U.S. patent application number 15/536296 was filed with the patent office on 2017-12-21 for sandwich assay design for small molecules.
This patent application is currently assigned to Siemens Healthcare Diagnostics Inc.. The applicant listed for this patent is Siemens Healthcare Diagnostics Inc.. Invention is credited to Manoj Sharma, Tie Q. Wei, Yi Feng Zheng.
Application Number | 20170362305 15/536296 |
Document ID | / |
Family ID | 56127406 |
Filed Date | 2017-12-21 |
United States Patent
Application |
20170362305 |
Kind Code |
A1 |
Zheng; Yi Feng ; et
al. |
December 21, 2017 |
SANDWICH ASSAY DESIGN FOR SMALL MOLECULES
Abstract
Methods are disclosed of designing antibodies for a sandwich
assay for a small molecule having a molecular weight of about 500
to about 2,000. The method comprises preparing a first antibody
that binds to the small molecule, and preparing a second antibody
that binds to the small molecule at a portion of the small molecule
other than a portion to which the first antibody binds. The second
antibody is prepared from an immunogen that comprises a
predetermined portion of the small molecule. The antibodies may be
employed in sandwich assays for the small molecule.
Inventors: |
Zheng; Yi Feng; (Wilmington,
DE) ; Wei; Tie Q.; (Wilmington, DE) ; Sharma;
Manoj; (Hockessin, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Siemens Healthcare Diagnostics Inc. |
Tarrytown |
NY |
US |
|
|
Assignee: |
Siemens Healthcare Diagnostics
Inc.
Tarrytown
NY
|
Family ID: |
56127406 |
Appl. No.: |
15/536296 |
Filed: |
December 11, 2015 |
PCT Filed: |
December 11, 2015 |
PCT NO: |
PCT/US15/65237 |
371 Date: |
June 15, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62093118 |
Dec 17, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/566 20130101;
C07K 16/00 20130101; C07K 16/44 20130101; C07D 497/04 20130101;
G01N 33/9493 20130101; C07K 2317/10 20130101; C07K 2317/54
20130101 |
International
Class: |
C07K 16/00 20060101
C07K016/00; G01N 33/566 20060101 G01N033/566; G01N 33/94 20060101
G01N033/94 |
Claims
1. A method of designing antibodies for a sandwich assay for a
small molecule having a molecular weight of about 500 to about
2,000, the method comprising: (a) preparing a first antibody that
binds to the small molecule, and (b) preparing a second antibody
that binds to the small molecule in a portion of the small molecule
other than a portion to which the first antibody binds, wherein the
second antibody is prepared from an immunogen that comprises a
predetermined portion of the small molecule.
2. The method according to claim 1 wherein the first antibody is
prepared from an immunogen that comprises a portion of the small
molecule other than the predetermined portion.
3. The method according to claim 1 wherein the predetermined
portion of the small molecule is obtained by modification of the
small molecule to alter a spatial conformation of the small
molecule.
4. The method according to claim 1 wherein the predetermined
portion of the small molecule is a compound that consists
essentially of the predetermined portion.
5. The method according to claim 1 wherein the small molecule is a
macrolide.
6. The method according to claim 1 wherein the small molecule is an
immunosuppressant drug.
7. The method according to claim 1 wherein one or both of the first
antibody and the second antibody are monoclonal antibodies.
8. A method of determining a presence and/or amount of a small
molecule having a molecular weight of about 500 to about 2,000 in a
sample suspected of containing the small molecule, the method
comprising: (a) providing in combination in a medium: (i) the
sample, (ii) the first antibody for the small molecule according to
claim 1, and (iii) the second antibody for the small molecule
according to claim 1, (b) incubating the medium under conditions
for binding of the first antibody and the second antibody to the
small molecule, and (c) examining the medium for the presence of an
immunocomplex comprising the small molecule, the first antibody and
the second antibody, the presence and/or amount of the
immunocomplex indicating the presence and/or amount of the small
molecule in the sample.
9. The method according to claim 8 wherein the small molecule is an
immunosuppressant drug.
10. A method of designing antibodies for a sandwich assay for a
small molecule having a molecular weight of about 500 to about
2,000, the method comprising: (a) preparing a first monoclonal
antibody that binds to a portion of the small molecule, and (b)
preparing a second monoclonal antibody that binds to the small
molecule at a portion of the small molecule other than the portion
to which the first monoclonal antibody binds, wherein the second
monoclonal antibody is prepared from an immunogen that comprises
the small molecule that is modified at the portion of the small
molecule to which the first monoclonal antibody binds.
11. The method according to claim 10 wherein the first monoclonal
antibody is prepared from an immunogen that comprises a portion of
the small molecule other than the portion to which the second
monoclonal antibody binds.
12. The method according to claim 10 wherein the derivatized
portion of the small molecule alters a spatial conformation of the
small molecule.
13. The method according to claim 10 wherein the small molecule is
an immunosuppressant drug.
14. The method according to claim 13 wherein the immunosuppressant
drug is sirolimus.
15. The method according to claim 14 wherein the derivatized
portion of the sirolimus molecule comprises a triene.
16. A method of determining a presence and/or amount of a small
molecule having a molecular weight of about 500 to about 2,000 in a
sample suspected of containing the small molecule, the method
comprising: (a) providing in combination in a medium: (i) the
sample, (ii) the first monoclonal antibody for the small molecule
according to claim 10, and (iii) the second monoclonal antibody for
the small molecule according to claim 10, (b) incubating the medium
under conditions for binding of the first monoclonal antibody and
the second monoclonal antibody to the small molecule, and (c)
examining the medium for the presence of an immunocomplex
comprising the small molecule, the first monoclonal antibody and
the second monoclonal antibody, the presence and/or amount of the
immunocomplex indicating the presence and/or amount of the small
molecule in the sample.
17. The method according to claim 16 wherein the small molecule is
an immunosuppressant drug.
18. A method of designing antibodies for a sandwich assay for
sirolimus, the method comprising: (a) preparing a first monoclonal
antibody that binds to sirolimus, and (b) preparing a second
monoclonal antibody that binds to sirolimus in a portion of
sirolimus other than the portion to which the first monoclonal
antibody binds, wherein the second monoclonal antibody is prepared
from an immunogen that is modified at the portion of sirolimus to
which the first antibody binds.
19. The method according to claim 18 wherein the modified portion
of sirolimus comprises a triene.
20. A method of determining a presence and/or amount of a sirolimus
in a sample suspected of containing sirolimus, the method
comprising: (a) providing in combination in a medium: (i) the
sample, (ii) the first monoclonal antibody for sirolimus according
to claim 18, and (iii) the second monoclonal antibody for sirolimus
according to claim 18, (b) incubating the medium under conditions
for binding of the first monoclonal antibody and the second
monoclonal antibody to sirolimus, and (c) examining the medium for
the presence of an immunocomplex comprising sirolimus, the first
monoclonal antibody and the second monoclonal antibody, the
presence and/or amount of the immunocomplex indicating the presence
and/or amount of sirolimus in the sample.
Description
[0001] The subject application claims benefit under 35 USC
.sctn.119(e) of U.S. Provisional Application No. 62/093,118, filed
Dec. 17, 2014. The entire contents of the above-referenced patent
application are hereby expressly incorporated herein by
reference.
BACKGROUND
[0002] The invention relates to compounds, methods and kits for the
determination of small molecules, in samples, such as patient
samples, known or suspected to contain one or more of such small
molecules. In some aspects the invention relates to sandwich assays
for small molecules such as, for example, immunosuppressant
drugs.
[0003] The body relies upon a complex immune response system to
distinguish self from non-self. At times, the body's immune system
must be controlled in order to either augment a deficient response
or suppress an excessive response. For example, when organs such as
kidney, heart, heart-lung, bone marrow and liver are transplanted
in humans, the body will often reject the transplanted tissue by a
process referred to as allograft rejection.
[0004] In treating allograft rejection, the immune system is
frequently suppressed in a controlled manner with drug therapy.
Immunosuppressant drugs are therapeutic drugs that are carefully
administered to transplant recipients in order to help prevent
allograft rejection of non-self tissue. Immunosuppressive drugs can
be classified as follows: glucocorticoids, cytostatics, antibodies,
drugs acting on immunophilins, and other drugs such as interferons,
opiates INF binding proteins, mycophenolate, FTY720 and the like. A
particular class of immunosuppressant drugs comprises those drugs
that act on immunophilins. Immunophilins are an example of
high-affinity, specific binding proteins having physiological
significance. Two distinct families of immunophilins are presently
known: cyclophilins and macrophilins, the latter of which
specifically bind, for example, tacrolimus or sirolimus.
[0005] Two most commonly administered immunosuppressive drugs to
prevent organ rejection in transplant patients are cyclosporine
(CSA) and FK-506 (FK or tacrolimus). Another drug that finds use as
an immunosuppressant in the United States and other countries is
sirolimus, also known as rapamycin. Derivatives of sirolimus are
also useful as immunosuppressants. Such derivatives include, for
example, everolimus, and the like.
[0006] The side effects associated with some immunosuppressant
drugs can be controlled in part by carefully controlling the level
of the drug present in a patient. Therapeutic monitoring of
concentrations of immunosuppressant drugs and related drugs in
blood is required to optimize dosing regimes to ensure maximal
immunosuppression with minimal toxicity. Although immunosuppressant
drugs are highly effective immunosuppressive agents, their use must
be carefully managed because the effective dose range is often
narrow and excessive dosage can result in serious side effects. On
the other hand, too little dosage of an immunosuppressant can lead
to tissue rejection. Because distribution and metabolism of an
immunosuppressant drug can vary greatly between patients and
because of a wide range and severity of adverse reactions, accurate
monitoring of the drug level is essential.
[0007] There is, therefore, a continuing need to develop fast and
accurate diagnostic methods to measure levels of small molecules
such as, for example, immunosuppressant drugs or derivatives
thereof in patients. The methods should be capable of being fully
automated and should selectively detect the parent molecule while
minimizing inaccuracies resulting from the cross-reactivity of its
metabolites or from constituents in a sample suspected of
containing the small molecule.
SUMMARY
[0008] Some examples in accordance with the principles described
herein are directed to methods of designing antibodies for a
sandwich assay for a small molecule having a molecular weight of
about 500 to about 2,000. The method comprises preparing a first
antibody that binds to the small molecule, and preparing a second
antibody that binds to the small molecule at a portion of the small
molecule other than a portion to which the first antibody binds.
The second antibody is prepared from an immunogen that comprises a
predetermined portion of the small molecule.
[0009] Some examples in accordance with the principles described
herein are directed to methods of determining a presence and/or
amount of a small molecule having a molecular weight of about 500
to about 2,000 in a sample suspected of containing the small
molecule. The sample, a first antibody for the small molecule as
described above, and a second antibody for the small molecule as
described above are provided in combination in a medium, which is
incubated under conditions for binding of the first antibody and
the second antibody to the small molecule. The medium is examined
for the presence of an immunocomplex comprising the small molecule,
the first antibody and the second antibody, the presence and/or
amount of the immunocomplex indicating the presence and/or amount
of the small molecule in the sample.
[0010] Some examples in accordance with the principles described
herein are directed to methods of designing antibodies for a
sandwich assay for a small molecule having a molecular weight of
about 500 to about 2,000. A first monoclonal antibody that binds to
a portion of the small molecule is prepared. A second monoclonal
antibody that binds to the small molecule at a portion of the small
molecule other than the portion to which the first monoclonal
antibody binds is also prepared from an immunogen that comprises
the small molecule that is derivatized at the portion of the small
molecule to which the first monoclonal antibody binds. The
antibodies may be employed in methods of determining a presence
and/or amount of a small molecule having a molecular weight of
about 500 to about 2,000 in a sample suspected of containing the
small molecule.
[0011] Some examples in accordance with the principles described
herein are directed to methods of designing antibodies for a
sandwich assay for sirolimus. A first monoclonal antibody that
binds to sirolimus is prepared. A second monoclonal antibody that
binds to sirolimus at a portion of sirolimus other than the portion
to which the first monoclonal antibody binds is also prepared from
an immunogen that is derivatized at the portion of sirolimus to
which the first antibody binds. The antibodies may be employed in
methods of determining a presence and/or amount of sirolimus in a
sample suspected of containing sirolimus.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The drawings provided herein are not to scale and are
provided for the purpose of facilitating the understanding of
certain examples in accordance with the principles described herein
and are provided by way of illustration and not limitation on the
scope of the appended claims.
