reagent in a
ligand-detection solution, which comprises the reagent and an
appropriate buffer, the ligand-detection reagent is incubated with
the sample. If present, the target ligand will displace the ligand
analog from the ligand-binding antibody almost immediately,
preferably less than 5 minutes. The ligand-detection reagent may be
illuminated with an appropriate wavelength, before, during or after
the reagent has been added to the sample. Alternatively, the
reagent may be illuminated continuously from the time of formation
to a time point after the reagent has been added to the sample.
[0210] This particular method also allows for the detection of
enzymes that modify phosphorylated biomolecules, such as kinase and
phosphatase enzymes, See Example 39.
[0211] Current commercial kinase and phosphatase assays are often
time-consuming and require many steps such as electrophoresis,
centrifugation, ELISA or immunoprecipitation. The present invention
provides methods for the rapid, sensitive, and non-radioactive
detection of a variety of selected kinases and phosphatases and
provides, in addition, methods that are well suited for
high-throughput screening. The kinase and phosphatase assays of the
present invention also permit the screening of inhibitors and
activators of, for example, tyrosine kinases and, in addition, also
permit the monitoring and the purification of kinase and
phosphatase enzymes. The enzyme substrate may be on a solid- or
semi solid matrix such as an array including Hydrogel slides or
present in a solution. The methods of the present invention are
particularly advantageous for the monitoring of kinase and
phosphatase activity in solution wherein the additional step adding
the substrate to a matrix is not necessary. After the formation of
the ligand-detection reagent the reagent is added along with enzyme
substrate and enzyme to an appropriate buffer, such as kinase
buffer comprising 50 mM Tris, pH 7.5, 10 mM MgCl.sub.2, 1 mM EGTA,
0.01% Brij 35, 2 mM DTT and 500 .mu.M ATP. The sample is typically
continually illuminated and monitored or at set intervals for a
period of time to determine the presence of kinase activity by the
addition of phosphate groups to tyrosine, threonine or serine
residues on an appropriate substrate. We have demonstrated that ATP
does not displace the phosphotyramide ligand analog and therefore
the observed detectable signal is related directly to the
displacement of the ligand analog by the phosphorylated enzyme
substrate, See Example 38.
[0212] In addition to a solution based assay, the present methods
are also preferred for assay systems that employ immobilized enzyme
substrate. In this instance, detection of the enzyme substrate on
the array makes the methods of the invention far more sensitive
than any known assays for kinases and phosphatases and use of
fluorescence for detection on the array permits a higher density of
labeling than is possible with radiochemical detection.
[0213] In addition to detecting a target ligand as an end point,
the present ligand-detection reagents and methods for determining
the presence of a target ligand in a sample can be employed to
detect and monitor enzymes that directly or indirectly modify the
target ligand.
[0214] The sample to be used with the present methods and
compositions is defined to include any material that may contain a
target to which an antibody has affinity for. Typically the sample
is biological in origin and comprises tissue, cell or a population
of cells, cell extracts, cell homogenates, purified or
reconstituted proteins, recombinant proteins, bodily and other
biological fluids, viruses or viral particles, prions, subcellular
components, or synthesized proteins. Possible sources of cellular
material used to prepare the sample of the invention include
without limitation plants, animals, fungi, bacteria, archae, or
cell lines derived from such organisms. The sample can be a
biological fluid such as whole blood, plasma, serum, nasal
secretions, sputum, saliva, urine, sweat, transdermal exudates,
cerebrospinal fluid, or the like. Alternatively, the sample may be
whole organs, tissue or cells from an animal. Examples of sources
of such samples include muscle, eye, skin, gonads, lymph nodes,
heart, brain, lung, liver, kidney, spleen, solid tumors,
macrophages, mesothelium, and the like.
[0215] Prior to combination with the immuno-labeled complexes or
ligand-detection reagents, the sample is prepared in a way that
makes the target (ligand), which is determined by the end user, in
the sample accessible to the immuno-labeled complexes or
ligand-binding antibody. Typically, the samples used in the
invention are comprised of tissue, cells, cell extracts, cell
homogenates, purified or reconstituted proteins, recombinant
proteins, biological fluids, or synthesized proteins. Large
macromolecules such as immuno-labeled complexes tend to be
impermeant to membranes of live biological cells. Treatments that
permeabilize the plasma membrane, such as electroporation, shock
treatments, or high extracellular ATP, can be used to introduce the
immuno-labeled complexes into cells. Alternatively, the
immuno-labeled complexes or ligand-detection reagents can be
physically inserted into cells, e.g. by pressure microinjection,
scrape loading, patch-clamp methods, or phagocytosis. However, the
desired target (ligand) may require purification or separation
prior to addition of the immuno-labeled complexes or
ligand-detection reagent, which will depend on the way the
antigenic determinants are contained in the sample. For example,
when the sample is to be separated on a SDS-polyacrylamide gel the
sample is first equilibrated in an appropriate buffer, such as a
SDS-sample buffer containing Tris, glycerol, DTT, SDS, and
bromophenol blue.
[0216] When the sample contains purified target materials, the
purified target materials may still be mixtures of different
materials. For example, purified protein or nucleic acid mixtures
may contain several different proteins or nucleic acids.
Alternatively, the purified target materials may be electrophoresed
on gels such as agarose or polyacrylamide gels to provide
individual species of target materials that may be subsequently
blotted onto a polymeric membrane or detected within the gel
matrix. Preparation of a sample containing purified nucleic acids
or proteins generally includes denaturation and neutralization. DNA
may be denatured by incubation with base (such as sodium hydroxide)
or heat. RNA is also denatured by heating (for dot blots) or by
electrophoresing in the presence of denaturants such as urea,
glyoxal, or formaldehyde, rather than through exposure to base (for
Northern blots). Proteins are denatured by heating in combination
with incubation or electrophoresis in the presence of detergents
such as sodium dodecyl sulfate. The nucleic acids are then
neutralized by the addition of an acid (e.g., hydrochloric acid),
chilling, or addition of buffer (e.g., Tris, phosphate or citrate
buffer), as appropriate.
[0217] Preferably, the preparation of a sample containing purified
target (ligand) materials further comprises immobilization of the
target materials on a solid or semi-solid support. Purified nucleic
acids are generally spotted onto filter membranes such as
nitrocellulose filters or nylon membranes in the presence of
appropriate salts (such as sodium chloride or ammonium acetate) for
DNA spot blots. Alternatively, the purified nucleic acids are
transferred to nitrocellulose filters by capillary blotting or
electroblotting under appropriate buffer conditions (for Northern
or Southern blots). To permanently bind nucleic acids to the filter
membranes, standard cross-linking techniques are used (for example,
nitrocellulose filters are baked at 80.degree. C. in vacuum; nylon
membranes are subjected to illumination with 360 nm light). The
filter membranes are then incubated with solutions designed to
prevent nonspecific binding of the nucleic acid probe (such as BSA,
casein hydrolysate, single-stranded nucleic acids from a species
not related to the probe, etc.) and hybridized to probes in a
similar solution. Purified proteins are generally spotted onto
nitrocellulose or nylon filter membranes after heat and/or
detergent denaturation. Alternatively, the purified proteins are
transferred to filter membranes by capillary blotting or
electroblotting under appropriate buffer conditions (for Western
blots). Nonspecifically bound probe is washed from the filters with
a solution such as saline-citrate or phosphate buffer. Filters are
again blocked, to prevent nonspecific adherence of immuno-labeled
complexes. Finally, samples are mixed with immuno-labeled complexes
or ligand-detection reagents. Nonspecifically bound immuno-labeled
complexes or ligand-binding antibodies are typically removed by
washing.
[0218] When the sample contains cellular nucleic acids (such as
chromosomal or plasmid-borne genes within cells, RNA or DNA viruses
or mycoplasma infecting cells, or intracellular RNA) or proteins,
preparation of the sample involves lysing or permeabilizing the
cell, in addition to the denaturation and neutralization already
described. Cells are lysed by exposure to agents such as detergent
(for example sodium dodecyl sulfate, Tween, sarkosyl, or Triton),
lysozyme, base (for example sodium, lithium, or potassium
hydroxide), chloroform, or heat. Cells are permeabilized by
conventional methods, such as by formaldehyde in buffer.
[0219] As with samples containing purified target (ligand)
materials, preparation of the sample containing cellular target
materials typically further comprises immobilization of the target
materials on a surface such as a solid or semi-solid matrix. The
targets may be arrayed on the support in a regular pattern or
randomly. These supports include such materials as slides,
polymeric beads including latex, optical fibers, and membranes. The
beads are preferably fluorescent or nonfluorescent polystyrene, the
slides and optical fibers are preferably glass or plastic, and the
membrane is preferably poly(vinylidene difluoride) or
nitrocellulose. Thus, for example, when the sample contains lysed
cells, cells in suspension are spotted onto or filtered through
nitrocellulose or nylon membranes, or colonies of cells are grown
directly on membranes that are in contact with appropriate growth
media, and the cellular components, such as proteins and nucleic
acids, are permanently bound to filters as described above.
Permeabilized cells are typically fixed on microscope slides with
known techniques used for in situ hybridization and hybridization
to chromosome "squashes" and "spreads," (e.g., with a reagent such
as formaldehyde in a buffered solution). Alternatively, the samples
used may be in a gel or solution.
[0220] In a particular aspect of the invention, the sample
comprises of cells in a fluid, such as ascites, hybridoma
supernatant, or serum, wherein the presence or absence of the
target in such cells is detected by using an automated instrument
that sorts cells according to the detectable fluorescence response
of the detectable moieties in the immunolabeling complexes bound to
such cells, such as by fluorescence activated cell sorting (FACS).
For methods using flow cytometry a cell population typically
comprises individually isolated cells that have been isolated from
other proteins and connective tissue by means well known in the
art. For example, lymphocyte cells are isolated from blood using
centrifugation and a density gradient. The cells are washed and
pelleted and the labeling solution added to the pelleted cells.
[0221] At any time after addition of the immuno-labeled complex or
ligand-detection reagent to the sample, the sample is illuminated
with a wavelength of light selected to give a detectable optical
response, and observed with a means for detecting the optical
response. Equipment that is useful for illuminating the reporter
molecule and/or label of the present invention includes, but is not
limited to, hand-held ultraviolet lamps, mercury arc lamps, xenon
lamps, lasers and laser diodes. These illumination sources are
optically integrated into laser scanners, fluorescent microplate
readers or standard or microfluorometers. The degree and/or
location of signal, compared with a standard or expected response,
indicates whether and to what degree the sample possesses a given
characteristic, i.e. desired target.
[0222] The optical response is optionally detected by visual
inspection, or by use of any of the following devices: CCD camera,
video camera, photographic film, laser-scanning devices,
fluorometers, photodiodes, quantum counters, epifluorescence
microscopes, scanning microscopes, flow cytometers, fluorescence
microplate readers, or by means for amplifying the signal such as
photomultiplier tubes. Where the sample is examined using a flow
cytometer, examination of the sample optionally includes sorting
portions of the sample according to their fluorescence
response.
[0223] When an indirectly detectable label is used then the step of
illuminating typically includes the addition of a reagent that
facilitates a detectable signal such as colorimetric enzyme
substrate. Radioisotopes are also considered indirectly detectable
wherein an additional reagent is not required but instead the
radioisotope must be exposed to X-ray film or some other mechanism
for recording and measuring the radioisotope signal. This can also
be true for some chemiluminescent signals that are best observed
after expose to film.
III. Kits of the Invention
[0224] Suitable kits for preparing an immuno-labeled complex or
ligand-detection reagent and for detection of a target (ligand) in
a sample also form part of the invention. Such kits can be prepared
from readily available materials and reagents and can come in a
variety of embodiments. The contents of the kit will depend on the
design of the assay protocol or reagent for detection or
measurement. Generally, the kits will contain instructions,
appropriate reagents and labels, and solid supports, as needed.
Typically, instructions include a tangible expression describing
the reagent concentration or at least one assay method parameter
such as the relative amounts of reagent and sample to be admixed,
maintenance time periods for reagent/sample admixtures,
temperature, buffer conditions and the like to allow the user to
carry out any one of the methods or preparations described
above.
[0225] A preferred kit of the present invention comprises: a) a
labeling solution comprising a labeling reagent that is
independently attached to one or more labels and b) a solution
comprising a capture reagent. A preferred embodiment of this kit
provides a labeling reagent that is anti-Fc Fab fragment, protein G
or protein G complexed with albumin. In a more particular
embodiment of this kit, the capture component is purified mouse IgG
or non-immune mouse serum and the albumin is human albumin, bovine
serum albumin, or ovalbumin. In a more preferred embodiment the
albumin is ovalbumin. The labeling solution is either a homogenous
mixture of labeling reagents or comprises a pooled subset of
labeling reagents. Alternatively the kit comprises a panel of
labeling reagent subsets that can be used to make a subset of
immuno-labeled complexes.
[0226] Additionally the kits may comprise one or more additional
components that include (a) stains for characterization of cellular
organelles, cell viability, or cell proliferation state, (b) enzyme
substrates or (c) enzyme conjugates such as avidin-HRP.
[0227] A wide variety of kits and components can be prepared
according to the present invention, depending upon the intended
user of the kit and the particular needs of the user. It is
understood by one skilled in the art, that any of the labeling
reagents contemplated by the present invention can be used to in a
labeling solution to be included in a kit. The labeling reagents
are not intended to be limited to only the described preferred
embodiments.
[0228] In one aspect of invention a kit for the detection of a
target ligand comprises a ligand analog, a labeling reagent and
optionally a ligand-binding antibody. In a preferred embodiment the
kit comprises a labeling reagent is preferably anti-Fc Fab fragment
or anti-kappa Fab fragment and is bound to a fluorophore or
quenching moiety. In another embodiment the kit comprises a
ligand-binding antibody that has affinity for a phosphorylated
biomolecule and an appropriately matched ligand analog that is
selected from the group consisting of a phosphotyramide,
phosphoserine, a phosphotyrosinamide, a phosphoethanolamine, a
phosphorylated kinase peptide substrate, a phosphatase substrate,
or a phosphorylated peptide.
[0229] A wide variety of kits and components can be prepared
according to the present invention, depending upon the intended
user of the kit and the particular needs of the user.
