Note: Descriptions are shown in the official language in which they were submitted.
W0 94/24566 PCT/US94/03885
2137785
IMMUNOASSAY AND TEST KIT
FOR THROMBIN-ANTITHROMBIN lll COMPLEX
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates an immunoassay for thrombin-
5 anliLhro",bin lll complex which comprises antibodies that specifically bind to the complex in a human fiuid sample.
2. Background
General agreement exists that blood co~glJI~tion or c!otting includes
10 three essential steps: 1) a complex of subst~nces called prothrombin
activator is formed e.~. in ~esl.onse to rupture of a blood vessel or damage
to blood itself; 2) the proll,rombin activator catalyzes the conversion of
p,oli,ro,nbin to thrombin; and 3) lhloll)bil1 acts as an enzyme to activate
elet~ and to convert fibrogen into fibrin threads that enmesh platelets
15 blood cells and plas"~a to form the clot itself.
The enzymatic activity of thrombin can be regulated in various ways
including the formation of an inactive complex with alllilhro~bin lll known
as the thrombin-antithrombin lll complex or the ~TAT complex~.
20 AnliUIlo"~l~in 111 or UATIII" is a proteinase inhibitor in plasma that complexes
with several serine proteinases in addition to thrombin. Thus ATIII is known
to form complexes with serine protease factors Xlla Xla Xa and IXa.
It is desi,a~le to measure the amount of TAT complex in a human
25 fluid sample. Determination of levels of the TAT complex will provide an
WO 941245~K PCT/US94/03885
.
2 131~ 8~ - 2 -
indication of coagulation activity of a patient and aid in the assessment of
thrombotic risk and other disorders such as disseminated intrav~scul~r
co~gul~tion.
An assay for TAT complex levels in human plasma should provide a
selective assessment of TAT complex concentrations, rather than a
measurement of the concentration of total complexes of ATIII in a plasma
sample, e.g. complexes of ATIII with factors Xlla, Xla, Xa and IXa in addition
to the TAT complex. Such measurement of the "total" complexes of ATIII
provides an indication of the equilibrium of several reactions, some of which
may reflect conditions other than those associated with particular TAT
complex levels. For instance, contact activation of factors Xll and Xl may
lead predominantly to activation of the complement and kinin systems,
therefore Xlla-ATIII and Xla-ATIII complexes may not be indicative of
thrombotic risk.
It thus would be desirable to have a means for determining levels of
the TAT complex in a human fluid sample. It would be particularly desirable
to have an assay for the concentration of TAT complex in a human fluid
sample where the assay provided a selective assessment of concenL,alioils
of the TAT complex in a test sample, rather than a measurement of the
sample's total complexes of ATIII.
SUMMARY OF THE INVENTION
The present invention provides an immunoassay for the detection
and measurement of concenl,alion of a human thrombin-anLiLl,rombin lll
(TAT) complex in a human fluid sample. The assay comprises contact of a
human fluid sample with at least two antibodies or immunoreactive
fragments thereof, preferably monoclonal antibodies or reactive fragments
wo 94~ 2 1 3 7 7 8 S ~/US94103885
thereof, wherein at least one of the antibodies specifically binds to the TAT
complex in the fluid sample. Preferably each~ of the antibodies of the assay
specifically binds to the TAT complex. As use~ herein, "specifically binds to
the TAT complex" or other similar phrase indicates that the antibody or
5 antibodies bind to the TAT complex but exhibit substantially or essentially
no cross-reactivity with free (i.e., not complexed) antithrombin lll or
antithrombin lll complexes of other plasma factors, specifically factor Xa-
antithrombin lll complex and factor IXa-antithrombin lll complex. Preferably
the mixture of antibodies of the assay bind to different epitopes of the TAT
10 complex, i.e. the antibodies are not competitive. Additionally, the antibodies
of the immunoassay of the invention preferably are contacted
simultaneously with a human fluid sample so that only a single incubation
and wash step is employed. Other aspects of the invention are disclosed
infra.
