U.S. patent application number 11/603425 was filed with the patent office on 2007-05-24 for molecular conjugate.
Invention is credited to Julia Ashworth-Sharpe, Christopher Bieniarz, Casey A. Kernag, Jerome W. Kosmeder, Mark Lefever.
Application Number | 20070117153 11/603425 |
Document ID | / |
Family ID | 37793952 |
Filed Date | 2007-05-24 |
United States Patent
Application |
20070117153 |
Kind Code |
A1 |
Bieniarz; Christopher ; et
al. |
May 24, 2007 |
Molecular conjugate
Abstract
A method is disclosed for making a conjugate of two molecules
using a hydrazide thiol linker. In a particular working embodiment,
an Fc-specific antibody-enzyme conjugate is made using the method
and demonstrated to provide exceptional staining sensitivity and
specificity in immunohistochemical and in situ hybridization
assays.
Inventors: |
Bieniarz; Christopher;
(Tucson, AZ) ; Ashworth-Sharpe; Julia; (Tucson,
AZ) ; Kernag; Casey A.; (Tucson, AZ) ;
Kosmeder; Jerome W.; (Tucson, AZ) ; Lefever;
Mark; (Tucson, AZ) |
Correspondence
Address: |
VENTANA MEDICAL SYSTEMS, INC.;ATTENTION: LEGAL DEPARTMENT
1910 INNOVATION PARK DRIVE
TUCSON
AZ
85755
US
|
Family ID: |
37793952 |
Appl. No.: |
11/603425 |
Filed: |
November 21, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60739794 |
Nov 23, 2005 |
|
|
|
Current U.S.
Class: |
435/7.1 ;
530/409; 564/148; 977/902 |
Current CPC
Class: |
C07C 323/60 20130101;
C12Y 301/03001 20130101; C07C 323/22 20130101; G01N 33/532
20130101; A61K 47/6801 20170801; A61K 47/6803 20170801; C07C 319/02
20130101; G01N 33/581 20130101; C12N 9/96 20130101; A61K 47/6815
20170801; C07C 327/28 20130101; A61K 39/44 20130101; G01N 33/535
20130101; C12N 9/16 20130101; C07K 16/00 20130101; C07C 319/02
20130101; C07C 323/12 20130101; C07C 319/02 20130101; C07C 323/60
20130101 |
Class at
Publication: |
435/007.1 ;
530/409; 977/902; 564/148 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C07K 14/47 20060101 C07K014/47; C07C 323/39 20060101
C07C323/39 |
Claims
1. A method for forming a conjugate of two or more molecules,
comprising: reacting a hydrazide thiol linker with a first molecule
having a hydrazide-reactive group to form a thiolated first
molecule, wherein reacting the hydrazide thiol linker with the
first molecule comprises reacting under conditions where a thiol
group of the hydrazide thiol linker is substantially present in its
neutral acid form; and reacting the thiolated first molecule with a
second molecule, the second molecule having a thiol-reactive group,
to form the conjugate.
2. The method of claim 1, wherein the first molecule comprises a
specific binding molecule and the second molecule comprises a
detectable label.
3. The method of claim 1, wherein reacting under conditions where
the thiol group of the hydrazide thiol linker compound is
substantially present in its neutral acid form comprises reacting
at a pH of less than about 7.
4. The method of claim 1, wherein reacting under conditions where
the thiol group of the hydrazide thiol linker compound is
substantially present in its neutral acid form comprises reacting
from about pH=3 to about pH=7.
5. The method of claim 1, wherein reacting under conditions where
the thiol group of the hydrazide thiol linker compound is
substantially present in its neutral acid form comprises reacting
from about pH=4 to about pH=6.
6. The method of claim 1, wherein the hydrazide-reactive group of
the first molecule comprises an aldehyde group.
7. The method of claim 6, wherein the first molecule comprises a
glycosylated molecule and the aldehyde group is introduced to the
first molecule by oxidation of a glycosylated portion of the first
molecule.
8. The method of claim 7, wherein the glycosylated molecule
comprises an antibody and the aldehyde group is introduced to an Fc
portion of the antibody.
9. The method of claim 1, wherein the thiol-reactive group of the
second molecule comprises a maleimide group introduced to the
second molecule.
10. The method of claim 9, wherein the maleimide group introduced
to the second molecule is introduced using a NHS-PEG-maleimide
linker.
11. The method of claim 9, wherein the second molecule comprises a
detectable label.
12. The method of claim 1, wherein the hydrazide thiol linker
compound is one or more of MBH, MBCH, MAMBH, THMBH, BTAL, BTHL,
TAGD, THGD, a PEG-based hydrazide thiol linker, a multifunctional
hydrazide thiol linker, a PEG-based multifunctional hydrazide thiol
linker, and a polyacrylamide hydrazide linker.
13. The method of claim 12, wherein the hydrazide thiol linker
compound is one or more of MBH, MBCH or a mercapto-dPEG-hydrazide
linker.
14. The method of claim 1, wherein the hydrazide thiol linker
compound has the formula: ##STR35## wherein n=1, 2 or 3; and
R.sub.1 is H, --CONHNH.sub.2, or --CO--A--CONHNH.sub.2, where A is
a divalent group having between 1 and 100 carbon atoms.
15. The method of claim 1, wherein the hydrazide thiol linker
compound has the formula: ##STR36## wherein m=2 to 50, R.sub.2 is
H, --CONHNH.sub.2, or --CO--A--CONHNH.sub.2, where A is a divalent
group having between 1 and 100 carbon atoms, and X and Y are
independently a bond or a divalent group having 1 to 20
carbons.
16. A conjugate prepared according to the method of claim 1.
17. A conjugate comprising an antibody covalently bonded to a
detectable label through a hydrazide thiol linker, wherein the
hydrazide thiol linker is one or more of MBH, MBCH, MAMBH, THMBH,
BTAL, BTHL, TAGD, THGD, a PEG-based hydrazide-thiol linker, a
multifunctional hydrazide thiol linker, a PEG-based multifunctional
hydrazide thiol linker, and a polyacrylamide hydrazide linker,
wherein a hydrazide group of the linker is covalently bonded to an
Fc region of the antibody.
18. The conjugate of claim 17, wherein the hydrazide thiol linker
comprises a PEG-based hydrazide thiol linker.
19. The conjugate of claim 18, wherein the PEG-based hydrazide
thiol linker comprises a mercapto-dPEG-hydrazide linker.
20. The conjugate of claim 17, wherein the hydrazide thiol linker
comprises MBH or MBCH.
21. The conjugate of claim 17, wherein the detectable label
comprises an enzyme, a fluorescent molecule, a hapten or a
fluorescent nanoparticle.
22. The conjugate of claim 21, wherein the detectable label
comprises an enzyme.
23. The conjugate of claim 22, wherein the detectable label
comprises an enzyme selected from the group consisting of alkaline
phosphatase and horseradish peroxidase.
24. The conjugate of claim 23, wherein the detectable label
comprises alkaline phosphatase.
25. The conjugate of claim 24, wherein the alkaline phosphatase
comprises cross-linked alkaline phosphatase.
26. The conjugate of claim 17, wherein the antibody comprises an
anti-hapten antibody.
27. The conjugate of claim 17, wherein the antibody comprises and
anti-antibody antibody.
28. A hydrazide thiol linker having the formula: ##STR37## wherein
R.sub.1 is H or --CONHNH.sub.2 and n=1, 2 or 3.
29. A kit comprising a hydrazide thiol linker having the formula:
##STR38## wherein R.sub.1 is H or --CONHNH.sub.2 and n=1, 2 or 3;
and instructions for performing the method of claim 1.
30. A kit comprising a hydrazide thiol linker having the formula:
H.sub.2N--NH--CO--(CH.sub.2--CH.sub.2--O).sub.t--CH.sub.2--CH.sub.2--SH
where t=2 to 50; and instructions for performing the method of
claim 1.
Description
RELATED APPLICATION DATA
[0001] This claims the benefit of U.S. Provisional Patent
Application No. 60/739,794, filed Nov. 23, 2005, which application
is incorporated by reference herein.
FIELD
[0002] The present invention relates to molecular conjugates,
linkers for making such conjugates, methods for making the
conjugates and the linkers, and methods of using the conjugates.
More particularly, the present invention relates to Fc-specific
antibody conjugates, hydrazide thiol linkers for preparing
Fc-specific conjugates, methods for making Fc-specific conjugates,
and methods of using Fc-specific antibody conjugates.
BACKGROUND
[0003] A wide variety of methods have been developed for linking
molecules together to form conjugates. Of particular interest are
biomolecular conjugates that are typically prepared to combine the
functionalities of the joined molecules into one construct. One
type of biomolecular conjugate combines a biomolecule that
specifically binds to another molecule (such as a nucleic acid, an
antibody, a lectin or an avidin) and a detectable label (such as a
fluorescent label, fluorescent nanoparticle or an enzyme).
[0004] Conjugates of antibodies and detectable labels (antibody
conjugates) can be used in immunoassays for detecting specific
target molecules in biological samples. The antibody portion of
such conjugates specifically binds to a target in the sample and
the detectable label is utilized to provide a detectable signal
that indicates the presence/and or location of the target. One type
of conjugate that has become widely used, especially for
immunohistochemical analysis, is a conjugate of an antibody and an
enzyme (antibody-enzyme conjugate). A detectable signal is
generated by adding a substrate to the sample under conditions
where the enzyme portion of the antibody-enzyme conjugate converts
the substrate to a detectable product (such as a colored,
different-colored or fluorescent product) at the site where the
antibody portion is bound to its target.
[0005] Antibody conjugates are typically prepared using coupling
reagents that are characterized by having at least two reactive
groups, one of which is reacted with a functional group on the
antibody and the other of which is reacted with a functional group
on the detectable label. However, coupling can lead to inactivation
of either or both of the antibody and the detectable label. In
particular, coupling can deactivate antibody-enzyme conjugates
through steric effects or because the coupling reagents react with
functional groups located on portions of the antibody and/or enzyme
that are critical for their specificity and/or catalytic activity.
Furthermore, some coupling schemes lead to conjugates that have
reduced water solubility.
[0006] Coupling schemes that can provide antibody-enzyme conjugates
with reduced impairment of antibody specificity and/or enzyme
activity are desirable and enable greater sensitivities to be
achieved in immunochemical assays such as immunohistochemical
assays. Greater sensitivity is of particular importance for
automated processes where additional amplification steps are
undesirable.
SUMMARY
[0007] A molecular conjugate that includes a hydrazide thiol linker
is disclosed. In one embodiment, an antibody-detectable label
conjugate is provided including a hydrazide thiol linker covalently
bonded to the Fc portion of the antibody. The Fc-specific conjugate
of this embodiment provides improved detection sensitivity, thereby
making immunohistochemical detection of a target molecule more
amenable to automation and high-throughput applications.
[0008] Also disclosed is a method for preparing a conjugate using a
hydrazide thiol linker. In one embodiment, a protecting group for a
thiol group of the linker is not needed because the linker is
reacted with a first molecule under conditions where the thiol
group is substantially present in its neutral acid form and thus
substantially unreactive. Under such conditions, a covalent bond
can be formed between a hydrazide group of the linker compound and
a first molecule while substantially preserving the thiol group for
subsequent reaction with a thiol-reactive group of a second
molecule.
[0009] Hydrazide thiol linkers and methods for making hydrazide
thiol linkers also are disclosed. In addition, methods are
described for using a disclosed conjugate to detect a target
molecule in a sample such as a tissue section or cytology sample.
The methods of detecting a target molecule can be readily automated
due to the improved sensitivity exhibited by the disclosed
conjugates. In certain embodiments, multiplexed assays using the
disclosed conjugates are provided, for example, multiplexed assays
employing disclosed antibody conjugates having fluorescent
molecules or fluorescent nanoparticles as the detectable label.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a series of images showing staining patterns for
detection of Kappa in tonsil tissue using a disclosed Fc-specific
antibody-alkaline phosphatase conjugate and using a
streptavidin-alkaline phosphatase conjugate.
[0011] FIG. 2 is a series of images showing staining patterns for
detection of Lambda in tonsil tissue using a disclosed Fc-specific
antibody-alkaline phosphatase conjugate and using a
streptavidin-alkaline phosphatase conjugate.
[0012] FIG. 3 is a series of images showing staining patterns for
detection of CMV in lung tissue using a disclosed Fc-specific
antibody-alkaline phosphatase conjugate and using a
streptavidin-alkaline phosphatase conjugate.
[0013] FIG. 4 is a series of images showing staining patterns for
detection of EBER in spleen tissue using a disclosed Fc-specific
antibody-alkaline phosphatase conjugate and using a
streptavidin-alkaline phosphatase conjugate.
[0014] FIG. 5 is a series of images showing staining patterns for
detection of HPV in CaSki xenograft tissue using a disclosed
Fc-specific antibody-alkaline phosphatase conjugate and using a
streptavidin-alkaline phosphatase conjugate.
[0015] FIG. 6 is a series of images showing staining patterns for
detection of HPV in HeLa xenograft tissue using a disclosed
Fc-specific antibody-alkaline phosphatase conjugate and using a
streptavidin-alkaline phosphatase conjugate.
[0016] FIG. 7 is a pair of images showing staining patterns for
detection of HPV in SiHa xenograft tissue using a disclosed
Fc-specific antibody-alkaline phosphatase conjugate and using a
streptavidin-alkaline phosphatase conjugate.
[0017] FIG. 8 is a series of images showing staining patterns for
detection of HPV in cytology samples using a disclosed Fc-specific
antibody-alkaline phosphatase conjugate and using a
streptavidin-alkaline phosphatase conjugate.
[0018] FIG. 9 is a pair of images showing staining patterns for
detection of actin in muscle tissue using a disclosed Fc-specific
antibody-alkaline phosphatase conjugate and using a
streptavidin-alkaline phosphatase conjugate.
[0019] FIG. 10 is a series of images showing a comparison of the
sensitivity of disclosed antibody-enzyme conjugates with each other
and with antibody-body enzyme conjugates prepared by other
methods.
DETAILED DESCRIPTION OF SEVERAL ILLUSTRATIVE EMBODIMENTS
[0020] Further aspects of the invention are illustrated by the
following non-limiting descriptions and examples, which proceed
with respect to the abbreviations and terms below.