[0013] FIG. 1 is a chemical formula for sirolimus (I).
[0014] FIG. 2 is the chemical formula of FIG. 1 with numbering and
depicting portions of the molecule to which monoclonal antibodies
can be prepared in accordance with the principles described
herein.
[0015] FIG. 3 is a reaction scheme for the preparation of adducts
at the triene area of sirolimus in an example in accordance with
the principles described herein.
[0016] FIG. 4 is a reaction scheme for the preparation of a PTAD
adduct at the triene area of sirolimus in another example in
accordance with the principles described herein.
[0017] FIG. 5 is a reaction scheme for the preparation of oxime
derivatives of sirolimus in another example in accordance with the
principles described herein.
[0018] FIG. 6 is a reaction scheme for the preparation of an
immunogen from an oxime derivative of sirolimus of FIG. 5 in
another example in accordance with the principles described
herein.
[0019] FIG. 7 is a reaction scheme for the preparation of oxime
derivatives of sirolimus PTAD adducts of FIG. 4 in another example
in accordance with the principles described herein.
[0020] FIG. 8 is a reaction scheme for the preparation of an
immunogen from an oxime derivative of sirolimus of FIG. 7 in
another example in accordance with the principles described
herein.
[0021] FIG. 9 is a reaction scheme for the preparation of
immunogens from an oxime derivative of a carboxylated PTAD adduct
of sirolimus in another example in accordance with the principles
described herein.
DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS
General Discussion
[0022] The present inventors have discovered that monoclonal
antibodies can be designed that specifically bind simultaneously to
separate portions of small molecules. This discovery is surprising
because small molecules are haptens, which are relatively small
molecules (molecular weight (daltons) less than about 2000, or less
than about 1500, or less than about 1000) and are not considered to
have more than one site to which an antibody can bind. In
accordance with the principles described herein, at least two
different antibodies can be prepared, which bind to separate
portions of a small molecule at the same time.
[0023] The term "small molecule" refers to a molecule having a
molecular weight of about 150 to about 2,000, or about 150 to about
1,500, or about 150 to about 1,000, or about 150 to about 500, or
about 300 to about 2,000, or about 300 to about 1,500, or about 300
to about 1,000, or about 500 to about 2,000, or about 500 to about
1,500, or about 500 to about 1,000, for example. For the most part,
small molecules, which are sometimes referred to as haptens, do not
elicit an immune response unless linked to large molecule or
immunogenic carrier that does illicit an immune response in order
to raise antibodies. Haptens are compounds capable of binding
specifically to corresponding antibodies, but do not themselves act
as immunogens (or antigens) for preparation of the antibodies.
[0024] The phrase "antibody for a small molecule" refers to an
antibody that binds specifically to the small molecule and does not
bind to any significant degree to other substances that would
distort the analysis for the small molecule. Furthermore, the
antibody for the small molecule binds specifically to a certain
domain of the small molecule. Specific binding involves the
specific recognition of one of two different molecules, or two
different domains of a small molecule, for the other compared to
substantially less recognition of other molecules or other domains
of a small molecule. On the other hand, non-specific binding
involves non-covalent binding between molecules that is relatively
independent of specific surface structures. Non-specific binding
may result from several factors including hydrophobic interactions
between molecules.
[0025] A small molecule, to which examples in accordance with the
principles described herein may be applied, has spatially separate
binding domains for the antibodies. The small molecule may be
linear or it may comprise one or more rings, for example, two
rings, or three rings, or four rings, or five rings, or more. The
binding domains on the small molecule should be separated such that
two different antibodies can bind simultaneously to the small
molecule without interfering with the binding of each other to form
a three-member complex (or immunocomplex) wherein each antibody
binds to an extent necessary so that a sufficiently stable complex
is formed comprising the two antibodies and the small molecule. The
complex is considered sufficiently stable when the complex remains
intact during an assay so that the complex can be detected and the
amount of the complex accurately reflects the amount of a small
molecule analyte in a sample. The stable complex permits an
accurate and sensitive assay for the small molecule analyte. In
some examples, the binding domains for the different antibodies on
a linear small molecule should be separated by at least 5 carbon
atoms, or at least 6 carbon atoms, or at least 7 carbon atoms, or
at least 8 carbon atoms, or at least 9 carbon atoms, or at least 10
carbon atoms, for example. In some examples, the binding domains
for the different antibodies on a small molecule that comprises one
or more rings should be separated by at least 3 carbon atoms, or at
least 4 carbon atoms, or at least 5 carbon atoms, or at least 6
carbon atoms, or at least 7 carbon atoms, or at least 8 carbon
atoms, for example.
[0026] In some examples the small molecule comprises at least one
large ring, which is a 15-50 membered ring, or a 15-45 membered
ring, or a 15-40 membered ring, or a 15-35 membered ring, or a
15-30 membered ring, or a 15-25 membered ring, or a 15-20 membered
ring, or a 20-50 membered ring, or a 20-45 membered ring, or a
20-40 membered ring, or a 20-35 membered ring, or a 20-30 membered
ring, or a 20-25 membered ring, or a 25-50 membered ring, or a
25-45 membered ring, or a 25-40 membered ring, or a 25-35 membered
ring, or a 25-30 membered ring, or a 30-50 membered ring, or a
30-45 membered ring, or a 30-40 membered ring, for example. The
atoms forming the ring are primarily carbon and may also include,
but are not limited to, oxygen, nitrogen and sulfur, for example.
The large ring may also comprise 1-5, or 1-4, or 1-3 or 1-2, or
2-5, or 2-4, or 2-3, small rings, which are 5-7 membered rings or
5-6 membered rings. Some of the atoms of the small rings may be
part of the large ring.
[0027] In some examples, the small molecule comprises a three
dimensional conformation with one or more unique chemical
functional groups that allow different antibodies to be prepared
where at least two different antibodies can approach and bind to
different binding domains on the small molecule without interfering
with one another. The unique chemical functional groups include, by
way of illustration and not limitation, carbon-carbon double bonds,
carbon-carbon triple bonds, carbonyl groups, imine groups, carboxyl
groups, hydroxyl groups, amine groups, amide groups, ester groups
and ether groups, for example, and combinations of two or more of
the above. The chemical functional groups may be unconjugated or
conjugated. The term "conjugated" refers to contiguous atoms that
have available p-orbitals such that electrons can be delocalized
over the contiguous atoms. Examples of conjugated atoms include,
but are not limited to, one, two, three, four, or five or more
conjugated carbon-carbon double bonds (vinyl groups); one, two,
three, four, or five or more conjugated carbon-carbon triple bonds;
a combination of one, two, three, four, or five or more conjugated
carbon-carbon double bonds, carbon-carbon triple bonds, imine
groups, carbonyl groups, and anions, for example. In some examples,
the chemical functional groups provide a particular spatial
conformation for the small molecule.
[0028] The chemical functional groups serve at least two purposes
in accordance with the principles described herein. They may be
employed to prepare a first antibody that specifically binds to a
binding domain of the small molecule at which the chemical
functional group is located. In addition, they may be employed for
modification of the small molecule at the area of the small
molecule comprising the chemical functional group and using the
modified small molecule as part of an immunogen to raise a second
antibody against a binding domain other than the binding domain to
which the first antibody binds. In some examples, modification of
the area of the small molecule comprising the chemical functional
group alters a three dimensional conformation of the area to
further promote the formation of antibodies at binding domains of
the small molecule other than the area comprising the chemical
functional group.
[0029] In some examples the small molecule is a macrolide. In some
examples the macrolide is an immunosuppressant drug. The term
"immunosuppressant drugs" includes those that act on immunophilin
such as, but not limited to, cyclosporin (including cyclosporin A,
cyclosporin B, cyclosporin C, cyclosporin D, cyclosporin E,
cyclosporin F, cyclosporin G, cyclosporin H, cyclosporin I),
tacrolimus (FR-900506, FK506, PROGRAF.RTM.), sirolimus (rapamycin,
RAPAMUNE.RTM.), and derivatives of the above such as, but not
limited to, Everolimus (RAD, CERTICAN.RTM.), for example.
[0030] As mentioned above, some examples in accordance with the
principles described herein are directed to methods of designing
antibodies for a sandwich assay for a small molecule having a
molecular weight of about 500 to about 2,000. The method comprises
preparing a first antibody that binds to a portion or domain of the
small molecule, and preparing a second antibody that binds to the
small molecule at a portion or domain of the small molecule other
than a portion or domain to which the first antibody binds. The
second antibody is prepared from an immunogen that comprises a
predetermined portion or domain of the small molecule.
[0031] The phrase "immunogen that comprises a predetermined portion
or domain of the small molecule" refers to a compound that
comprises a portion of the small molecule other than the portion of
the small molecule to which the first antibody binds. The compound
may be the small molecule that has been modified to enhance the
ability of preparing an antibody that binds to a portion of the
small molecule other than the portion to which the first antibody
binds. On the other hand, the compound may be a compound other than
the small molecule that comprises the portion of the small molecule
other than the portion to which the first antibody binds. In some
examples, the predetermined portion of the small molecule is
obtained by modification of the small molecule to alter a spatial
conformation of the small molecule. In some examples, the
predetermined portion of the small molecule is a compound that
consists essentially of the predetermined portion. In either case,
the compound is linked to an immunogenic carrier for use in
preparing antibodies in accordance with the principles described
herein.
[0032] Preparation of monoclonal antibodies that simultaneously
bind to two different domains on a small molecule enables the use
of such antibodies in sandwich assays in which the small molecule
is simultaneously bound by the two different antibodies to form an
immunocomplex. The ability to perform sandwich assays on small
molecules enhances the sensitivity of an assay for the small
molecule. In addition, in the case of sandwich assays involving one
monoclonal antibody bound to a support, the assay may be conducted
in the presence of impurities and interfering substances of a
sample because the support can be separated from the sample and
washed after small molecule has been allowed to bind to the
monoclonal antibody of the support but before introduction of the
second monoclonal antibody.
[0033] Antibodies may include a complete immunoglobulin or fragment
thereof, which immunoglobulins include the various classes and
isotypes, such as IgA, IgD, IgE and IgM, for example. Fragments
thereof may include Fab, Fv and F(ab').sub.2, Fab', for example. In
addition, aggregates, polymers, and conjugates of immunoglobulins
or their fragments can be used where appropriate so long as binding
affinity for a particular molecule is maintained.
[0034] Antibodies in accordance with the principles described
herein may be prepared by techniques including, but not limited to,
immunization of a host and collection of sera (polyclonal),
preparing continuous hybrid cell lines and collecting the secreted
protein (monoclonal) or cloning and expressing nucleotide sequences
or mutagenized versions thereof coding at least for the amino acid
sequences required for specific binding of natural antibodies, for
example.
[0035] Monoclonal antibodies can be prepared by techniques that are
well known in the art such as preparing continuous hybrid cell
lines and collecting the secreted protein (somatic cell
hybridization techniques). Monoclonal antibodies may be produced
according to the standard techniques of Kohler and Milstein, Nature
265:495-497, 1975. Reviews of monoclonal antibody techniques are
found in Lymphocyte Hybridomas, ed. Melchers, et al.
Springer-Verlag (New York 1978), Nature 266: 495 (1977), Science
208: 692 (1980), and Methods of Enzymology 73 (Part B): 3-46
(1981).
[0036] In another approach for the preparation of antibodies, the
sequence coding for antibody binding sites can be excised from the
chromosome DNA and inserted into a cloning vector, which can be
expressed in bacteria to produce recombinant proteins having the
corresponding antibody binding sites. This approach involves
cloning and expressing nucleotide sequences or mutagenized versions
thereof coding at least for the amino acid sequences required for
specific binding of natural antibodies.