DETAILED DESCRIPTION OF THE INVENTION
The immunoassay of the invention comprises reaction of at least two
antiho~ies with a human fluid sample, wherein at least one of the antibodies
specifically binds to said TAT complex. rleferably the antibodies are
20 monoclonal antibodies and both of said two antibodies of the assay
specifically bind to the TAT complex. The antibody or antibodies of the
assay that specifically bind to the TAT complex preferably exhibit less than
about 3% cross-reactivity, in an assay such as that described in Example 7
below or similar assay with a human plasma sample, with ATIII, factor Xa-
25 a"lilhr~",bin lll complex and factor IXa-antithrombin lll complex. More
pre~erably the antibody or antibodies of the invention exhibit less than about
1% cross-reactivity with ATIII and such ATIII complexes. Still more
preferably the antibody or antibodies of the invention exhibit less than about
0.2%, or even less than about 0.1% cross-reactivity with antithrombin lll and
WO 94/24566 . PCT/USg4/03885
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such ATIII complexes in an assay as described in Example 7 below or
similar assay using a human plasma sample. It is also preferred that
antibodies of the invention exhibit su~ch~lack of cross-reactivity with free (not
complexed) thrombin, i.e. the antibodies of the invention preferably exhibit
5 less than about 3% cross-reactivity with free thrombin in reaction with a
human plasma sample, more preferably exhibit less than about 1% cross-
reactivity with thrombin, and still more preferably exhibit less than about
0.2%, or even less than about 0.1% cross-reactivity with free thrombin upon
contact reaction of the antibody with a human plasma sample. In particular,
10 it was found that specifically preferred monoclonal antibodies of the
invention upon contact with a human fluid sample bind to human TAT
complex and exhibited less than about 0.005% cross-reactivity with
thrombin, less than about 0.005% cross-reactivity with antithrombin lll, less
than about 0.005% cross-reactivity with factor IXa-antithrombin lll complex
15 and less than about 0.02% cross-reactivity with factor Xa-antithrombin lll
complex. "Antibody", "antibody of the invention~ or other similar term as
used herein includes a whole immunoglobulin as well as antigenic binding
fragments or immunoreactive fragments which specifically bind to the TAT
complex, including Fab, Fab', F(ab')2 and F(v).
If only one antibody of the immunoassay specifically binds to the TAT
complex, the other antibody of the assay should bind to the TAT complex
but also may exhibit cross-reactivity, e.g. with anLi~hrombin lll or other
complexes of a"lill,ro,t,bin lll. Thus references herein to an antibody that
25 "binds" to TAT complex, rather than specifically binds thereto, indicates that
the antibody binds to the human TAT complex and also may exhibit cross-
reactivity with antithrombin lll, factor Xa-antithrombin lll complex and/or
factor IXa-antithrombin lll complex. Antibodies, including monoclonal
antibodies, that bind to the TAT complex and also exhibit cross-reactivity
wo 94~ 2 1 3 7 7 8 5 pcTlus94lo388s
with anlill,ro",bin lll and complexes thereof have been reported. See J.
Dawes, et al., Thrombosis Research, 36:397 (1984); S. Asakura, et al.,
Bioch. et Bio~hy. Acta, 952:37 (1988); Z. Hrkal, Hybridoma, 10(5):633
(1991); and European Published Patent Application No.0391433A2. In this
S embodiment of the invention, preferably the antibody that specifically binds
to the TAT complex is used as a carrier antibody and is immobilized on a
substrate in a sandwich-type assay, and the cross-reactive antibody is used
as the labeled conjugate, although the cross-reactive antibody also can be
used as the carrier antibody and the antibody that specifically binds to the
10 TAT complex can be used as the labeled conjugate.
The human fluid sample used in the assay of the invention can be
any sample that contains the TAT complex, e.g. blood or urine. Typically a
plasma sample is employed.
Antibodies of the invention can be prepared by techniques generally
known in the art, and are typically generated to a purified sample of a TAT
complex of human plasma. The antibodies also can be generated from an
immunogenic peptide that comprises one or more epitopes of the TAT
20 complex that are not exhibited by free ATIII or thrombin.
More particularly, antibodies can be prepared by immunizing a
mamrnal with a purified sample of a TAT complex, or an immunogenic
peptide as discussed above, alone or complexed with a carrier. Suitable
25 ma~,n,als include typical laboratory animals such as sheep, goats, rabbits,
guinea pigs, rats and mice. Rats and mice, especially mice, are preferred
for obtaining monoclonal antibodies. The antigen can be ad",i,-i;,tered to
the mammal by any of a number of suitable routes such as subcutaneous,
intraperitoneal, intravenous, intramuscular or intracutaneous injection.
wo 94~SC6 PCTnUS94/~ ~5
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Preferably immunization is by subcutaneous, intraperitoneal, or intravenous
injection. The optimal immunizing interval, immunizing dose, etc. can vary
within relatively wide ranges and can be :determined empirically based on
this disclosure. Typical procedures involve injection of the antigen several
times over a number of weeks. Antibodies are collected from serum of the
immunized animal by standard techniques and screened to find antibodies
specific for the TAT complex. Monoclonal antibodies can be produced in
cells which produce antibodies and those cells used to generate
monoclonal antibodies by using standard fusion techniques for forming
hybridoma cells. See G. Kohler, et al., Nature, 256:456 (197~). Typically
this involves fusing an antibody producing cell with an immortal cell line
such as a myeloma cell to produce the hybrid cell. Alternatively,
monoclonal antibodies can be produced from cells by the method of Huse,
et al., Science. 256:1275 (1989).