I. Abbreviations
[0021] Ab--antibody
[0022] (Ab-AP)--antibody-alkaline phosphatase conjugate
[0023] AP--alkaline phosphatase
[0024] BSA--bovine serum albumin
[0025] CMV--cytomegalovirus
[0026] EBER--Epstein-Barr virus early RNA
[0027] DL--detectable label
[0028] Fc--fragment crystallizable
[0029] HRP--horseradish peroxidase
[0030] IHC--immunohistochemistry
[0031] ISH--in situ hybridization
[0032] MAL--maleimide
[0033] MBCH--mercaptobutyric acid carbohydrazide
[0034] MBH--mercaptobutyric acid hydrazide
[0035] NHS--N-hydroxy-succinimide
[0036] PEG--polyethylene glycol
[0037] SBM--specific binding molecule
II. Terms
[0038] The terms "a," "an" and "the" include both singular and
plural referents unless the context clearly indicates
otherwise.
[0039] The term "amination" as used herein refers to reaction of a
carbonyl group of an aldehyde or a ketone with an amine group,
wherein an amine-containing compound such as an amine or a
hydrazide reacts with the aldehyde or ketone to first form a Shiff
base that can then reversibly rearrange to a more stable form, or
optionally be reduced to prevent reversal of the reaction.
"Reductive amination" conditions include addition of a reducing
agent, more typically addition of a mild reducing agent such as
sodium cyanoborohydride or one of its co-geners, for example,
sodium triacetoxyborohydride. Other mild reducing agents that can
be employed include various amine boranes.
[0040] The term "antibody" collectively refers to an immunoglobulin
or immunoglobulin-like molecule (including IgA, IgD, IgE, IgG and
IgM, and similar molecules produced during an immune response in
any organism, for example, in mammals such as humans, goats,
rabbits and mice), or a fragment thereof, that specifically binds
to a target (or a group of highly similar targets) to the
substantial exclusion of binding to other molecules. In some
embodiments, an antibody specifically binds to a target with a
binding constant that is at least 10.sup.3 M.sup.-1 greater,
10.sup.4 M.sup.-1 greater or 10.sup.5 M.sup.-1 greater than a
binding constant for other molecules in a sample. In other
embodiments, an antibody has a Kd value for binding to an antigenic
determinant (such as a hapten or epitope) that is on the order of
10.sup.-6 M or lower, such as 10.sup.-9 M or lower, or even
10.sup.-12 M or lower. Kd values can, for example, be determined by
competitive ELISA (enzyme-linked immunosorbent assay) or using a
surface-plasmon resonance device such as the Biacore T100, which is
available from Biacore, Inc., Piscataway, N.J. Antibody fragments
include proteolytic antibody fragments [such as F(ab').sub.2
fragments, Fab' fragments, Fab'-SH fragments and Fab fragments as
are known in the art], recombinant antibody fragments (such as sFv
fragments, dsFv fragments, bispecific sFv fragments, bispecific
dsFv fragments, diabodies, and triabodies as are known in the art),
and camelid antibodies (see, for example, U.S. Pat. Nos. 6,015,695;
6,005,079; 5,874,541; 5,840,526; 5,800,988; and 5,759,808).
Antibodies include both monoclonal and polyclonal antibody
preparations. Although an antibody of a disclosed conjugate can
specifically bind any particular molecule or any particular group
of highly similar molecules, in particular embodiments, the
antibody comprises an anti-hapten antibody (which can, for example,
be used to detect a hapten-labeled probe sequence directed to a
nucleic acid sequence of interest). In particular embodiments, the
antibody comprises an anti-antibody antibody that can be used as a
secondary antibody in an immunoassay. For example, the antibody can
comprise an anti-IgG antibody such as an anti-mouse IgG antibody,
an anti-rabbit IgG antibody or an anti-goat IgG antibody.
[0041] The phrase "conditions where a thiol group of a hydrazide
thiol linker is substantially present in its neutral acid form"
refers to conditions, such as conditions of pH, wherein less than
about 1% of the thiol group (--SH; the protonated neutral acid
form) of the linker is present in its conjugate base form (--S--;
unprotonated, negatively charged form). For example, under such
conditions less than about 0.1%, less than about 0.01%, or even
less than about 0.001% of the linker can be in the conjugate base
form. Conditions where the thiol group of the hydrazide thiol
linker compound is substantially present in its neutral acid form
include a pH of less than about 7, for example, a pH of less than
about 6 such as a pH of less than about 5.5. In particular
embodiments, such conditions include a range of pHs, for example,
from a pH of about 3 to a pH of about 7, from a pH of about 4 to a
pH of about 7, from a pH of about 4 to a pH of about 6, from a pH
of about 4.5 to a pH of about 5.5, or any sub-range of each of
these ranges. In other embodiments, the upper limit of the pH range
in which a thiol group of a particular linker is substantially
present in its neutral acid form (less than 1% of the thiol group
being present as the conjugate base form) can be higher than 7,
such as a pH of 8. One of ordinary skill in the art can readily
determine an upper limit to the pH range in which a given thiol
group will be substantially present in the neutral acid form using
the Henderson-Hasselbach equation and a pKa value for a thiol group
of the linker. In yet other embodiments, a thiol group of a
particular linker can be substantially present in its neutral acid
form in a solvent system for which an accurate pH cannot be
determined, and one of ordinary skill in the art will recognize
that solvent systems that are less polar than water may help keep
the thiol group in its neutral acid form at higher apparent pHs.
Alternatively, an experimental determination of whether under
particular conditions a thiol group of a linker is substantially
present in its neutral acid form can be made by determining whether
the thiol will reduce a disulfide bond present in another molecule.
For example, a determination can be made of the number of free
thiol groups (for example, using Ellman's reagent) introduced into
a molecule having disulfides (such as an irnmunoglobulin) by
contact with the linker under the particular conditions of pH (or
estimated pH for non-aqueous systems). Addition of an excess of the
hydrazide thiol linker (such as a 50-fold excess or more) over a
period of time (such as an hour or more) can be followed by the
determination of the average number of free thiols introduced into
the molecule. If free thiols are generated to a substantial degree
(such as greater than an average of two thiols introduced per
immunoglobulin molecule), it shows that the thiol of the linker is
not substantially present in it neutral acid form under the tested
conditions. For example, at a pH of about 7, a one hundred-fold
excess of the linker MBH relative to an immunoglobulin will produce
an average of about 2 thiols per immunoglobulin molecule. At a
lower pH of 5, a thousand-fold excess of the MBH linker will
produce, on average, substantially less than 1 thiol per
immunoglobulin in 24 hours. These results demonstrate that for the
linker MBH, the thiol group is substantially present in its neutral
acid form at a pH of about 7 or lower, since as pH is lowered, the
equilibrium between the neutral acid form and its conjugate base is
shifted more towards the neutral acid form.
[0042] A "conjugate" refers to two or more molecules (and/or
materials such as nanoparticles) that are covalently linked into a
larger construct. In some embodiments, a conjugate includes one or
more biomolecules (such as peptides, nucleic acids, proteins,
enzymes, sugars, polysaccharides, lipids, glycoproteins, and
lipoproteins) covalently linked to one or more other molecules,
such as one or more other biomolecules. In other embodiments, a
conjugate includes one or more specific-binding molecules (such as
antibodies and nucleic acid sequences) covalently linked to one or
more detectable labels (such as fluorescent molecules, fluorescent
nanoparticles, haptens, enzymes and combinations thereof).
[0043] A "detectable label" is a molecule or material that can
produce a detectable (such as visually, electronically or
otherwise) signal that indicating the presence and/or concentration
of the label in a sample. When conjugated to a specific binding
molecule, the detectable label can be used to locate and/or
quantify the target to which the specific binding molecule is
directed. Thereby, the presence and/or concentration of the target
in a sample can be detected by detecting the signal produced by the
detectable label. A detectable label can be detected directly or
indirectly, and several different detectable labels conjugated to
different specific-binding molecules can be used in combination to
detect one or more targets. For example, a first detectable label
such as a hapten conjugated to a nucleic acid probe or antibody
specific to a target can be detected indirectly through the use of
a second detectable label that is conjugated to a molecule that
specifically binds the first detectable label. Multiple detectable
labels that can be separately detected can be conjugated to
different specific binding molecules that specifically bind
different targets to provide a multiplexed assay that can provide
simultaneous detection of the multiple targets in a sample. A
detectable signal can be generated by any known or yet to be
discovered mechanism including absorption, emission and/or
scattering of a photon (including radio frequency, microwave
frequency, infrared frequency, visible frequency and ultra-violet
frequency photons). Detectable labels include colored, fluorescent,
phosphorescent and luminescent molecules and materials, catalysts
(such as enzymes) that convert one substance into another substance
to provide a detectable difference (such as by converting a
colorless substance into a colored substance or vice versa, or by
producing a precipitate or increasing sample turbidity), haptens
that can be detected through antibody-hapten binding interactions
using additional detectably labeled antibody conjugates, and
paramagnetic and magnetic molecules or materials. Particular
examples of detectable labels include enzymes such as horseradish
peroxidase, alkaline phosphatase, acid phosphatase, glucose
oxidase, .beta.-galactosidase, .beta.-glucuronidase or
.beta.-lactamase; fluorescent molecules such as fluoresceins,
coumarins, BODIPY dyes, resorufins, and rhodamines (many additional
examples of fluorescent molecules can be found in The Handbook--A
Guide to Fluorescent Probes and Labeling Technologies, Molecular
Probes, Eugene, Oreg.); nanoparticles such as quantum dots
(obtained, for example; from QuantumDot Corp, Invitrogen
Nanocrystal Technologies, Hayward, Calif.; see also, U.S. Pat. Nos.
6,815,064, 6,682,596 and 6,649,138, each of which patents is
incorporated by reference herein); metal chelates such as DOTA and
DPTA chelates of radioactive or paramagnetic metal ions like
Gd.sup.3+; and liposomes, for example, liposomes containing trapped
fluorescent molecules. Where the detectable label includes an
enzyme, a detectable substrate such as a chromogen, a fluorogenic
compound, or a luminogenic compound can be used in combination with
the enzyme to generate a detectable signal (A wide variety of such
compounds are commercially available, for example, from Invitrogen
Corporation, Eugene Oreg.). Particular examples of chromogenic
compounds include diaminobenzidine (DAB), 4-nitrophenylphospate
(pNPP), fast red, bromochloroindolyl phosphate (BCIP), nitro blue
tetrazolium (NBT), BCIP/NBT, fast red, AP Orange, AP blue,
tetramethylbenzidine (TMB), 2,2'-azino-di-[3-ethylbenzothiazoline
sulphonate] (ABTS), o-dianisidine, 4-chloronaphthol (4-CN),
nitrophenyl-.beta.-D-galactopyranoside (ONPG), o-phenylenediamine
(OPD), 5-bromo-4-chloro-3-indolyl-.beta.-galactopyranoside (X-Gal),
methylumbelliferyl-.beta.-D-galactopyranoside (MU-Gal),
p-nitrophenyl-.alpha.-D-galactopyranoside (PNP),
5-bromo-4-chloro-3-indolyl-.beta.-D-glucuronide (X-Gluc),
3-amino-9-ethyl carbazol (AEC), fuchsin, iodonitrotetrazolium
(INT), tetrazolium blue and tetrazolium violet. Alternatively, an
enzyme can be used in a metallographic detection scheme.
Metallographic detection methods include using an enzyme such as
alkaline phosphatase in combination with a water-soluble metal ion
and a redox-inactive substrate of the enzyme. The substrate is
converted to a redox-active agent by the enzyme, and the
redox-active agent reduces the metal ion, causing it to form a
detectable precipitate. (See, for example, co-pending U.S. patent
application Ser. No. 11/015,646, filed Dec. 20, 2004, PCT
Publication No. 2005/003777 and U.S. Patent Application Publication
No. 2004/0265922; each of which is incorporated by reference
herein). Metallographic detection methods include using an
oxido-reductase enzyme (such as horseradish peroxidase) along with
a water soluble metal ion, an oxidizing agent and a reducing agent,
again to for form a detectable precipitate. (See, for example, U.S.
Pat. No. 6,670,113, which is incorporated by reference herein).
Haptens are small molecules that are specifically bound by
antibodies, although by themselves they will not elicit an immune
response in an animal and must first be attached to a larger
carrier molecule such as a protein or a poly-nucleic acid to
generate an immune response. Examples of haptens include
di-nitrophenol, biotin, digoxigenin, and fluorescein. Additional
examples of oxazole, pyrazole, thiazole, nitroaryl, benzofuran,
triperpene, urea, thiourea, rotenoid, coumarin and cyclolignan
haptens are disclosed in co-pending U.S. Provisional Patent
Application No., 60/856133, filed Nov. 1, 2006, which is
incorporated by reference herein.
[0044] The term "Fc-specific conjugate" as used herein refers to a
conjugate of an immunoglobulin (or fragment thereof) in which a
second molecule (such as a detectable label) is covalently bonded
to the glycosylated portion of the immunoglobulin (or a fragment of
an immunoglobulin that retains the glycosylated portion). The
glycosylated portion of an immunoglobulin is found in the
Fc-region, which is a region of an immunoglobulin that is located
on the heavy chains of the immunoglobulin at positions outside of
the portion of the immunoglobulin that is responsible for the
specific binding activity of the imrnunoglobulin.
[0045] The term "hydrazide group" refers to a hydrazide group
(--CO--NH--NH.sub.2); a carbohydrazide group
(--NH--NH--CO--NH--NH.sub.2); a semicarbazide group
(--NH--CO--NH--NH.sub.2); a thiosemicarbazide group
(--NH--CS--NH--NH.sub.2); a thiocarbazide group
(--NH--NH--CS--NH--NH.sub.2); a carbonic acid dihydrazine group
(--NH--CO--NH--NH--CO--NH--NH.sub.2) or a sulfur containing
derivative thereof; or a hydrazine carboxylate group
(--O--CO--NH--NH.sub.2) or a sulfur-containing derivative
thereof.
[0046] The term "hydrazide-reactive group" refers to a group of
atoms that can react with and form a covalent bond to a hydrazide
group. Aldehyde and ketone groups are examples of
hydrazide-reactive groups. Hydrazide-reactive groups can be an
intrinsic part of a molecule or can be introduced to a molecule.
One method for introducing an aldehyde group (a hydrazide-reactive
group) into polysaccharides and glycoproteins (including
antibodies) is by oxidation such as periodate-mediated oxidation of
vicinal diols. In addition, double bonds in unsaturated fatty acids
and ceramides can be converted to diols by osmium tetroxide and
then oxidized by periodate to aldehydes. Furthermore, N-terminal
serine and threonine residues of peptides and proteins can be
selectively oxidized by periodate to aldehyde groups, permitting
selective modification of certain proteins such as corticotrophin
and .beta.-lactamase. Modification of periodate-oxidized antibodies
does not typically inactivate the antibody. Varying the
concentration of sodium periodate during the oxidation reaction
gives some specificity with regard to the types of sugar residues
that are modified. For example, sodium periodate at a concentration
of 1 mM at 0.degree. C. typically cleaves only at the adjacent
hydroxyls between carbon atoms 7, 8 and 9 of sialic acid residues.