[0037] In one approach for the production of monoclonal antibodies,
a first step includes immunization of an antibody-producing animal
such as a mouse, a rat, a goat, a sheep, or a cow with an immunogen
in accordance with the principles described herein. Immunization
can be performed with or without an adjuvant such as complete
Freund's adjuvant or other adjuvants such as monophosphoryl lipid A
and synthetic trehalose dicorynomycolate adjuvant. A next step
includes isolating spleen cells from the antibody-producing animal
and fusing the antibody-producing spleen cells with an appropriate
fusion partner, typically a myeloma cell, such as by the use of
polyethylene glycol or other techniques. Typically, the myeloma
cells used are those that grow normally in hypoxanthine-thymidine
(HT) medium but cannot grow in hypoxanthine-aminopterin-thymidine
(HAT) medium, used for selection of the fused cells. A next step
includes selection of the fused cells, typically by selection in
HAT medium. A next step includes screening the cloned hybrids for
appropriate antibody production using immunoassays such as
enzyme-linked immunosorbent assay (ELISA) or other immunoassays
appropriate for screening.
[0038] An antibody (prepared from an immunogen in accordance with
the principles described herein) with the requisite specificity may
be selected by screening methodologies, which include, by way of
illustration and not limitation, ELISA, dot blots, Western
analysis, and Surface Plasmon Resonance, for example. In this
manner an antibody is obtained that binds to a domain of a small
molecule of interest and does not bind to any detectable degree to
other domains of the small molecule or to other molecules that are
not of interest in a particular assay. In some examples in
accordance with the principles described herein, an antibody that
binds to a domain of a small molecule of interest has a binding
affinity for the domain of the small molecule of interest of about
10.sup.7 to about 10.sup.14 liters/mole, or about 10.sup.7 to about
10.sup.11 liters/mole, or about 10.sup.7 to about 10.sup.12
liters/mole, or about 10.sup.8 to about 10.sup.14 liters/mole, or
about 10.sup.8 to about 10.sup.11 liters/mole, or about 10.sup.8 to
about 10.sup.12 liters/mole, for example. The phrase "any
detectable degree" means that the antibody that specifically binds
to a domain of a small molecule of interest has a binding affinity
for other domains of the small molecule of interest or for other
molecules that are not of interest of less than about 10.sup.4
liters/mole, or less than about 10.sup.3 liters/mole, or less than
about 10.sup.2 liters/mole, or less than about 10 liters/mole, for
example.
[0039] The term "immunogenic carrier" means a group or moiety
which, when conjugated to a hapten and injected into a mammal or
otherwise employed as an immunogen, induces an immune response and
elicits production of antibodies that bind to the hapten.
Immunogenic carriers are also sometimes referred to as antigenic
carriers. In some examples in accordance with the principles
described herein, immunogens comprising immunogenic carriers,
including poly(amino acid) and non-poly(amino acid) immunogenic
carriers, linked to a small molecule at a particular position are
synthesized and used to prepare antibodies in accordance with the
principles described herein.
[0040] The molecular weight range (in Daltons) for poly(amino
acids) that are immunogenic carriers is about 5,000 to about
10,000,000, or about 20,000 to about 600,000, or about 25,000 to
about 250,000 molecular weight, for example. Poly(amino acid)
immunogenic carriers include proteins such as, for example,
albumins, serum proteins, e.g., globulins, ocular lens proteins and
lipoproteins. Illustrative proteins include, but are not limited
to, bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH),
egg ovalbumin, and bovine gamma-globulin (BGG), thyroglobulin,
ovalbumin or fibrinogen, for example. In one illustrative example,
the protein is KLH; in another illustrative example, the protein is
BSA. Non-poly(amino acid) immunogenic carriers include
polysaccharides, nucleic acids and particles (biologic and
synthetic materials). A wide variety of immunogenic carriers are
disclosed in Davalian, et al., U.S. Pat. No. 5,089,390, column 4,
line 57 to column 5, line 5, which is incorporated herein by
reference.
[0041] As mentioned above, the immunogenic carrier may be a
polysaccharide, which is a high molecular weight polymer of
monosaccharides that may be prepared naturally or synthetically and
usually involves repeated condensations of monosaccharides.
Examples of polysaccharides are starches, glycogen, cellulose,
carbohydrate gums, such as gum arabic, agar, and so forth. The
polysaccharide can also contain poly(amino acid) residues and/or
lipid residues.
[0042] As mentioned above, in some examples in accordance with the
principles described herein, the immunogenic carrier may be linked
to the small molecule at a predetermined position on the small
molecule by means of a linking group. In some examples, the linking
group may comprise about 2 to about 50 atoms, or 4 to about 30
atoms, not counting hydrogen and may comprise a chain of from 2 to
about 30 atoms, or 3 to about 20 atoms, each independently selected
from the group normally consisting of carbon, oxygen, sulfur,
nitrogen, and phosphorous. Part or all of the linking group may be
a portion of the molecule being linked to the small molecule such
as, but not limited to, an amino acid residue on a poly(amino
acid), for example. In some examples, the linking group comprises
an oxime functionality.
[0043] The number of heteroatoms in the linking group may be in the
range from 0 to about 20, or 1 to about 15, or about 2 to about 10.
The linking group may be aliphatic or aromatic. When heteroatoms
are present, oxygen is normally present as oxo or oxy, bonded to
carbon, sulfur, nitrogen or phosphorous, nitrogen is normally
present as nitro, nitroso or amino, normally bonded to carbon,
oxygen, sulfur or phosphorous; sulfur is analogous to oxygen; while
phosphorous is bonded to carbon, sulfur, oxygen or nitrogen,
usually as phosphonate and phosphate mono- or diester. Common
functionalities in forming a covalent bond between the linking
group and the molecule to be conjugated are alkylamine, amidine,
thioamide, ether, urea, thiourea, guanidine, azo, thioether and
carboxylate, sulfonate, and phosphate esters, amides and
thioesters. One specific embodiment of a linking group comprising
heteroatoms is an oxime functionality as mentioned above.
[0044] For the most part, when a linking group has a linking
functionality (functionality for reaction with a moiety) such as,
for example, a non-oxocarbonyl group including nitrogen and sulfur
analogs, a phosphate group, an amino group, alkylating agent such
as halo or tosylalkyl, oxy (hydroxyl or the sulfur analog,
mercapto) oxocarbonyl (e.g., aldehyde or ketone), or active olefin
such as a vinyl sulfone or .alpha.-, .beta.-unsaturated ester,
these functionalities are linked to amine groups, carboxyl groups,
active olefins, alkylating agents, e.g., bromoacetyl. Where an
amine and carboxylic acid or its nitrogen derivative or phosphoric
acid are linked, amides, amidines and phosphoramides are formed.
Where mercaptan and activated olefin are linked, thioethers are
formed. Where a mercaptan and an alkylating agent are linked,
thioethers are formed. Where aldehyde and an amine are linked under
reducing conditions, an alkylamine is formed. Where a ketone or
aldehyde and a hydroxylamine (including derivatives thereof where a
substituent is in place of the hydrogen of the hydroxyl group) are
linked, an oxime functionality (.dbd.N--O--) is formed. Where a
carboxylic acid or phosphate acid and an alcohol are linked, esters
are formed. Various linking groups are well known in the art; see,
for example, Cautrecasas, J. Biol. Chem. (1970) 245:3059.
Sirolimus as a Specific Example
[0045] The following specific description is by way of illustration
of, and not as a limitation on, the scope of the present invention.
Selection of immunosuppressant drugs, and sirolimus in particular,
is also by way of illustration and not limitation as the present
invention has general application to detection of any small
molecule that has spatially separated regions to which antibodies
can be raised and to which such raised antibodies will bind
specifically during an assay for the compound.
[0046] Monoclonal antibodies may be prepared that bind to separate
portions of the sirolimus molecule (FIG. 1). The separate portions
to which the monoclonal antibodies bind may be determined, for
example, by cross-reactivity studies using, for example,
metabolites of sirolimus, or modified sirolimus.
[0047] Referring to FIG. 2, by way of illustration and not
limitation, three potential binding domains on the sirolimus
molecule are indicated as D1, D2 and D3. Binding domain D1 extends
approximately from ring atom 15 to ring atom 21 and includes a
triene moiety from ring atom 17 to ring atom 22. Binding domain D2
extends approximately from the methyl group on atom 11 to the
methoxy group on atom 39. Binding domain D3 extends from the methyl
group on ring atom 25 to atom 41. In one example in accordance with
the principles described herein, a first antibody is prepared that
binds to D1 of the sirolimus molecule. A second antibody is
prepared that binds to sirolimus at a portion of the small molecule
other than a portion to which the first antibody binds, which in
this example is either domain D2 or domain D3 of the sirolimus
molecule. The second antibody is prepared from an immunogen that
comprises a predetermined portion of the small molecule.
[0048] In one example the predetermined portion of the sirolimus
molecule is obtained by modification of the sirolimus molecule to
alter a spatial conformation of the sirolimus molecule. In the
example shown by way of illustration and not limitation (FIG. 3),
the sirolimus molecule is modified in the triene area (D1) to yield
modified sirolimus compound IIA or IIB, or a mixture thereof,
which, when linked to an immunogenic carrier, may be used to
prepare an immunogen to raise antibodies that bind specifically to
D2 or D3 of I.
##STR00001##
wherein:
[0049] R.sup.8 and R.sup.9 are each independently H, non-bulky
organic radical or a bulky organic radical, or are taken together
to form a double bond to O or CH.sub.2;
[0050] R.sup.10 and R.sup.11 are each independently H, non-bulky
organic radical or a bulky organic radical, or are taken together
to form a double bond to O or CH.sub.2;
[0051] R.sup.12 is H, non-bulky hydrocarbyl, or a bulky organic
radical;
[0052] wherein, in one example in accordance with the principles
described herein, at least one of R.sup.8, R.sup.9, R.sup.10,
R.sup.11 or R.sup.12 is a bulky organic radical;
[0053] p is 1, 2 or 3;
[0054] a is 0 or 1; and
[0055] D is N, O, or CH, with the proviso that a is 0 when D is
O.
[0056] The term "hydrocarbyl" refers to an organic radical that
consists solely of carbon and hydrogen. A hydrocarbyl group may be
unsaturated or it may contain one or more carbon-carbon double
bonds or one or more carbon-carbon triple bonds or a mixture
thereof. The term "hydrocarbyl" includes alkyl, alkenyl and
alkynyl.
[0057] The phrase "bulky organic radical" refers to an organic
radical that exhibits a large molecular size for its weight. The
bulky organic radical hinders the ability of a specific binding
member to bind to an area of a molecule that comprises the bulky
organic radical. The phrase "bulky hydrocarbyl" refers to a
hydrocarbyl group that exhibits a large molecular size for its
weight such as, for example, that exhibited by an alkyl group that
is branched or cyclic.
[0058] The phrase "non-bulky organic radical" refers to an organic
radical that does not exhibit a large molecular size for its
weight. The non-bulky organic radical does not hinder to a
significant degree the ability of an antibody to bind to an area of
a molecule that comprises the non-bulky organic radical. The phrase
"non-bulky hydrocarbyl" refers to a hydrocarbyl group that does not
exhibit a large molecular size for its weight such as that
exhibited by a straight chain alkyl group.
[0059] The term "alkyl" refers to an organic radical that consists
solely of single-bonded carbon and hydrogen in either a straight,
branched, or cyclic configuration. The number of carbon atoms in
the organic radical is 1 to 50, or 1 to 40, or 1 to 30, or 1 to 25,
or 1 to 20, or 1 to 15, or 1 to 10, or 1 to 5, or 2 to 50, or 2 to
40, or 2 to 30, or 2 to 25, or 2 to 20, or 2 to 15, or 2 to 10, or
2 to 5, or 5 to 50, or 5 to 40, or 5 to 30, or 5 to 25, or 5 to 20,
or 5 to 15, or 5 to 10. The term "lower alkyl" refers to alkyl
wherein the number of carbon atoms in the organic radical is 1 to
10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to
4, or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or 2 to 8, or 2 to
7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10, or 3 to
9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or 4 to
10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to
10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to
9, or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8, or 8 to
10, or 8 to 9, or 9 to 10.
[0060] Bulky hydrocarbyl includes branched chain hydrocarbyl and
cyclic hydrocarbyl. Bulky branched chain hydrocarbyl has branching
at or near the carbon atom that is attached to another molecule.
Examples of bulky branched chain alkyl include, but are not limited
to, sec-butyl, tert-butyl, triethylmethyl, diethylmethyl,
tripropylmethyl and dipropylmethyl, for example. Cyclic alkyl is
alkyl comprising one or more rings. Examples of cyclic alkyl
include, but are not limited to, cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl and norbornyl, for example.
Examples of non-bulky alkyl include, but are not limited to,
methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl and octyl, for
example.