One suitable protocol provides for intraperitoneal immunization of a
mouse with a composition comprising purified TAT complex conducted over
a period of about two to seven months. Spleen ceils then can be removed
from the immunized mouse. Sera from the immunized mouse is assayed
for titers of antibodies specific for TAT complex prior to excision of spleen
cells. The excised mouse spleen cells are then fused to an appropriate
homogenic or heterogenic (preferably homogenic) Iymphoid cell line having
a marker such as hypoxanthine-guanine phosphoribosyltransferase
deficiency (HGPRr) or thymidine kinase deficiency (TK-). Preferably a
myeloma cell is employed as the Iymphoid cell line. Myeloma cells and
spleen cells are mixed together, e.g. at a ratio of about 1 to 4 myeloma
cells to spleen cells. The cells can be fused by the polyethylene glycol
(PEG) method. See G. Kohler, et al., Nature, supra. The thus cloned
hybridoma is grown in a culture medium, e.g. RPMI-1640. See G. E. More,
wo g4~ 2 1 3 7 7 8 S ~/US94103885
et al., Journal of American Medical Association, 199:549 (1967).
Hybridomas, grown after the fusion procedure, are screened such as by
radioimmunoassay or enzyme immunoassay for secretion of antibodies that
bind specifically to the TAT complex, e.g. antibodies are selected that bind
5 to the TAT complex, but not to ATIII. Preferably an ELISA is employed for
the screen. Hybridomas that show positive results upon such screening
can be ex~,anded and cloned by limiting dilution method. Further screens
are preferably performed to select antibodies that can bind to TAT complex
in solution as well as in a human fluid sample.
Antibodies of the invention preferably show an affinity constant with
the TAT complex of greater than about 1 x 104 L/M, more preferably
greater than about 1 x 105 L/M, still more preferably greater than about 1 x
108 L/M, such constants determined by the method disclosed in Friguet, et
al., J. Immunol. Methods, 77:305 (1985).
Preferred antibodies of the invention are highly sensitive for TAT
complex in a human fluid sample. For example, it was found that preferred
antibodies detected with precision a concenl,alion of TAT complex of about
20 0.55 ng or greater per ml of plasma sample with recovery of spiked material
being essentially 100%.
Immunoassays of the invention generally exhibit good precision. For
example, the following results were found for preferred assays of the
25 invention: within assay values for control levels I and ll for n-80 of 3.9% CV
and 6.8% respectively; and between assay precision values for n=80 of
9.3% and 12.2% CV respectively.
WO 94/245CC PC~/US94tQ~885
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Competitive assays have shown that preferred antibodies of the
invention are at least substantially or essentially non-competitive, i.e. two ormore antibodies of the invention show little or no binding interference
among themselves when reacted simultàneously with a sample of TAT
5 complex. By use of such antibodies in an immunoassay, a test sample can
be incub~ted simultaneously (i.e., single incubation step) with both the
bound capture antibody and labeled conjugate antibody of the assay. This
is distinct from prior assays that utilize separate incubation and wash steps
for each of the capture and conjugate antibodies, e.g. to avoid binding
10 interference between the antibodies.
It has also been found that preferred antibodies of the invention can
bind to TAT complex of a human fluid sample, particularly a human plasma
sample, that is not diluted, or is diluted only by the solution containing the
15 TAT complex antibody. In particular, it has been found that preferred
antibodies of the present invention can bind to TAT complex of a human
fluid sample where the fluid sample is diluted only by an equal volume or
less of a diluent. This is distinct from at least some prior antibodies that arereported to bind to TAT complex, but will only do so when contacted with a
20 ~JIas",a sample that is diluted to a significant extent, e.g. when a plasma or
other test sample is diluted 100 times or more. Such dilution is undesirable
as it can add to the complexity of a TAT complex assay that utilizes such
antibodies.
Specifically preferred monoclonal antibodies of the invention include
D2M, C72, C44-T and SB46, which are mouse monoclonal antibodies that
specifically bind to the TAT complex. Hybridomas expressing such
monoclonals have been deposited with the American Type Culture
Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852 and
wo g4124566 2 1 3 778 5
given Accession No. _ (TAT-4-C44-T-3:ST; expresses C44-T); Accession
No. _tTAT-5-B46-4; expresses 5B46); Accession No. _ (TAT-4-C72E;
expresses C72); Accession No. _(TAT-1-D2M; expresses D2M).