Oxidizing polysaccharides using 10 mM or greater concentrations of
sodium periodate results in oxidation of sugar residues other than
sialic acid, thereby creating many aldehydes on a given
polysaccharide. A suitable general protocol is described by
Hermanson, "Bioconjugate Techniques," Academic Press, San Diego,
1996, ISBN 0-12-342336-8, which is incorporated by reference
herein. Another method for introducing aldehydes into biomolecules
is through the use of specific sugar oxidases, for example,
galactose oxidase, which is an enzyme that oxidizes terminal
galactose residues to aldehydes, particularly in glycoproteins.
When galactose residues are penultimate to sialic acid residues,
neuramidase can be used to remove the sialic acid residue and
expose galactose as the terminal residue. A protocol for using a
combination of neuramidase and galactose oxidase to oxidize
galactose residues to provide a reactive aldehyde group is provided
in Hermanson, "Bioconjugate Techniques," Academic Press, San Diego,
1996, ISBN 0-12-342336-8, which is incorporated by reference
herein. Aldehydes also can be introduced to a molecule by reacting
an amine group of a molecule with an NHS-aldehyde such as
succinimidyl p-formylbenzoate (SFB) or
succinimidylp-formylphenoxyacetate (SFPA) (Invitrogen Corp.,
Eugene, Oreg.). Alternatively, bis-aldehyde compounds such as
glutaraldehyde can be used to modify an amine group to provide an
aldehyde group. Again, suitable protocols are provided in
Hermanson, "Bioconjugate Techniques," Academic Press, San Diego,
1996, ISBN 0-12-342336-8, which is incorporated by reference
herein.
[0047] The term "hydrazide thiol linker" refers to a molecule
including one or more hydrazide groups and one or more thiol groups
(--SH) joined covalently through one or more linking atoms. The
hydrazide group(s) and thiol group(s) of a hydrazide thiol linker
can be joined through one or more of various groups of atoms
including methylene groups (--CH.sub.2--), branched alkylene
groups, additional hydrazide groups, aromatic groups,
heteroaromatic groups, alicyclic groups, polyalkylene glycol groups
(such as ethylene oxide groups; --O--CH.sub.2--CH.sub.2--), amide
groups (--CONH--), amine groups (--NH--), ether groups (--O--), and
combinations thereof. A "PEG-based hydrazide thiol linker" refers
to a linker including 1 or more ethylene glycol groups as part of
its structure. A "multifunctional hydrazide thiol linker" refers to
a branched linker having at least one hydrazide group, at least one
thiol group, and at least one additional reactive group, such as an
additional hydrazide group, an additional thiol group, or any other
group useful for preparing molecular conjugates. In some
embodiments, a PEG-based hydrazide thiol linker comprises a
discrete PEG (dPEG) linker, which can be made from dPEG starting
materials such as those disclosed in U.S. Patent Application
Publication No. 20060020134, and can be purchased from Quanta
Biodesign (Powell, Ohio). Examples of additional reactive groups
that can be included in a polyfunctional hydrazide-thiol linker
include maleimide groups and active esters, such as
N-hydroxysuccinimide esters, and hydroxy groups (--OH). Additional
examples of reactive groups can be found in Hernanson,
"Bioconjugate Techniques," Academic Press, San Diego, 1996, ISBN
0-12-342336-8, which is incorporated by reference herein.
[0048] The term "sample" refers to any liquid, semi-solid or solid
substance (or material) in or on which a target can be present. In
particular, a sample can be a biological sample or a sample
obtained from a biological material. Examples of biological samples
include tissue samples and cytology samples.
[0049] The term "specific binding molecule" refers to a molecule
that specifically binds to a second molecule. "Specifically binds"
means that the specific-binding molecule binds to the second
molecule to the substantial exclusion of other molecules that are
present in a sample (for example, the binding constant of the
specific-binding molecule is at least 10.sup.2 M.sup.-1 greater,
10.sup.3 M.sup.-1 greater, 10.sup.4 M.sup.-1 greater or 10.sup.5
M.sup.-1 greater than a binding constant for other molecules in the
sample). Examples of specific binding molecules include nucleic
acids, receptors, antibodies, enzymes, lectins and avidins.
Examples of specific-binding interactions in which specific binding
molecules can participate include formation of duplexes and
triplexes of nucleic acid sequences, receptor-ligand interactions
(such as folate-folate receptor interactions), antibody-antigen
interactions, enzyme-substrate interactions, lectin-sugar reactions
and avidin-biotin interactions (such as streptavidin-biotin
interactions).
[0050] The term "target" refers to any molecule for which the
presence, location and/or concentration is or can be determined.
Examples of target molecules include proteins, nucleic acid
sequences, and haptens, such as haptens covalently bonded to
nucleic acid sequences or proteins. Target molecules are typically
detected using one or more conjugates of a specific binding
molecule and a detectable label.
[0051] The term "thiol-reactive group" refers to an atom or atoms
that can react with and form a covalent bond with a thiol group. A
thiol reactive group can be an intrinsic part of a molecule or can
be introduced to the molecule through reaction with one or more
other molecules. Examples of thiol-reactive groups include
non-polymerizable Michael acceptors, haloacetyl groups (such as
bromoacetyl and iodoacetyl groups), alkyl halides, maleimides,
aziridines, acryloyl groups, vinyl sulfones, benzoquinones,
aromatic groups that can undergo nucleophilic substitution such as
fluorobenzene groups (such as tetra and pentafluorobenzene groups),
and disulfide groups such as pyridyl disulfide groups and thiols
activated with Ellman's reagent. Additional examples of each of
these types of groups will be apparent to those skilled in the art.
Further examples and information regarding reaction conditions and
methods for exchanging one type of reactive group for another to
add a thiol-reactive group are provided in Hermanson, "Bioconjugate
Techniques," Academic Press, San Diego, 1996, ISBN 0-12-342336-8,
which is incorporated by reference herein. In a particular
embodiment, a heterobifunctional linker molecule is attached to a
molecule to introduce a thiol-reactive group. For example, a linker
having a maleimide group and an N-Hyroxysuccinimide (NHS) group can
be attached to an amine group on a molecule through the NHS group,
thereby providing the molecule with a thiol-reactive maleimide
group that can be reacted with a thiol group on another molecule
(such as one introduced using a hydrazide thiol linker and the
disclosed method) to form a conjugate.
III. Overview
[0052] One of ordinary skill in the art will recognize that the
disclosed method can be used to join any combination of molecules
having functional groups that can react with a hydrazide thiol
linker. The non-limiting description that follows focuses on
antibody conjugates, and more particularly, on antibody-enzyme
conjugates, but should not be construed as a limitation on the
scope of the invention. Although the specifically disclosed
conjugates are antibody-enzyme conjugates, conjugates between other
biomolecules (such as nucleic acid sequences) and other detectable
labels (such as haptens, fluorescent labels, fluorescent
nanoparticles and fluorescent proteins, such as green fluorescent
protein) are contemplated and fall within the scope of the
disclosure.
[0053] Accordingly, in one aspect, a method is disclosed for
forming a conjugate of two or more molecules. The method includes
reacting a hydrazide thiol linker with a first molecule (such as an
antibody) having a hydrazide-reactive group (such as an aldehyde)
to form a thiolated first molecule. The reaction is carried out
under conditions where a thiol group of the hydrazide thiol linker
is substantially present in its neutral acid (protonated) form. The
thiolated first molecule can then be reacted with a second molecule
having a thiol-reactive group (such as a maleimide group introduced
to the second molecule) to form the conjugate. In a particular
embodiment, the reaction of the first molecule with the linker is
carried out at a pH from about pH=4 to about pH=7. In other
particular embodiments, the hydrazide thiol linker can be a
PEG-based hydrazide thiol linker, a multifunctional hydrazide thiol
linker, or a PEG-based multifunctional hydrazide thiol linker.
[0054] In another embodiment, the method can be used to covalently
join a specific binding molecule to a detectable label. In a more
particular embodiment, the method can be used to link a first
molecule having a glycosylated portion to another molecule. In this
embodiment, the glycosylated portion is first oxidized to generate
an aldehyde group that can be reacted with a hydrazide thiol
linker. In an even more particular embodiment, the glycosylated
first molecule can be an antibody that has a glycosylated Fc
region. An Fc-specific thiolated antibody is formed by reaction
with a hydrazide thiol linker, and the Fc-specific thiolated
antibody can be reacted with a detectable label having a
thiol-reactive group.
[0055] In another aspect, a variety of hydrazide thiol linkers and
methods for making the same are provided as outlined in the
Synthetic Overview and specific Examples that follow. A further
aspect is a conjugate prepared with a disclosed linker. In an
additional aspect, a kit is disclosed that includes a disclosed
linker and instructions for performing the disclosed method for
making a conjugate. Also disclosed are methods for using disclosed
conjugates to detect a target in a sample.
IV. Synthetic Overview
[0056] A. Preparation of Hydrazide thiol Linkers
[0057] Although any hydrazide thiol linker can be used in the
disclosed method of making a conjugate, in one embodiment, a
hydrazide thiol linker can be provided by reacting a thiolactone
with hydrazine, carbohydrazide or a dihydrazide according to Scheme
1 below, wherein n=1, 2 or 3, R.sub.1 is H, --CONHNH.sub.2, or
--CO--A--CONHNH.sub.2, where A is a divalent group having between 1
and 100 carbon atoms that can be interrupted by one or more
heteroatoms (for example, O, N or S), and can be substituted, for
example, with one or more alkyl, hydroxyl, alkoxy, acyl, carboxy,
halogen, sulfonate, oxo, phosphonate and/or amine groups. In more
particular embodiments, A is a divalent group consisting of 1-10
methylene groups (--CH.sub.2--) and/or 1-24 ethylene oxide
(--CH--CH.sub.2--O--) groups. In even more particular embodiments,
A is a divalent group consisting of 1-6 methylene groups or 4-12
ethylene oxide groups. ##STR1##
[0058] A wide variety of hydrazide thiol linkers that can be used
in the disclosed method also can be provided according to scheme 2
below. In this scheme, Z is a divalent group having from 1 to 100
carbon atoms, wherein the divalent group can be interrupted by one
or more heteroatoms (for example, O, N or S), and can be
substituted, for example, with one or more hydroxyl, alkoxy, acyl,
carboxy, halogen, sulfonate, oxo, phosphonate and/or amine groups.
In more particular embodiments, Z is a divalent group consisting of
1-10 methylene groups (--CH.sub.2--) and/or 1-24 ethylene oxide
(--CH--CH.sub.2--O--) groups. In even more particular embodiments,
Z is a divalent group consisting of 1-6 methylene groups or 4-12
ethylene oxide groups. R.sub.2 is H, --CONHNH.sub.2, or
--CO--A--CONHNH.sub.2, where A is a divalent group having between 1
and 100 carbon atoms that can be interrupted by one or more
heteroatoms (for example, O, N or S), and can be substituted, for
example, with one or more alkyl, hydroxyl, alkoxy, acyl, carboxy,
halogen, sulfonate, oxo, phosphonate and/or amine groups.
##STR2##
[0059] In some embodiments, a PEG-based hydrazide thiol linker that
can be used in the disclosed method is provided and is prepared
according to scheme 3. In scheme 3, m=2 to 50; R.sub.3 is H,
--CONHNH.sub.2, or --CO--A--CONHNH.sub.2, where A is a divalent
group having between 1 and 100 carbon atoms that can be interrupted
by one or more heteroatoms (for example, O, N or S), and can be
substituted, for example, with one or more alkyl, hydroxyl, alkoxy,
acyl, carboxy, halogen, sulfonate, oxo, phosphonate and/or amine
groups; and X and Y are independently a bond or a divalent group
having 1 to 20 carbon atoms. In more particular embodiments, A is a
divalent group consisting of 1-10 methylene groups (--CH.sub.2--)
and/or 1-24 ethylene oxide (--CH--CH.sub.2--O--) groups. In even
more particular embodiments, A is a divalent group consisting of
1-6 methylene groups or 4-12 ethylene oxide groups. The X and Y
divalent groups can be interrupted by one or more heteroatoms (for
example, O, N or S), and can be substituted, for example, with one
or more alkyl, hydroxyl, alkoxy, acyl, carboxy, halogen, sulfonate,
oxo, phosphonate and/or amine groups. In more particular
embodiments, X and Y are independently a bond or
--(CH.sub.2).sub.p-- where p=1 to 3. The carbodiimide used in the
coupling reaction can be any carbodiimide that provides the desired
coupling according to the scheme. Examples of suitable
carbodiimides include DCC (N,N'-dicyclohexylcarbodiimide), and DIC
(N,N'-diisopropylcarbodiimide). In a working embodiment that is
discussed below, DCC is used to accomplish the coupling.
##STR3##
[0060] In other embodiments, a multifunctional hydrazide thiol
linker that can be used in the disclosed method is provided.
Schemes 4a, 4b, 4c, and 4d below show general methods for preparing
multifunctional linkers from homocysteine, lysine, glutamic acid
and homoserine, respectively. In Schemes 4a, 4b, 4c and 4d, D is a
divalent group having from 1 to 100 carbon atoms, wherein the
divalent group can be interrupted by one or more heteroatoms (for
example, O, N or S), and can be substituted, for example, with one
or more alkyl, hydroxyl, alkoxy, acyl, carboxy, halogen, sulfonate,
oxo, phosphonate and/or amine groups. In more particular
embodiments, D is a divalent group consisting of 1-10 methylene
groups (--CH.sub.2--) and/or 1-24 ethylene oxide
(--CH--CH.sub.2--O--) groups. In even more particular embodiments,
D is a divalent group consisting of 1-6 methylene groups or 4-12
ethylene oxide groups. Also in Schemes 4a, 4b, 4c and 4d, R.sub.4
is H, --CONHNH.sub.2, or --CO--A--CONHNH.sub.2, where A is a
divalent group having between 1 and 100 carbon atoms that can be
interrupted by one or more heteroatoms (for example, O, N or S),
and can be substituted, for example, with one or more alkyl,
hydroxyl, alkoxy, acyl, carboxy, halogen, sulfonate, oxo,
phosphonate and/or amine groups. In more particular embodiments, A
is a divalent group consisting of 1 -10 methylene groups
(--CH.sub.2--) and/or 1-24 ethylene oxide (--CH--CH.sub.2--O--)
groups. In even more particular embodiments, A is a divalent group
consisting of 1-6 methylene groups or 4-12 ethylene oxide groups.