[0061] The term "alkenyl" refers to a hydrocarbyl group having
hydrocarbon chains of the number of carbon atoms specified above of
either a straight- or branched-configuration and having at least
one carbon-carbon double bond, which may occur at any point along
the hydrocarbon chain, examples of which include, but are not
limited to, ethenyl, propenyl, butenyl, pentenyl, dimethyl
pentenyl, for example.
[0062] The term "alkynyl" refers to a hydrocarbyl group having
hydrocarbon chains of the number of carbon atoms specified above
containing at least one carbon-carbon triple bond, including, but
not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, and
2-butynyl, for example.
[0063] The term "lower hydrocarbyloxy" refers to a hydrocarbyl
group that is an organic radical of the number of carbon atoms
designated above of either a straight, branched or cyclic
configuration wherein the organic radical includes an ether oxygen
for linking a hydrocarbyl group to a parent compound.
[0064] The term "lower alkoxy" refers to an organic radical of the
number of carbon atoms designated above of either a straight,
branched or cyclic configuration wherein the organic radical
includes an ether oxygen for linking an alkyl group to a parent
compound.
[0065] As used herein, the term "aryl" refers to an organic radical
derived from an aromatic hydrocarbon by the removal of one atom and
containing one or more aromatic rings such as, but not limited to,
1 to 5 aromatic rings, or 1 to 4 aromatic rings, or 1 to 3 aromatic
rings, or 1 to 2 aromatic rings, or 2 to 4 aromatic rings, or 2 to
3 aromatic rings, for example. Examples of aryl include, but are
not limited to, phenyl (from benzene), naphthyl (from naphthalene),
and anthracyl (from anthracene), for example. The aryl radical may
be substituted or unsubstituted. "Substituted aryl" refers to aryl
groups that comprise one or more substituents such as, but not
limited to, a bulky hydrocarbyl, a non-bulky hydrocarbyl, a
functional group (e.g., chloro, bromo, iodo, fluoro, nitro and
sulfone), for example.
[0066] As used herein, "arylhydrocarbyl" refers to an organic
radical having a lower hydrocarbyl group to which is attached an
aryl group. As used herein, "aralkyl" refers to an organic radical
having a lower alkyl group to which is attached an aryl group such
as, but not limited to, benzyl, phenethyl, 3-phenylpropyl and
1-naphthylethyl, for example.
[0067] An immunogenic carrier may be linked to IIA or IIB or both
through a substituent at ring atom 26 or a substituent at ring atom
32, or both, employing a linking group such as that described
above. In one example, an oxime functionality may be formed from
the carbonyl group at ring atom 26 or the carbonyl at ring atom 32,
or both. The linking group for linking to an immunogenic carrier
may be introduced into I either prior to or after modification of I
at the triene functionality. In another example, one of R.sup.8,
R.sup.9, R.sup.10, R.sup.11 or R.sup.12 can be modified to
incorporate a linking group for linking an immunogenic carrier to
the modified sirolimus molecule.
[0068] In one example the predetermined portion of the sirolimus
molecule is obtained by modification of the sirolimus molecule to
alter a spatial conformation of the sirolimus molecule. In the
example shown by way of illustration and not limitation, the
sirolimus molecule is modified in the triene area (D1) with
4-phenyl-1,2,4-triazole-3,5-dione (PTAD) to yield modified
sirolimus compound IIIA or IIIB (FIG. 4), or a mixture thereof,
which, when linked to an immunogenic carrier, may be used to
prepare an immunogen to raise antibodies that bind specifically to
D2 or D3 of sirolimus (I).
##STR00002##
[0069] Similar to the discussion above with respect to IIA and IIB,
an immunogenic carrier may be linked to IIIA or IIB or both through
a substituent at ring atom 26 or a substituent at ring atom 32, or
both, employing a linking group such as that described above. In
one example, an oxime functionality may be formed from the carbonyl
group at ring atom 26 or the carbonyl group at ring atom 32, or
both. The linking group for linking to an immunogenic carrier may
be introduced into I either prior to or after modification of I at
the triene functionality. In another example, one of R.sup.8,
R.sup.9, R.sup.10, R.sup.11 or R.sup.12 is a functionality that may
be modified to incorporate a linking group for linking an
immunogenic carrier to the modified sirolimus molecule.
Preparation of Compounds
[0070] Examples of methods of preparing compounds for preparation
of antibodies in accordance with the principles described herein
are described, by way of illustration and not limitation, with
reference to FIG. 3. Other approaches may be employed to form the
compounds consistent with the principles described herein.
Referring to FIG. 3, sirolimus (I) is combined with cyclic reagent
IV under conditions for carrying out a Diels-Alder addition
reaction. The conditions include using an anhydrous non-polar
organic medium such as, but not limited to, methylene chloride,
toluene, hexane, nitrobenzene and carbon tetrachloride, for
example; or a polar organic medium such as, but not limited to,
ethanol, acetonitrile and phosphonium tosylates, and aqueous
mixtures thereof, for example. The reaction is conducted at a
temperature of about 15.degree. C. to about 40.degree. C., or about
20.degree. C. to about 30.degree. C., or about room temperature
(about 22.degree. C. to about 24.degree. C.) for a period of about
15 minutes to about 45 minutes or about 30 minutes and then at a
temperature of about 50.degree. C. to about 100.degree. C., or
about 50.degree. C. to about 80.degree. C., or about the reflux
temperature of the non-polar organic solvent for a period of about
30 minutes to about 90 minutes, or about 45 minutes to about 75
minutes, or about 60 minutes. The resulting product is purified by
one or more techniques such as, but not limited to, evaporation,
recrystallization, and chromatography such as, for example, thin
layer chromatography (TLC), high performance liquid chromatography
(HPLC), reverse phase liquid chromatography (RPLC), high turbulence
liquid chromatography (HTLC), gas chromatography, for example. The
product is a mixture of two isomers represented by compounds IIA
and IIB in FIG. 3, which may be employed together or may be
separated by one or more techniques for separating positional
isomers such as, but not limited to, liquid chromatography (TLC,
HPLC, RPLC, HTLC), and gas chromatography, for example.
[0071] A particular example of a method of preparing compounds in
accordance with the principles described herein is described, by
way of illustration and not limitation, with reference to FIG. 4.
Other approaches may be employed to form the compounds consistent
with the principles described herein. Referring to FIG. 4,
sirolimus (I) is combined with cyclic reagent PTAD under conditions
for carrying out a Diels-Alder addition reaction. The conditions
include using an anhydrous non-polar organic medium such as, but
not limited to, methylene chloride, toluene, hexane, nitrobenzene
and carbon tetrachloride, for example; or a polar organic medium
such as, but not limited to, ethanol, acetonitrile and phosphonium
tosylates, and aqueous mixtures thereof, for example. The reaction
is conducted at a temperature of about 15.degree. C. to about
40.degree. C., or about 20.degree. C. to about 30.degree. C., or
about room temperature (about 22.degree. C. to about 24.degree. C.)
for a period of about 15 minutes to about 45 minutes or about 30
minutes and then at a temperature of about 50.degree. C. to about
100.degree. C., or about 50.degree. C. to about 80.degree. C., or
about the reflux temperature of the non-polar organic solvent for a
period of about 30 minutes to about 90 minutes, or about 45 minutes
to about 75 minutes, or about 60 minutes. The resulting product is
purified by one or more techniques such as, but not limited to,
evaporation, liquid chromatography such as, for example, TLC, HPLC,
RPLC, and HTLC, and gas chromatography, for example. The product is
a mixture of two isomers represented by compounds IIIA and IIIB in
FIG. 4, which may be employed together or may be separated by one
or more techniques for separating positional isomers such as, but
not limited to, liquid chromatography (TLC, HPLC, RPLC, HTLC) and
gas chromatography, for example.
[0072] An example of the preparation of an immunogen in accordance
with the principles described herein, by way of illustration and
not limitation, is set forth in FIGS. 5 and 6. Referring to FIG. 5,
sirolimus (I) is reacted with aminooxyacetic acid to form a mixture
of oximes of the Formula IVa (representing formation of an oxime at
C-26) and IVb (representing formation of an oxime at C-32). The
reaction is carried out in an organic solvent such as, for example,
an alcohol (e.g., methanol or ethanol), under conditions for
forming an oxime. In some examples the temperature during the
reaction is about 10.degree. C. to about 30.degree. C., or about
15.degree. C. to about 25.degree. C. The time period of the
reaction is about 1 hour to about 30 hours, or about 2 hours to
about 24 hours. Compounds IVa and IVb may be separated or the
mixture of compounds IVa and IVb may be employed in the next step
of the preparation of an immunogen. Separation of IVa and IVb may
be carried out by, but not limited to, chromatography (TLC, HPLC,
RPLC, HTLC) and gas chromatography, for example.
[0073] FIG. 6 depicts, by way of illustration and not limitation,
formation of an immunogen from compound IVa. A poly(amino)acid
immunogenic carrier (R.sup.5 precursor) is combined with compound
of IVa to form a compound of the formula Va. The reaction is
carried out in an aqueous buffered medium at a pH of about 5.0 to
about 7.0, or about 5.5 to about 6.5, or about 6. An activation
agent or coupling for facilitating the reaction of the carboxylic
acid functionality of Va with an amine group of the R.sup.5
precursor is included in the reaction medium. Such coupling agents
include, but are not limited to,
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC),
N-hydroxysuccinimide (NHS), or
N,N,N',N'-tetramethyl-O--(N-succinimidyl)uronium tetrafluoroborate,
or combinations of two or more of the above. The reaction is
carried out under conditions for forming an amide. In some
examples, the reaction medium is an aqueous medium, which may be
solely water or may include from 0.1 to about 40 volume percent of
a cosolvent, which may be a polar organic solvent such as for
example, an amine (e.g., dimethylformamide (DMF)); an alcohol
(e.g., ethanol); or an ether (e.g., furan), for example. In some
examples the temperature during the reaction is about 15.degree. C.
to about 25.degree. C. The time period of the reaction is about 3
hours to about 24 hours, or about 4 hours to about 20 hours, or
about 4 hours to about 10 hours, for example. In some examples, by
way of illustration and not limitation, the R.sup.5 precursor is a
protein such as BSA or KLH, for example. An immunogen may also be
prepared from compound IVb in a similar manner to that described
above for the preparation of immunogen Va. As mentioned above, a
mixture of compounds IVa and IVb may also be used to prepare a
mixture of immunogens.
[0074] FIG. 7 depicts a reaction scheme for the preparation of
oximes VIa and VIb from compounds IIIa and IIIb. The reaction is
carried out in a manner similar to that described above for FIG. 5.
Compound VIa is separated from the mixture of VIa and VIb and is
treated (FIG. 8) to prepare immunogens VIIa and VIIb in a manner
similar to that described above for the preparation of immunogen Va
as discussed above with regard to FIG. 6.
[0075] Another example of the preparation of immunogens in
accordance with the principles described herein is set forth in
FIG. 9. Sirolimus (I) is reacted with a carboxyl derivative of PTAD
to give a mixture of compounds VIIIa and VIIIb. The conditions of
the reaction are similar to those described above for the
preparation of PTAD adducts of sirolimus (I), the details of which
are set forth above with reference to FIG. 4. The mixture of
compounds VIIIa and VIIIb is treated (FIG. 9) to prepare immunogens
IXa and IXb in a manner similar to that described above for the
preparation of immunogen Va as discussed above with regard to FIG.
6.
Preparation of Antibodies for Sandwich Assay for Sirolimus
[0076] In one example, by way of illustration and not limitation, a
first monoclonal antibody is prepared that binds to a portion of
sirolimus represented by domain region D1. This first monoclonal
antibody may be prepared using compound Va (R.sup.5 is BSA), for
example, as an immunogen in the methods of antibody production
described in detail above. A second monoclonal antibody is prepared
that binds to a portion of sirolimus represented by domain region
D2. The second monoclonal antibody may be prepared using, for
example, compound VIIa or VIIb (R.sup.5 is BSA in both) or a
mixture of both as an immunogen for antibody preparation in methods
described above. Examination of the sirolimus structure by
three-dimensional analysis reveals the conformation of regions D1
and D2.