IV. Applications
[0230] The instant invention has useful applications in basic
research, high-throughput screening, immunohistochemistry,
fluorescence in situ hybridization (FISH), microarray technology,
flow cytometry, diagnostics, and medical therapeutics. The
invention can be used in a variety of assay formats for diagnostic
applications in the disciplines of microbiology, immunology,
hematology and blood transfusion, tissue pathology, forensic
pathology, and veterinary pathology. The invention is particularly
useful in the characterization and selection of optimized
antibodies from hybridoma supernatants. Additionally, the invention
can be used to deliver therapeutics to a specific target. In
general, the current invention provides a versatile and convenient
method to enhance any assay that uses an antibody as part of its
detection methodology.
[0231] The instant invention can be used to study biological
phenomena, such as, for example, cell proliferation, signal
transduction in cells, or apoptosis. For illustration purposes only
and not limitation, one could study thymidine analog
5-bromo-2'-deoxyuridine (BrdU) incorporation. BrdU is a marker for
both cell proliferation and apoptosis, as it is readily
incorporated into newly synthesized DNA that has progressed through
the S-phase of the cell cycle and also into DNA break sites by
deoxynucleotidyl transferase (TdT). Anti-BrdU antibodies are used
to detect cells marked by BrdU incorporation. By being able to
directly label the anti-BrdU antibodies, the current invention
provides a convenient method to allow for detection of the
incorporated BrdU by conventional immunohistochemistry or
fluorescence, depending on detection method required.
[0232] Additionally, the current invention has the advantage of
allowing staining for multiple targets in one cocktail, thereby
reducing the need for more samples or processing steps per
experiment. This is particularly important when analyzing precious
samples (e.g., pediatric samples, leukocytes isolated from
biopsies, rare antigen-specific lymphocytes and mouse tissues that
yield a small number of cells). Although it is currently possible
to simultaneously measure up to 11 distinct fluorescent colors
through a convoluted series of novel developments in flow cytometry
hardware, software, and dye chemistry, the use of these advances
has been severely limited by the lack of commercial availability of
spectrally distinct directly labeled primary and secondary
antibodies. Although labeled secondary antibodies directed at
individual isotype-specific targeting antibodies (e.g., anti-IgG,
isotype antibodies) exist, it is not possible to use this type of
labeled antibody to detect more than one of the same isotype of an
antibody (e.g., an IgG.sub.1 isotype antibody) in a single sample
due to cross-reactivity. The current invention overcomes these
limitations by providing for a convenient and extremely versatile
method of rapidly labeling either small or large quantities of any
primary antibody including primary antibodies of the same isotype
to be used in, for example, multicolor flow cytometry and on
Western blots. This advance in multicolor systems has a number of
advantages over current two- and three-color flow cytometric
measurements. For example, no combination of one-color stains can
accurately enumerate or be used to isolate CD3.sup.+ CD4.sup.+
CD8.sup.- T cells (excluding, for example CD3.sup.+ CD4.sup.+
CD8.sup.+ T cells and small CD4.sup.+ monocytes). The use of cell
membrane markers to study leukocyte composition in blood and tissue
serves as an example of an analytical monoclonal antibody
application, particularly in combination with flow cytometry. It is
also the example most relevant to studies of the immune system,
because the cellular composition of blood and lymphoid tissue
provides a `window`, allowing the analysis and monitoring of the
immune system.
[0233] The methods of the invention can also be used in
immunofluorescence histochemistry. This technique involves the use
of antibodies labeled with fluorophores to detect substances within
a specimen. The pathologist derives a great deal of information of
diagnostic value by examining thin sections of tissue in the
microscope. Tissue pathology is particularly relevant to, for
example, the early diagnosis of cancer or premalignant states, and
to the assessment of immunologically mediated disorders, including
inflammation and transplant rejection. The problems associated with
immunofluorescence histochemistry, however, stem from the
limitations of the methods currently available for use in such
application. For example, directly labeling an antibody can result
in antibody inactivation and requires a relatively large of amount
of antibody and time to do the conjugation. It is also expensive
and impractical to prepare directly labeled antibodies having
variable degrees of label substitution. Similarly, indirect
labeling of an antibody has problems, such as lack of secondary
antibody specificity, and reliance upon primary antibody
differences, including antibody isotypes and available
fluorophores, to do multicolor labeling. Secondary antibody
labeling is not practical where the primary antibody is from the
same species or of the same isotypes. Combinations of fluorophores
or other detectable labels on the same target-binding antibody,
which can be readily prepared in multiple mixtures by the methods
on this invention, greatly increase the number of distinguishable
signals in multicolor protocols. Lack of secondary antibody
specificity arises when the specimen containing the targeted moiety
and target-binding antibody are from homologous species. For
example, BrdU-labeled DNA in rodent tissue is detected by
immunohistochemical staining. The target-binding antibody is
conventionally mouse anti-BrdU, and the detecting antibody system
uses an anti-mouse immunoglobulin antibody, labeled with
fluorescein. Because there is homology between mouse immunoglobulin
and immunoglobulins from a number of rodent species (for example,
rats, mice, hamsters, etc.), the detecting antibody not only binds
to the target-binding antibody, but also nonspecifically binds to
immunoglobulin in the tissue. The current invention eliminates this
problem by pre-forming the immunolabeling complex and allows for a
simple, rapid and convenient method to proceed with labeling with
two, three or more fluorescent antibodies in one experiment. Very
significantly, it can always be used with primary antibodies of
either the same or different isotype, and always on tissue of the
same or similar species as the primary antibody.
[0234] The instant invention also has application in the field of
microarrays. Microarray technology is a powerful platform for
biological exploration (Schena (Ed.), Microarray Biochip
Technology, (2000)). Many current applications of arrays, also
known as "biochips," can be used in functional genomics as
scientists seek characteristic patterns of gene expression in
different physiopathological states or tissues. A common method
used in gene and protein microarray technology involves the use of
biotin, digoxigenin (DIG), or dinitrophenyl (DNP) as an epitope or
a "tag" such as an oligohistidine, glutathione transferase,
hemagglutinin (HA), or c-myc. In this case a detectably labeled
anti-biotin, anti-DIG, anti-DNP, anti-oligohistidine,
anti-glutathione transferase, anti-HA, or anti-c-myc is used as the
detection reagent. The instant invention allows for the use of
multiple fluorophore- or enzyme-labeled antibodies, thereby greatly
expanding the detection modalities and also providing for enhanced
multiplexing and two-dimensional analysis capabilities.
[0235] Similarly, the invention can be used with protein
microarrays and on Western blots. Protein microarrays can provide a
practical means to characterize patterns of variation in hundreds
of thousands of different proteins in clinical or research
applications. Antibody arrays have been successfully employed that
used a set of 115 antibody/antigen pairs for detection and
quantitation of multiple proteins in complex mixtures (Haab et al.,
Genome Biology, 2, 4.1 (2001)). However, protein microarrays use
very low sample volumes, which historically have significantly
limited the use of antibody technology for this application. The
invention of the application readily overcomes this limitation and
provides a means to label antibodies with the fluorescent dyes
using a very low sample volume and to automate formation of the
staining complex and the staining process.
[0236] The present invention also provides a means for the specific
detection, monitoring, and/or treatment of disease and contemplates
the use of immunolabeling complexes to detect the presence of
particular targets in vitro. In such immunoassays, the sample may
be utilized in liquid phase, in a gel, or bound to a solid-phase
carrier, such as an array of fluorophore-labeled microspheres
(e.g., U.S. Pat. Nos. 5,981,180 and 5,736,330). For example, a
sample can be attached to a polymer, such as aminodextran, in order
to link the sample to an insoluble support such as a polymer-coated
bead, plate, or tube. For instance, but not as a limitation, using
the methods of the present invention in an in vitro assay,
antibodies that specifically recognize an antigen of a particular
disease are used to determine the presence and amounts of this
antigen.
[0237] Likewise, the immunolabeling complexes of the present
invention can be used to detect the presence of a particular target
in tissue sections prepared from a histological specimen.
Preferably, the tissue to be assayed will be obtained by surgical
procedures, e.g., biopsy. The excised tissue will be assayed by
procedures generally known in the art, e.g. immunohistochemistry,
for the presence of a desired target that is recognized by an
immunolabeling complex, as described above. The tissue may be fixed
or frozen to permit histological sectioning. The immunolabeling
complex may be labeled, for example with a dye or fluorescent
label, chemical, heavy metal or radioactive marker to permit the
detection and localization of the target-binding antibody in the
assayed tissue. In situ detection can be accomplished by applying a
detectable immunolabeling complex to the tissue sections. In situ
detection can be used to determine the presence of a particular
target and to determine the distribution of the target in the
examined tissue. General techniques of in situ detection are well
known to those of ordinary skill. See, for example, Ponder, "Cell
Marking Techniques and Their Application," in MAMMALIAN
DEVELOPMENT: A PRACTICAL APPROACH, Monk (ed.), 115 (1987).
[0238] For diagnosing and classifying disease types, tissues are
probed with an immuno-labeled complex, as defined above, that
comprises a target-binding antibody to a target antigen associated
with the disease, e.g., by immunohistochemical methods. Where the
disease antigen is present in body fluids, such immuno-labeled
complexes comprising a target-binding antibody to the disease
antigen are preferably used in immunoassays to detect a secreted
disease antigen target.
[0239] Detection can be by a variety of methods including, for
example, but not limited to, flow cytometry and diagnostic imaging.
When using flow cytometry for the detection method, the use of
microspheres, beads, or other particles as solid supports for
antigen-antibody reactions in order to detect antigens or
antibodies in serum and other body fluids is particularly
attractive. Flow cytometers have the capacity to detect particle
size and light scattering differences and are highly sensitive
fluorescence detectors. Microfluidic devices provide a means to
perform flow-based analyses on very small samples.
[0240] Alternatively, one can use diagnostic imaging. The method of
diagnostic imaging with radiolabeled antibodies is well known. See,
for example, Srivastava (ed.), RADIOLABELED MONOCLONAL ANTIBODIES
FOR IMAGING AND THERAPY, Plenum Press (1988); Chase, "Medical
Applications of Radioisotopes," in REMINGTON'S PHARMACEUTICAL
SCIENCES, 18.sup.th Edition, Gennaro et al. (eds.) Mack Publishing
Co., 624 (1990); and Brown, "Clinical Use of Monoclonal
Antibodies," in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al. (eds.),
Chapman & Hall, 227 (1993). This technique, also known as
immunoscintigraphy, uses a gamma camera to detect the location of
gamma-emitting radioisotopes conjugated to antibodies. Diagnostic
imaging is used, in particular, to diagnose cardiovascular disease
and infectious disease.
[0241] Thus, the present invention contemplates the use of
immuno-labeled complexes to diagnose cardiovascular disease. For
example, immuno-labeled complexes comprising anti-myosin antibodies
can be used for imaging myocardial necrosis associated with acute
myocardial infarction. Immuno-labeled complexes comprising
antibodies that bind platelets and fibrin can be used for imaging
deep-vein thrombosis. Moreover, immuno-labeled complexes comprising
antibodies that bind to activated platelets can be used for imaging
atherosclerotic plaque.
[0242] Immuno-labeled complexes of the present invention also can
be used in the diagnosis of infectious diseases. For example,
immuno-labeled complexes comprising antibodies that bind specific
bacterial antigens can be used to localize abscesses. In addition,
immuno-labeled complexes comprising antibodies that bind
granulocytes and inflammatory leukocytes can be used to localize
sites of bacterial infection. Similarly, the immuno-labeled
complexes of the present invention can be used to detect signal
transduction in cells, the products of signal transduction, and
defects, inhibitors, and activators of signal transduction.
[0243] Numerous studies have evaluated the use of antibodies for
scintigraphic detection of cancer. Investigations have covered the
major types of solid tumors such as melanoma, colorectal carcinoma,
ovarian carcinoma, breast carcinoma, sarcoma, and lung carcinoma.
Thus, the present invention contemplates the detection of cancer
using immuno-labeled complexes comprising antibodies that bind
tumor markers (targets) to detect cancer. Examples of such tumor
markers include carcinoembryonic antigen, .alpha.-fetoprotein,
oncogene products, tumor-associated cell surface antigens, and
necrosis-associated intracellular antigens. In addition to
diagnosis, antibody imaging can be used to monitor therapeutic
responses, detect recurrences of a disease, and guide subsequent
clinical decisions and surgical procedures. In vivo diagnostic
imaging using fluorescent complexes that absorb and emit light in
the near infrared (such as those of the Alexa Fluor 700 and Alexa
Fluor 750 dyes) is also known.
EXAMPLES
[0244] The following examples describe specific aspects of the
invention to illustrate the invention and to provide a description
of the methods for those of skill in the art. The examples should
not be construed as limiting the invention, as the examples merely
provide specific methodology useful in understanding and practicing
the invention.
Example 1
Preparation of Fc Antigen
[0245] Purified mouse and rabbit IgG was fragmented with the
proteolytic enzyme papain (CURRENT PROTOCOLS IN CELL BIOLOGY,
16.4.1-16.4.10 (2000)). A 12 mL solution of mouse IgG was prepared
at .about.2 mg/mL in phosphate-buffered saline (PBS). A solution
containing 0.1 mg of papain in digestion buffer (PBS, 0.02 M EDTA,
0.02 M cysteine) was added to the antibody and allowed to react at
37.degree. C. for 16 hours. The digestion was terminated by the
addition 20 .mu.L of 0.3 M iodoacetamide in PBS. The fragments were
dialyzed against 2 L of PBS for 16 hours at 4.degree. C. The Fc
fragment was purified on a protein G-Sepharose CL-4B column. The
bound fraction containing the Fc fragment was eluted from the
column using 50-100 mM glycine/HCl buffer, pH 2.5-2.8. The eluate
was collected in 1 mL fractions. The pH of the protein fractions
was immediately raised to neutral by addition of 100 .mu.L of
either 500 mM phosphate or Tris buffer, pH 7.6, to each 1 mL
fraction. The solution was then loaded onto a Sephacryl S-200
Superfine size-exclusion column and fractions corresponding to a
molecular weight of .about.50 kDa were collected and analyzed by
SDS-PAGE and HPLC.