##STR4## ##STR5## ##STR6## ##STR7##
[0061] In other particular embodiments, a PEG-based multifunctional
hydrazide thiol linker that can be used in the disclosed method is
provided. Schemes 5a, 5b and 5c illustrate general synthetic
schemes that can be used to provide such linkers. In these schemes,
p=2 to 50 and R.sub.5 is H, --CONHNH.sub.2, or
--CO--A--CONHNH.sub.2, where A is a divalent group having between 1
and 100 carbon atoms that can be interrupted by one or more
heteroatoms (for example, O, N or S), and can be substituted, for
example, with one or more alkyl, hydroxyl, alkoxy, acyl, carboxy,
halogen, sulfonate, oxo, phosphonate and/or amine groups. In more
particular embodiments, A is a divalent group consisting of 1-10
methylene groups (--CH.sub.2--) and/or 1-24 ethylene oxide
(--CH--CH.sub.2--O--) groups. In even more particular embodiments,
A is a divalent group consisting of 1-6 methylene groups or 4-12
ethylene oxide groups. R.sub.7 can be H, alkyl or a protecting
group. ##STR8## ##STR9## ##STR10##
[0062] B. Preparation of Fc-Specific Antibody Conjugates
[0063] In one embodiment, a conjugate including a hydrazide thiol
linker comprises a conjugate of an antibody and a detectable label.
In a particular embodiment, the conjugate comprises an Fc-specific
conjugate of an antibody and a detectable label. In a more
particular embodiment, the conjugate comprises an Fc-specific
conjugate of an antibody and an enzyme such as alkaline
phosphatase. Scheme 6 illustrates a method of adding a hydrazide
thiol linker to an antibody in an Fc-specific manner. ##STR11##
[0064] In Scheme 6, an antibody having a glycosylated Fc-portion is
site-specifically oxidized to generate one or more aldehyde groups
in the sugar moieties of the glycosylated Fc-portion. The aldehyde
group(s) is (are) then reacted with a hydrazide thiol linker under
conditions where the thiol group of the hydrazide thiol linker is
substantially protonated (substantially in its neutral acid form).
Under such conditions, the hydrazide group of the linker is
covalently bonded to the Fc-portion of the antibody while leaving
the thiol group substantially unreacted (such as substantially
unreacted with disulfide linkages in the antibody) and thus
retained for later reaction with a second molecule having a
thiol-reactive group such as a detectable label having a
thiol-reactive group. The reaction desirably includes further
reaction with a mild reductant (an example of a reductive
amination) to form a more stable hydrazone. Coupling of the
thiolated antibody with a detectable label having a thiol-reactive
group (such as a maleimide group) is illustrated in Scheme 7.
##STR12##
V. EXAMPLES
[0065] The following non-limiting examples of working embodiments
are provided to further illustrate certain aspects of the
invention.
Example 1
Synthesis of Mercaptobutyric Acid Hydrazide (MBH)
[0066] In a particular working embodiment, a hydrazide thiol linker
was prepared from .gamma.-butyrothiolactone according to Scheme 8.
##STR13## In particular, to a stirred solution of hydrazine
monohydrate (2.43 ml, 50 mmol) was slowly added
.gamma.-butyrothiolactone (0.43 ml, 5 mmol). After 4 hours the
excess hydrazine was removed in vacuo. The crude product was
purified by flash chromatography (SiO.sub.2, 1:19 MeOH/MeCN) to
give the desired product as a colorless oil. Yield: 599 mg (89%):
.sup.1H NMR (250 MHz, CDCl.sub.3) .delta. 7.56 (s, 1H), 3.89 (s,
2H), 2.56-2.47 (q, J=6.9 Hz, 2H), 2.28-2.22 (t, J=7.0 Hz, 2H),
1.94-1.83 (p, J=7.0 Hz, 2H), 1.35-1.29 (t, J=8.0 Hz, 1H); .sup.13C
NMR (62.9 MHz, CDCl.sub.3) .delta. 173.02, 32.38, 29.16, 23.94;
ESI-HRMS m/z 135.05955 (M+H.sup.+, C.sub.4H.sub.11N.sub.2OS calc'd
135.05921).
Example 2
Synthesis of Mercaptobutyric Acid Carbohydrazide (MBCH)
[0067] In another particular working embodiment, a carbohydrazide
thiol linker was prepared from .gamma.-butyrothiolactone according
to Scheme 9. ##STR14## In particular, .gamma.-butyrothiolactone
(0.43 ml, 5 mmol) was diluted in acetonitrile (5 ml) and then
slowly added to a solution of carbohydrazide (2.25 g, 25 mmol) in
deionized water (5 ml). The reaction was stirred at 40.degree. C.
for 18 h, and then concentrated in vacuo. The crude product was
removed by filtration with acetonitrile and flash chromatography
(SiO.sub.2, 1:19 MeCN/MeOH ) to give the product as a white solid.
Yield: 672 mg (70%): .sup.1H NMR (250 MHz, D.sub.2O) .delta.
2.62-2.56 (t, J=7.1 Hz, 2H), 2.47-2.41 (t, J=7.4 Hz, 2H), 1.98-1.87
(m, 2H); .sup.13C NMR (62.9 MHz, D.sub.2O) .delta. 179.14, 163.94,
34.86, 31.74, 25.91; ESI-HRMS m/z 215.05818 (M+Na.sup.+,
C.sub.5H.sub.12N.sub.4NaO.sub.2S calc'd 215.25787).
Example 3
Synthesis of Mercapto-dPEG.sub.4-Hydrazide
[0068] In yet another particular working embodiment, a PEG-based
hydrazide thiol linker was prepared according to Scheme 10 to
provide a mercapto-dPEG hydrazide. ##STR15##
[0069] Acetyl-S-dPEG.sub.4.TM.-NHS ester (Quanta Biodesign, Powell,
Ohio; 580 mg, 1.38 mmol) was slowly added to anhydrous hydrazine
(10 ml), and was stirred for 18 h at ambient temperature. The
reaction was concentrated in vacuo to give the crude product. Flash
chromatography (SiO.sub.2, 199:1 MeCN/AcOH) gave the product as a
colorless oil. Yield: 240 mg (59%): .sup.1H NMR (250 MHz,
CDCl.sub.3) .delta. 8.04 (s, 1H), 3.88 (s, 2H), 3.68-3.52 (m, 17H),
2.65-2.60 (t, J=6.3 Hz, 2H), 2.43-2.39 (t, J=5.8 Hz, 2H); .sup.13C
NMR (62.9 MHz, CDCl.sub.3) .delta. 171.94, 72.74, 70.52, 70.49,
70.38, 70.15, 70.09, 66.72, 35.17, 24.12; ESI-HRMS m/z 319.13073
(M+Na.sup.+, C.sub.11H.sub.24N.sub.2NaO.sub.5S calc'd 319.13036).
An acetyl-S-dPEG.sub.8.TM.-NHS ester also is commercially available
from Quanta Biodesign, (Powell, Ohio). In general, a
mercapto-dPEG-hydrazide can have the formula
H.sub.2N--NH--CO--(CH.sub.2--CH.sub.2--O).sub.t--CH.sub.2--CH.sub.2--SH,
where t=2 to 50.
Example 4
Synthesis of Conjugates of IgG and Alkaline Phosphatase
[0070] An Fc-specifically thiolated immunoglobulin was prepared
according to Scheme 11. ##STR16## Specifically, to a solution of
polyclonal antibody (1.5 ml, 3.0 mg/ml) was added sodium periodate
(0.5 ml, 10 mg/ml in deionized water) for a final periodate
concentration of 11.7 mM. The reaction solution was rotated for 2
hours before being passed through a PD-10 desalting column (0.1 M
NaOAc, 1 mM EDTA, pH=5.0) to remove excess periodate. A hydrazide
thiol linker (MBH, AMBH, MBCH or Mercapto-dPEG.sub.4-hydrazide) was
added in a 1000-fold molar excess to the antibody followed by
sodium cyanoborohydride (3.14 mg, 50 .mu.mol), and the reaction was
rotated for a period of 18 h before being concentrated to a final
volume of 1 ml. Size exclusion chromatography (Superdex 200; 0.1 M
NaOAc, pH=5.0) gave the purified thiolated antibody. The number of
thiols was quantitated through a modified Ellman's assay (see, for
example, Hermanson, "Bioconjugate Techniques," Academic Press, San
Diego, 1996, ISBN 0-12-342336-8, which is incorporated by reference
herein). This procedure yielded an average of 3-5 thiol groups per
antibody.
[0071] Reaction of a hydrazide thiol linker with an aldehyde group
introduced to the Fc region of an immunoglobulin is advantageously
performed at a mildly acidic pH, for example, a pH between 4 and 6
such as a pH near 5. Without wishing to be bound by theory, it is
likely that at such mildly acidic pHs the aldehyde group is
electrophilically activated by protonation of the aldehyde oxygen
and, concurrently, the hydrazide group (pKa of about 4) is not
substantially protonated and remains highly nucleophilic, thereby
facilitating the reaction between the aldehyde group and the
hydrazide group. Since such mildly acidic conditions also represent
conditions where the sulfur of the thiol group is substantially
protonated (substantially present in its neutral acid form) and
thus unable to react with disulfides linking the heavy and light
chains of an immunoglobulin, the reaction is facile and yet less
likely to disrupt immunoglobulin structure. Furthermore, a free
thiol group is maintained for further reaction to form a
conjugate.
[0072] Thiol-reactive maleimide groups were introduced to alkaline
phosphatase according to Scheme 12. ##STR17## Specifically,
alkaline phosphatase (Biozyme, San Diego, Calif.), which was
received in a reactive buffer containing Tris, was passed through a
PD-10 column in order to exchange the AP into a non-reactive buffer
(0.1 M sodium phosphate, 0.1 M sodium chloride, 1 mM magnesium
chloride, 0.1 mM zinc chloride, pH=7.5). Then, to a solution of
alkaline phosphatase (0.8 ml, 17.5 mg/ml) a 100-fold excess of
NHS-dPEG.sub.12-MAL (Quanta Biodesign, Powell, Ohio) was added and
the reaction was rotated for a period of 1 h. Size exclusion
chromatography (Superdex 200; 0.1 M Tris, 1 mM MgCl.sub.2, 0.1 mM
ZnCl.sub.2, pH 7.5) yielded the purified maleimido-alkaline
phosphatase. The number of maleimides was quantitated using a
modified Ellman's assay (see, for example, Hermanson, "Bioconjugate
Techniques," Academic Press, San Diego, 1996, ISBN 0-12-342336-8,
which is incorporated by reference herein), and on average 17-25
maleimide groups were introduced to each alkaline phosphatase
enzyme.
[0073] The final conjugation of the thiolated Ab and the reactive
AP was then performed at a pH above 7, which in this instance
allowed for fast formation of a conjugate by reaction of the thiol
on the Ab (present to a greater extent in the conjugate base
thiolate form at higher pHs) and the thiol-reactive maleimide group
introduced to alkaline phosphatase. Scheme 10 below depicts the
final conjugation of the thiolated Ab and the thiol-reactive AP.
##STR18## Specifically, the purified maleimido-alkaline phosphatase
was combined with the purified thiolated antibody in a 1:1 molar
ratio and rotated for a period of 18 h. Size exclusion
chromatography (Superdex 200; 0.1 M Tris, 1 mM MgCl.sub.2, 0.1 mM
ZnCl.sub.2, pH =7.5) gave the purified conjugate which was diluted
to an A.sub.280=0.0087 into a 1:1 dilution of Stabilzyme.TM. AP
enzyme-stabilizing diluent (SurModics, Eden Prairie, Minn.) and
analyzed on tissue as described in the examples that follow. The
resulting conjugates showed unprecedented staining sensitivities in
a variety of tissues, as shown in the Examples that follow.
[0074] The synthesis of the Ab-AP conjugate according to this
procedure produces a 1:1 conjugate having a median molecular weight
of approximately 270 kDa. This is true regardless of the antibody
used to prepare the conjugate (such as goat anti-mouse IgG, goat
anti-rabbit IgG and rabbit anti-DNP antibodies). The crude
chromatograms, obtained after conjugation show overlap between
product and starting material (median molecular weight of 145 kDa),
which can be taken into consideration during the purification
process.
Example 5
Detection of Kappa in Tonsil Tissue
[0075] In this example, the performance of an Ab-AP conjugate
prepared using MBH according to the procedure of Example 4 was
evaluated for its detection sensitivity in an in-situ hybridization
(ISH) assay. The procedure utilized was adapted from a standard ISH
protocol available on a BenchMark.RTM. automated slide staining
instrument (Ventana Medical Systems, Inc., Tucson, Ariz.). The
automated staining protocol was as follows.
[0076] A paraffin-embedded tonsil tissue sample on a slide was
heated to 75.degree. C. for 4 minutes and treated twice with
EZPrepl.TM. volume adjust (Ventana Medical Systems, Inc., Tucson,
Ariz.) at 75.degree. C. before application of LiquidCoverslip.TM.
(Ventana Medical Systems, Inc., Tucson, Ariz.) with EZPrep.TM.
volume adjust. After 4 minutes at 75.degree. C., the slide was
rinsed and EZPrep.TM. volume adjust was added along with
LiquidCoverslip.TM. to de-paraffinize the tissue at 76.degree. C.
for 4 minutes. Liquid coverslip was dispensed to cover the EZ-Prep.
The slide was then heated to 90.degree. C. for 4 minutes and rinsed
before cooling to 37.degree. C. ISH-Protease 1 (100 .mu.l, Ventana
Medical Systems, Inc., Tucson, Ariz.) was added, incubated for 2
minutes, and then rinsed, followed by the addition of a
fluorescein-labeled kappa nucleic acid probe (100 .mu.l,
INFORM.RTM. Kappa, Ventana Medical Systems, Inc., Tucson, Ariz.).