[0077] In another example, by way of illustration and not
limitation, a first monoclonal antibody is prepared that binds to a
portion of sirolimus represented by domain region D1. This first
monoclonal antibody may be prepared using compound Va (R.sup.5 is
KLH), for example, as an immunogen in the methods of antibody
production described in detail above. A second monoclonal antibody
is prepared that binds to a portion of sirolimus represented by
region D3. The second monoclonal antibody may be prepared using,
for example, compound IXa or IXb (R.sup.5 is KLH in both) or a
mixture of both as an immunogen for antibody preparation in methods
described above. Examination of the sirolimus structure by
three-dimensional analysis reveals the conformation of regions D1
and D3.
General Description of Assays for a Small Molecule
[0078] As mentioned above, examples in accordance with the
principles described herein enable a sandwich assay for the
determination of a small molecule in a sample suspected of
containing the small molecule. In the discussion below, an
immunosuppressant drug is used as an example, by way of
illustration and not limitation, of a small molecule as defined
herein. In the sandwich assay, two monoclonal antibodies are
employed, each of which bind at the same time to separate regions
of the immunosuppressant drug molecule to form an immunocomplex.
Detection of the immunocomplex permits the determination of the
immunosuppressant drug in the sample.
[0079] The sample to be tested is usually a biological sample. The
phrase "biological sample" refers to any biological material such
as, for example, body fluid, body tissue, body compounds and
culture media. The sample may be a solid, semi-solid or a fluid (a
liquid or a gas) from any source. In some embodiments the sample
may be a body excretion, a body aspirant, a body excisant or a body
extractant. The body is usually that of a mammal and in some
embodiments the body is a human body. Body excretions are those
substances that are excreted from a body (although they also may be
obtained by excision or extraction) such as, for example, urine,
feces, stool, vaginal mucus, semen, tears, breath, sweat, blister
fluid and inflammatory exudates. Body excisants are those materials
that are excised from a body such as, for example, skin, hair and
tissue samples including biopsies from organs and other body parts.
Body aspirants are those materials that are aspirated from a body
such as, for example, mucus, saliva and sputum. Body extractants
are those materials that are extracted from a body such as, for
example, whole blood, plasma, serum, spinal fluid, cerebral spinal
fluid, lymphatic fluid, synovial fluid and peritoneal fluid. In
some examples the sample is whole blood, plasma or serum.
[0080] Prior to the assay, or in some instances during the assay,
the sample may be subjected to one or more pretreatments to lyse
cells and/or to release immunosuppressant drug from endogeneous
binding substances. Lysing cells may be accomplished by use of a
hemolytic agent, which is a compound or mixture of compounds that
disrupts the integrity of the membranes of red blood cells thereby
releasing intracellular contents of the cells. Hemolytic agents
include, but are not limited to, non-ionic detergents, anionic
detergents, amphoteric detergents, low ionic strength aqueous
solutions (hypotonic solutions), bacterial agents, and antibodies
that cause complement dependent lysis, for example.
[0081] Non-ionic detergents that may be employed as the hemolytic
agent include both synthetic detergents and natural detergents.
Examples of synthetic detergents include TRITON.TM. X-100,
TRITON.TM. N-101, TRITON.TM. X-114, TRITON.TM. X-405, TRITON.TM.
SP-135, TWEEN.RTM. 20 (polyoxyethylene (20) sorbitan monolaurate),
TWEEN.RTM. 80 (polyoxyethylene (20) sorbitan monooleate),
DOWFAX.RTM., ZONYL.RTM., pentaerythrityl palmitate, ADOGEN.RTM.
464, ALKANOL.RTM. 6112 surfactant, allyl alcohol
1,2-butoxylate-block-ethoxylate HLB 6, BRIJ.RTM., ethylenediamine
tetrakis(ethoxylate-block-propoxylate) tetrol, IGEPAL.RTM.,
MERPOL.RTM., poly(ethylene glycol),
2-[ethylKheptadecafluorooctyl)sulfonyl]amino] ethyl ether,
polyethylene-block-poly(ethylene glycol), polyoxyethylene sorbitan
tetraoleate, polyoxyethylene sorbitol hexaoleate, TERGITOL.RTM.
NP-9, GAFAC.RTM. (RHODAFAC.RTM., an alkyl polyoxyethylene glycol
phosphate ester such as, for example,
alpha-dodecyl-omega-hydroxypoly(oxy-1,2-ethanediyl) phosphate), and
EP110.RTM. and the like. Naturally-occurring detergents that may be
employed as the hemolytic agent include, for example, saponins,
sodium or potassium neutralized fatty acid, neutralized
phospholipids, diacylglycerol, neutralized phosphatidyl serine,
phosphatidate, neutralized phosphatidyl ethanoliamin, phosphatidyl
choline, phosphatidyl inositol, phosphatidylcholine, bile salt,
unesterified cholesterol, neutralized sphingosine, ceramide, and
the like. Combinations of one or more synthetic detergents or one
or more naturally occurring detergents and combinations of
synthetic detergents and naturally occurring detergents may also be
employed.
[0082] The nature and amount or concentration of hemolytic agent
employed depends on one or more of the nature of the sample, the
nature of the immunosuppressant drug, the nature of the rest of the
reagent components, and the reaction conditions, for example. The
amount of the hemolytic agent is at least sufficient to cause lysis
of red blood cells to release contents of the cells. In some
examples the amount of the hemolytic agent is about 0.0001% to
about 0.5%, about 0.001% to about 0.4%, about 0.01% to about 0.3%,
about 0.01% to about 0.2%, about 0.1% to about 0.3%, about 0.2% to
about 0.5%, or about 0.1% to about 0.2%, for example (percent is
weight/volume).
[0083] The releasing agent is a compound or mixture of compounds
that displaces the immunosuppressant drug from endogenous binding
moieties. The releasing agent can, and does in many instances,
displace metabolites of the immunosuppressant drug from endogenous
binding moieties. In many examples the releasing agent has high
binding affinity to the endogenous binding proteins so that it
readily displaces the immunosuppressant drug, and its metabolites
where desired, from endogenous binding proteins. In addition, the
releasing agent does not bind to any significant degree to a
monoclonal antibody for the drug that is used in an assay. By the
phrase "does not bind to any significant degree" is meant that the
extent of binding should be low enough so that an accurate assay
for the drug may be carried out. The releasing agent, therefore,
may be any moiety, either a single compound or a mixture of
compounds, which accomplishes the desired result of displacement
with no significant binding to an assay antibody.
[0084] In some examples the releasing agent is an analog, including
structural analogs, of the immunosuppressant drug. An
immunosuppressant drug analog is a modified drug that can displace
the analogous immunosuppressant drug from a binding protein but
does not compete to any substantial degree for a monoclonal
antibody for the immunosuppressant drug. The modification provides
means to join an immunosuppressant drug analog to another molecule.
In an example, the immunosuppressant drug analog may be, for
example, the immunosuppressant drug conjugated to another molecule
through a linking group. For immunosuppressant drugs that comprise
a hydroxy or carboxylic acid functionality, the releasing agent may
be an ester of the immunosuppressant drug, which has a high binding
affinity for endogenous binding proteins relative to the
immunosuppressant drug to be detected and which has no significant
binding affinity for an antibody for the immunosuppressant drug.
For example, in a determination for tacrolimus, an ester of
tacrolimus may be employed as the releasing agent so long as it
meets the above requirements. A structural analog is a moiety that
has the same or similar structural or spatial characteristics as
the immunosuppressant drug such that the structural analog
accomplishes the same or similar result as the analog of the
immunosuppressant drug. The structural analog may be, for example,
another compound that is related to the immunosuppressant drug. For
example, in a determination for tacrolimus, an ester of sirolimus
may be employed as the releasing agent. The ester may be, for
example, a carbamate, a carbonate, an ester of a C.sub.1 to C.sub.6
carboxylic acid, and the like. See, for example, U.S. Pat. No.
7,186,518, the relevant disclosure of which is incorporated herein
by reference. Other examples of releasing agents include
[Thr.sub.2, Leu.sub.5, D-Hiv.sub.8, Leu.sub.10]-cyclosporin A for
cyclosporin A, FK506 for sirolimus, sirolimus for FK506, and the
like. See, for example, U.S. Pat. No. 6,187,547, the relevant
disclosure of which is incorporated herein by reference.
[0085] The concentration of the releasing agent in the medium is
that sufficient to achieve the desired result of displacing the
immunosuppressant drug, and in some instances the metabolites of
the immunosuppressant drug, from endogenous binding moieties to
render the drug and metabolites accessible for binding to an
antibody for the drug as discussed above. The amount or
concentration of the releasing agent employed depends on one or
more of the nature of the sample, the nature of the
immunosuppressant drug, the nature of the drug metabolites, the
nature of other reagent components, and the reaction conditions,
for example. In some embodiments the amount of the releasing agent
is about 0.000001% to about 0.5%, about 0.0001% to about 0.4%,
about 0.001% to about 0.3%, about 0.01% to about 0.2%, about 0.1%
to about 0.3%, about 0.2% to about 0.5%, about 0.1% to about 0.2%,
and so forth (percent is weight/volume).
[0086] The assay is an immunoassay, which may be performed either
without separation (homogeneous) or with separation (heterogeneous)
of any of the assay components or products. The homogeneous or
heterogeneous assays are carried out in an aqueous buffered medium
at a moderate pH, generally that which provides optimum assay
sensitivity. The aqueous medium may be solely water or may include
from 0.1 to about 40 volume percent of a cosolvent. The pH for the
medium will usually be in the range of about 4 to about 11, or in
the range of about 5 to about 10, or in the range of about 6.5 to
about 9.5. The pH will usually be a compromise between optimum
binding of the monoclonal antibodies and the immunosuppressant
drug, and the pH optimum for other reagents of the assay such as
members of the signal producing system, for example.
[0087] Various buffers may be used to achieve the desired pH and
maintain the pH during the determination. Illustrative buffers
include borate, phosphate, carbonate, tris, barbital and the like.
The particular buffer employed is not critical to this invention,
but in an individual assay one or another buffer may be preferred.
Various ancillary materials may be employed in the above methods.
For example, in addition to buffers the medium may comprise
stabilizers for the medium and for the reagents employed.
Frequently, in addition to these additives, proteins may be
included, such as albumins; organic solvents such as formamide;
quaternary ammonium salts; polyanions such as dextran sulfate;
surfactants, particularly non-ionic surfactants; binding enhancers,
e.g., polyalkylene glycols; for example.
[0088] One or more incubation periods may be applied to the medium
at one or more intervals including any intervals between additions
of various reagents mentioned above. The medium is usually
incubated at a temperature and for a time sufficient for binding of
various components of the reagents to occur. Moderate temperatures
are normally employed for carrying out the method and usually
constant temperature, preferably, room temperature, during the
period of the measurement. Incubation temperatures range from about
5.degree. to about 99.degree. C., or about 15.degree. C. to about
70.degree. C., or about 20.degree. C. to about 45.degree. C. The
time period for the incubation is about 0.2 seconds to about 6
hours, or about 2 seconds to about 1 hour, or about 1 to about 5
minutes. The time period depends on the temperature of the medium
and the rate of binding of the various reagents, which is
determined by the association rate constant, the concentration, the
binding constant and dissociation rate constant. Temperatures
during measurements range from about 10.degree. C. to about
50.degree. C., or from about 15.degree. C. to about 40.degree.
C.
[0089] The concentration of immunosuppressant drug analyte that may
be assayed generally varies from about 10.sup.-5 to about
10.sup.-17 M, or from about 10.sup.-6 to about 10.sup.-14 M.
Considerations, such as whether the assay is qualitative,
semi-quantitative or quantitative (relative to the amount of
analyte present in the sample), the particular detection technique
and the concentration of the analyte normally determine the
concentrations of the various reagents.
[0090] The concentrations of the various reagents in the assay
medium will generally be determined by the concentration range of
interest of the immunosuppressant drug analyte. However, the final
concentration of each of the reagents is normally determined
empirically to optimize the sensitivity of the assay over the
range. That is, a variation in concentration of analyte that is of
significance should provide an accurately measurable signal
difference. Considerations such as the nature of a signal producing
system and the nature of the immunosuppressant analyte normally
determine the concentrations of the various reagents.
[0091] While the order of addition may be varied widely, there will
be certain preferences depending on the nature of the assay. The
simplest order of addition is to add all the materials
simultaneously and determine the effect that the assay medium has
on the signal as in a homogeneous assay. Alternatively, the
reagents can be combined sequentially. Optionally, an incubation
step may be involved subsequent to each addition as discussed
above.