Example 2
Production of Anti-Fc Antibodies
[0246] Polyclonal antibodies specific for the Fc region of an
antibody were raised in goats against the purified FC region of an
antibody from a different species (Example 1). Methods of
immunizing animals are well known in the art, and suitable
immunization protocols and immunogen concentrations can be readily
determined by those skilled in the art (Current Protocols in
Immunology 2.4.1-9 (1995); ILAR Journal 37, 93 (1995)). Briefly,
individual goats were immunized with purified mouse Fc or purified
rabbit Fc fragments. The initial immunization in 50% Freund's
complete adjuvant (1000 .mu.g conjugate (half subcutaneous, half
intramuscularly)) was followed by 500 .mu.g conjugate per goat in
Freund's incomplete adjuvant two and four weeks later and at
monthly intervals thereafter. Antibodies were purified from serum
using protein A-Sepharose chromatography. Antibodies against mouse
Fc isotypes can be prepared by starting with isotype-selected mouse
Fc antigens. Rabbits have a single Fc isotype. Characterization of
the selectivity and cross-reactivity of isotype-specific antibodies
is by standard techniques, including HPLC.
Example 3
Preparation of Fab Fragments
[0247] Fragmentation of the goat anti-(mouse Fc) antibody to the
monovalent Fab fragment was carried out using the proteolytic
enzyme, papain, as described in Example 1. Following dialysis
against PBS, the Fab fragment was purified on a protein A-Sepharose
CL-4B column. The unbound fraction containing the Fab fragment and
the papain was collected. This solution was then loaded onto a
Sephacryl S-200 Superfine size-exclusion column and fractions
corresponding to a molecular weight of .about.50 kDa were collected
and analyzed by SDS-PAGE. The Fab fragments of goat anti-(rabbit
Fc) can be prepared similarly.
Example 4
Preparation of the Labeled Antibody Immunoglobulin-Binding Protein
or the Non-Antibody Immunoglobulin-Binding Peptide and Protein
Conjugates in Homogeneous Solution
[0248] Conjugates of antibody immunoglobulin-binding protein or the
non-antibody immunoglobulin-binding peptides or proteins with low
molecular weight dyes and haptens such as biotin or digoxigenin are
typically prepared from succinimidyl esters of the dye or hapten,
although reactive dyes and haptens having other protein-reactive
functional groups are also suitable. The typical method for protein
conjugation with succinimidyl esters is as follows. Variations in
molar ratios of dye-to-protein, protein concentration, time,
temperature, buffer composition and other variables that are well
known in the art are possible that still yield useful
conjugates.
[0249] A protein solution of the Fab fragment of goat anti-(rabbit
Fc), goat anti-(mouse Fc), protein A, protein G, or protein L or an
immunoglobulin-binding peptide (e.g., a peptide identified by
screening a library of peptides) is prepared at .about.10 mg/mL in
0.1 M sodium bicarbonate (pH .about.8.3). The labeling reagents are
dissolved in a suitable solvent such as DMF at .about.10 mg/mL.
Predetermined amounts of the labeling reagents are added to the
protein solution with stirring. A molar ratio of 10 moles of dye to
1 mole of protein is typical, though the optimal amount can be
varied with the particular labeling reagent, the protein being
labeled and the protein's concentration. The optimal ratio was
determined empirically. When optimizing the fluorescence yield and
determining the effect of degree of substitution (DOS) on the
conjugate's brightness, it is typical to vary the ratio of reactive
dye to protein over a several-fold range. The reaction mixture is
incubated at room temperature for a period that is typically one
hour or on ice for several hours. The dye-protein conjugate is
typically separated from unreacted reagents by size-exclusion
chromatography, such as on BIO-RAD P-30 resin equilibrated with
PBS. The initial, protein-containing band is collected and the DOS
is determined from the absorbance at the absorbance maximum of each
fluorophore, using the extinction coefficient of the free
fluorophore. The DOS of nonchromophoric labels, such as biotin, is
determined as described in Haugland (Haugland et al., Meth. Mol.
Biol. 45, 205 (1995); Haugland, Meth. Mol. Biol. 45, 223 (1995);
Haugland, Meth. Mol. Biol. 45, 235 (1995); Haugland, Current
Protocols in Cell Biol. 16.5.1-16.5.22 (2000)). Using the above
procedures, conjugates of goat anti-(mouse Fc) and goat
anti-(rabbit Fc) were prepared with several different Alexa Fluor
dyes, with Oregon Green dyes, with biotin-X succinimidyl ester,
with desthiobiotin-X succinimidyl ester, with succinimidyl
3-(2-pyridyldithio)propionate (SPDP) and with succinimidyl
trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (SMCC).
[0250] Some dye conjugates of protein A and protein G, including
those of some Alexa Fluor dyes, are commercially available, such as
from Molecular Probes. Inc. (Eugene, Oreg.). The interspecies
specificity and approximate affinity of some other non-antibody
immunoglobulin-binding proteins bind to segments of a target
antibody, such as that of protein A and protein G are known
(Langone, Adv. Immunol. 32, 157 (1982); Surolia et al., Trends
Biochem. Sci. 7, 74 (1982); Notani et al., J. Histochem. Cytochem.
27, 1438 (1979); Goding, J. Immunol. Meth. 20, 241 (1978); J.
Immunol. Meth. 127, 215 (1990); Bjorck et al., J. Immunol. 133, 969
(1984)).
[0251] In addition, labeling reagents (goat Fab anti-(mouse Fc),
goat Fab anti-(mouse lambda light chain), goat Fab anti-(mouse
kappa light chain), protein A, protein G, protein L, lectins,
single-chain fragment variable antibodies (ScFv)) conjugated to the
detectable labels of R-phycoerythrin (R-PE), allophycocyanin (APC),
tandem conjugates of phycobiliproteins with chemical dyes including
several Alexa Fluor dyes, horseradish peroxidase (HRP), Coprinus
cinereus peroxidase, Arthromyces ramosus peroxidase, glucose
oxidase and alkaline phosphatase (AP) were or can be prepared by
standard means (Haugland et al., Meth. Mol. Biol. 45, 205 (1995);
Haugland, Meth. Mol. Biol. 45, 223 (1995); Haugland, Meth. Mol.
Biol. 45, 235 (1995); Haugland, Current Protocols in Cell Biol
16.5.1-16.5.22 (2000)). Fusion proteins, such as of protein G or
protein A with detectable labels such as luciferin, aequorin,
green-fluorescent protein and alkaline phosphatase are also known
that are suitable for practice of the invention (Sun et al., J.
Immunol. Meth. 152, 43 (1992); Eliasson et al., J. Biol. Chem. 263,
4323 (1988); Eliasson et al., J. Immunol. 142, 575 (1989)).
[0252] Immunoglobulin heavy and light chains, like most secreted
and membrane bound proteins, are synthesized on membrane-bound
ribosomes in the rough endoplasmic endoplasmic reticulum where
N-linked glycosylation occurs. The specificity of lectins for
carbohydrates, including N-linked glycoproteins, is also known (EY
laboratories, Inc. Lectin Conjugates Catalog, 1998).
Example 5
Preparation of the Labeled Antibody Immunoglobulin-Binding Protein
or the Non-Antibody Immunoglobulin-Binding Peptide and Protein
Conjugates While Bound to an Affinity Matrix
[0253] Unlabeled Fab fragment for goat anti-(mouse Fc) (prepared as
in Example 3) was bound to agarose-immobilized mouse IgG for one
hour. Following a wash step with bicarbonate buffer, pH 8.3, the
complex of immobilized IgG and unlabeled Fab was labeled for one
hour at room temperature with the succinimidyl ester of the
amine-reactive label. Unconjugated dye was eluted with bicarbonate
buffer, and then the covalently labeled Fab fragment was eluted
with 50-100 mM glycine/HCl buffer, pH 2.5-2.8. The eluate was
collected in 1 mL fractions. The pH of the protein fractions was
immediately raised to neutral by addition of 100 .mu.L of either
500 mM phosphate or Tris buffer, pH 7.6, to each 1 mL fraction.
Variations of the reagent concentrations, labeling times, buffer
composition, elution methods and other variables are possible that
can yield equivalent results. Conjugates of the Fab fragment of
goat anti-(rabbit Fc) and of protein G and protein A are prepared
similarly.
Example 6
Comparison of the Alexa Fluor 488 Dye-Labeled Fab Fragments of Goat
Anti-(Mouse Fc) Prepared as in Example 4 and as in Example 5
[0254] Conjugates of the Fab fragment of goat anti-(mouse Fc) with
the Alexa Fluor 488 succinimidyl ester were separately prepared, as
described in Examples 4 and 5. The conjugates had estimated degrees
of substitution of .about.1.9 (labeled as in Example 4) and
.about.3.0 (labeled as in Example 5), respectively, and virtually
identical absorption and emission spectral maxima. When excited at
488 nm, conjugates prepared using the fragment prepared as
described in Example 5 were about 3.2-times more fluorescent than
using the fragments that were prepared in Example 4 (FIG. 8) as
detected by flow cytometry when bound to CD3 on Jurkat T cells.
Similar results were observed with other dyes.
Example 7
Preparation of a Labeling Reagent from Protein G and Albumins
[0255] Native protein G has a high affinity binding (nanomolar)
site for albumins, in particular ovalbumin. Equal weights of
protein G and Texas Red ovalbumin (Molecular Probes. Inc.) were
dissolved in PBS, pH 7.5. After one hour, the resulting complex was
separated on a Sephacryl S-200 Superfine size-exclusion column and
analyzed by SDS-PAGE and HPLC. Alternatively, the protein G is
combined with a labeled albumin while the protein G is immobilized
on any of the several immunoglobulins to which it binds, and the
excess labeled albumin is washed away preceding elution of the
albumin-labeled protein G complex from the matrix.
Example 8
Preparation of an Immunolabeling Complex on a Very Small Scale
[0256] Submicrogram quantities of a target-binding antibody were
complexed with submicrograms of a labeling reagent in varying molar
ratios of between about 1:1 and 1:20 to prepare an immunolabeling
complex that was suitable for staining a sample. For instance, 0.1
.mu.g of mouse monoclonal anti-.alpha.-tubulin in 1 .mu.L PBS with
0.1% BSA was complexed with 0.5 .mu.g of the Alexa Fluor 488
dye-labeled Fab fragment of goat anti-(mouse Fc) (prepared as in
Example 4) or with 0.1 .mu.g of the Alexa Fluor 488 dye-labeled Fab
fragment of goat anti-(mouse Fc) (prepared as in Example 5) in 5
.mu.L of PBS for 10 minutes at room temperature. The immunolabeling
complex can be used immediately for staining tubulin in fixed-cell
preparations (Example 16) or any excess unbound Alexa Fluor 488
dye-labeled Fab fragment of goat anti-(mouse Fc) in the
immunolabeling complex can be captured with non-immune mouse IgG
(Example 9) for combination with other antibody conjugates,
including those of targeting antibodies that have been directly
conjugated to other labels. Rabbit antibodies were labeled
similarly using labeled goat anti-(rabbit Fc). Labeling of
targeting antibodies with a labeled protein A, protein L, protein
G, protein G complexed with a labeled albumin, or other
immunoglobulin-binding peptides or proteins proceeds similarly. In
the case of a mouse (or rat) monoclonal antibody, it is preferred
to use a labeled protein that is selective for the specific isotype
of the primary antibody (e.g. anti-(mouse IgG.sub.1) for a mouse
IgG.sub.1 isotype primary antibody). Although some cross-reactivity
for other mouse (or rat) isotypes was observed using a goat
antibody that was selective for mouse IgG.sub.1 isotype monoclonal
antibodies, routine and optimal use for labeling unmatched mouse
isotypes required greater amounts of immunolabeling complexes and
was somewhat less reliable.
Example 9
Capturing Excess Immunoglobulin-Binding Protein by a Capturing
Component
[0257] Immunolabeling complexes were prepared as described in
Example 8. To the immunolabeling complex was added to each tube 25
.mu.L of a 14.1 mg/mL stock solution of unlabeled mouse IgG to
capture excess immunolabeling complexes. As shown in FIG. 1, not
all of the immunoglobulin-binding protein was necessarily complexed
with the target-binding antibody to form an immunolabeling complex.
Consequently, particularly for applications in which labeling
complexes of multiple primary antibodies from the same species
(e.g. mouse monoclonal antibodies) or crossreacting species (e.g.
mouse and human antibodies, FIG. 2, Table 1) were to be used
simultaneously or sequentially, it is necessary to quench or
otherwise remove any excess immunoglobulin-binding protein by use
of a capturing component or by other means to avoid inappropriate
labeling of the sample. The most effective capturing component to
capture excess immunoglobulin-binding protein is one that contains
the binding site of the targeting agent. For instance, whole mouse
IgG or mouse serum was shown to be an effective and inexpensive
reagent when the immunoglobulin-binding protein was bound to a
segment of a mouse monoclonal antibody. The mouse IgG was added in
excess to the amount of immunoglobulin-binding protein and
incubated for a period of approximately 1-5 minutes, or longer.
[0258] It is preferred to prepare the immunolabeling complex and
then add the capturing component shortly before the experiment. The
rapid quenching effect permits this to be done within minutes of
performing labeling of the sample by the immunolabeling complex. If
desired, the excess capturing component can be removed following
labeling of the sample by a simple wash step. Alternatively,
fixation of the stained sample by aldehyde-based fixatives or other
reagents or methods subsequent to incubation with the
immunolabeling complex can provide permanent immobilization of the
immunolabeling complex on its target in the sample. As an
alternative to adding a soluble capturing component to the
immunolabeling complex, the capturing component can be immobilized
on an insoluble matrix such as agarose and the immunolabeling
complex contacted with that matrix. A preferred matrix when
labeling mouse antibodies to mouse antigens is mouse IgG
immobilized on agarose. Excess labeled anti-rabbit antibodies can
be captured using rabbit IgG that is free in solution or
immobilized. Alternatively, the immunolabeling complex can be
separated from any capturing component by chromatographic or
electrophoretic means.
Example 10
HPLC Analysis of a Labeling Complex
[0259] In order to analyze the success and extent of complex
formation of the labeling reagent with the target-binding antibody,
size exclusion HPLC of the samples was performed. For instance, a
complex of Alexa Fluor 488 dye-labeled goat Fab anti-(mouse Fc)
with a monoclonal mouse anti-tubulin in molar ratios of
approximately 1:1, 3:1, 5:1 and 10:1. These were separated by
analytical HPLC using a BioSep S-3000 column and eluting with 0.1 M
NaP.sub.i, 0.1 M NaCl, pH 6.8, at a flow rate of 0.25 mLs/min. An
example of the separation using the 5:1 molar ratio (FIG. 6)
demonstrates that, using this molar ratio, formation of the labeled
complex is essentially quantitative.