After a 4 minute incubation, the slide was heated to 85.degree. C.
for 12 minutes, then cooled to 47.degree. C. and incubated for a
further 64 minutes. The slide was rinsed four times before the
addition of a mouse anti-fluorescein primary antibody (100 .mu.l,
Ventana Medical Systems, Inc., Tucson, Ariz.), which was incubated
for 20 minutes, and then rinsed twice. At this point either a
secondary antibody (for further amplification) was automatically
added or the Ab-AP conjugate was manually added or added
automatically from a dispenser. For the slides that were amplified,
a rabbit anti-mouse antibody (100 .mu.l, Ventana Medical Systems,
Inc., Tucson, Ariz.) was added, incubated for 8 minutes and then
the slide was rinsed twice. In either case, once the AP-Ab
conjugate (goat anti-rabbit or rabbit anti-mouse IgG conjugate, for
samples with and without the secondary antibody, respectively; 100
.mu.l) was applied to the slide, the slide was incubated for 16
minutes and the slide was rinsed twice. Application of iView.TM.
Blue Enhance enhancer (100 .mu.l, Ventana Medical Systems, Inc.,
Tucson, Ariz.) was followed by incubation for 4 minutes and
application of both iView.TM. Blue NBT (100 .mu.l, Ventana Medical
Systems, Inc., Tucson, Ariz.) and iView.TM. Blue BCIP (100 .mu.l,
Ventana Medical Systems, Inc., Tucson, Ariz.). BCIP, which is a
substrate of alkaline phosphatase generates an insoluble dark
blue/purple precipitate, and NBT enhances the color of the BCIP.
The slide was then incubated for a period of 32 minutes, rinsed
twice, and Counterstain NFR (100 .mu.l, Ventana Medical Systems,
Inc., Tucson, Ariz.) was added. After incubation with the
counterstain for 6 minutes, the slide was again rinsed and taken
off of the instrument. The slide was treated with a detergent wash
before dehydration with the series ethanol, acetone then xylene. A
coverslip was added to the slide and the slide was viewed and
photographed through a microscope. A negative control slide that
was not treated with the kappa probe also was prepared in a similar
fashion.
[0077] For comparison, a reference tonsil tissue sample was stained
using a similar procedure employing a SA-AP conjugate for detection
of the kappa probe (the procedure included addition of the
secondary antibody as above followed by extra amplification steps,
wherein automated addition of a biotinylated anti-IgG antibody was
performed instead of application of the Ab-AP conjugate, which was
then followed by addition of the SA-AP conjugate) The use of
biotin-labeled antibodies and SA-AP conjugates is an industry
standard for detection in automated ISH staining methods and served
as a reference on which to determine the relative performance of
the Ab-AP conjugate. A negative control slide that was not treated
with the kappa probe also was prepared in a similar fashion using
SA-AP detection. Coverslips were added to the slides and the slides
were viewed and photographed at 40.times. through a brightfield
microscope.
[0078] FIG. 1 is a set of photomicrographs comparing the desired
staining and background staining observed for kappa ISH detection
in tonsil tissue using the antibody-alkaline phosphatase conjugate
and the SA-AP conjugate. In FIG. 1A the staining of kappa without
amplification afforded by the secondary antibody and using the
disclosed antibody conjugate is shown. FIG. 1B shows a negative
control slide treated with the conjugate. FIG. 1C shows the
staining of kappa using the SA-AP, and FIG. 1D shows the negative
control for the same. A comparison of FIGS. 1A and 1C demonstrates
more defined staining by the antibody conjugate (even though fewer
amplification steps were used), and a comparison of FIGS. 1B and 1D
demonstrates the lower background provided by the antibody
conjugate. These results illustrate the superiority of the
non-biotin detection scheme enabled by the antibody conjugate.
Example 6
Detection of Lambda in Tonsil Tissue
[0079] The automated staining process described in Example 5 (with
the exceptions that the fluorescein-labeled nucleic acid probe used
was specific for Lambda; INFORM.RTM. Lamdba, Ventana Medical
Systems, Inc., Tucson, Ariz.; and ISH Protease 1 was incubated for
4 minutes) was used to assess the performance of an Ab-AP conjugate
for detection of Lambda in tonsil tissue. The Ab-AP conjugate was
used without a secondary antibody amplification step and was
prepared using MBH as described in Example 4. For comparison, a
reference slide was prepared using the SA-AP conjugate detection
scheme described in Example 4.
[0080] The results are presented in FIG. 2. Specifically, FIGS. 2A
and 2B show the staining pattern obtained using the Ab-AP conjugate
with and without (negative control) the addition of the Lambda
specific nucleic acid probe, respectively. FIGS. 2C and 2D show the
staining pattern obtained using the SA-AP conjugate with and
without (negative control) addition of the Lambda probe,
respectively. A comparison of FIGS. 2A and 2C shows that the
staining pattern obtained using the Ab-AP conjugate is at least as
intense as that seen with the SA-AP conjugate, despite the process
used for the Ab-AP conjugate involving one less amplification step.
A comparison of FIGS. 2B and 2D demonstrates that there is much
less background staining (evidenced by darker overall staining of
the tissue) by the Ab-AP conjugate. Again, these results
demonstrate the advantageous reduction of background seen when the
disclosed Ab-AP conjugate is employed.
Example 7
Detection of CMV in Lung Tissue
[0081] The automated staining process described in Example 5 (with
the exceptions that the fluorescein-labeled nucleic acid probe used
was specific for CMV; INFORM.RTM. CMV, Ventana Medical Systems,
Inc., Tucson, Ariz.; and ISH Protease 1 was incubated for 4
minutes) was used to assess the performance of an Ab-AP conjugate
for detection of CMV in lung tissue. The Ab-AP conjugate was used
without a secondary antibody amplification step and was prepared
using MBH as described in Example 4. For comparison, a reference
slide was prepared using the SA-AP conjugate detection scheme
described in Example 4.
[0082] The results are presented in FIG. 3. Specifically, FIG. 3A
shows the staining pattern obtained using the Ab-AP conjugate in
the presence of the probe, FIG. 3B shows the staining pattern
obtained using the Ab-AP conjugate in the absence of the probe,
FIG. 3C shows the staining pattern obtained using the SA-AP
conjugate in the presence of the probe, and FIG. 3D shows the
staining pattern using the SA-AP conjugated in the absence of the
probe. A comparison of FIG. 3A and 3C shows that staining with the
Ab-AP conjugate is more defined than and at least as intense
(despite having one less amplification step) as the staining
provided by the SA-AP conjugate. Furthermore, less background
staining is seen for the Ab-AP conjugate. The reduction in
background provided by the Ab-AP conjugate also is evident from a
comparison of FIGS. 3B and 3D.
Example 8
Detection of EBER in Spleen Tissue
[0083] The automated staining process described in Example 5 (with
the exceptions that the fluorescein-labeled nucleic acid probe used
was specific for EBER ; INFORM.RTM. EBER, Ventana Medical Systems,
Inc., Tucson, Ariz.; and ISH Protease 1 was incubated for 4
minutes) was used to assess the performance of an Ab-AP conjugate
for detection of EBER in spleen tissue. The Ab-AP conjugate was
used without a secondary antibody amplification step and was
prepared using MBH as described in Example 4. For comparison, a
reference slide was prepared using the SA-AP conjugate detection
scheme described in Example 4.
[0084] The results are presented in FIG. 4. Specifically, FIG. 4A
shows the staining pattern obtained using the Ab-AP conjugate in
the presence of the probe, FIG. 4B shows the staining pattern
obtained using the Ab-AP conjugate in the absence of the probe,
FIG. 4C shows the staining pattern obtained using the SA-AP
conjugate in the presence of the probe, and FIG. 4D shows the
staining pattern using the SA-AP conjugated in the absence of the
probe. A comparison of FIG. 4A and 4C shows that staining with the
Ab-AP conjugate is more defined than and at least as intense
(despite having one less amplification step) as the staining
provided by the SA-AP conjugate. Furthermore, less background
staining is seen for the Ab-AP conjugate. The reduction in
background provided by the Ab-AP conjugate also is evident from a
comparison of FIGS. 3B and 3D
Example 9
Detection of HPV in Tissue Xenografts
[0085] In this example, the performance of an Ab-AP conjugate that
was prepared using MBH according to the procedure of Example 4 was
assessed, in part, to determine if it provided enough sensitivity
to permit a further reduction in the number of steps needed to
detect HPV sequences by ISH. The results show that it is possible
to achieve a reduction in the number of steps needed for detection,
thereby making the disclosed Ab-AP conjugate very useful for an
automated process where a reduction in the number of steps leads to
a significant reduction in processing time and concomitantly, assay
cost.
[0086] The three detection schemes presented below as Schemes
14-16, were performed in an automated or semi-automated fashion. In
each of these schemes, a DNP-labeled nucleic acid probe that
specifically binds to at least a portion of an HPV nucleic acid
sequence is first added to the sample. The subsequent steps
depicted in these schemes are steps used to detect the presence of
the probe bound to HPV nucleic acid. ##STR19## ##STR20##
[0087] In Scheme 14, an anti-DNP antibody is first bound to the
probe. An anti-IgG antibody is then added (first amplification
step). In a second amplification step, a biotinylated anti-IgG
antibody is added. An SA-AP conjugate is added, which binds to the
biotinylated antibody, and staining is accomplished through
addition of a chromogenic substrate that is acted upon by AP. In
scheme 15, the second amplification step is eliminated, and an
anti-IgG antibody conjugated to AP is added prior to staining
rather than an SA-AP conjugate. In scheme 16, both amplification
steps are eliminated and the DNP-labeled probe is directly detected
by an anti-DNP antibody conjugated to AP.
[0088] HPV detection in a variety of cell lines grown in xenografts
in SCID mice was performed according to the following procedure
adapted from a standard ISH protocol for the BenchMark.RTM.
automated staining instrument (Ventana Medical Systems, Inc,
Tucson, Ariz.). Paraffin-embedded tissue on a slide was heated to
75.degree. C. for 4 minutes and then treated twice with EZPrep.TM.
volume adjust (Ventana Medical Systems, Inc., Tucson, Ariz.) at
75.degree. C. before application of Liquid Coverslip.TM. (Ventana
Medical Systems, Inc., Tucson, Ariz.) with EZPrep.TM. volume
adjust. After 4 minutes at 75.degree. C., the slide was rinsed and
EZPrep.TM. volume adjust was added to de-paraffinize the tissue at
76.degree. C. for 4 minutes. Liquid Coverslip was dispensed to
cover the EZPrep.TM.. Cell Conditioner #2, cell conditioning
solution (Ventana Medical Systems, Inc., Tucson, Ariz.) was added,
the slide warmed to 90.degree. C., and incubated for 8 minutes.
This was followed by another application of Cell Conditioner #2 and
yet another incubation at 90.degree. C. for 12 minutes. The slide
was rinsed with Reaction Buffer (Ventana Medical Systems, Inc.,
Tucson, Ariz.), cooled to 37.degree. C. and ISH-Protease 3 (100
.mu.l, Ventana Medical Systems, Inc., Tucson, Ariz.) was added.
After an incubation of 4 minutes, the slide was rinsed three times
before the application of a hybridization buffer (iView.TM. Plus
HybReady.TM. Solution, 100 .mu.l, Ventana Medical Systems, Inc.,
Tucson, Ariz.) and was incubated for 4 minutes. Addition of a
DNP-labeled HPV nucleic acid probe (HPV HR Probe, 200 .mu.l,
Ventana Medical Systems, Inc., Tucson, Ariz.) was followed by an
incubation of 4 minutes at 37.degree. C., 12 minutes at 95.degree.
C. and 124 minutes at 52.degree. C. The slide was then rinsed twice
and warmed to 72.degree. C. This last step was repeated two more
times before cooling the slide down to 37.degree. C., and then,
depending upon the detection scheme followed, such slides were
treated in an automated or semi-automated fashion in one of three
ways.
[0089] In one instance, as depicted in Scheme 14, an
iView.TM.+Anti-DNP (100 .mu.l, Ventana Medical Systems, Inc,
Tucson, Ariz.) primary antibody was applied, and incubated for 20
minutes. The slide was then rinsed twice before the addition of the
iView.TM.+Amp (100 .mu.l, Ventana Medical Systems, Inc, Tucson,
Ariz.) secondary antibody. Incubation of the conjugate occurred for
8 minutes before rinsing the slide. The addition of
iVIEW.TM.+Biotin-Ig (100 .mu.l, Ventana Medical Systems, Inc,
Tucson, Ariz.) was followed by a 12 minute incubation and addition
of the iVIEW.TM.+SA-AP (100 .mu.l, Ventana Medical Systems, Inc,
Tucson, Ariz.). The slide was rinsed three times before the
application of the iview.TM.+Enhancer (100 .mu.l, VMSI) which was
followed by incubation for 4 minutes and application of both
iView.TM.+NBT (100 .mu.l, Ventana Medical Systems, Inc, Tucson,
Ariz.) and iView.TM.+BCIP (100 .mu.l, Ventana Medical Systems, Inc,
Tucson, Ariz.). The slide was then incubated for a period of 24
minutes, rinsed three times, and Counterstain NFR (100 .mu.l,
Ventana Medical Systems, Inc, Tucson, Ariz.) was added. After
incubation with the counterstain for 4 minutes, and slide was
rinsed three more times and taken off of the instrument. The slide
was treated with detergent wash before dehydration with a series of
ethanol, acetone and then xylene. A coverslip was applied to the
slide and then the slide was viewed and photographed through a
brightfield microscope.
[0090] In another instance, as depicted in Scheme 15, a rabbit
anti-DNP primary antibody was added (iView.TM. Plus anti-DNP
primary antibody, 100 .mu.l, Ventana Medical Systems, Inc., Tucson,
Ariz.). The primary antibody was incubated for 20 minutes and the
slide was then rinsed twice before the manual addition (this step
can also be automated to make the procedure fully automated) of the
anti-rabbit IgG antibody conjugated to alkaline phosphatase (100
.mu.l). Incubation of the conjugate occurred for 16 minutes before
rinsing the slide four times. Application of the iView.TM. Plus
Enhancer (100 .mu.l, Ventana Medical Systems, Inc., Tucson, Ariz.)
was followed by incubation for 4 minutes and application of both
NBT and BCIP for color development (iView.TM. Plus NBT and
iView.TM. Plus BCIP, 100 .mu.l, Ventana Medical Systems, Inc.,
Tucson, Ariz.). The slide was then incubated for a period of 24
minutes, rinsed three times, and Counterstain NFR (100 .mu.l,
Ventana Medical Systems, Inc., Tucson, Ariz.) was added. After
incubation with the counterstain for 4 minutes, and slide was
rinsed three more times and taken off of the instrument. The slide
was treated with a detergent wash before dehydration with ethanol,
acetone and xylene. Following application of a cover slip, the
slide was viewed through a microscope and photographed at 40.times.
using a brightfield microscope.