[0092] In the assays discussed above, one or more labels are
employed wherein the label is usually part of a signal producing
system ("sps"). The nature of the label is dependent on the
particular assay format. An sps usually includes one or more
components, at least one component being a detectable label, which
generates a detectable signal that relates to the amount of bound
and/or unbound label, i.e. the amount of label bound or not bound
to the immunosuppressant drug being detected or to an agent that
reflects the amount of the immunosuppressant drug to be detected.
The label is any molecule that produces or can be induced to
produce a signal, and may be, for example, a fluorescer, a
radiolabel, an enzyme, a chemiluminescer or a photosensitizer.
Thus, the signal is detected and/or measured by detecting enzyme
activity, luminescence, light absorbance or radioactivity, as the
case may be.
[0093] Suitable labels include, by way of illustration and not
limitation, enzymes such as .beta.-galactosidase, alkaline
phosphatase, glucose-6-phosphate dehydrogenase ("G6PDH") and
horseradish peroxidase; ribozyme; a substrate for a replicase such
as QB replicase; promoters; dyes; fluorescers, such as fluorescein,
isothiocyanate, rhodamine compounds, phycoerythrin, phycocyanin,
allophycocyanin, o-phthaldehyde, and fluorescamine; complexes such
as those prepared from CdSe and ZnS present in semiconductor
nanocrystals known as Quantum dots; chemiluminescers such as
isoluminol; sensitizers; coenzymes; enzyme substrates; radiolabels
such as .sup.125I, .sup.131I, .sup.14C, .sup.3H, .sup.57 Co and
.sup.75Se; particles such as latex particles, carbon particles,
metal particles including magnetic particles, e.g., chrome
particles, and the like; metal sol; crystallite; liposomes; cells,
etc., which may be further labeled with a dye, catalyst or other
detectable group. Suitable enzymes and coenzymes are disclosed in
Litman, et al., U.S. Pat. No. 4,275,149, columns 19-28, and
Boguslaski, et al., U.S. Pat. No. 4,318,980, columns 10-14;
suitable fluorescers and chemiluminescers are disclosed in Litman,
et al., U.S. Pat. No. 4,275,149, at columns 30 and 31; which are
incorporated herein by reference.
[0094] The label can directly produce a signal and, therefore,
additional components are not required to produce a signal.
Numerous organic molecules, for example fluorescers, are able to
absorb ultraviolet and visible light, where the light absorption
transfers energy to these molecules and elevates them to an excited
energy state. This absorbed energy is then dissipated by emission
of light at a second wavelength. Other labels that directly produce
a signal include radioactive isotopes and dyes.
[0095] Alternately, the label may need other components to produce
a signal, and the signal producing system would then include all
the components required to produce a measurable signal. Such other
components may include substrates, coenzymes, enhancers, additional
enzymes, substances that react with enzymic products, catalysts,
activators, cofactors, inhibitors, scavengers, metal ions, and a
specific binding substance required for binding of signal
generating substances. A detailed discussion of suitable signal
producing systems can be found in Ullman, et al., U.S. Pat. No.
5,185,243, columns 11-13, incorporated herein by reference.
[0096] The label or other sps members or one or more of the
monoclonal antibodies can be bound to a support. A monoclonal
antibody may be bound to a solid support in any manner known in the
art, provided only that the binding does not substantially
interfere with the ability to bind with a region of the
immunosuppressant drug. In some examples, the label or other sps
member or the monoclonal antibody may be coated or covalently bound
directly to the solid phase or may have layers of one or more
carrier molecules such as poly(amino acids) including proteins such
as serum albumins or immunoglobulins, or polysaccharides
(carbohydrates) such as, for example, dextran or dextran
derivatives. Linking groups may also be used to covalently couple
the solid support and the moiety to be coupled. The linking group
may be one as described above for the linking of immunogen to an
immunosuppressant drug molecule. Other methods of binding to a
support may also be employed. For instance, a solid support may
have a coating of a binder for a small molecule such as, for
example, avidin or an antibody, where a small molecule such as,
e.g., biotin or a hapten, can be bound to the moiety to be coupled
or vice versa. The binding of components to the surface of a
support may be direct or indirect, covalent or non-covalent and can
be accomplished by well-known techniques, commonly available in the
literature. See, for example, "Immobilized Enzymes," Ichiro
Chibata, Halsted Press, New York (1978) and Cautrecasas, J. Biol.
Chem., 245:3059 (1970).
[0097] The support may be comprised of an organic or inorganic,
solid or fluid, water insoluble material, which may be transparent
or partially transparent. The support can have any of a number of
shapes, such as particle, including bead, film, membrane, tube,
well, strip, rod, planar surfaces such as, e.g., plate, and
DENDRIMERS, for example. Depending on the type of assay, the
support may or may not be suspendable in the medium in which it is
employed. Examples, by way of illustration and not limitation, of
suspendable supports are polymeric materials such as latex, lipid
bilayers or liposomes, oil droplets, cells and hydrogels, and
magnetic particles, for example. Other support compositions include
polymers, such as nitrocellulose, cellulose acetate, poly (vinyl
chloride), polyacrylamide, polyacrylate, polyethylene,
polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate,
poly(ethylene terephthalate), nylon, poly(vinyl butyrate), for
example; either used by themselves or in conjunction with other
materials.
[0098] The support may be a particle. The particles should have an
average diameter of at least about 0.02 microns and not more than
about 100 microns. In some embodiments, the particles have an
average diameter from about 0.05 microns to about 20 microns, or
from about 0.3 microns to about 10 microns. The particle may be
organic or inorganic, swellable or non-swellable, porous or
non-porous, preferably of a density approximating water, generally
from about 0.7 g/mL to about 1.5 g/mL, and composed of material
that can be transparent, partially transparent, or opaque. The
particles can be biological materials such as cells and
microorganisms, e.g., erythrocytes, leukocytes, lymphocytes,
hybridomas, streptococcus, Staphylococcus aureus, and E. coli,
viruses, for example. The particles can also be particles comprised
of organic and inorganic polymers, liposomes, latex particles,
magnetic or non-magnetic particles, phospholipid vesicles,
chylomicrons, lipoproteins, and the like. In some examples, the
particles are chrome particles or latex particles.
[0099] The polymer particles can be formed of addition or
condensation polymers. The particles will be readily dispersible in
an aqueous medium and can be adsorptive or functionalizable so as
to permit conjugation to a monoclonal antibody for an
immunosuppressant drug, either directly or indirectly through a
linking group. The linking group may be one as described above for
the linking of immunogens to an immunosuppressant drug molecule.
The particles can also be derived from naturally occurring
materials, naturally occurring materials that are synthetically
modified, and synthetic materials. Among organic polymers of
particular interest are polysaccharides, particularly cross-linked
polysaccharides, such a agarose, which is available as Sepharose,
dextran, available as Sephadex and Sephacryl, cellulose, starch,
and the like; addition polymers, such as polystyrene, polyvinyl
alcohol, homopolymers and copolymers of derivatives of acrylate and
methacrylate, particularly esters and amides having free hydroxyl
functionalities, and the like.
[0100] The label and/or other sps member may be bound to one or
both of the two different monoclonal antibodies. Bonding of the
label to the sbp member may be accomplished by chemical reactions
that result in replacing a hydrogen atom of the label with a bond
to the monoclonal antibody or may include a linking group between
the label and the monoclonal antibody. The linking group may be one
as described above for the linking of immunogens to an
immunosuppressant drug molecule. Other sps members may also be
bound covalently to the monoclonal antibodies. For example, two sps
members such as a fluorescer and quencher can each be bound,
respectively, to the monoclonal antibodies where the fluorescer is
bound to one of the monoclonal antibodies and a quencher is bound
to the other of the monoclonal antibodies. When the two different
monoclonal antibodies bind to the immunosuppressasnt drug, the
formation of a sandwich complex brings the fluorescer and quencher
in close proximity, thus permitting the quencher to interact with
the fluorescer to produce a signal. Methods of conjugation are well
known in the art. See, for example, Rubenstein, et al., U.S. Pat.
No. 3,817,837, incorporated herein by reference.
[0101] Enzymes of particular interest as label proteins are redox
enzymes, particularly dehydrogenases such as glucose-6-phosphate
dehydrogenase, lactate dehydrogenase, etc., and enzymes that
involve the production of hydrogen peroxide and the use of the
hydrogen peroxide to oxidize a dye precursor to a dye. Particular
combinations include, but are not limited to, saccharide oxidases,
e.g., glucose and galactose oxidase, or heterocyclic oxidases, such
as uricase and xanthine oxidase, coupled with an enzyme which
employs the hydrogen peroxide to oxidize a dye precursor, that is,
a peroxidase such as horse radish peroxidase, lactoperoxidase, or
microperoxidase. Additional enzyme combinations are known in the
art. When a single enzyme is used as a label, other enzymes may
find use such as hydrolases, transferases, and oxidoreductases,
preferably hydrolases such as alkaline phosphatase and
beta-galactosidase. Alternatively, luciferases may be used such as
firefly luciferase and bacterial luciferase.
[0102] Illustrative co-enzymes that find use include NAD[H],
NADP[H], pyridoxal phosphate, FAD[H], FMN[H], etc., usually
coenzymes involving cycling reactions. See, for example, U.S. Pat.
No. 4,318,980, the disclosure of which is incorporated herein by
reference.
[0103] Activation of a signal producing system depends on the
nature of the signal producing system members. For those members of
a signal producing system that are activated with light, the member
is irradiated with light. For members of signal producing systems
that are on the surface of a particle, addition of a base may
result in activation. Other activation methods will be suggested to
those skilled in the art in view of the disclosures herein. For
some signal producing systems, no agent for activation is necessary
such as those systems that involve a label that is a radioactive
label, an enzyme, and so forth. For enzyme systems addition of a
substrate and/or a cofactor may be necessary.
[0104] The examination for presence and amount of the signal also
includes the detection of the signal, which is generally merely a
step in which the signal is read. The signal is normally read using
an instrument, the nature of which depends on the nature of the
signal. The instrument may be a spectrophotometer, fluorometer,
absorption spectrometer, luminometer, chemiluminometer,
actinometer, photographic instrument, and the like. The presence
and amount of signal detected is related to the presence and amount
of the sirolimus compound present in a sample. Temperatures during
measurements may range from about 10.degree. to about 70.degree.
C., or from about 20.degree. to about 45.degree. C., or from about
20.degree. to about 25.degree. C. In one approach standard curves
are formed using known concentrations of the analytes to be
screened. As discussed above, calibrators and other controls may
also be used.
[0105] The phrase "measuring the amount of an immunosuppressant
drug" refers to the quantitative, semi-quantitative and qualitative
determination of the immunosuppressant drug. Methods that are
quantitative, semi-quantitative and qualitative, as well as all
other methods for determining the immunosuppressant drug, are
considered to be methods of measuring the amount of the
immunosuppressant drug. For example, a method, which merely detects
the presence or absence of the immunosuppressant drug in a sample
suspected of containing the immunosuppressant drug, is considered
to be included within the scope of the present disclosure. The
terms "detecting" and "determining," as well as other common
synonyms for measuring, are contemplated within the scope of the
present disclosure.
[0106] In one example in accordance with the principles described
herein, one of the monoclonal antibodies specific for a region of
an immunosuppressant drug is bound to a support and the other of
the monoclonal antibodies that is specific for a region of the
immunosuppressant drug that is spatially separated from the region
of the immunosuppressant drug to which the other monoclonal
antibodies binds is bound to an sps member such as, for example, a
label. The sample suspected of containing the immunosuppressant
drug is combined in a suitable medium with the two conjugated
monoclonal antibodies and the medium is incubated. Then, the medium
is examined for the one or both of the presence and amount of an
immunocomplex formed by the two different monoclonal antibodies and
the immunosuppressant drug from the sample. The support may or may
not be separated from the medium prior to the examination. The
presence and/or amount of the immunocomplex is determined by
determining the presence and/or amount of the label in the medium
or on the support.