Example 11
Cross-Reactivity of Goat Fab Anti-(Mouse Fc) to Other Species of
IgG
[0260] Microplates were equilibrated overnight with IgG from a
mouse or non-mouse species, and then further blocked with BSA.
Variable amounts of the biotinylated Fab fragment of goat
anti-(mouse Fc) were added to each well and allowed to bind. After
washing, streptavidin-HRP and the Amplex Red peroxidase substrate
were added. HRP activity was detected by the addition of
H.sub.2O.sub.2 using the Amplex Red Peroxidase Assay Kit (Molecular
Probes, Inc., Eugene, Oreg.). Reactions containing 200 .mu.M Amplex
Red reagent, 1 U/mL HRP and 1 mM H.sub.2O.sub.2 (3% solution) in 50
mM sodium phosphate buffer, pH 7.4, were incubated for 30 minutes
at room temperature. Fluorescence was measured with a fluorescence
microplate reader using excitation at 560.+-.10 nm and fluorescence
detection at 590.+-.10 nm. Background fluorescence, determined for
a no-H.sub.2O.sub.2 control reaction, was subtracted from each
value (Table 1 and FIG. 2). Table 1 shows that the goat anti-(mouse
Fc) antibody because of the highly conserved structure of the Fc
region of an antibody it can be used to complex other non-mouse
antibodies, including rat, and human antibodies. The goat
anti-mouse IgG antibody reaction with mouse antibody was set at
100% and the crossreacting antibodies were expressed as a
percentage compared the mouse on mouse data. The data in Table 1
show that the Fab fragment of the goat anti-(mouse Fc) antibody of
the current invention does not strongly bind to the goat or sheep
Fc domain; however, one skilled in the art could generate
antibodies that will react with the goat and sheep Fc domain or the
Fc domain of any other species. Biotinylated Fab goat anti-(mouse
Fc) was used in this example because it provided a convenient
method to quantitate the amount of crossreactivity in a
conventional method but it could have been accomplished using a
fluorophore Fab labeled goat anti-(mouse Fc). It was demonstrated
by HPLC (as in Example 10) that Alexa Fluor 488 dye-labeled goat
anti-(rabbit Fc) bound to rabbit primary antibodies. TABLE-US-00004
TABLE 1 Cross-reactivity of goat anti-mouse IgG antibody with other
non-mouse antibodies. Species Crossreactivity % Fluorescence Mouse
++++ 100 Rat +++ 80.7 Human ++ 66.7 Rabbit + 16.9 Goat - 6.5 Sheep
- 5.7
Example 12
Determination of the Optimal Molar Ratio of Immunoglobulin-Binding
Protein to Target Antibody Using a Microplate Assay
[0261] To 1.6 .mu.g of mouse monoclonal anti-biotin (MW
.about.145,000) in 8.0 .mu.L PBS was added varying amounts of the
Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc)
(MW .about.50,000) (prepared as in Example 4) to form an
immunolabeling complex. After equilibration for 20 min, a 100 .mu.L
aliquot was added to a 96-well microplate coated with biotinylated
BSA. After 30 minutes, the plates were washed and the residual
fluorescence was quantitated using a fluorescence microplate reader
using excitation at 485+/-10 nm and detecting emission at
530+/-12.5 nm. As shown in FIG. 3, a molar ratio of the Alexa Fluor
488 dye-labeled Fab fragment of goat anti-(mouse Fc) to the
anti-biotin between 5 to 20 was sufficient to form appreciably
detectable complexes (FIG. 3; fluorescence quantitated, performed
in triplicate (circles); control experiments performed but without
adding the primary anti-biotin antibody (solid squares)). A molar
ratio of about 5 to about 10 was preferred for this pair of
immunoglobulin-binding protein and target antibody. This ratio can
be varied somewhat to increase or decrease the signal or to affect
the consumption of valuable reagents. The weight ratio of
immunoglobulin-binding protein to target-binding antibody is
particularly affected by the actual molecular weight of the
immunoglobulin-binding protein.
[0262] For instance, equal weights of the dye-labeled goat Fab
anti-(mouse Fc) (prepared as in Example 5) and an intact mouse
primary antibody, which corresponds to an approximately 3 to 1
molar ratio, usually yields suitable labeling complexes.
Fluorescence intensity (or enzymatic activity) of the
immunolabeling complex is readily adjusted by a corresponding
adjustment of the amount of labeled Fab fragment used.
[0263] Similar analyses of the ratio for other labeling reagents
(including those of labeled protein A, protein G, protein L,
IgG-binding peptides and antibodies to other segments of the
primary antibody), and for conjugates of labels other than Alexa
Fluor 488 dye (including enzymes in combination with the
appropriate enzyme substrates) are done essentially as described in
this example.
Example 13
Dissociation Rate of the Immunolabeling Complex
[0264] A pre-equilibrated immunolabeling complex was prepared from
50 .mu.g of an Alexa Fluor 488 dye-labeled Fab fragment of goat
anti-(mouse Fc) and 15 .mu.g of an anti-biotin monoclonal antibody
(mAb). The immunolabeling complex was rapidly diluted with
capturing component sufficient to give a 6.2 molar excess over the
anti-biotin mAb. At various times, an aliquot was taken and added
to a microplate well containing an excess of biotinylated BSA.
After 30 minutes, the plates were washed and the remaining
fluorescence was quantitated. Displacement of the labeling reagent
from the target-binding antibody through exchange was measured by
any time-dependent decrease in fluorescence in the microplate well.
For example the fragments prepared as described in Example 4 had 68
percent fragments bound to the target-binding antibody after 30
minutes compared to 87 percent of bound fragments that were
prepared according to Example 5. One hour showed a similar
decrease, 56 percent and 68 percent respectively. The labeling
reagent was shown to undergo a stable interaction with the
target-binding antibody, with a lifetime for half exchange under
these conditions of 3.5 hours. Dissociation rates were measured for
labeling reagent prepared according to Example 4 and for labeling
reagent prepared according to Example 5, demonstrating the greater
stability of immunolabeling complexes made using the labeling
reagents prepared according to Example 5.
Example 14
Protocol for Staining Cultured Cells with a Single Immunolabeling
Complex
[0265] Culturable cells, such as bovine pulmonary artery
endothelial cells (BPAEC), were grown on a 22.times.22 mm glass
coverslip. The cells were fixed for 10 minutes using 3.7%
formaldehyde in DMEM with fetal calf serum (FCS) at 37.degree. C.
The fixed cells were washed 3 times with PBS. The cells were
permeabilized for 10 min with 0.02% Triton X-100 in PBS, washed
3.times. with PBS and blocked for 30 min with 1% BSA in PBS.
Variations of the cell type and cell preparation, fixation, and
permeabilization methods, including methods for antigen retrieval,
are well known to scientists familiar with the art. An
immunolabeling complex was prepared as described in Example 8. The
immunolabeling complex was added directly to the fixed and
permeabilized cells in an amount sufficient to give a detectable
signal if there is a binding site for the primary antibody present
in the sample. After an incubation period that was typically 10-60
minutes (usually about 15-30 minutes), the cells were washed with
fresh medium and the labeling was evaluated by methods suitable for
detection of the label. Staining by the immunolabeling complex can
be additionally preceded, followed by or combined with staining by
additional reagents, such as DAPI, which yields blue-fluorescent
nuclei.
Example 15
Protocol for Staining Cultured Cells with Multiple Immunolabeling
Complexes
[0266] Cells were fixed and permeabilized as described in Example
14. Multiple immunolabeling complexes were individually prepared
from a variety of labeling reagents, according to the procedure
described in Example 8. The multiple immunolabeling complexes were
either used individually or sequentially to stain the cells,
according to the procedure described in Example 14, or two or more
immunolabeling complexes were formed then co-mixed in a single
staining solution and used to simultaneously stain the sample. The
optimal method for cell fixation and permeabilization and the best
ratio for combination of the immunolabeling complexes are typically
determined by preliminary experimentation using single
immunolabeling complexes or multiple immunolabeling complexes used
in combination. A first immunolabeling complex was prepared from an
Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc)
and mouse monoclonal anti-.alpha.-tubulin, a second immunolabeling
complex was prepared from an Alexa Fluor 568 dye-labeled Fab
fragment of goat anti-(mouse Fc) and mouse monoclonal anti-vimentin
(anti-vimentin was an ascites fluid preparation) and a third
immunolabeling complex was prepared from an Alexa Fluor 647
dye-labeled Fab fragment of goat anti-(mouse Fc) and mouse
monoclonal anti-cdc6 peptide antibody (Molecular Probes). Aliquots
of the three different immunolabeling complexes were combined and
used to stain BPAE cells for 30 minutes, washed with fresh medium
and observed by fluorescence microscopy using optical filters
appropriate for the three dyes. In this example, some cells showed
cytoplasmic staining by the anti-vimentin antibody, nuclear
staining by the anti-cdc6 peptide antibody and staining of mitotic
spindles by the anti-.alpha.-tubulin antibody, indicative of a cell
in mitosis. Staining by the immunolabeling complexes was
additionally preceded, followed by or combined with staining by
additional reagents, such as Alexa Fluor 350 phalloidin, which
yielded blue-fluorescent actin filaments in the above example.
[0267] The immunolabeling complexes that are used in combination do
not have to be targeted toward antibodies from the same species.
For instance, complexes of Alexa Fluor 488 dye-labeled goat
anti-(mouse IgG.sub.1 Fc) with a mouse IgG.sub.1 monoclonal
target-binding antibody and an Alexa Fluor 594 dye-labeled goat
anti-(rabbit Fc) with a rabbit primary target-binding antibody can
be prepared and used in combined staining protocols.
Example 16
Protocol for Staining Tissue with a Single Immunolabeling
Complex
[0268] A mouse intestine cryosection (University of Oregon
histology core facility), a cross-section of about 16 .mu.m
thickness, was mounted on a slide. The intestine was perfused and
fixed with 4% formaldehyde prior to dissection, embedding, and
sectioning. The tissue section was rehydrated for 20 minutes in
PBS. An immunolabeling complex was prepared as described in Example
8. Briefly, 0.1 .mu.g of mouse monoclonal anti-cdc6 peptide (a
nuclear antigen) in 1 .mu.L PBS with 0.1% BSA was complexed with
0.5 .mu.g of the Alexa Fluor 350 dye-labeled Fab fragment of goat
anti-(mouse IgG.sub.1 Fc) (prepared as in Example 4) in 5 .mu.L of
PBS for 10 minutes at room temperature. Excess Fab fragment of goat
anti-(mouse IgG.sub.1 Fc) was captured with 25 .mu.L of a 14.1
mg/mL stock of unlabeled mouse IgG. The tissue was permeabilized
with 0.1% Triton X-100 for 10 min. The tissue was washed two times
with PBS and was blocked in 1% BSA for 30 min. The immunolabeling
complex was added directly to the tissue for 30 minutes and washed
three times in PBS. The sample was mounted in Molecular Probes'
Prolong antifade mounting medium and observed by fluorescence
microscopy using optical filters appropriate for the Alexa Fluor
350 dye. Results showed that the mouse monoclonal anti-cdc6 peptide
immunolabeling complex showed specific nuclear labeling in the
mouse intestine tissue section. Variations of the tissue type and
tissue preparation, fixation and permeabilization methods, mounting
methods, including methods for antigen retrieval, are well known to
scientists familiar with the art.
Example 17
Staining of a Tissue Target in Combination with Tyramide Signal
Amplification (TSA)
[0269] Mouse brain cryosections were labeled with a pre-formed
complex of horseradish peroxidase (HRP)-labeled goat anti-(mouse
IgG.sub.1 Fc) antibody and a mouse IgG, monoclonal anti-(glial
fibrillary acidic protein (GFAP)) prepared essentially as in
Example 8 using a molar ratio of labeling reagent to monoclonal
antibody of 3. Staining of the mouse tissues was essentially as in
Example 16. The staining localization and intensity was compared to
that of (a) goat anti-mouse IgG HRP conjugate and mouse anti-GFAP,
(b) the Alexa Fluor 488 dye-labeled Fab fragment of goat
anti-(mouse IgG.sub.1 Fc) antibody complex of mouse anti-GFAP, (c)
Alexa Fluor 488 goat anti-mouse IgG secondary antibody and mouse
anti-GFAP, and (d) a direct conjugate of the Alexa Fluor 488 dye
with mouse anti-GFAP. The HRP-conjugated probes were incubated with
Alexa Fluor 488 tyramide using TSA Kit #2 (Molecular Probes, Inc.)
according to standard procedures. The tissue staining patterns in
each case were similar and consistent with the expected staining
pattern of mouse anti-GFAP and staining was essentially free of
nonspecific background. The relative fluorescence intensities of
staining measured by digital imaging were sequentially: 541
relative intensity units for the HRP-goat anti-(mouse IgG.sub.1 Fc)
complex of mouse anti-GFAP and (using the combinations indicated by
the letters above): (a) 539, (b) 234, (c) 294, and (d) 255 relative
intensity units.
Example 18
Staining of Live Cells by Multiple Immunolabeling Complexes
[0270] A first immunolabeling complex was prepared from an Alexa
Fluor 488 dye-labeled Fab fragment of goat anti-(mouse IgG.sub.1
Fc) and mouse monoclonal anti-(human CD8), a second immunolabeling
complex was prepared from an R-phycoerythrin-conjugated Fab
fragment of goat anti-(mouse IgG.sub.1 Fc) and mouse anti-(human
CD3), and a third immunolabeling complex was prepared from an Alexa
Fluor 647 dye-labeled Fab fragment of goat anti-(mouse IgG.sub.1
Fc) and mouse anti-(human CD4). The complexes were prepared as
described in Example 8 and were each blocked with 20 .mu.g (1.3
.mu.L of 14.1 .mu.g/mL) of mouse IgG for 10 minutes at room
temperature. The first immunolabeling complex was added to 100
.mu.L of whole blood and incubated for 15 min. The cells were
washed with PBS and 280.5 .mu.L of the second immunolabeling
complex was added and incubated for 15 min. The cells were again
washed, and 46.2 .mu.L of the third labeling complex was added and
incubated for 15 min. After the final incubation, the red blood
cells were lysed with cell-lysis buffer. The cells were resuspended
in 1% formaldehyde/PBS and analyzed on a FACS Vantage flow
cytometer using a 488 nm argon-ion laser for excitation of the
first and second immunolabeling complexes and a 633 nm red He--Ne
laser for excitation of the third immunolabeling complex (FIGS. 5a,
5b). The emission band pass filters used for selective detection of
the dyes are 525+/-10 nm for the Alexa Fluor 488 (CD8), 585+/-21 nm
for R-PE (CD3) and 675+/-10 nm for the Alexa Fluor 647 dye (CD4).