[0091] In yet another instance, as depicted in scheme 16, the slide
was treated directly with an alkaline phosphatase rabbit anti-DNP
conjugate (100 .mu.l). The slide was incubated for 20 minutes and
was then rinsed twice before the application of the
iView.TM.+Enhancer (100 .mu.l, Ventana Medical Systems, Inc,
Tucson, Ariz.). This was followed by incubation for 4 minutes and
simultaneous application of both iView.TM.+NBT (100 .mu.l, Ventana
Medical Systems, Inc, Tucson, Ariz.) and iView.TM.+BCIP (100 .mu.l,
Ventana Medical Systems, Inc, Tucson, Ariz.). The slide was then
incubated for a period of 24 minutes, rinsed three times, and
Counterstain NFR (100 .mu.l, Ventana Medical Systems, Inc, Tucson,
Ariz.) was added. After incubation with the counterstain for 4
minutes, and slide was rinsed three more times and taken off of the
instrument. The slide was treated to a detergent wash before serial
dehydration with ethanol, acetone and xylene. A coverslip was added
to slide and it was viewed and photographed at 40.times. using a
brightfield microscope.
[0092] FIGS. 5-7 show the results of HPV detection in three
different xenograft tissue types. In FIGS. 5A, 5B and 5C, the
staining patterns for HPV detection in CaSki xenograft tissue
according to each of Schemes 14, 15 and 16 are shown, respectively.
In FIGS. 6A, 6B and 6C, the staining patterns for HPV detection in
HeLa xenograft tissue according to each of Schemes 14, 15 and 16
are shown, respectively. In FIGS. 7A and 7B, the staining patterns
for single copy HPV detection (indicated by arrows) in SiHa
xenograft tissue according to each of Schemes 14 and 15 are shown,
respectively.
[0093] A comparison of FIGS. 5A and 5B demonstrates that the
staining intensity provided by detection according to Scheme 15 is
greater than that provided according to Scheme 14 even though
Scheme 15 includes two fewer amplification steps. FIG. 5C
demonstrates that HPV detection can be accomplished without
amplification using direct detection (Scheme 16) with an Ab-AP
conjugate prepared according to Example 4. A comparison of FIGS. 6A
and 6B also demonstrates that the staining intensity provided by
detection according to Scheme 15 is greater than that provided
according to Scheme 14, even though Scheme 15 includes two fewer
steps of amplification. FIG. 6C demonstrates that HPV detection can
be accomplished without amplification using direct detection
(scheme 16) with an Ab-AP conjugate prepared according to Example
4. A comparison of FIGS. 7A and 7B shows that even single copies of
HPV nucleic acid sequences can be detected with the detection
process of Scheme 15. Overall, the results demonstrate that the
superior sensitivity exhibited by a disclosed Fc-specific Ab-AP
facilitates automated detection by reducing the number of steps
needed to detect HPV in tissue samples. The reduction of the number
of steps between Schemes 14 and 15 can reduce the total automated
staining process time by 15% (from 6.5 hrs to 5.5 hrs). Further
reductions in process time can be realized by use of Scheme 16.
[0094] While a DNP labeled probe and specific types of antibodies
were described in this example, one of ordinary skill in the art
will appreciate that many other haptens (such as fluorescein,
digoxigenin and biotin) can be used to label nucleic acid sequences
and that the use of multiple nucleic acid probes to different
targets, each having a different hapten label, can be used to
permit multiplexed detection (such as with different detection
antibodies conjugated to different fluorescent nanoparticles that
emit light of various different wavelengths). Furthermore, one of
ordinary skill in the art will recognize that antibodies of other
types and from other species than those described, other detectable
labels, and other reagents for generating a detectable signal can
be used in similar assays to detect other targets.
Example 10
Detection of HPV in Liquid-Based Preparations
[0095] Slides for the liquid-based prep HPV assay were prepared
using the ThinPrep.RTM. 2000 System slide preparation system (Cytyc
Corporation, Marlborough, Mass.). Cells obtained through vaginal
scraping are placed within a methanol-based, buffered preservative
solution (ThinPrep.RTM. PreservCyt Solution, Cytyc Corporation,
Marlborough, Mass.) and then layered onto the glass slide by the
instrument.
[0096] The following is an adapted procedure from the Ventana
BenchMark.RTM. Instrument: the liquid based prep slide was heated
to 65.degree. C. for 12 minutes followed by an additional 4 minutes
at 75.degree. C. and rinsed twice with Reaction buffer (Ventana
Medical Systems, Inc, Tucson, Ariz.; 1.2 ml) at 75.degree. C.
before application of the liquid cover slip (Ventana Medical
Systems, Inc, Tucson, Ariz.). The slide was then rinsed with 0.9 ml
of Rinse Buffer (Ventana Medical Systems, Inc, Tucson, Ariz.)
followed by the application of Cell Conditioner #2 cell
conditioning solution (Ventana Medical Systems, Inc, Tucson, Ariz.)
and the slide was warmed to 90.degree. C. and incubated for 16
minutes. The slide was rinsed with Reaction Buffer, cooled to
37.degree. C. and ISH-Protease 3 (100 .mu.l, Ventana Medical
Systems, Inc, Tucson, Ariz.) was added. After an incubation of 4
minutes, the slide was rinsed three times before the application of
iView.TM.+HybReady (100 .mu.l, Ventana Medical Systems, Inc,
Tucson, Ariz.) which was incubated for 4 minutes. Addition of HPV
HR Probe (200 .mu.l, Ventana Medical Systems, Inc, Tucson, Ariz.)
was followed by an incubation of 4 minutes at 37.degree. C., 12
minutes at 95.degree. C. and 124 minutes at 52.degree. C. The slide
was then rinsed twice and warmed to 72.degree. C. This last step
was repeated two more times before cooling the slide down to
37.degree. C. and adding iView.TM.+Anti-DNP (100 .mu.l, Ventana
Medical Systems, Inc, Tucson, Ariz.).
[0097] For standard SA-AP detection (according to Scheme 14 above),
the primary antibody was incubated for 20 minutes and the slide was
then rinsed twice before the addition of the i.TM.VIEW+Amp
secondary antibody (Ventana Medical Systems, Inc, Tucson, Ariz.,
100 .mu.l). Incubation of the antibody occurred for 8 minutes
before rinsing. Then, the i.TM.VIEW+Biotin-IgG antibody conjugate
(Ventana Medical Systems, Inc, Tucson, Ariz., 100 .mu.l) was added
followed by a 12 minute incubation and rinse step. Lastly, the
iVIEW.TM.+SA-AP conjugate (Ventana Medical Systems, Inc, Tucson,
Ariz., 100 .mu.l ) was added and after an 8 minute incubation, the
slide was rinsed three times with Reaction Buffer. For detection
using the Ab-AP conjugate as the secondary antibody (according to
Scheme 15 above), the primary antibody was incubated for 20 minutes
and the slide was then rinsed twice before the addition of the
AP-IgG conjugate (100 .mu.l). Incubation of the conjugate occurred
for 8 minutes before rinsing three times with Reaction Buffer. For
direct detection of the labeled probe using the Ab-AP conjugate,
the conjugate was incubated for 20 minutes before the slide was
rinsed three times with Reaction Buffer.
[0098] In all three cases, the steps above were followed by
application of iVIEW+Enhancer (100 .mu.l, Ventana Medical Systems,
Inc, Tucson, Ariz.) was followed by incubation for 4 minutes and
application of both iVIEW.TM.+NBT (100 .mu.l, Ventana Medical
Systems, Inc, Tucson, Ariz.) and iVIEW.TM.+BCIP (100 .mu.l, Ventana
Medical Systems, Inc, Tucson, Ariz.). The slide was then incubated
for a period of 24 minutes, rinsed three times, and Counterstain
NFR (100 .mu.l, Ventana Medical Systems, Inc, Tucson, Ariz.) was
added. After incubation with the counterstain for 4 minutes, and
slide was rinsed three more times and taken off of the instrument.
The slide was treated with a detergent wash before dehydration with
ethanol, acetone and xylene and subsequent application of a cover
slip to the slide, after which the slide was viewed through a
microscope.
[0099] A comparison of FIGS. 8A and 8B shows that detection
according to Scheme 15 (see, Example 9), using an Ab-AP conjugate
that was prepared using MBH according to the procedure of Example
4, provides more intense staining than provided by detection using
an SA-AP conjugate according to Scheme 14 (see, Example 9). A
comparison of FIGS. 8B and 8C demonstrates that direct detection
using an anti-DNP Ab-AP conjugate according to Scheme 16 (see,
Example 9) provides a signal that is comparable to the signal
provide by an SA-AP conjugate according to Scheme 14. These results
again demonstrate that the detection sensitivity provided by an
Fc-specific Ab-AP conjugate according to Example 4 permits a
reduction in the number of steps needed to provide adequate
signals, thereby facilitating automation.
Example 11
Detection of Actin in Muscle Tissue
[0100] In this example, immunohistochemical detection of a protein
target (actin) using an Ab-AP conjugate prepared as described in
Example 4 with an MBH linker was compared to the performance of a
SA-AP conjugate.
[0101] The following is the adapted procedure from the Ventana
BenchMark.RTM. Instrument: the paraffin coated tissue on the slide
was heated to 75.degree. C. for 4 minutes and treated twice with
EZPrep.TM. volume adjust (Ventana Medical Systems, Inc, Tucson,
Ariz.) at 75.degree. C. before application of the liquid cover slip
(Ventana Medical Systems, Inc, Tucson, Ariz.) with EZPrep.TM.volume
adjust. After another 4 minutes at 76.degree. C., the slide was
rinsed and Depar volume adjust (Ventana Medical Systems, Inc,
Tucson, Ariz.) was added along with liquid cover slip to
de-paraffinize the tissue. The slide was then cooled to 42.degree.
C. for 2 minutes, before reaching the final temperature of
37.degree. C. The primary antibody was then added (100 .mu.l,
anti-muscle actin, Ventana Medical Systems, Inc, Tucson, Ariz.) and
the slide incubated at 37.degree. C. for 16 minutes. The slide was
then rinsed twice and the alkaline phosphatase conjugated goat
anti-mouse material (100 .mu.l) was added and incubated 37.degree.
C. for 16 minutes. The slide was rinsed once before the
simultaneous addition of Enhanced V-Red Enhancer (100 .mu.l,
Ventana Medical Systems, Inc, Tucson, Ariz.) and Enhance Naphthol
(100 .mu.l, Ventana Medical Systems, Inc, Tucson, Ariz.), and the
slide was again incubated at 37.degree. C. for 4 minutes. This was
followed by the addition of Enhance Fast Red A (100 .mu.l, Ventana
Medical Systems, Inc, Tucson, Ariz.) an 8 minute incubation and the
addition of Enhance Fast Red B (100 .mu.l, Ventana Medical Systems,
Inc, Tucson, Ariz.) with a final 8 minute incubation. After
development of the stain, the slide was treated with a detergent
wash before dehydration with ethanol, acetone and xylene and
subsequent application of a cover slip to the slide, after which
the slide was viewed through a microscope.
[0102] The results are presented in FIG. 9. Specifically, FIG. 9A
shows that detection using the Ab-AP conjugate and a single
amplification step is superior to detection using an SA-AP
conjugate and two amplification steps (FIG. 9B). These results
again demonstrate the superior detection sensitivity provided by
Fc-specific antibody conjugates according to the disclosure.
Example 12
Variation of Antibody Linker Length and Type
[0103] In this example, the effect of linker length and type on
conjugate composition and staining characteristics was determined.
Several conjugates were prepared according to the method of Example
4, but using a variety of hydrazide thiol linkers, specifically,
conjugates prepared using a thio-PEG.sub.4-hydrazide linker, a
mercaptobutyric acid hydrazide (MBH) linker, and a mercaptobutyric
acid carbohydrazide (MBCH) linker. These conjugates were compared
to each other and to a conjugate prepared through generation of
thiols by reduction of immunoglobulin disulfides, specifically an
Ab-AP conjugate prepared by the method described in co-pending U.S.
Provisional Patent Application No. 60/675759 that involves
generation of thiols via DTT reduction followed by conjugations
using a PEG-based maleimide-NHS bifunctional linker. Also for
comparison, a commercially available acetamidomercaptobutyric acid
hydrazide (AMBH, Invitrogen, Eugene, Oreg.) linker was used in the
method of Example 4 to generate an Ab-AP conjugate. In addition, an
Ab-AP conjugate prepared with a maleimido-hydrazide (EMCH;
N[.epsilon.-Maleimidocaproic acid]hydrazide, Pierce Biotechnology,
Rockford, Ill.) using the manufacturer's instructions was prepared
and used in the staining protocol for comparison. Furthermore, the
Fc-specific conjugation method described in U.S. Pat. No. 5,191,066
employing cystamine was used to provide Fc-specific Ab-AP conjugate
for comparison.
[0104] Ellman's Assay results showed that between 3-5 thiols/Ab
were added to an immunoglobulin through addition with the MBH and
PEG-based hydrazide thiol linkers, 5-7 thiols/Ab for the AMBH and
MBCH linkers, and 8-12 thiols/Ab for the DTT reduction method.
After coupling of the thiols introduced or generated in the
immunoglobulin to maleimide-derivatized AP, size exclusion
chromatograms were obtained.
[0105] Size exclusion chromatograms were obtained using an AKTA
Purifier LC (GE Biosciences, Uppsala, Sweden) using a Superdex
10/300 200 GL column and 0.1 M Tris, 1 mM MgCl.sub.2, 0.1 mM
ZnCl.sub.2, pH=7.5 as the mobile phase. The flow rate was held at 1
ml/min in all cases. From the size exclusion chromatograms, it was
determined that the best yield of conjugate was obtained using
AMBH. However, it began to precipitate out of solution when stored
at 2-8.degree. C. for 48 hours. The other linkers all yielded
conjugates having similar size exclusion profiles.
[0106] FIG. 10 compares the staining as outlined in Example 6 of
Kappa on tonsil tissue using the conjugates as the secondary
antibody. FIG. 10A shows the staining pattern seen for an Ab-AP
conjugate prepared with EMCH. FIG. 10B shows the staining pattern
seen for the Fc-specific cystamine method of U.S. Pat. No.