[0107] In one particular example, a capture assay is employed. In
this assay format, one monoclonal antibody is covalently bound to a
magnetic particle such as, for example, a chrome (chromium dioxide)
particle. The sample is incubated with these particles to allow the
immunosuppressant drug in the sample to bind to the monoclonal
antibody on the magnetic particle. Subsequently, a second
monoclonal antibody conjugated to an enzyme such as, for example,
.beta.-galactosidase, is incubated with the magnetic particles.
After application of a magnet and washing of the magnetic
particles, the amount of enzyme that is bound to the magnetic
particles is measured and is directly related to the presence
and/or amount of the immunosuppressant drug in the sample. In this
approach substrate of the reporter enzyme is added to the final
reaction container, and the enzyme activity is measured
spectrophotometrically as a change in absorbance over time.
[0108] In an alternative approach, the magnetic particle reagent is
added in an excess amount, i.e., an amount greater than that
required to bind all of the immunosuppressant drug that might be
present in the sample. Then, a magnet is applied to separate the
magnetic particles from the medium and the magnetic particles are
washed and resuspended in assay medium. The enzyme conjugated to
the second monoclonal antibody is added and the medium is incubated
followed by signal determination as described above.
[0109] In another example, by way of illustration and not
limitation, chemiluminescent particles are employed, which comprise
the chemiluminescent compound associated therewith such as by
incorporation therein or attachment thereto. One of the monoclonal
antibodies for the immunosuppressant drug is bound to the particles
such as through the intermediacy of a polysaccharide coating the
particles. The other monoclonal antibody that binds to the
immunosuppressant drug is part of a biotin conjugate. Streptavidin
is conjugated to a second set of particles having a photosensitizer
associated therewith. The chemiluminescent particles are mixed with
a sample suspected of containing the immunosuppressant drug and the
photosensitizer particles. The reaction medium is incubated to
allow the particles to bind to the immunosuppressant drug by virtue
of the binding of the monoclonal antibodies to the
immunosuppressant drug. Then, the medium is irradiated with light
to excite the photosensitizer, which is capable in its excited
state of activating oxygen to a singlet state. Because the
chemiluminescent compound of one of the sets of particles is now in
close proximity to the photosensitizer by virtue of the presence of
the immunosuppressant drug, it is activated by singlet oxygen and
emits luminescence. The medium is then examined for the presence
and/or the amount of luminescence or light emitted, the presence
thereof being related to the presence and/or amount of the
immunosuppressant drug in a sample.
Kits for Conducting Assays
[0110] The reagents for conducting a particular assay may be
present in a kit useful for conveniently performing an assay for
the determination of a small molecule such as, for example, an
immunosuppressant drug analyte. In one example, a kit comprises in
packaged combination reagents for analyzing for the analyte, the
nature of which depend upon the particular assay format. The
reagents may include, for example, one or more monoclonal
antibodies in accordance with the principles described herein,
which may be conjugated to a label or a support. The reagents may
each be in separate containers or various reagents can be combined
in one or more containers depending on the cross-reactivity and
stability of the reagents. The kit can further include other
separately packaged reagents for conducting an assay such as
additional binding members and ancillary reagents.
[0111] The relative amounts of the various reagents in the kits can
be varied widely to provide for concentrations of the reagents that
substantially optimize the reactions that need to occur during the
present method and further to optimize substantially the
sensitivity of the assay. Under appropriate circumstances one or
more of the reagents in the kit can be provided as a dry powder,
usually lyophilized, including excipients, which on dissolution
will provide for a reagent solution having the appropriate
concentrations for performing a method or assay. The kit can
further include a written description of a method in accordance
with the present embodiments as described above.
[0112] The phrase "at least" as used herein means that the number
of specified items may be equal to or greater than the number
recited. The phrase "about" as used herein means that the number
recited may differ by plus or minus 10%; for example, "about 5"
means a range of 4.5 to 5.5. The designation "first" and "second"
is completely arbitrary and is not meant to suggest any order or
ranking among any members of a group to which the above language
pertains such as, for example, "first and second monoclonal
antibodies" or "first monoclonal antibody" and "second monoclonal
antibody."
[0113] The following examples further describe the specific
embodiments of the invention by way of illustration and not
limitation and are intended to describe and not to limit the scope
of the invention. Parts and percentages disclosed herein are by
volume unless otherwise indicated.
EXAMPLES
[0114] All chemicals were purchased from the Sigma-Aldrich Company
(St. Louis Mo.) unless otherwise noted.
[0115] Testing was carried out using the DIMENSION.RTM. RxL
analyzer, available from Siemens AG, Newark Del. The instrument was
employed using enzymatic detection system with sandwich immunoassay
format. In the embodiment of the sandwich method used herein and
discussed in more detail below, binding between a labeled antibody
(Ab) conjugated to an enzyme (conjugate) and sirolimus drug (SIRO)
in patient samples and subsequent binding of the resulting
immunocomplex with a capture antibody on chrome particles
determined the amount of sirolimus in the patient samples. The
unbound tag antibody enzyme conjugate was removed automatically by
3-4 mix/wash and magnetic separation cycles. The enzymatic activity
from conjugate remaining on the chrome particles was measured and
was directly proportional to the amount of sirolimus in the patient
sample.
Definitions
[0116] mg=milligram
[0117] g=gram(s)
[0118] ng=nanogram(s)
[0119] mL=milliliter(s)
[0120] .mu.L=microliter(s)
[0121] mmol(s)=millimole(s)
[0122] .mu.mol=micromolar
[0123] .degree. C.=degrees Centigrade
[0124] min=minute(s)
[0125] sec=second(s)
[0126] hr=hour(s)
[0127] w/v=weight to volume
[0128] v/v=volume to volume
[0129] TLC=thin layer chromatography
[0130] HPLC=high performance liquid chromatography
[0131] UV=ultraviolet
[0132] EtOAc=ethyl acetate
[0133] MeOH=methanol
[0134] DMF=dimethylformamide
[0135] DI=deionized
[0136] THF=tetrahydrofuran
[0137] NHS=N-hydroxysuccinimide
[0138] DCC=N,N-dicyclohexyl carbodiimide
[0139] BSA=bovine serum albumin
[0140] BGG=bovine gamma globulin
[0141] MS=mass spectrometry
[0142] SIRO=sirolimus
[0143] rotovap=rotary evaporator
Example 1
Preparation of Compounds
Preparation of C-32-Sirolimus and C-26-Sirolimus Oximes (IVa and
IVb) (FIG. 5)
[0144] To a solution of Sirolimus (I) (653.6 mg, 0.715 mmol) and
carboxymethoxyamine hemihydrochloride (234.4 mg, 2.14 mmol) in MeOH
(20 mL) was added sodium acetate (181.8 mg, 3.1 mmol). The reaction
mixture was stirred at room temperature (23.degree. C.) overnight
(18 hr) under a nitrogen atmosphere. TLC analysis indicated that
the reaction was completed. (TLC, Silica gel plate,
CH.sub.2Cl.sub.2/MeOH=9/1). CH.sub.2Cl.sub.2 (80 mL) and DI water
(20 mL) was added to the mixture, which was stirred 10 min. The
CH.sub.2Cl.sub.2 layer was separated. The aqueous layer was
extracted with CH.sub.2Cl.sub.2 (3.times.30 mL). The combined
CH.sub.2Cl.sub.2 solutions were washed with DI water (2.times.40
mL), were dried over Na2SO4, were filtered and were concentrated on
a rotovap to give a mixture of C-32-Sirolimus and C-26-Sirolimus
oximes (IVa and IVb, 622 mg).
Isolation of C-26-Sirolimus Oxime (IVa) (FIG. 5)
[0145] An optimal TLC condition (silica gel,
EtOAc/Hexanes/MeOH=5/2/1, R.sub.f C-32-oxime=0.59, R.sub.f
C-26-oxime=0.51) for the separation of C-32-Sirolimus and
C-26-Sirolimus oximes was developed and applied successfully in an
BIOTAGE.RTM. ISOLERA.TM. One Flash Chromatography System (John
Morris Scientific, Chatswood, NSW). A mixture of C-32-Sirolimus and
C-26-Sirolimus oximes (IVa and IVb, 622 mg) was dissolved in
CH.sub.2Cl.sub.2 (5 mL). The CH.sub.2Cl.sub.2 solution was eluted
to a cartridge (silica, 50 g SNAP Ultra) associated with the
BIOTAGE.RTM. ISOLERA.TM. One Flash Chromatography System. The
system was run with mixed solvent in a flow rate of 25 mL/min. All
the collected fractions from the cartridge were checked by TLC
(EtOAc/Hexanes/MeOH=5/2/1). Base on TLC analysis, the more polar
pure fractions (R.sub.f C-26-oxime=0.51) were combined and
concentrated to give C-26-Sirolimus oximes (IVa) (197 mg). HPLC-UV
analysis of this compound indicated a purity of 95%.
Preparation of C-26-Sirolimus Oxime-BSA Conjugate (Va) (R.sup.5=BSA
in FIG. 6)
[0146] To a solution of IVa (167.97 mg, 0.17 mmol) in THF/DMF (8 mL
THF, 0.4 mL DMF), NHS (41.8 mg, 0.35 mmol) and DCC (70.9 mg, 0.34
mmol) was added. The reaction mixture was stirred at room
temperature under a nitrogen atmosphere and the product NHS ester
is slightly less polar than compound IVa in TLC analysis. A white
solid formed during the reaction was filtered and then washed with
EtOAc. After solvent was removed, the reaction mixture was
re-dissolved in EtOAc and filtered; evaporation of solvent afforded
a slight yellow solid, which was held under high vacuum for 1
hr.
[0147] The activated hapten NHS ester (slight yellow solid) was
dissolved in DMF (1 mL) and the solution was added dropwise to a
BSA (120 mg) in phosphate buffered saline (PBS) buffer (0.1 M
NaH.sub.2PO.sub.4/Na.sub.2HPO.sub.4, pH 8) (14 mL) in an ice bath.
After stirring for 1 hr at room temperature, pH of the solution was
adjusted to pH 8 with NaOH (1N) and the mixture was stirred in a
cold room (4.degree. C.) overnight. The BSA conjugate was purified
through an equilibrated SEPHADEX.RTM. G-25 column (C26.times.70)
with PBS buffer (0.1 M NaH.sub.2PO.sub.4/Na.sub.2PO.sub.4, pH 7),
and eluted with same PBS buffer. A UV detector at 280 nm was used
to monitor the eluted fractions from the column. A clean separation
between BSA conjugate and the unconjugated hapten IVa was observed.
Fractions containing BSA conjugate (Va) were pooled to a total of
57 mL, and the concentration of the Va was determined to be 2.52
mg/mL by the BCA Protein Concentration Assay (Pierce Biotechnology,
Rockford Ill.).
Preparation of Diels-Alder adduct of
4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) and sirolimus (I)
[0148] Reference is made to FIG. 4. A solution of PTAD (38 mg,
0.217 mmols) in anhydrous CH.sub.2Cl.sub.2 (1 ml) was added to a
solution of sirolimus (I) (200 mg, 0.219 mmols) in anhydrous
CH.sub.2Cl.sub.2 (7 ml) at room temperature (24.degree. C.). The
characteristic red color of PTAD disappeared. The reaction mixture
was stirred at room temperature for 30 minutes and refluxed under
nitrogen at 60.degree. C. for 60 min. TLC analysis of the mixture
showed that very small amount of sirolimus remained. (TLC
conditions: Hexane/ethyl acetate/MeOH=30/65/5 (v/v)). Then, 5 mg of
PTAD was added to the reaction mixture. The mixture was stirred at
24.degree. C. for 30 min. TLC analysis of the mixture again
demonstrated that all sirolimus was consumed. The light red color
of PTAD remained in the reaction indicating an excess of PTAD. Most
of the CH.sub.2Cl.sub.2 was evaporated by rotary evaporation. The
residue solution (0.5 ml) was applied to a preparative TLC plate
(20.times.20 cm, 2000 micron; Analtech, Newark Del.). The plate was
developed with the same solvent system as above (Hexane/ethyl
acetate/MeOH=30/65/5 (v/v)). The silicon band containing product
was collected and extracted with MeOH/CH.sub.2Cl.sub.2 (1/9; v/v;
40 ml.times.3) three times. The combined organic extracts were
evaporated and the residue was dried in high vacuum for 16 hr. This
gave a mixture of the desired pure PTAD-sirolimus Diels-Alder
adducts Ma and IIIb (220 mg, 92% yield) as a white solid. HPLC
region-isomers ratio of IIIa/IIIb was 86/14; HPLC-MS (ES):
MNa+1111.5; 1H-NMR (CDCl.sub.3) 7.62 (1H); 7.46 (3H); 7.37 (1H);
5.98 (1H); 5.84 (1H); 5.55 (1H); 3.4 (s, 3H); 3.35 (s, 3H); 3.15
(s, 3H); 0.72 (q, 1H).