FIGS. 5a and 5b show that the instant invention can be used in a
3-color immunophenotyping experiment using peripheral blood
lymphocytes. CD3-positive T cells were stained with the
R-phycoerythrin-conjugated Fab fragment of goat anti-(mouse Fc) and
mouse anti-(human CD3), upper left (UL) quadrant, FIG. 5a.
CD4-positive cells, a T cell subset, are identified using Alexa
Fluor 647 dye-labeled Fab fragment of goat anti-(mouse IgG.sub.1
Fc) and mouse anti-(human CD4), UL quadrant, FIG. 5b and
CD8-positive T cells, a T cell subset, were identified using Alexa
Fluor 488 dye-labeled Fab fragment of goat anti-(mouse IgG.sub.1
Fc) and mouse monoclonal anti-(human CD8), lower right (LR)
quadrant, FIG. 5b.
[0271] Exposed antigens of live cells, including cultured cells and
cells from biological fluids such as blood and cerebrospinal fluid
can be simultaneously or sequentially stained by combinations of
immunolabeling complexes, including antibodies to the same target
labeled with two or more separately detectable
immunoglobulin-binding proteins.
Example 19
The Dye-Labeled Fab Fragment of Goat Anti-(Mouse Fc) can be
Utilized for the Combinatorial Labeling of Primary Antibodies, to
Generate a Multitude of Colored Targets
[0272] A first immuno-labeled complex was made by combining 2.5
.mu.g Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse
IgG, Fc) with 0.5 .mu.g mouse anti-human CD3 (Caltag at 200
.mu.g/mL), according to the procedure described in Example 4. A
second immunolabeling complex was made by combining 5.0 .mu.g Alexa
Fluor 647 dye-labeled Fab fragment of goat anti-(mouse IgG.sub.1
Fc) with 0.5 .mu.g mouse anti-human CD3, according to the procedure
in Example 4. Each complex was separately incubated at room
temperature for 5 minutes, and each complex was then separately
combined with an excess of mouse IgG (14.1 mg/mL) for min at room
temperature to capture excess unbound dye-labeled Fab fragments.
The two immunolabeling complexes were then added in different
percentage combinations (see Table 2) to 100 .mu.L of washed
heparinized blood. The cells were incubated with the respective
combinations of complexes for 20 min on ice. The red blood cells
were then lysed with a cell-lysis buffer. The cells were
resuspended in 1% formaldehyde/PBS and analyzed on a FacVantage
flow cytometer using a 488 nm argon 633 HeNe laser for excitation
and a 530+/-10 nm band pass emission filter (FL1), and a 640 long
pass filter (FL4). Five samples of different combined percentages
(Table 2) were compared by flow cytometry, with signals being
collected in FL1 and FL4. To determine the percentage of cells
detected with each type of emission, the FL1 and FL4 intensities
for each percentage combination were normalized by dividing the FL1
and FL4 channel intensities for such combinations by the
intensities of the 100% Alexa Fluor 488 dye- and 100% Alexa Fluor
647 dye-labeled cells, respectively. TABLE-US-00005 TABLE 2
Theoretical versus recovered dye-labeled Fab fragment of goat
anti-(mouse IgG.sub.1 Fc) combinatorial experiment. Recovered
percentage of Recovered measured cells Experimentally mixed
percentage of Experimentally mixed labeled with Alexa percentage of
cells measured cells percentage of cells Fluor 647 dye- labeled
with Alexa labeled with Alexa labeled with Alexa labeled Fab Fluor
488 dye-labeled Fluor 488 dye-labeled Fluor 647 dye-labeled
fragment of goat Fab fragment of goat Fab fragment of goat Fab
fragment of goat anti-(mouse IgG.sub.1 anti-(mouse IgG.sub.1 Fc)
anti-(mouse IgG.sub.1 Fc) anti-(mouse IgG.sub.1 Fc) Fc) 100% 100%
0% 0% 75% 81% 25% 14% 50% 63% 50% 38% 25% 35% 75% 73% 0% 0% 100%
100%
Example 20
The Immunolabeling Complex can be Used to Detect Antigens on a
Western Blot
[0273] Bovine heart mitochondria were isolated (Hanson et al.,
Electrophoresis 22, 950 (2001)). The isolated mitochondria were
resuspended to .about.10 mg/mL in 100 mM Tris-HCl, pH 7.8, 1 mM
phenylmethylsulfonyl fluoride (a protease inhibitor), 2% SDS and
insoluble material was removed by centrifugation for 10 minutes at
10,000.times.g in a tabletop centrifuge. The protein concentration
of the lysate was checked by the BCA assay (Pierce, Rockford,
Ill.). Samples for gel electrophoresis were prepared by mixing
lysate, water, and loading buffer to the appropriate concentrations
(final concentration of loading buffer in samples: 58 mM Tris/HCl,
10% glycerol, 2% SDS, 0.02 mg/mL bromphenol blue, 50 mM DTT, pH
8.6). The samples were then heated to 90.degree. C. for 5 minutes
before loading on the gel and separated on a 13% SDS-PAGE gel.
Two-fold serial dilution of the extracts ranging from 8 .mu.g of
extract down to 0.03 .mu.g were loaded on the SDS-PAGE gel. The
proteins were transferred to PVDF membrane for 1.5 hours using a
semi-dry transfer system according to manufacturer's directions
(The W.E.P. Company, Concord, Calif.). The PVDF membrane was
blocked for 1 hour in 5% milk.
[0274] Immunolabeling complexes were made with mouse monoclonal
antibodies that recognize two different mitochondrial proteins.
Alexa Fluor 647 dye-labeled Fab fragment of goat anti-(mouse IgG,
Fc) (5 .mu.L of a 1 mg/mL stock, prepared as in Example 4) was
incubated with 21 .mu.L (0.88 mg/mL) mouse anti-(CV-alpha) and
Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse
IgG.sub.1 Fc) (5 .mu.L of a 1 mg/mL stock, prepared as in Example
4) was incubated with 19 .mu.L (0.88 mg/mL) mouse anti-(CIII-core2)
(Molecular Probes, Eugene, Oreg.). Following a 30 minute
incubation, 25 .mu.L of a 14.1 mg/mL stock of unlabeled mouse IgG
was added to each tube. The immunolabeling complexes were then
mixed together and brought up to 5 mL in 5% milk. The blot was
incubated with the mixture of immunolabeling complexes for 1 hour
at room temperature. The blot was washed twice for 5 seconds each
with PBST (PBS with 0.1% Tween) and once with PBST for 15 minutes.
The blot was air dried and imaged on an EG&G Wallac Imager with
the appropriate filters. The Western blot revealed two distinct
bands of the appropriate molecular weight. The Western blot also
showed that no cross-labeling of the antibodies occurred and the
detection limit was 125 ng.
Example 21
High-Throughput Screening of Hybridomas for Identifying High
Affinity and High IgG Producers
[0275] Microplate wells containing both a fluorescent labeled
antigen of one fluorescent color label and fluorescently labeled
Fab fragments of goat anti-(mouse Fc) of a different fluorescent
color made by the method described in Example 4 and 5. Hybridoma
supernatant is harvested and added to the wells. If the hybridoma
are producing the desired antibody, i.e. antibodies that bind to
the labeled antigen, polarization of the florescence corresponding
to the labeled antigen will allow visualization of those wells
containing antigen specific antibody. In addition, the amount of
IgG that the hybridomas produce, can be simultaneously identified
by polarization of the fluorescence corresponding to the labeled
Fab fragments. This method thus allows for both quantitation of the
amount of antibody present in a specific amount of hybridoma
supernatant and the affinity of the monoclonal antibodies for the
antigen.
Example 22
Synthesis of Phosphorylethanolamine Ligand Analog, Compound 2
[0276] To an orange solution of BODIPY FL succinimidyl ester
(Molecular Probes 2184, 200 mg, 0.51 mmol) in 20 mL anhydrous
tetrahydrofuran was added a solution of ethanolamine (36
.quadrature.L, 0.6 mmol) in 1 mL dioxane. The resulting cloudy
orange mixture was stirred at room temperature for 3 hours, and
concentrated in vacuo. The residue was purified by flash
chromatography on silica gel using 10% methanol in chloroform as
eluant to give the corresponding ethanolamine amide of BODIPY FL as
0.17 g (99%) of an orange powder: .sup.1H NMR (CD.sub.2Cl.sub.2)
.quadrature. 7.19 (s, 1H), 6.97 (d, 1H), 6.34 (d, 1H), 6.20 (s,
1H), 6.08 (br s, 1H), 3.65 (t, 2H), 3.36 (m, 2H), 3.26 (t, 2H),
2.66 (t, 2H), 2.57 (s, 3H), 2.30 (s, 3H); LRMS m/z 335 (335 calcd
for C.sub.16H.sub.20N.sub.3O.sub.2BF.sub.2). ##STR5##
[0277] To a solution of BODIPY FL succinimidyl ester (Molecular
Probes 2184, 50 mg, 0.13 mmol) in 5 mL dioxane was added a solution
of O-phosphorylethanolamine (27 mg, 0.19 mmol) in 2 mL of 0.5 M
triethylammonium bicarbonate. The resulting solution was kept at
room temperature for 40 minutes and then concentrated to dryness.
Water was twice evaporated from the residue, which was purified by
chromatography on Sephadex LH-20 using water as eluant. Pure
product fractions were pooled and lyophilized to give Compound 2 as
an orange powder. ##STR6##
Example 23
Synthesis of Phosphotyramide Ligand Analog, Compound 4
[0278] To a solution of
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid
(Molecular Probes 6103, 0.10 g, 0.30 mmol) in 15 mL anhydrous THF
under argon was added oxalyl chloride (78 .quadrature.L, 0.89 mmol)
and one drop of DMF. The volatiles were removed in vacuo after 15
minutes of stirring, leaving a residue of
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3, 5-dipropionyl
chloride. This bis-acid chloride was dissolved in 15 mL anhydrous
THF, and the resulting solution added dropwise to a solution of
4-aminophenol (98 mg, 0.90 mmol) and diisopropylethylamine (0.16
mL, 0.90 mmol) in 10 mL anhydrous THF with stirring. The resulting
green-orange mixture was stirred at room temperature for 3 h and
then quenched with 10% citric acid (75 mL). The resulting mixture
was extracted with ethyl acetate (2.times.50 mL). The extract was
washed with brine (1.times.), dried over sodium sulfate, and
concentrated to an orange residue. Flash chromatography using
methanol in chloroform gave Compound 3 as an orange powder: LCMS
m/z 518 (518 calcd for C.sub.27H.sub.25N.sub.4O.sub.4BF.sub.2).
##STR7##
[0279] To a solution of
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionyl chloride
(0.059 mmol) in 2 mL methylene chloride was added a solution of
O-phosphoryl-4-aminophenol disodium salt (34 mg, 0.15 mmol) in 5 mL
DMF/0.2 mL acetic acid. The resulting mixture was stirred at room
temperature for two hours and then evaporated to dryness. Toluene
was evaporated from the residue, which was purified by
chromatography on Sephadex LH-20 using water as eluant to give
Compound 4 as an orange powder. ##STR8##
Example 24
Synthesis of a Fluorogenic Phosphotyramide Ligand Analog, Compound
5
[0280] A 0.05M solution of
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionyl chloride
in anhydrous dioxane is added dropwise to a 0.1M solution of 2.5 eq
O-phosphotyramine disodium salt in water (pH 8-9) with stirring.
After stirring at room temperature overnight, the volatiles are
removed in vacuo. The residue is purified by chromatography on
Sephadex LH-20 using water as eluant to give Compound 5 as an
orange powder. ##STR9##
Example 25
Synthesis of Phosphotyramide Ligand Analog, Compound 6
[0281] To a solution of O-phosphoryl-4-aminophenol disodium salt
(0.27 mmol) in 30 mL anhydrous DMF was added diisopropylethylamine
(0.23 mL, 1.3 mmol) and a solution of
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-5,7-dimethyl-3-propionyl
chloride in anhydrous dichloromethane (15 mL). The resulting
mixture was stirred at room temperature overnight, then evaporated
to dryness. The residue was dissolved in 1:1 methanol/water and
then loaded onto a Sephadex LH-20 column, followed by gravity
elution with water. Pure product fractions were combined and
lyophilized to give Compound 6 as 10 mg of an orange powder.
##STR10##
Example 26
Synthesis of Phosphotryamide Ligand Analog, Compounds 7-21 and
39
[0282] First, the intermediate O-phsphotyramine synthesized wherein
the amine group was protected as t-BOC and the phosphorylation was
done with POCl.sub.3 and N,N-diisopropylethylamine in CHCl.sub.3.
The t-BOC group was removed by HCl in aqueous solution.
Specifically this was accomplished wherein a suspension of tyramine
(1.0 g, 7.29 mmol) in 50 ml of chloroform was added
N,N-diisopropylethylamine (1.3 ml, 7.59 mmol) followed by addition
of di-tert-butyl carbonate (1.60 g, 7.34 mmol) and the mixture was
stirred at room temperature for 3 hours. The resulting reaction
mixture was washed with 1% HCl (1.times.50 ml), water (2.times.50
ml) and then separated. Organic layer is dried over
Na.sub.2SO.sub.4, filtered and concentrated under vacuum to give
N-t-BOC protected intermediate. This intermediate (1.50 g, 6.33
mmol) is dissolved in 50 ml of chloroform and was added
N,N-diisopropyl-ethylamine (1.09 ml, 6.36 mmol) and phosphorus
oxychloride (580 .mu.l, 6.34 mmol). After stirring at room
temperature for 2 hours, all the chloroform was removed under
vacuum and H.sub.2O (10 ml) was added, stirred at room temperature
for 2 hours. The resulting aqueous solution was subjected to 2H-2O
column by elution with water. From the combined desired fractions,
O-Phosphotyramine (0.52 g) is obtained. (Rf=0.40 (silica gel, 20%
water is acetonitrile)). ##STR11##
[0283] Next the succinimidyl ester version of the dye to
O-Phosphotyramine was added to give Compound 7. This is
accomplished wherein a solution of O-Phosphotyramine (3 mg, 0.01
mmol) and triethylamine (5 .mu.l, 0.04 mmol) in 500 .mu.l of water
is added a solution of Dye-carboxylic acid, succinimidyl ester
(6-isomer) (5 mg, 0.01 mmol) in 500 .mu.l of DMF and the mixture is
stirred at room temperature for 2 hours. To the reaction mixture is
added ethyl acetate (10 ml) and stirred at room temperature for 5
minutes. The upper ethyl acetate layer is removed by decantation.