5,191,066. FIG. 10C shows the staining pattern seen for the Ab-AP
conjugate prepared by a DTT reduction method according U.S. patent
application Ser. No. 11/413,418, filed Apr. 27, 2006. that utilized
a dPEG-based bifunctional linker FIG. 10D shows the staining
pattern seen for the Ab-AP conjugated prepared with the
commercially available AMBH linker using the disclosed method of
Fc-specific conjugation. FIG. 10E shows the staining pattern seen
for the Ab-AP conjugate prepared according to the disclosed
Fc-specific conjugation method employing the disclosed MBH linker
of Example 1. FIG. 10F shows the staining pattern seen for the
Ab-AP conjugate prepared according to the disclosed Fc-specific
conjugation method employing the disclosed dPEG.sub.4 hydrazide
thiol linker of Example 3. FIG. 10G shows the staining pattern seen
for the Ab-AP conjugate prepared according to the disclosed
Fc-specific conjugation method employing the disclosed MBCH
hydrazide thiol linker of Example 2. A comparison of the staining
patterns reveals the following trend for the staining intensity
provided by the conjugates:
EMCH<Cystamine<AMBH<MBCH<PEG4=DTT<MBH The images
illustrate the superior sensitivity that can be achieved by
Fc-specific conjugation of enzymes using the disclosed method and
various disclosed and commercially available hydrazide thiol
linkers. The disclosed method also yields superior conjugates to
the cystamine Fc-specific method and coupling with EMCH. Only the
DTT-mediated method of conjugation provides conjugates that give
similar specificity and sensitivity.
Example 13
Variation of MBH Linker Excess
[0107] In this example, the dependence of conjugate composition and
staining characteristics on the excess of hydrazide thiol linker
was determined. Synthesis of AP-IgG conjugates with MBH linker was
carried out following the procedure of Example 4, however the molar
excess of the MBH linker was varied from a five thousand-fold
excess to a fifty-fold excess. The results from the Ellman's Assay
showed the following number of thiols/Ab: 5000.times.-9-15;
1000.times.-7-10; 500.times.-3-5; 100.times.-2-4; 50.times.-1-3.
Analysis of the conjugates (5000.times., 1000.times., 500.times.,
100.times., and 50.times.) after reaction with the
maleimide-derivatized Ab was performed by size exclusion
chromatography and showed that the conjugates synthesized using a
larger excess of linker had a higher overall yield. However, the
tissue staining (anti-mouse--muscle, muscle actin;
anti-rabbit--skin, S100) for each of these conjugates showed that
the 500.times. had the most intense stain with the lowest amount of
background.
Example 14
Variation of Alkaline Phosphatase Linker Length/Type
[0108] In this example, the dependence of conjugate composition and
staining characteristics on the length and type of linker used to
add thiol-reactive groups to alkaline phosphatase was determined.
Synthesis of AP-IgG conjugates with MBH linker was carried out
following the procedure of Example 4, but the following linkers
were used to activate alkaline phosphatase for reaction with the
thiolated antibody: LC-SMCC (Pierce, Rockford, Ill),
MAL-dPEG.sub.8-NHS ester (Quanta Biodesign, Powell, Ohio),
MAL-dPEG.sub.4-NHS ester (Quanta Biodesign, Powell, Ohio) and
MAL-dPEG.sub.12-NHS ester (Quanta Biodesign, Powell, Ohio). Each of
these linkers was reacted with AP in a hundred-fold excess, in a
buffer system (0.1 M sodium phosphate. 0.1 M NaCl, 1 mM MgCl.sub.2,
0.1 mM ZnCl.sub.2, pH=7.5) for 1 hour. The LC-SMCC had to be
dissolved in dimethylformamide (DMF) and added to the AP, but not
exceeding 10% total volume of DMF in buffer. Ellman's Assay showed
a maleimide incorporation of 20/AP for the PEG.sub.12 and LC-SMCC
linkers, 27/AP for the PEG.sub.8 linker and 30/AP for the PEG.sub.4
linker. After coupling to an Fc-thiolated antibody (made with MBH),
size exclusion chromatograms were obtained upon purification. The
PEG.sub.12 linker gave the highest conjugate yield, followed by the
PEG.sub.8, LC-SMCC, and the PEG.sub.4 linkers. The tissue staining
(anti-mouse--muscle, muscle actin; anti-rabbit--skin, S100)
mirrored the conjugate yield with the PEG.sub.12 conjugate giving
the most intense staining.
Example 15
Variation of NHS-PEG.sub.12-MAL Linker Excess
[0109] In this example, the dependence of conjugate composition and
staining characteristics on excess of NHS-PEG.sub.12-MAL linker
used to add thiol-reactive groups to alkaline phosphatase was
determined. Syntheses of an AP-IgG conjugates according to the
method of Example 4 was performed where the molar excess of a
MAL-dPEG.sub.12-NHS ester linker was varied from a five
hundred-fold excess to a twenty-five-fold excess.
[0110] Ellman's Assay results showed maleimide incorporation of:
500.times.-34 maleimides; 250.times.-29 maleimides;
100.times.-18-20 maleimides; 50.times.-17 maleimides; 25.times.-15
maleimides. Analysis of the conjugates (500.times., 250.times.,
100.times., 50.times., and 25.times.), after reaction with the
Fc-thiolated Ab, using size exclusion chromatography showed that
the conjugates synthesized using a larger excess of linker had a
larger yield and a higher percentage of maleimide incorporation.
Tissue staining (anti-mouse--muscle, muscle actin;
anti-rabbit--skin, S100) for each of the conjugates showed that use
of 100.times. maleimide gave the sharpest, most intense
staining.
Example 16
Variation of AP/Ab Molar Ratios
[0111] In this example, the dependence of conjugate composition and
staining characteristics on the ratio of the thiolated antibody
(prepared with an MBH-linker) to the maleimide-derivatized AP
(NHS-PEG.sub.12-MAL linker) in the final reaction was determined.
The following ratios (Antibody/AP) were used: 2:1, 1:1, 1:2, and
1:3. The profiles of size exclusion chromatographs showed that
maximum yield was obtained when the molar ratio was 2 AP: 1 Ab.
However, the tissue staining of the conjugates (anti-mouse--muscle,
muscle actin; anti-rabbit--skin, S100) demonstrated that the best
signal-to-noise ratio was seen with the 1:1 conjugate.
Example 17
Synthesis of Cross-Linked AP
[0112] Alkaline phosphatase is a dimeric protein, and its stability
can be increased by cross-linking the enzyme to help prevent
dissociation of the dimer. Alkaline phosphatase was cross-linked
using the following procedure. Alkaline phosphatase (Biozyme, San
Diego, Calif.; 17.5 mg, 0.125 .mu.mol) was exchanged into a
different buffer from that in which it was received (0.1 M sodium
phosphate, 0.1 M sodium chloride, 1.0 mM magnesium chloride, 0.1 mM
zinc chloride, pH=7.5) and added to reconstituted, pre-oxidized,
aldehyde-activated dextran (Avg. Molecular Wt. 40,000; Pierce
Biotechnologies, Rockford, Ill.; 5 mg, 0.125 .mu.mol) in the
presence of sodium cyanoborohydride (1.6 mg, 25 .mu.mol). The
reaction mixture was then rotated for a period of one hour at room
temperature. Excess aldehydes were quenched by ethanolamine (151
.mu.l, 2.5 mmol) followed by addition of more sodium
cyanoborohydride (157.1 mg, 2.5 mmol). The reaction mixture was
rotated for an additional one hour. The cross-linked AP was
isolated by size exclusion chromatography using an Akta Purifier
(GE Biosciences, Uppsala, Sweden) equipped with a Superdex 200 GL
10/300 column (GE Biosciences, Uppsala, Sweden). The flow rate was
1 ml/min and the aqueous mobile phase was 0.1 M sodium phosphate,
0.1 M sodium chloride, 1.0 mM magnesium chloride, 0.1 mM zinc
chloride, at pH=7.5. The number of amines remaining after the
reaction was quantitated using a fluoraldehyde assay (Protein Assay
Technical Handbook, Pierce Biotechnology, Rockford, Ill.), and on
average 8-12 amines remained following cross-linking. The
cross-linked AP was attached to MAL-dPEG.sub.12-NHS ester (which
reacted with the remaining amines) and was conjugated to an
Fc-thiolated antibody as described in Example 4 to generate a
conjugate including a cross-linked AP enzyme. Stability studies
showed that cross-linking improved the stability of the conjugate
in an avidin-containing diluent (Ventana Medical Systems, Inc,
Tucson, Ariz.; P/N 95130). Specifically, at 45.degree. C., the
total loss of staining intensity on the 3.sup.rd day for the
conjugate with the cross-linked AP was 50%, whereas in the same
diluent and at the temperature, a conjugate prepared with a
non-crosslinked AP lost 95% of its staining intensity on the
1.sup.st day.
[0113] Alternative methods for cross-linking AP to increase its
stability are provided in Bieniarz et al., Bioconj. Chem., 9:
390-398, 1998, Bieniarz et al., Bioconj. Chem., 9: 399-402, 1998,
and U.S. Pat. No. 5,789,219. These methods also can be used to
cross-link alkaline phosphatase enzymes for use in a disclosed
conjugate.
Example 18
Analytical SDS PAGE of Alkaline Phosphatase Conjugates
[0114] In this example, the Fc-specificity of the conjugation
method of Example 4 is demonstrated by polyacrylamide gel
electrophoresis under denaturing conditions. Six different
preparations of the conjugate, 3 prepared with an anti-mouse IgG
antibody and 3 prepared with an anti-rabbit IgG antibody were
analyzed. Briefly, five to 20 .mu.l of a 100-200 .mu.g per ill
solution of each conjugate was mixed with 4.times. LDS gel loading
buffer (Invitrogen, Carlsbad, Calif.), and 2-Mercaptoethanol was
added to a final concentration of 1 mM. The sample mixture was
moderately heated at 48-50.degree. C. for 5 minutes. This
temperature was chosen to minimize dissociation of the covalent
linkage between the enzyme and the antibody, while still permitting
dissociating the light and heavy chains of the antibody portion of
the conjugate by the 2-Mercaptoethanol. Each sample was then cooled
and added to different wells of a polyacrylamide gel (either a
1.0-mm-thick, pre-formed NuPAGE.TM. 4-20% polyacrylamide Bis Tris
gel or a NuPAGE.TM. 3-8% polyacrylamide Tris acetate gel from
Invitrogen, Carlsbad, Calif.). The molecular weight standards used
were pre-stained Multimark.TM. and Mark12 wide range.TM. standards,
both of which were purchased from Invitrogen (Carlsbad, Calif.).
Electrophoresis was carried out at 70 mA for 60 to 90 minutes at
room temperature using a Novex XCell II cassette system
(Invitrogen, Carlsbad, Calif.). The running buffer was MES-SDS or
Tris Acetate-SDS buffer, for the 3-8% and 4-20% gels, respectively.
Gels were removed from the cassettes and washed twice in deionized
water for 5 minutes in order to remove the SDS and buffer. The
SDS-PAGE gels were then fixed in ethanol/water/acetic acid
[40:50:10 (v:v:v)] for 1 hour at room temperature and stained with
Coomassie Blue R-250 dissolved in methanol/water/acetic acid
[50:40:10 (v:v:v), Sigma-Aldrich, St. Louis, Mo.]. The gels were
stained for a minimum of 2 hours to a maximum of overnight by
gentle rocking at room temperature. De-staining was carried out in
the same manner as staining. The de-staining solution was identical
to the staining solution minus the dye. Gels were dried using an
Invitrogen gel drying kit (Invitrogen, Carlsbad, Calif.). Analysis
of the gels clearly showed for each of the conjugates the presence
of a band at a molecular weight corresponding to the light chain of
the antibody. Also, for each conjugate, there was a substantial
absence of bands corresponding to the molecular weight of the heavy
chain and of alkaline phosphatase. Instead, a series of bands at
higher molecular weights showed that the alkaline phosphatase was
selectively bound to the heavy chain of the IgGs for each
conjugate. Since the heavy chain of an immunoglobulin includes the
Fc region, the results showed the Fc-site specific nature of the
conjugation.
Example 19
Synthesis of an Fc-Specific Antibody-HRP Conjugate
[0115] In this example, preparation of an Fc-specific antibody
conjugate including a PEG-based hydrazide thiol linker is
described. Thiol-reactive maleimide groups were added to
horseradish peroxidase as follows. To a 4 mL amber vial was added
7.8 mg (15.2 .mu.mol, 100 eq.) of MAL-dPEG.sub.4.TM. NHS ester
(Quanta Biodesign, Powell, Ohio), followed by horseradish
peroxidase (HRP; Pierce Biotechnology, Rockford, Ill.; 0.25 ml, 25
mg/ml in 0.1 M Na.sub.3PO.sub.4, 0.15 M NaCl, pH=7.5). The vial was
rotated in the dark at ambient temperature for 1 hour before being
purified by size exclusion chromatography using an Akta Purifier
equipped with a Superdex 200 column (GE Biosciences, Uppsala,
Sweden) using an aqueous buffer solution (0.1 M Na.sub.3PO.sub.4,
0.15 M NaCl, pH=7.5). HRP containing fractions were pooled to give
a solution of HRP-PEG.sub.4-maleimide. The HRP concentration was
determined from the A.sub.280 of the solution
(.epsilon..sub.280=0.652 ml cm.sup.-1 mg.sup.-1) and the number of
maleimides was quantitated through a modified Ellman's assay to be
between 6 and 8 maleimides per enzyme.
[0116] The purified maleimido-horseradish peroxidase was combined
with a purified thiolated antibody (according to Example 4,
prepared using an MBH linker) in a 3:1 molar ratio and rotated for
a period of 18 h. Size exclusion chromatography (Superdex 200; 0.1
M Na.sub.3PO.sub.4, 0.15 M NaCl, pH=7.5) gave the purified
conjugate which was diluted to an A.sub.280=0.0375 into Avidin
Diluent with B5 Blocker (Ventana Medical Systems, Inc., Tucson,
Ariz.) and analyzed on tissue. A comparison of staining of prostate
specific antigen on prostate tissue using the HRP conjugate of this
Example to an HRP conjugate prepared by DTT reduction of the
immunoglobulin as described in U.S. Provisional Patent Application,
No. 60/675,759 showed that the HRP conjugate of this Example
exhibited slightly less background than the DTT-prepared HRP
conjugate, but also exhibited slightly less staining intensity.