Preparation of Oximes of Compounds IIIa and IIIb (FIG. 7)
[0149] Oximes VIa and VIb are prepared from Compounds Ma and IIIb
in a manner similar to that described above for the Preparation of
C-32-Sirolimus and C-26-Sirolimus Oximes (IVa and IVb) of FIG.
5.
Preparation of BSA Conjugates (VIIa and VIIb) (R.sup.5=BSA in FIG.
8)
[0150] Oxime VIa is isolated from the above mixture of VIa and VIb
in a manner similar to that described above for the isolation of
IVa of FIG. 5. BSA conjugates VIIa and VIIb are prepared from VIa
in a manner similar to that described above for the preparation of
BSA conjugate Va.
Preparation of KLH Conjugates (VIIa and VIIb) (R.sup.5=KLH in FIG.
8)
[0151] Oxime VIa is isolated from the above mixture of VIa and VIb
in a manner similar to that described above for the isolation of
IVa of FIG. 5. KLH conjugates VIIa and VIIb are prepared from VIa
in a manner similar to that described above for the preparation of
BSA conjugate Va.
Preparation of Monoclonal Antibody that Binds to Domain D3 of
Sirolimus
[0152] Monoclonal antibodies that bind to separate portions of the
sirolimus molecule are prepared as follows. The immunogen is KLH
conjugates VIa and VIb prepared as described above. This immunogen
is used to immunize Balb/c mice. The first immunization is 25 .mu.g
in a volume of 200 .mu.al with monophosphoryl lipid A and synthetic
trehalose dicorynomycolate adjuvant (RIBI MPL+TDM Emulsion, RIBI
ImmunoChem Research Inc., Hamilton Mont.) intraperitoneally. Five
weeks later a boost immunization is given with 25 .mu.g of the
immunogen in 200 .mu.l of monophosphoryl lipid A and synthetic
trehalose dicorynomycolate adjuvant intraperitoneally.
Subsequently, after another 8 weeks, a prefusion boost is given of
the 25 .mu.g of the immunogen in 200 .mu.l of Hanks' Balanced Salt
Solution intravenously and intraperitoneally.
[0153] Three days later, fusion is performed by standard methods
using a nonsecreting murine myeloma designated P3x63-AG8.653.
Cloning is carried out by standard methods.
[0154] The clones are screened by the following reverse ELISA
immunoassay procedure according to the following protocol. Plates
are coated with polyclonal goat anti-mouse IgG (IgG+IgA+IgM) (Zymed
Laboratories, South San Francisco Calif.) at 5 .mu.g/ml in
phosphate buffered saline at 100 .mu.l per well. Plate coating is
performed for 2 hours or more at room temperature or overnight at
about 4.degree. C. The plates are then flicked dry and blocked with
300 .mu.l per well of blocking buffer diluent (0.5% bovine serum
albumin, 0.05% TWEEN.RTM. 20 in PBS). Plate blocking is performed
by incubation for 15 minutes or more at room temperature with plate
shaking. The plates are then flicked dry. The monoclonal antibody
to be screened is then added to each well as follows: 50 .mu.l per
well of blocking buffer diluent was added along with 50 .mu.l per
well culture supernatant transferred from the corresponding well in
the fusion growth plate. Incubation is for about 1 hour at room
temperature with shaking. The plate is washed using a TITERTECK
PLUS.RTM. plate washer with S20 stacker with the washing buffer
being PBS with 0.05% TWEEN.RTM. 20. An enzyme conjugate of
sirolimus covalently coupled to glucose-6-phosphate dehydrogenase
diluted in blocking buffer diluent to 1:4000 is added at 100 .mu.l
per well. Incubation is performed for about 1 hour at room
temperature with shaking. The plate is then washed and a
chromogenic solution is added at a volume of 100 .mu.l per well.
The chromogenic solution contains 0.593 mM p-iodonitrotetrazolium
violet, 0.02 M NAD, 0.033 M glucose-6-phosphate, 0.055 M Tris,
0.02% sodium azide, and a 1:4000 dilution of diaphorase (lipoyl
dehydrogenase). BSA is present at 1% (vol/vol) of a 5% w/vol BSA
solution. BSA is used to help prevent rapid precipitation of
reduced p-iodonitrotetrazolium violet.
[0155] From the screening a hybridoma producing a suitable
monoclonal antibody that binds to domain D3 of sirolimus is
selected.
[0156] Preparation of Hemolytic Pretreatment Solution.
[0157] This pretreatment solution contains 5 .mu.g/mL of FK506, 6.8
mg/mL PIPES.TM. 1.5 sodium salt, 0.3 mg/mL EDTA Disodium, 1.0 mg/mL
Saponin, 0.2% PROCLIN.RTM. 300, 0.024 mg/mL Neomycin sulfate and
0.99 mg/mL NaN3, pH 6.5. The FK506 concentration in the final
reaction mixture is 1.1 .mu.g/mL.
Example 2
Determination of Sirolimus Using Automated Chrome Particle Sandwich
Assay
[0158] Preparation of Anti-Sirolimus
F(Ab').sub.2-.beta.-Galactosidase Conjugate Using a Monoclonal
Antibody that Binds to Domain D3 of Sirolimus.
[0159] Monoclonal anti-sirolimus antibody that binds to domain D3
of sirolimus (prepared as described above in Example 1) is
fragmented to F(ab').sub.2 using lysyl-endopeptidase (Wako,
Richmond, Va.) digestion and then is conjugated to
.beta.-galactosidase using a standard heterobifunctional SMCC
(succinimidyl
trans-4-(N-maleimidylmethyl)cyclohexane-1-carboxylate) linker
according to known techniques. The antibody conjugate solution
contains approximately 2.0 .mu.g/mL anti-sirolimus
antibody-.beta.-galactosidase conjugate, 30 mg/mL protease free
bovine serum albumin, 0.126 mg/mL MgCl.sub.2, 0.03 mL/mL of
ethylene glycol, 24.5 mg/mL HEPES, 38.5 mg/mL Na HEPES, 50 mg/mL
NaCl and beta-gal mutein (inactivated beta-galactosidase), pH
7.8.
[0160] Magnetic Chrome Particle Preparation.
[0161] Chrome particles (immunoassay solid phase) are prepared by
conjugating a monoclonal antibody that binds to domain D1 of
sirolimus (prepared as described above in Example 1 using as an
immunogen C-26-Sirolimus Oxime-BSA Conjugate (Va) (R.sup.5=BSA in
FIG. 6)) to glutaraldehyde coated chromium dioxide particles. The
chrome reagent contains chrome particles and 60.4 mg/mL trehalose
dihydrate and 7.2 mg/mL polyethylene glycol (PEG) 8000. Three
chrome particle concentrations, namely 5, 2.5, and 1.67 mg/mL, are
used in the study.
[0162] Sandwich Sirolimus Assay.
[0163] The principle and operation of the Sandwich assay for
sirolimus is as follows: A whole blood sample (50 .mu.L) containing
sirolimus is combined with a hemolytic pretreatment reagent
prepared as described above in a reaction vessel on the
DIMENSION.RTM. RxL analyzer. The whole blood is sampled from a
standard cup by first mixing the blood with the ultrasonic sample
probe. The mixing of whole blood sample with the pretreatment
solution ensures the hemolysis of the whole blood and the
displacement of the protein-bound sirolimus molecules from their
binding domains.
[0164] Anti-sirolimus F(ab').sub.2-.beta.-galactosidase conjugate
prepared using the monoclonal antibody that binds to the D3 domain
of sirolimus (50 .mu.L) is added to the reaction vessels and the
mixture is held for a period of time (35 sec) and at a temperature
of 43.degree. C. to allow sirolimus, if present, to react with the
antibody enzyme conjugate. Chrome particles with immobilized
monoclonal antibody that binds to domain D1 of sirolimus are added
(50 .mu.L) to the reaction vessels and are allowed to bind the
anti-sirolimus F(ab').sub.2-.beta.-galactosidase complex to form a
sandwich. This reaction mixture is incubated for 14 min at a
temperature of 43.degree. C. before the automated magnetic
separation, mix and wash cycles begin on the DIMENSION.RTM.
instrument. A total of 4 separation/wash cycles are employed to
remove the unbound anti-sirolimus F(ab').sub.2-.beta.-galactosidase
conjugate and debris from sample. The automated chrome washes are
conducted on board using Chemistry Wash solution at pH 8.0 in HEPES
buffer, both of which were provided for the DIMENSION.RTM.
Heterogeneous Immunoassay Module. The washed chrome particles are
then re-suspended in the Chemistry Wash solution by ultrasound
mixing and a portion (54 .mu.L) of the suspended chrome particles
are transferred to a photometric cuvette to mix with a
.beta.-galactosidase substrate solution (chlorophenol
red-.beta.-D-galactopyranoside, or CPRG). The sirolimus bound to
the anti-sirolimus F(ab').sub.2-.beta.-galactosidase conjugate on
the chrome particle surface is detected by measuring the enzymatic
rate of the conjugate in the presence of CPRG. The rate for each
reaction vessel is measured bichromatically at 577 and 700 nm. The
results indicate successful detection of sirolimus.
Example 3
Determination of Sirolimus Using Automated ELISA Sandwich Assay
[0165] Sandwich Enzyme-Linked Immunosorbent Assay (ELISA) for
Sirolimus.
[0166] The following steps are employed: Step 1: 50 .mu.L of
purified monoclonal antibody that binds to domain D1 of sirolimus
(prepared as described above in Example 1 using as an immunogen
C-26-Sirolimus Oxime-BSA Conjugate (Va) (R.sup.5=BSA in FIG. 6))
(10 .mu.g/mL in PBS) is coated on ELISA plates overnight at
4.degree. C. Plates are washed using MILLI-Q.RTM. water (Millipore
Corporation, Billerica Mass.) containing 0.05% TWEEN.RTM. 20. Step
2: 200 .mu.L of PCT Blocker solution (0.5% Casein (milk protein) in
phosphate buffer containing 0.05% TWEEN.RTM. 20) is added to each
well and the media are incubated at room temp for 30 min. Plates
are washed using MILLI-Q.RTM. water containing 0.05% TWEEN.RTM. 20.
Step 3: 50 .mu.L of desired concentration of sirolimus diluted in
PBS is added to the respective wells and the media are incubated at
room temperature for 30 min. Plates are washed using MILLI-Q.RTM.
water containing 0.05% TWEEN.RTM. 20. Sirolimus drug concentrations
tested are 0, 0.01, 0.02, 0.04, 0.08, 0.16, 0.31, 0.63, 1.25, 2.50,
5.0 and 10.0 ng/mL, respectively. Step 4: The anti-sirolimus
F(ab).sub.2-.beta.-galactosidase conjugate prepared using the
monoclonal antibody that binds to the D3 domain of sirolimus
(prepared in a manner similar to that described above) (1:300
diluted in PCT Blocker solution) is added and the media are
incubated at room temperature for 30 min. Plates are washed using
MILLI-Q.RTM. water containing 0.05% TWEEN.RTM. 20. Step 5:
.beta.-galactosidase substrate solution (chlorophenol
red-.beta.-D-galactopyranoside, or CPRG) is added to each well (100
.mu.L/well). Step 6: The wells are read in plate reader at 577 nm
every minute for 20 min. The results indicate successful detection
of sirolimus.
[0167] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0168] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
Furthermore, the foregoing description, for purposes of
explanation, used specific nomenclature to provide a thorough
understanding of the invention. However, it will be apparent to one
skilled in the art that the specific details are not required in
order to practice the invention. Thus, the foregoing descriptions
of specific embodiments of the present invention are presented for
purposes of illustration and description; they are not intended to
be exhaustive or to limit the invention to the precise forms
disclosed. Many modifications and variations are possible in view
of the above teachings. The embodiments were chosen and described
in order to explain the principles of the invention and its
practical applications and to thereby enable others skilled in the
art to utilize the invention.
* * * * *