The resulting aqueous layer is treated again with ethyl acetate (10
ml) and decanted. The remaining aqueous residue is purified by
preparative TLC eluting with 20% H2O in acetonitrile. Obtained 4.0
mg of a pure desired product. ##STR12##
[0284] This synthesis scheme was followed to produce Compounds
8-21, wherein the same starting material was used but different
reporter molecule compounds with a succinimidyl ester reactive
group were conjugated to the O-Phosphotyramine intermediate. It is
appreciated that numerous phosphotryamide ligand analogs can be
made using this synthesis scheme wherein the desired reporter
molecule with an appropriate reactive group such as succinimidyl
ester is conjugated to the phosphotyramide moiety. In this instance
the following compounds are not intended to be limiting. ##STR13##
##STR14## ##STR15##
Example 27
Synthesis of Homolog of Phosphotyramide Ligand Analogs, Compound
22-33 and 40
[0285] The following compounds were made with the same starting
material but different reporter molecule compounds with a
succinimidyl ester reactive group. First, the phosphotyramide
intermediate was synthesized wherein a solution of mono N-t_BOC
ethylenediamine hydrochloride (1.0 g, 4.84 mmol) and triethylamine
(1.35 ml, 9.66 mmol) in 100 ml of dichloromethane is added
N-succinimidyl 3-(4-hydroxyphenyl)propionate (1.27 g, 4.82 mmol)
and the mixture is stirred at room temperature for 6 hours. It is
washed with 0.5% HCl (1.times.100 ml) and then with water
(2.times.100 ml). The separed organic layer is dried over
Na.sub.2SO.sub.4, filtered and concentrated under vacuum to give
1.0 g of an off-white solid. The resulting solid is dissolved in 30
ml of dichloromethane and cooled with and ice-water bath. To this
ice-water cooled solution is added triethylamine (540 .mu.l, 3.82
mmol), followed by addition of phosphorus oxychloride (340 .mu.l,
3.71 mmol). After stirring at room temperature for 1 hour, all the
solvent is removed under vacuum. To the resulting residue is added
a solution of sodium bicarbonate (84 mg, 1 mmol) in 10 ml of water
and stirred at room temperature overnight. From the combined
desired fractions, homolog of O-Phosphotyramine is obtained.
##STR16##
[0286] Next the succinimidyl ester version of the dye to homolog of
O-Phosphotyramine was added to give Compound 22. This is
accomplished wherein a solution of the homolog of O-Phosphotyramine
(3 mg, 0.01 mmol) and triethylamine (5 .mu.l, 0.04 mmol) in 500
.mu.l of water is added a solution of Dye-carboxylic acid,
succinimidyl ester (6-isomer) (5 mg, 0.01 mmol) in 500 .mu.l of DMF
and the mixture is stirred at room temperature for 2 hours. To the
reaction mixture is added ethyl acetate (10 ml) and stirred at room
temperature for 5 minutes. The upper ethyl acetate layer is removed
by decantation. The resulting aqueous layer is treated again with
ethyl acetate (10 ml) and decanted. The remaining aqueous residue
is purified by preparative TLC eluting with 20% H2O in
acetonitrile. Obtained 4.0 mg of a pure desired product.
##STR17##
[0287] It is appreciated that numerous homologs of phosphotryamide
ligand analogs can be made using this synthesis scheme wherein the
desired reporter molecule with an appropriate reactive group such
as succinimidyl ester is conjugated to the phosphotyramide moiety.
In this instance the following compounds are not intended to be
limiting. Compounds 23-33 and 40 were made using this synthesis
scheme. ##STR18## ##STR19## ##STR20##
Example 28
Synthesis of Phosphotryosinamide Ligand Analogs, Compounds 34-38
and 41-42
[0288] The following compounds were made with the same starting
material but different reporter molecule compounds with a
succinimidyl ester reactive group. First, the phosphotryosinamide
intermediate was synthesized wherein a solution of L-tyrosinamide
(0.75 g, 4.17 mmol) and triethylamine (640 .mu.l, 4.59 mmol) in 20
ml of THF was added di-tert-butyl dicarbonate (1.0 g, 4.59 mmol).
After stirring at room temperature for 4 hours, 100 ml of
chloroform was added and the mixture was washed with water
(2.times.100 ml). The separated organic layer is dried over
Na.sub.2SO.sub.4 and concentrated under vacuum to give 1 g of a
white solid. This solid is dissolved in 30 ml of THF. To this
solution is added di-tert-butyl diethylphosphoramidite (1.0 g, 3.93
mmol), followed by addition of tetrazole (750 mg, 10.70 mmol).
After stirring at room temperature for 2 hours, a solution of
3-chloroperbenzoic acid (930 mg, 5.39 mmol) in 10 ml of
dichloromethane was added while the reaction mixture was stirred
under ice water bath. After stirring at room temperature for 1
hour, a 10% solution of sodium bisulfite in water (50 ml) was added
and stirred at room temperature for 20 minutes. It was then
extracted with chloroform (2.times.100 ml) and washed with 10%
sodium bisulfite (2.times.100 ml) followed by washing with 10%
sodium bicarbonate (1.times.100 ml). The separated organic layer
was dried over sodium sulfate and concentrated under vacuum to give
a crude fully protected intermediate. This crude intermediate was
purified by column chromatography (silica gel) eluting with 5%
methanol in chloroform to give 1.8 g of a protected intermediate.
This is dissolved in 10 ml of TFA and stirred at room temperature
overnight. All the TFA is removed under vacuum and the resulting
residue is dissolved in about 2 ml of water and subjected to LH-20
column eluting with water. From the combined desired fractions 400
mg of a product is obtained as a white powder. ##STR21##
[0289] Next the succinimidyl ester version of a dye was added to
phosphotryosinamide to give Compound 34. This is accomplished
wherein a solution of the phosphotryosinamide compound (3 mg, 0.01
mmol) and triethylamine (5 .mu.l, 0.04 mmol) in 500 .mu.l of water
is added to a solution of Dye-carboxylic acid, succinimidyl ester
(6-isomer) (5 mg, 0.01 mmol) in 500 .mu.l of DMF and the mixture is
stirred at room temperature for 2 hours. To the reaction mixture is
added ethyl acetate (10 ml) and stirred at room temperature for 5
minutes. The upper ethyl acetate layer is removed by decantation.
The resulting aqueous layer is treated again with ethyl acetate (10
ml) and decanted. The remaining aqueous residue is purified by
preparative TLC eluting with 20% H2O in acetonitrile. Obtained 4.0
mg of a pure desired product. ##STR22##
[0290] The following compounds were made with the same starting
material but different reporter molecule compounds with a
succinimidyl ester reactive group. It is appreciated that numerous
phosphotryosinamide ligand analogs can be made using this synthesis
scheme wherein the desired reporter molecule with an appropriate
reactive group, such as succinimidyl ester, is conjugated to the
phosphotryosineamide moiety. In this instance the following
compounds are not intended to be limiting. ##STR23## ##STR24##
Example 29
Synthesis of Phosphoserine Ligand Analog, Compound 43
[0291] O-phosphorylserine, N-methylamide, N'-acylated with the
6-isomer of carboxy-2',7'-difluorofluorescein (B750-82-GEE45). The
pH of a 0.10 M solution of O-phosphorylserine-N-methylamide was
raised to 8.0 with aqueous sodium carbonate. A 100 .mu.L aliquot
(0.01 mmol) of this solution was added to a solution of Oregon
Green 488 succinimidyl ester, 6-isomer (Molecular Probes 6149, 5.0
mg, 0.01 mmol) in 1.0 mL dioxane. The resulting mixture was stirred
at room temperature for 4 hours, then filtered and lyophilized to
afford Compound 43 as an orange powder. ##STR25##
[0292] It is appreciated that other phosphoserine ligand analogs
can be made using a similar synthetic scheme wherein a different
reporter group with an appropriate reactive group such as
succinimidyl ester is used to make numerous phosphoserine ligand
analogs with various reporter groups. In this instance, Compound 43
is not intended to be limiting.
Example 30
Detection of Digoxigenin Employing a Ligand-Binding
Antibody-Labeling Reagent-Ligand Analog Ternary Complex
[0293] To form a ligand-binding antibody labeling reagent complex,
mouse monoclonal anti-Digoxigenin antibody (Roche product #1333062)
in 50 mM MOPS buffer, pH 7.2, was mixed in a 1:5 ratio with a Fab
fragment of goat-anti-mouse kappa chain labeled with a quenching
moiety (QSY 9) (Molecular Probes, Inc.). The degree of QSY 9
labeling on the Fab fragment was 1.7, determined by absorbance. The
ligand-binding antibody+labeling reagent complex (zero to 100 nm
final concentration/zero to 500 nM final concentration) was
serially diluted two-fold in 90 .mu.l buffer down a black, 96-well
flat-bottom microplate precoated with 1% (w/v) bovine serum
albumin. As a control, the ligand-binding antibody
(anti-Digoxigenin antibody) was diluted in the plate in 90 .mu.l
buffer. After the serial dilution, 10 .mu.l of the ligand analog
(BODIPY FL Digoxigenin, Molecular Probes Inc., B-23460) (50 nM
final) in 50 mM MOPS buffer, pH 7.2 was added to the same
wells.
[0294] The resulting fluorescence intensity was measured on a
Victor.sup.2 microplate reader (Wallac), 1 read/well for 1 sec each
at 50000 V gain, excitation 485+/-17.5 nm, emission 535+/-12.5 nm.
This demonstrates the ability of the quenching moiety on the
labeling reagent to diminish the fluorescent signal of the BODIPY
dye on the ligand analog when a ternary complex is formed. As the
amount anti-Digoxigenin/Fab fragment complex increases, the
fluorescence of the BODIPY-FL Digoxigenin decreases. After the
initial read, 1 .mu.l of 100 .quadrature.M ligand (unlabeled
Digoxigenin) (Sigma, catalog #D-9026) in 50 mM MOPS buffer, pH 7.2,
was added to all wells, and the resulting fluorescence intensity
was measured on the same instrument at the same settings.
[0295] These results demonstrate that the ligand (unlabeled
Digoxigenin) is capable of displacing the ligand analog to restore
the fluorescent signal generated by the BODIPY fluorophore. When
excess (1 .mu.M) unlabeled Digoxigenin is added, the fluorescence
quenching is partially relieved. See, FIG. 9.
Example 31
Binding of Ethanolamine Phosphate Ligand Analog (Compound 2) by
Anti-Akt Antibody
[0296] A 5 mM solution of BODIPY FL ethanolamine phosphate Compound
2 was made in water. A 2.5 mM solution of BODIPY FL ethanolamine
(Compound 1) was made in 50% (v/v) DMSO, See Example 1. Rabbit
anti-Akt polyclonal antibody (Cell Signaling Technology, catalog
#9611) was serially diluted in 5 .mu.l 50 mM Tris buffer, pH 7.5 in
a black, 384-well flat-bottom Packard ProxiPlate preblocked with
0.25% (v/v) Mowiol. After the antibody was serially diluted in the
plate, 5 .mu.l of either Compound 2 or Compound 1 was added to the
same wells. The final concentration of both compounds in the wells
was each 50 nM. The final antibody concentration in the wells was
zero to 250 nM. The fluorescence intensity was measured on an
EnVision microplate reader (Perkin Elmer), PMT 1 gain 155, PMT 2
gain 191, excitation light 76%, 100 flashes at 9 mM height. The
ligand analog, Compound 2, is slightly quenched by the Rabbit
anti-Akt polyclonal antibody when the ligand is bound by the
antibody.
Example 32
Fluorescence Enhancement of
bis(acetamidophenylphosphate)-Derivatized Dye Upon Binding to
Antiphosphotyrosine Antibody
[0297] Solutions of Compounds 3, 4 and 6 (100 nM) were prepared in
50 mM Tris-HCl, pH 7.5. 25 .quadrature.l aliquots of these
solutions were pipetted into the wells of a 384-well microplate. 25
.mu.l aliquots of a serially-diluted 1 .mu.M stock solution of
P-Tyr-100 (U.S. Pat. No. 6,441,140) antiphosphotyrosine monoclonal
antibody (Cell Signaling Technology, Beverly, Mass.) were added to
the wells. The resulting samples contained 50 nM test compound and
antibody concentrations ranging from 0.5 to 500 nM. Fluorescence
intensity of the samples was measured on an EnVision microplate
reader (PerkinElmer Life Sciences) using excitation/emission filter
settings of 480/535 nm. The bis(acetamidophenylphosphate) (Compound
4) exhibits fluorescence enhancement upon interaction with the
antibody, whereas the corresponding mono-substituted compound 6 and
the parent bis(acetamidophenol) (Compound 3) do not. See, FIG.
10.
Example 33
Competitive Immunoassay with Compound 15 as the Ligand Analog,
Anti-Phosphotyrosine Antibody as the Ligand-Binding Antibody and a
Phosphotyrosine Peptide as the Target Ligand
[0298] A 5 mM solution of Compound 15 was prepared in water. In 150
.quadrature.l kinase buffer (50 mM Tris, pH 7.5, 10 mM MgCl.sub.2,
1 mM EGTA, 0.01% Brij 35, 2 mM DTT, 100 .mu.M ATP) several separate
reactions were made containing: 1) 100 nM Compound 15; 2) 100 nM
Compound 14+100 nM P-Tyr-100 monoclonal anti-phosphotyrosine
antibody (Cell Signaling Technology, catalog #9411); 3) 100 nM
Compound 15+100 nM P-Tyr-100+10 .quadrature.M phospho-pp60 c-src
peptide (521-533) (TSTEPQY*QPGENL) from Bachem, catalog #H-3258; 4)
100 nM Compound 15+100 nM P-Tyr-100+10 .quadrature.M phospho-abl
peptide (EAIY*AAPFAKKK), custom peptide MPIJ6 from Anaspec. The
resulting fluorescence was measured using a Hitachi F-4500 cuvette
fluorimeter using 100 .mu.l cuvettes. The F-4500 was set on
`emission scan`, with an excitation at 470 nm, slit width of 5 nm,
emission scan from 485-650 nm, slit width 5 nm, and the PMT gain
was at 700 V.