Example 20
Multifunctional Hydrazide Thiol Linkers Derived From Amino
Acids
[0117] In some embodiments, multifunctional hydrazide thiol linkers
that can be used in the disclosed method are prepared from amino
acids and amino acid analogues according to schemes 4a, 4b, 4c and
4d above. In this example, synthetic routes to specific linkers are
outlined in the following schemes. In each of specific schemes 17a,
17b, 17c and 17d, an amino acid or amino acid analog
(Sigma-Aldrich, St. Louis, Mo.) is first reacted with
N-Succinimidyl S-Acetylthioacetate (SATA; Pierce Biotechnology,
Rockford, Ill.) in the presence of triethylamine (TEA). In scheme
17a, the product of this first reaction is reacted with hydrazine
to provide a multifunctional hydrazide thiol linker having one
hydrazide group and two thiol groups. In Scheme 17b,
carbodiimide-meditated coupling with DCC is used to form an NHS
active ester with the carboxylic acid functionality of the product
of the first reaction, followed by reaction with hydrazine to yield
another multifunctional hydrazide thiol linker having one hydrazide
group and two thiol groups. In Scheme 17c, as in 17b, NHS ester
formation using the product of the first reaction is followed by
reaction with hydrazine to yield a multifunctional hydrazide thiol
linker having two hydrazide groups and one thiol group. In Scheme
17d, the reaction with hydrazine yields a multifunctional hydrazide
thiol linker having one hydrazide group, one thiol group and one
hydroxyl group. ##STR21## ##STR22## ##STR23## ##STR24##
[0118] The products of Schemes 17a, 17b, 17c and 17d are,
respectively, 2-mercaptoacetamido-mercaptobutyric acid hydrazide
(MAMBH),
N,N'-(6-hydrazinyl-6-oxohexane-1,5-diyl)bis(2-mercaptoacetamide)
(BTAL),
N-(1,5-dihydrazinyl-1,5-dioxopentan-2-yl)-2-mercaptoacetamide
(TAGD) and
N-(1-hydrazinyl-4-hydroxy-1-oxobutan-2-yl)-2-mercaptoacetamide.
[0119] In a particular embodiment, MAMBH is synthesized as follows.
First S-Acetylthioacetamide homocysteine is prepared by preparing a
solution of triethylamine (0.15 ml, 1.1 mmol) in acetonitrile (10
ml) to which was added homocysteine hydrochloride (150 mg, 1.0
mmol). The resulting slurry was stirred for 5 minutes before the
addition of S-acetylthioacetate (250 mg, 1.1 mmol). The reaction
was stirred for 16 h at ambient temperature and then concentrated
in vacuo. Column chromatography (SiO.sub.2, 9:1
CH.sub.2Cl.sub.2/Et.sub.2O) resulted in the isolation of the
product as a colorless powder. Yield: 174 mg (75%): .sup.1H NMR
(250 MHz, CDCl.sub.3) .delta. 6.66 (bs, 1H), 4.51-4.41 (p, J=6.7
Hz, 1H), 3.63-3.50 (m, 2H), 3.36-3.18 (m, 2H), 2.88-2.80 (m, 1H),
2.38 (s, 3H), 2.01-1.88 (m, 1H); .sup.13C NMR (62.9 MHz,
CDCl.sub.3) .delta. 204.37, 195.38, 168.59, 59.51, 32.74, 31.43,
30.18, 27.43; ESI-HRMS m/z 256.00693 (M+Na.sup.+,
C.sub.8H.sub.11NNaO.sub.3S.sub.2 calcd 256.00780).
2-Mercaptoacetamido-mercaptobutyric acid hydrazide (MAMBH) is then
prepared by adding the S-acetylthioacetamide homocysteine (300 mg,
1.3 mmol) to hydrazine monohydrate (10 ml). The resulting slurry
was stirred for 16 h at ambient temperature at which time the
solution becomes homogeneous. The hydrazine was removed in vacuo
and the crude product was purified by reverse-phase flash
chromatography (15% C.sub.8 SiO.sub.2, 160:39:1 H.sub.2O/MeOH/AcOH)
to give the desired compound as a colorless oil. Yield: 207 mg
(72%): .sup.1H NMR (250 MHz, CD.sub.3OD) .delta. 4.52-4.46 (m, 1H),
3.23-3.21 (m, 2H), 2.59-2.52 (m, 2H), 2.10-2.01 (m, 2H); .sup.13C
NMR (62.9 MHz, CD.sub.3OD) .delta. 172.82, 172.43, 52.49, 37.72,
21.42, 20.49; ESI-HRMS m/z 246.03251 (M+Na.sup.+,
C.sub.6H.sub.13N.sub.3NaO.sub.2S.sub.2 calcd 246.03469).
[0120] Substitution of 6-Acetylthiohexanoic acid NHS ester for SATA
in schemes 17a, 17b and 17c yields the corresponding compounds
TMBH, BTHL and THGD that are shown below. 6-Acetylthiohexanoic acid
NHS ester has the following structure6-Acetylthiohexanoic acid NHS
ester can be prepared from by carbodiimide mediated coupling of
6-Acetylthiohexanoic acid with N-Hydroxysuccinimide. ##STR25##
Thiohexanamidomercaptobutyric acid hydrazide (THMBH) ##STR26##
Bisthiohexanamidohydrazidolysine (BTHL) ##STR27##
Thiohexamidoglutamnic acid dihydrazide (THGD)
[0121] 6-Acetylthiohexanoic acid NHS ester has the following
structure: ##STR28## and is prepared by carbodiimide mediated
coupling of 6-Acetylthiohexanoic acid with N-Hydroxysuccinimide
(both available from Sigma-Aldrich, St. Louis, Mo.).
[0122] One of ordinary skill in the art also will recognize that
PEG-based S-acetyl-thiocarboxylic acid derivatives can be
substituted for SATA in the schemes above to provide
multifunctional PEG-based linkers that be used in the disclosed
method of conjugation. For example, PEG-based multifunctional
hydrazide thiol linkers can be made by substituting a molecule of
the following formula for SATA in schemes 17a-d above: ##STR29##
wherein m=2 to 50. Compounds of this formula are commercially
available from Quanta Biodesign (Powell, Ohio), or can be prepared
from corresponding carboxylic acids.
Example 21
Multifunctional PEG-Based Hydrazide Thiol Linkers
[0123] In some embodiments, multifunctional PEG-based hydrazide
thiol linkers that can be used in the disclosed method are prepared
according to schemes 5a, 5b and 5c above. In this example,
synthetic routes to specific linkers are outlined in the following
schemes 18a, 18b and 18c. Specific protocols for the reactions also
are presented. Unless otherwise stated, reagents and solvents are
conventional and can be obtained, for example, from Sigma-Aldrich
(St. Louis, Mo.). ##STR30##
[0124] According to Scheme 18a, to a solution of 5.0 grams of Tris
in 10 ml water is added sodium carbonate (1.3 eq) followed by
phenoxy acetylchloride (1.2 eq), and the reaction is allowed to
stir on ice under nitrogen for 16 hours. The precipitated amino
protected product is then washed three times with water and dried
under vacuum. The pure compound resulting from this first reaction
is obtained by chromatography on a C18 silica based column eluted
with acetonitrile/H2O, 5-100% acetonitrile over 30 minutes.
Mesylate groups are then introduced by treating with triethylamine
(4.0 eq) and methane sulfonyl chloride (5.0 eq) in DMF. The DMF is
removed under vacuum, the residue taken in dry DCM and the salts
removed by filtration. Removal of the DCM under vacuum gives the
crude mesylate 2 which is used without further purification. To a
solution of the mesylate (0.3 eq) in dry DMF is added
HO-dPEG.sub.4.TM.-SATA (1.0 eq; Quanta Biodesign, Powell, Ohio) and
K.sub.2CO.sub.3 (1.5 eq) and the reaction allowed to stir under
nitrogen for 16 hours. The DMF is removed under vacuum, the residue
taken in dry DCM and salts are removed by filtration. Removal of
the DCM under vacuum followed by silica gel chromatography gives
the pegylated intermediate. The Pac protecting group is then
removed by treating with Pd/C in a mixture of EtAc/MeOH. The
semicarbizide is then elaborated by treating the resulting
intermediate first with carbonyl diimidizole (10 eq) followed by
hydrazine (100 eq). ##STR31##
[0125] According to Scheme 18b, to a solution of alcohol
(HO-PEG4-SATA, Quanta Biodesign, Powell, Ohio, 1.3 eq) in DCM is
added 1.5 eq of diazo-diisopropyldicarboxylate followed by 1.8 eq
of tributyl phosphine, and the reaction is stirred under dry
nitrogen for 30 minutes. To the resulting suspension is then added
1.0 eq of the phenol in DCM and the reaction allowed to stir under
dry nitrogen for 16 hrs. The phenol ether obtained after silica gel
chromatography is then taken in neat hydrazine and the solution is
microwaved to give the multi functional PEG-based hydrazide thiol
linker. ##STR32## ##STR33## According to Scheme 18c, to a solution
of 5.0 grams of Tris in 10 ml water is added potassium carbonate
(1.3 eq) followed by phenoxy acetylchloride (1.2 eq), and the
reaction is allowed to stir on ice under nitrogen for 16 hours. The
precipitated amino protected product is then washed three times
with water and dried under vacuum. The alkyne groups are then
introduced by treating with sodium hydride (3.0 eq) and propargyl
bromide (10 eq) in DMF to give the alkyne intermediate 1 after
silica gel chromatography. To a solution of HO-PEG.sub.4-SATA
(Quanta Biodesign, Powell, Ohio) in DCM is added methane sulfonyl
chloride (1.2 eq) followed by triethyl amine (1.4 eq), and the
reaction allowed to stir on ice under nitrogen for 16 hours. The
triethyl amine salt is then removed by filtration and the mesylate
product dried under vacuum. To a solution of the mesylated alcohol
in DCM is added sodium azide (1.2 eq), and the reaction allowed to
stir under nitrogen for 16 hours to afford the azide intermediate 2
after silica gel chromatography. To a 1:1 solution of
t-butanol/water containing copper sulfate (0.2 eq) and sodium
ascorbate (0.5 eq) is added one equivalent each of the intermediate
alkyne 1 and the intermediate azide 2. The reaction then stirred
under nitrogen for sixteen hours to afford the intermediate with
the protected nitrogen after silica gel chromatography. The
nitrogen protecting group is then removed by treating with 10% Pd/C
in a 1:1 mixture of ethyl acetate and methanol and the free amine
then obtained by an acid-base work up. To a solution of the free
amine in DCM is added carbonyl diimidizole (10 eq), and the
reaction stirred under nitrogen for four hours. The reaction is
then concentrated under vacuum and the residue taken into neat
hydrazine. The solution is then microwave at 100.degree. C. for 1
hour to give the multifunctional PEG-based hydrazide thiol
linker.
[0126] It will be readily apparent to one skilled in the art that
the PEG-based molecules can be replaced with other SATA alcohols in
these schemes to provide additional multifunctional PEG-based
hydrazide thiol linkers, and that PEG SATA alcohols of differing
lengths can be substituted as well.
Example 22
Synthesis of a Polyacrylamide Hydrazide Thiol Linker
[0127] In this example, a polymeric multivalent hydrazide thiol
linker is provided, which linker can be prepared according to
Scheme 19 below. ##STR34## In Scheme 19, x can be, for example,
100-500 and y can be, for example, 10-50. L represents a thiolating
reagent used to convert a portion of the hydrazide groups to thiol
groups. Polyacrylamide hydrazide (PAH) can be synthesized by the
method provided in published U.S. Patent Application No.
20050158770. Briefly, in a 100 mL round-bottom flask fitted with a
condenser, 20 mL polyacrylamide (1 mmol, 50% wt in water,
Sigma-Aldrich, Milwaukee, Wis.) is mixed with 10 mL distilled (DI)
water and 20 mL hydrazine monohydrate (420 mmol, Sigma-Aldrich,
Milwaukee, Wis.). The reaction is microwaved for 60 min. After
cooling to room temperature, the reaction is precipitated with an
equal volume of methanol, centrifuged and decanted. The residue is
taken up in 50 mL DI water and the precipitation repeated for a
total of three times. The final residue is dissolved in DI water
and lyophilized to give a fine, white hygroscopic powder. In an
appropriate solvent, the resulting PAH is reacted with a thiolating
agent, such as a thiol-dPEG-NHS ester (Quanta Biodesign, Powell,
Ohio) or Traut's reagent, to thiolate a portion (for example,
approximately 50-75%) of available hydrazides (z=5 to 40) and
provide a polymeric multifunctional hydrazide thiol linker that can
be used in the disclosed method. Additional thiolating reagents can
be found, for example, in Hermanson, "Bioconjugate Techniques,"
Academic Press, San Diego, 1996, ISBN 0-12-342336-8, which is
incorporated by reference herein.
[0128] It is possible to use/prepare the polyacrylamide hydrazide
thiol linker in one of two ways, either synthesize it first and use
the disclosed conjugation method, or first react PAH with one
molecule, thiolate the PAH, and then react the now thiolated first
molecule with a second molecule.
[0129] Although the principles of the present invention have been
described with reference to several illustrative embodiments, it
should be apparent to those of ordinary skill in the art that the
details of the embodiments may be modified without departing from
such principles. For example, although the detailed description has
focused on antibody-enzyme conjugates, the linkers and methods can
be used to prepare any type of conjugate including conjugates of
antibodies and other detectable labels such as nanoparticles (for
example, metal and semiconductor nanoparticles such as gold
nanoparticles and quantum dots, respectively), fluorescent
molecules, fluorogenic molecules, colored molecules, colorogenic
molecules, and paramagnetic constructs (such as chelates of
paramagnetic ions). Conjugates of antibodies for directed therapies
(for example, conjugates of antibodies with drug molecules, toxins
and radioactive constructs such as chelates of radioactive metal
ions) also are contemplated. Although, specific examples provided
show the use of hydrazide thiol linkers having hydrazide and
carbohydrazide groups, any "hydrazide group" can be substituted for
the hydrazide or carbohydrazide groups shown in both the disclosed
method and the disclosed conjugate. Furthermore, it should be
understood that while one or more of a single hydrazide thiol
linker can be used to form a conjugate, it is also possible to use
multiple different hydrazide thiol linkers to form a conjugate. The
disclosed conjugates can be used in any type of assay where a
specific binding molecule attached to a detectable label can be
used, for example, in any type of immunoassay in addition to the
illustrated immunohistochemical assays, or in any type of in situ
hybridization assay. Detection protocols can be performed manually
or in an automated fashion. Furthermore, the disclosed linkers also
can be used to modify surfaces for binding molecules to a
substrate, and such surface modification reactions can be performed
using the disclosed method. The present invention includes all
modifications, variations, and equivalents thereof as fall within
the scope and spirit of the following claims.
* * * * *