[0299] Addition of P-Tyr-100 antibody to the ligand analog
(Compound 15) significantly quenched the reporter molecule of the
ligand analog. Addition of either of two phosphotyrosine peptides
relieved almost all of the quenching, demonstrating both the
ability of the ligand-binding antibody to quench the ligand analog
when bound to the antibody and the ability of the target ligand
(phosphotyrosine peptide) to displace the ligand analog (Compound
15). See, FIG. 11
Example 34
Rapid Displacement of the Ligand Analog by the Target Ligand
[0300] A 5 mM solution of Compound 15 was prepared in water. In 150
.mu.l kinase buffer (50 mM Tris, pH 7.5, 10 mM MgCl.sub.2, 1 mM
EGTA, 0.01% Brij 35, 2 mM DTT, 100 .mu.M ATP) a reaction was made
containing 100 nM Compound 15+100 nM P-Tyr-100 monoclonal
anti-phosphotyrosine antibody (Cell Signaling Technology, catalog
#9411). The fluorescence was measured over time using a Hitachi
F-4500 fluorimeter (Ex 470, Em 510, slit width 5 nm for both
wavelengths, PMT gain 700 V). After 15 seconds, 10 .mu.M
phospho-pp60 c-src peptide (521-533) (TSTEPQY*QPGENL) from Bachem,
catalog #H-3258 was added.
[0301] The off rate of Compound 15 (ligand analog) was calculated
as 0.14 sec.sup.-1, demonstrating the ability of the target ligand
to displace the ligand analog very rapidly. See, FIG. 12.
Example 35
[0302] A 2 mM solution of Compound 34 was prepared in water. Two
working stocks were prepared in kinase buffer (50 mM Tris, pH 7.5,
10 mM MgCl.sub.2, 1 mM EGTA, 0.01% Brij 35, 2 mM DTT, 100 .mu.M
ATP): 1) 50 nM of Compound 34+100 nM P-Tyr-100 monoclonal
anti-phosphotyrosine antibody (Cell Signaling Technology, catalog
#9411); 2) 100 mM phospho-pp60 c-src peptide (521-533)
(TSTEPQY*QPGENL) from Bachem, catalog #H-3258. In two 384-well
microplates, 25 .mu.l of the 50 nM Compound 34+100 nM P-Tyr-100
complex was added to 96 wells in each plate. To the same wells, 25
.quadrature.l of either kinase buffer alone (48 wells each plate)
or the 100 .mu.M phospho-pp60 c-src peptide in kinase buffer (48
wells each plate) was added. The fluorescence was measured in a
Victor.sup.2 microplate reader (Wallac), 1 read/well for 0.2 sec
each at 30000V gain, excitation 485+/-17.5 nm, emission 535+/-12.5
nm.
[0303] The Z' statistic was calculated using equation 5 from Zhang,
J.-H., Chung, T., D., Y., and Oldenburg, K. R. (1999) A simple
statistical parameter for use in evaluation and validation of high
throughput screening assays. J. Biomol. Screen. 4, 67-73. The Z'
factor using data from both plates combined was calculated as
0.919, demonstrating the ability to differentiate between
background signal and signal generate when the target ligand is
bound by the antibody. In other words, there is a 4-fold increase
in fluorescent signal after the ligand analog is displaced by the
phosphorylated peptide.
Example 36
[0304] A 5 mM solution of Compound 15 was prepared in water. In
kinase buffer (50 mM Tris, pH 7.5, 10 mM MgCl.sub.2, 1 mM EGTA,
0.01% Brij 35, 2 mM DTT, 100 .quadrature.M ATP) 120 .mu.M
phospho-pp60 c-src peptide (521-533) (TSTEPQY*QPGENL) (Bachem,
catalog #H-3258) was serially diluted three-fold across a
microplate in 20 .mu.l volume. 20 .mu.l of 100 nM of Compound
15+200 nM P-Tyr-100 complex was added to all wells. The
fluorescence intensity was measured on an EnVision microplate
reader (Perkin Elmer), PMT 1 gain 155, PMT 2 gain 183, excitation
light 60%, 100 flashes at 9 mM height.
[0305] To generate the Z' scores in FIG. 7, the Z' factor was
calculated using equation 5 from Zhang, J.-H., Chung, T., D., Y.,
and Oldenburg, K. R. (1999) A simple statistical parameter for use
in evaluation and validation of high throughput screening assays.
J. Biomol. Screen. 4, 67-73. This demonstrates the large increase
in fluorescent signal after the ligand analog is displaced by the
target ligand.
Example 37
[0306] A 5 mM solution of Compound 15 was prepared in water. In
kinase buffer (50 mM Tris, pH 7.5, 10 mM MgCl.sub.2, 1 mM EGTA,
0.01% Brij 35, 2 mM DTT, 100 .mu.M ATP) 80 .quadrature.M solutions
of six different peptides or proteins were made: 1) phospho-pp60
c-src peptide (521-533) (TSTEPQY*QPGENL) (Bachem, catalog #H-3258);
2) non-phospho-pp60 c-src peptide (521-533) (TSTEPQYQPGENL)
(Bachem, catalog #H-3256); 3) phospho-DSIP peptide (WAGGDAS*GE)
(SynPep, catalog #3920); 4) phospho-RRA(pT)VA peptide (RRAT*VA)
(Sigma, catalog V248A); 5) beta-casein (Sigma, catalog #C6905); 6)
bovine serum albumin (Sigma, catalog #A7284). These peptides or
proteins were serially diluted 2-fold in 20 .mu.l in a 0.025%
Mowiol-blocked 384-well black Packard OptiPlate. 20 .quadrature.l
of a 2.times. mix of 50 nM of Compound 15+100 nM P-Tyr-100 (Cell
Signaling Technology, catalog #9411) complex in kinase buffer was
added to the wells. The fluorescence intensity was measured on an
EnVision microplate reader (Perkin Elmer), PMT 1 gain 155, PMT 2
gain 183, excitation light 60%, 100 flashes at 9 mM height.
[0307] The phospho-pp60 c-src peptide is the only peptide or
protein that significantly displaces the ligand analog (Compound
15), indicating that the reaction is specific for phosphotyrosine
residues. See, FIG. 13
Example 38
Displacement of Phosphotyramide Ligand Analog by Phosphotyrosine
Containing Peptide but not by ATP
[0308] A 5 mM solution of Compound 15 was prepared in water.
Separate reactions of 100 nM Compound 15+/-100 nM P-Tyr-100 (Cell
Signaling Technology, catalog #9411) complexes were made in kinase
buffer with various amounts of ATP (50 mM Tris, pH 7.5, 10 mM
MgCl.sub.2, 1 mM EGTA, 0.01% Brij 35, 2 mM DTT, with zero, 0.1,
0.25, 0.5, or 1 mM ATP). The fluorescence emission of each of the
ten solutions was measured using a Hitachi F-4500
spectrofluorometer (Ex 450, Em 510, slit width 5 nm for both
wavelengths, PMT gain 700 V). After the initial read, 10 .mu.M
phospho-pp60 c-src (10 .mu.M final concentration) peptide (521-533)
(TSTEPQY*QPGENL) (Bachem, catalog #H-3258) in kinase buffer was
added to the antibody:ligand reactions, and the resulting
fluorescence intensity was measured on the same instrument at the
same settings.
[0309] These data indicate that the assay is relatively insensitive
to ATP concentrations. See, FIG. 14.
Example 39
Detection of Kinase Activity
[0310] A 5 mM solution of Compound 15 was prepared in water. Abl
kinase (New England Biolabs, catalog #P6050S) was serially diluted
2-fold in 20 .quadrature.l kinase buffer (50 mM Tris, pH 7.5, 10 mM
MgCl.sub.2, 1 mM EGTA, 0.01% Brij 35, 2 mM DTT, 500 .mu.M ATP) in a
0.025% Mowiol-blocked 384-well black Packard OptiPlate. To the
wells containing the serial dilution, 20 .quadrature.l of either
400 .mu.g/ml poly (Glu:Ala:Tyr) 6:3:1 ratio (Sigma, catalog
#P-3899)+100 nM p-Tyr-100 antibody (Cell Signaling Technology,
catalog #9411)+50 nM Compound 15 in kinase buffer or 100 .mu.M abl
substrate peptide (custom peptide from AnaSpec, MPIJ-5
(EAIYAAPFAKKKC))+100 nM p-Tyr-100 antibody+50 nM Compound 15 in
kinase buffer was added. After a one hour incubation the
fluorescence intensity was measured on a Victor.sup.2 microplate
reader (Wallac), 1 read/well for 0.1 sec each at 20000 V gain,
excitation 450+/-3 nm, emission 510+/-20 nm.
[0311] The assay is capable of detecting Abl kinase activity
wherein the phosphorylated peptides displace the ligand analog
(Compound 15). The Abl kinase can phosphorylate both peptides,
though it is more effective at phosphorylating the abl substrate
peptide, MPIJ-5. See, FIG. 15.
Example 40
[0312] A 5 mM solution of Compound 15 was prepared in water. In
kinase buffer (50 mM Tris, pH 7.5, 10 mM MgCl.sub.2, 1 mM EGTA,
0.01% Brij 35, 2 mM DTT, 100 .mu.M ATP) prepared 150 .mu.l 333.3
.mu.M staurosporine (Sigma, catalog #S-4400). The 333.3 .mu.M
staurosporine stock was serially diluted three-fold in 15 .mu.l
kinase buffer in a 0.025% Mowiol-blocked 384-well black Packard
OptiPlate. To the same wells 10 .mu.l of either kinase buffer
alone, 10 .mu.l of 333 Units/ml abl kinase (New England Biolabs,
catalog #P6050S) in kinase buffer, or 10 .mu.l of 125 Units/ml src
kinase (Upstate Biotechnology, catalog #14-326) in kinase buffer
was added. The plate was centrifuged to ensure mixing and then
incubated for 20 minutes at 37.degree. C. To this, 25 .mu.l of
kinase buffer containing 200 nM Compound 15+200 nM p-Tyr-100
antibody (Cell Signaling Technology, catalog #9411)+1.2 .mu.M Fab
fragment of goat-anti-mouse antibody labeled with Alexa Fluor.RTM.
555 as a quenching moiety+250 .mu.g/ml poly (Glu:Tyr) 4:1 ratio
(Sigma, catalog #P-0275). The plate was centrifuged again then
incubated at 37.degree. C. while monitoring the fluorescence
intensity in a Victor.sup.2 microplate reader (Wallac), 1 read/well
for 0.1 sec each at 20000 V gain, excitation 450+/-3 nm, emission
510 +/-20 nm. After 45 minutes there was no increase in intensity
in the abl-kinase-containing wells, so abl peptide substrate
(custom peptide from AnaSpec, MPIJ-5 (EAIYAAPFAKKKC) was added to a
final concentration of 100 .mu.M, and the plate incubated for an
additional 50 minutes at 37.degree. C.
[0313] The assay can determine the IC.sub.50 of staurosporine, a
model tyrosine kinase inhibitor. This indicates that the assay can
be used to determine the IC.sub.50 of unknown/experimental tyrosine
kinase inhibitors. See, FIG. 16.
Example 41
[0314] A 4 mM stock of Compound 16 was made in 16% (v/v) DMSO.
Three different antibodies were diluted to 1 .mu.M in 50 mM MOPS,
pH 7.2: 1) p-Tyr-100 antibody (Cell Signaling Technology, catalog
#9411); 2) p-Tyr-69 antibody (BD Transduction Labs, catalog
#610430); 3) p-Tyr-20 antibody (BD Transduction Labs, catalog
#610000). A fourth antibody, the 4G10 antibody (Upstate
Biotechnology, catalog #05-321), was diluted to 400 nM in MOPS
buffer. The antibodies were serially diluted in a microplate in 25
.quadrature.l 50 mM MOPS, pH 7.2, buffer. 25 .mu.l 100 nM Compound
16 in MOPS buffer was added to all wells. The fluorescence
intensity was measured on an EnVision microplate reader (Perkin
Elmer), PMT 1 gain 155, PMT 2 gain 187, excitation light 53%, 100
flashes at 9 mM height.
[0315] This experiment indicates that the p-Tyr-100 antibody from
Cell Signaling Technology has the highest affinity for the ligand
analog (Compound 16). See, FIG. 17.
Example 42
O-Phosphotyrosine Causes Minimal Displacement of
Phosphotyramide-Dye Ligand Analogs from Antibody Binding Sites
[0316] Solutions containing 100 nM ligand analog Compound 15 or
ligand-detection complex (100 nM (B573-85-HCK)+100 nM P-Tyr-100
antiphosphotyrosine monoclonal antibody (Cell Signaling Technology,
Beverly, Mass.)) were prepared in 50 mM Tris-HCl pH 7.5, 10 mM
MgCl.sub.2, 0.01% Brij-35, 100 .mu.M ATP, 2 mM DTT, 1 mM EGTA.
Fluorescence emission spectra of these samples were recorded on a
Hitach F-4500 spectrofluorometer using an excitation wavelength of
470 nm. O-phosho-L-tyrosine (Sigma Chemical Co., St. Louis, Mo.) or
phosphotyrosine peptide (phospho-pp60 c-src (521-533);
TSTEPQY*QPGENL, Bachem California, Inc. Torrance, Calif.) at a
concentration of 10 .mu.M were added to samples of the detection
complex and the resulting change in fluorescence was measured.
Example 43
Method for Selecting Ligands for Use in Displacement Assay
[0317] TABLE-US-00006 TABLE 4 Ligands screened for efficacy in
FIGS. 14A-D Compound Number Ligand analog F.sub.max/F.sub.min** 15
Oregon Green 488 6-phosphotyramide 5.4 7 Oregon Green 514
phosphotyramide 4.0 8 5-FAM phosphotyramide 1.3 9 6-FAM
phosphotyramide 3.2 18 5-FITC phosphotyramide 1.7 19 6-FITC
phosphotyramide 1.6 **obtained from data in Figure B as the ratio
of the fluorescence intensity at the lowest antibody concentration
(F.sub.max) to that at the highest antibody concentration
(F.sub.min).
[0318] Solutions of test compounds (100 nM) were prepared were
prepared in 50 mM Tris-HCl pH 7.5, 10 mM MgCl.sub.2, 0.01% Brij-35,
500 .mu.M ATP, 2 mM DTT, 1 mM EGTA. 20 .mu.l aliquots of these
solutions were pipetted into the wells of a 384-well microplate. 20
.mu.l aliquots of a serially-diluted 0.8 .mu.M stock solution of
P-Tyr-100 antiphosphotyrosine monoclonal antibody (Cell Signaling
Technology, Beverly, Mass.) were added to the wells. The resulting
samples contained 50 nM of each ligand analog (Compounds 15, 7, 8,
9, 18 and 19) and antibody concentrations ranging from 0.8 to 500
nM. Fluorescence polarization and intensity of the samples was
measured on an EnVision microplate reader (PerkinElmer Life
Sciences) using excitation/emission filter settings of 480/535 nm.
Increasing fluorescence polarization as a function of antibody
concentration (A) provides confirmation of ligand analog binding to
the antibody. Ligand analogs exhibiting the largest possible signal
changes (B) upon antibody binding (i.e. largest value of
F.sub.max/F.sub.min see Table 4) are preferred for displacement
assays using a fluorescence intensity readout. Phosphotyrosine
peptide, target ligand, (phospho-pp60 c-src (521-533);
TSTEPQY*QPGENL, Bachem California, Inc. Torrance, Calif.) was then
added to all samples at a concentration of 10 .mu.M and the
fluorescence intensity and polarization measurements were repeated
(C, D). Preferred ligand analogs for displacement assays using a
fluorescence intensity readout exhibit large fluorescence intensity
upon phosphopeptide addition (i.e. C compared to B). Displacement
of the ligand analogs from antibody is confirmed by depolarization
of fluorescence (D compared to A). Based on these considerations,
xanthene dye-based ligand analogs with a phosphotyramide moiety
attached at the 6-position of the carboxyphenyl ring (e.g Compound
8) exhibit superior performance to the corresponding compounds
derivatized at the 5-position (e.g. Compound 9). See, FIG. 18.
Example 44
Comparison of Phosphotyramide Ligand Analog to Phosphotyrosinamide
Ligand Analog: Determination of Dissociation Constants for
Antibody-Ligand Complexes
[0319] Solutions of Compound 15 and Compound 34 (2 nM) were
prepared in 50 mM Tris-HCl pH 7.5, 10 mM MgCl.sub.2, 0.01% Brij-35,
500 .mu.M ATP, 2 mM DTT, 1 mM EGTA, 0.5 mg/ml bovine serum albumin.
25 .mu.l aliquots of these solutions were pipetted into the wells
of a 384-well microplate. 25 .mu.l aliquots of a serially-diluted
0.08 .mu.M stock solution of P-Tyr-100 anti-phosphotyrosine
monoclonal antibody (Cell Signaling Technology, Beverly, Mass.)
were added to the wells. The resulting samples contained 1 nM test
compound and antibody concentrations ranging from 0.02 to 40 nM.
Fluorescence intensities of triplicate samples at each antibody
concentration were measured on an EnVision microplate reader
(PerkinElmer Life Sciences) using excitation/emission filter
settings of 480/535 nm. The mean (n=3) fluorescence intensities
were plotted against the corresponding antibody concentrations.
Dissociation constants were determined from hyperbolic single-site
saturation binding functions fitted to the experimental data by
nonlinear regression analysis (SigmaPlot, Jandel Scientific Inc).
The dissociation constants obtained were 2.0 nM for Compound
15+P-Tyr-100 and 1.7 nM for Compound 34+P-Tyr-100.
Example 45
Detection of a Target Ligand
[0320] Compound 12 and Compound 13 were assessed for efficacy using
the methods described in Example 43. The results of this assessment
show large fluorescence polarization changes (FIG. 20A), indicating
binding of the ligands to the P-Tyr-100 antibody, but little or no
analytically useful fluorescence intensity change (FIG. 20B). The
experiment shown in FIG. 20C demonstrates that complexation of the
mouse monoclonal anti-phosphotyrosine antibody (P-Tyr-100) with a
secondary antibody labeled with a fluorescence resonance energy
transfer acceptor dye (Alexa Fluor 647 dye-labeled F(ab').sub.2
fragment of goat anti-mouse IgG; "labeling reagent") results in
quenching of the fluorescence of Compound 13 that is reversed up
addition of a phosphotyrosine-containing peptide. Samples
containing 100 nM of Compound 13 and additional components
identified in the figure legend were prepared in 50 mM Tris-HCl pH
7.5, 10 mM MgCl.sub.2, 0.01% Brij-35, 100 .mu.M ATP, 2 mM DTT, 1 mM
EGTA. 100 .mu.L volumes of these samples were transferred to
microcuvettes. The fluorescence emission spectrum was recorded for
each sample using a Hitachi F-4500 spectrofluorometer (excitation
wavelength=520 nm). See, FIG. 19.
Example 46
Flow Cytometric Assays
[0321] Coupling of antibodies to microspheres is a common
methodology utilized to develop assays for analytes detected using
flow cytometry instrumentation. Antibody-microsphere coupling
procedures are typically performed so as to preserve
antigen-binding activity. Hence, antibodies on microspheres will be
capable of binding the ligand analog. Upon exposure of
antibody-coupled microspheres to antigen, the ligand analog will be
displaced from the microsphere, and a signal decrease will be
observed in the flow cytometer. To generate flow cytometry assays
with signals that increase upon analyte detection, a bound labeling
reagent (anti-Fc antibody fragment) can be complexed with the
microsphere-coupled ligand-analog bound antibody. The observed
signal from the labeling reagent will be quenched, through FRET to
the ligand-analog. Upon exposure to the antibody-coupled
microsphere to antigen, the ligand analog will be displaced, and
the donor labeling reagent signal will increase.
Example 47
The Use Enzyme Amplified Detectable Signal with an Enzyme Cofactor
Conjugated Ligand Analog for a One Step Immunoassay
[0322] The fluorophore moiety of the ligand-detection-reagent is
replaced with an "enzyme-activating-factor" and then the signal
associated with a single antigen-binding event is enzymatically
amplified. For instance, the cofactor-ligand-analog is
pre-complexed with the antibody wherein the ligand analog is
displaced upon antigen binding. The types of cofactors utilized
are: NAD(P)H, ATP, GTP, cAMP, coenzyme-A, FADH, hematin, etc. Any
small molecule that can be coupled to an antigen and still retain
the ability to activate an enzyme-reaction are candidates for this
approach. Antigen target detection is performed in the presence of
the inactive (cofactor requiring) enzyme. The concentrations of the
reagents are established, such that while the
cofactor-ligand-analog is bound by antibody, the cofactor-requiring
enzyme is not able to bind cofactor (not effective at competing
with the antibody). Upon displacement of the cofactor-ligand-analog
from the antibody, the cofactor-requiring enzyme binds the
cofactor-ligand-analog and is activated. Any fluorogenic or
chromogenic substrate is utilized that can be coupled to the
activated enzyme and thereby significantly amplifying the single
antigen-binding event. In this manner, the requirement for a second
separate detection/amplification antibody to detect the antigen is
obviated.
Example 48
Efficacy of Secondary Antibody Labels as Fluorescence Resonance
Energy Transfer Acceptors
[0323] TABLE-US-00007 TABLE 5 Sample B. Sample A. 100 nM Sampl C.
100 nM Compound 100 nM Compound Dy lab I on Fab Compound 16 + 100
nM 16 + 100 nM P-Tyr- (DOS)** 16 P-Tyr-100 100 + 600 nM Fab
Unlabeled 100 51 51 Alexa Fluor 555 (3.8) 100 53 36 Alexa Fluor 594
(2.5) 100 49 48 Alexa Fluor 647 (2.8) 100 49 47 Tabulated values
are fluorescence intensities measured at 510 nm (excitation at 450
nm) expressed as percentages of the fluorescence intensity of 100
nM phosphotyramide ligand analog (Sample A) under the same
conditions. **DOS = degree of substitution i.e. the average number
of dye labels per antibody.
[0324] Solutions containing (A) 100 nM phosphotyramide ligand
analog, (B) 100 nM phosphotyramide ligand analog complexed with 100
nM P-Tyr-100 mouse monoclonal anti-phosphotyrosine antibody and (C)
100 nM phosphotyramide ligand analog complexed with 100 nM
P-Tyr-100 anti-phosphotyrosine antibody and 600 nM Fab fragments of
goat anti-mouse IgG were prepared in 50 mM Tris-HCl, pH 7.5.
Corresponding sets for solutions were prepared for Fab fragments
labeled with three different dyes and an unlabeled control.
Fluorescence intensities at 510 nm (excitation at 450 nm) were
measured on a Hitachi F-4500 spectrofluorometer and were expressed
as percentages of the intensity of the free phosphotyramide ligand
sample (Table 5). In all cases, binding of the ligand to the
P-Tyr-100 antibody resulted in an approximately 50% decrease of
fluorescence intensity. Addition of a labeled secondary antibody
produced a further decrease in intensity only in the case of the
Alexa Fluor 555 labeling dye, which has spectral characteristics
that are consistent with efficient fluorescence resonance energy
transfer from the BODIPY FL phosphotyramide ligand (Compound
16).
Example 49
The Use of Ligand-Detection Reagent with Protein Microarray
Assays
[0325] Antibodies are immobilized on a solid surface to form a
microarray. Antibody arrays of this type are often utilized for
analyte detection in a multi-step process. The microarrayed
antibody captures the antigen, and then a second detection antibody
is utilized to record the presence/absence of the captured antigen.
Utilizing ligand-detection-reagents, this multi-step process can be
eliminated, and antigen detection performed in the following
manner. Microarraying antibodies either pre-bound with
ligand-analog or subsequently exposed to ligand-ligand, will
generate a signal associated with the bound ligand. Exposure of
that antibody to a detectably distinct labeling reagent, generates
two independent signals associated with any given microarrayed
antibody spot. Exposure of the protein microarray to antigen
(target ligand) will cause displacement of the ligand-analog
signal, but will not alter the labeling reagent signal. For
instance, for phosphotyrosine detection, a high affinity binding
(yet not quenching) dye-antigens are utilized such as compound 12
or 13. Microarraying the antiphosphotyrosine-Compound 12 complexes
will yield "bright-spots" on the protein microarray when detected
with the appropriate excitation/emission filters. For graphical
representation purposes, these data can be psuedo-colored "green."
Pre-complexing the same antibodies with, for instance, a detectably
distinct labeling reagent will yield bright-spots when detected
with the appropriate excitation/emission filters. For graphical
representation purposes, these data can be psuedo-colored "red." In
the absence of antigen, the images psuedo-colored "red" and "green"
can be superimposed (overlay image) to generate a "yellow" spot,
indicative of the absence of antigen. Exposure of the antibody
microarray to target ligand will cause a displacement of the
"green" signal and not effect the "red" signal. Hence, a
single-step determination of the exposure of the antibody
microarray to target ligand can be quantitated by observing to what
degree the original "yellow-spots" change to "red-spots." For 100%
displacement, pure red-spots would be observed, and for 0% analyte
yellow-spots. Hence, protein-microarray spots are observed to
change from "yellow-to-red" upon antigen detection--in a 1-step
immunoassay detection scheme.
[0326] The reagents employed in the preceding examples are
commercially available or can be prepared using commercially
available instrumentation, methods, or reagents known in the art or
whose preparation is described in the examples. It is evident from
the above description and results that the subject invention is
greatly superior to the presently available methods for determining
the presence of a target in a biological sample. The subject
invention overcomes the shortcomings of the currently used methods
by allowing small quantities of antibodies to be labeled and in
unlimited media while maintaining specificity and sensitivity. The
examples are not intended to provide an exhaustive description of
the many different embodiments of the invention. Thus, although the
forgoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding,
those of ordinary skill in the art will realize readily that many
changes and modifications can be made thereto without departing
from the spirit or scope of the appended claims.
[0327] All publications, patents and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent or patent
application was specifically and individually indicated to be
incorporated by reference.
Sequence CWU 1
1
15 1 9 PRT Artificial phosphotyrosine ligands 1 Glu Asn Asp Tyr Ile
Asn Ala Ser Leu 1 5 2 11 PRT Artificial phosphotyrosine ligands 2
Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly 1 5 10 3 6 PRT
Artificial phosphotyrosine ligands 3 Asp Ala Asp Glu Tyr Leu 1 5 4
5 PRT Artificial phosphotyrosine ligands 4 Ile Tyr Gly Glu Phe 1 5
5 9 PRT Artificial phosphotyrosine ligands 5 Thr Glu Pro Glu Tyr
Gln Pro Gly Glu 1 5 6 6 PRT Artificial phosphotyrosine ligands 6
Asp Tyr Val Pro Met Leu 1 5 7 11 PRT Artificial phosphotyrosine
ligands 7 Glu Pro Gln Tyr Glu Glu Ile Pro Ile Tyr Leu 1 5 10 8 17
PRT Artificial phosphotyrosine ligands 8 Glu Gly Pro Trp Leu Glu
Glu Glu Glu Glu Ala Tyr Gly Trp Met Ser 1 5 10 15 Phe 9 13 PRT
Artificial phosphotyrosine ligands 9 Thr Ser Thr Glu Pro Gln Tyr
Gln Pro Gly Glu Asn Leu 1 5 10 10 12 PRT Artificial phosphotyrosine
ligands 10 Glu Ala Ile Tyr Ala Ala Pro Phe Ala Lys Lys Lys 1 5 10
11 9 PRT Artificial phosphotyrosine ligands 11 Trp Ala Gly Gly Asp
Ala Ser Gly Glu 1 5 12 12 PRT Artificial ABL Peptide 12 Glu Ala Ile
Tyr Ala Ala Pro Phe Ala Lys Lys Lys 1 5 10 13 13 PRT Artificial
CSRC Peptide 13 Thr Ser Thr Glu Pro Gln Tyr Gln Pro Gly Glu Asn Leu
1 5 10 14 9 PRT Artificial DSIP Peptide 14 Trp Ala Gly Gly Asp Ala
Ser Gly Glu 1 5 15 13 PRT Artificial TYR Peptide 15 Glu Ala Ile Tyr
Ala Ala Pro Phe Ala Lys Lys Lys Cys 1 5 10
* * * * *