Thus antibodies of the invention are particularly suitable for use in an
immunoassay for detecting and measuring the concen~lalion of TAT
complex in a human fluid sample. A particularly prefer,ed immunoassay of
the invention is a sandwich assay in which the capture antibody is
immobilized using known methods such as physical adsorption onto the
surface of a support. Suitable supports include, e.g., polymer materials
such as polypropylene or polystyrene, glass, metals, cross-linked dextran,
agarose, etc. The support may be in a variety of shapes such as a tray,
sphere, rods, test tube, etc. A tray having wells such as a miclolilre plate is
generally preferred.
The detectably labeled antibody of the assay is formed by coupling
the antibody to a reporter, e.g. a radioactive isotopes, an enzyme, a
fluorescent or luminescent reagent and the like. Typically used radioactive
isotopes '251, Tc99m and 3H. Known isotope labeling methods include
iactoperoxidase and Hunter-Bolton methods for t251 and reduction
methylation for 3H. See A. Bolton, et al., Biochem. J., 133:529-539 (1972).
Enzyme tags may include peroxidases, alkaline phosphatases, ~-D-
galactosidases, glucoseoxidases, and the like. Peroxidases are particularly
preferred. The peroxidases can be selected from those derived from
various sources, e.g. horseradish, pineapple, fig, sugarcane, java bean,
corn. Horseradish peroxidase is a particularly preferred material. Means
for covalent labeling of an antibody with such enzymes are known in the art.
Methods suitable for a specific enzyme and antibody can depend on
wo s4n4sc6 Pcrluss4lo388s
2~3r~ 5 10-
available reactive moieties on the enzyme and antibody and can be readily
asce,lained empirically.
``:
The assay of the invention is illustrated by the following protocol
5 using peroxidase as the labei of the conjugate antibody. A test sample, e.g.
a human plasma sample, is added to a TAT complex antibody (i.e., an
antibody that binds to the TAT complex) bound on a carrier such a
microtiter plate and the antibody-antigen reaction is conducted, followed by
addition of the peroxidase labeled TAT complex antibody conjugate
10 obtained as outlined above, and then a further antibody-antigen reaction
conducted. The antibody are- typically dissolved in solution prior to contact
with a test sample. Suitable diluents include those known in the art for use
in immunoassays. A specifically preferred solution for dissolving the
antibodies for contact with a test sample contai,)s 20 mmol Tris, 500 mmol
15 sodium chloride, 0.05 mg/ml mouse IgG and 5% BSA.
Preferably both the carrier and conjugate antibodies of the assay of
the invention specifically bind to the TAT complex, and the antibody bound
to the support, the human fluid sample and labeled antibody are incubated
20 together, followed by a single wash step to remove any unreacted labeled
antibody and the human plasma sample other than the reacted TAT
complex. Suitable washing agents include those known in the art for use in
immunoassays. A specifically preferred washing buffer solution contains
27.2 9/l imidazole, 17.5 9/l sodium chloride and 4 ml/liter Tween 20. If
25 necessary a substrate for peroxidase is added to the assay and the
reaction products are assayed for enzyme activity by measuring the
absorbance or fluorescence of the resulting substance. The detected
amount of bound labeled antibody is directly proportional to the
concentration of TAT complex in the assayed human plasma sample. Thus
wo g4~ 2 1 3 7 7 8 5 PCr/uss4lo388s
a qua"LiLa~ive determination of the TAT complex concentration in the plasma
test sample can be dete",~ined by comparison of the absorbance or
fluorescence of the test sample with absorbance values obtained from
standardized solutions that contain known amounts of TAT complex. It may
5 be desirable to prepare calibration curves from absorbance values obtained
from a number of standardized solutions to facilitate interpretation of values
obtained from a test sample.
A specifically preferred immunoassay of the invention was conducted
10 as follows. The capture monoclonal antibody and conjugate monoclonal
antibody labeled with horseradish peroxidase (HRP) is incubated together
with a human plasma sample at 37~C for 30 rninutes. The plate is then
washed and incubated for 15 minutes at room temperature with HRP
substrate and the bound conjugate is quantitated.
A preferred immunoassay of the invention was used to measure
plasma samples in individuals (n=15) with Disseminated Intravascular
Coagulation. Assay results showed TAT complex levels significantly
elevated in all but one patient plasma sample. Twelve of the individuals
20 tested had levels of TAT complex greater than 160 ng/ml.
TAT complex can be purified from a human plasma sample by use of
carrier complexes coupled with one or more antibodies of the invention and
hence the invention includes methods for obtaining purified TAT complex
25 using the antibodies of the invention and related apparatus. A suitable
purification procedure provides coupling an antibody of the invention on an
appro,c,riate carrier as is known in the art such as a gel or resin then
packing the carrier in a column and then eluting a sample solution
containing TAT complex through the column to selectively adsorb the TAT
WO g4/245C6 PCT/US94/03885
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complex. The antibody can be suitably coupled onto the carrier by known
methods, e.g. the cyanogen bromide method and glutaraldehyde method,
aqueous carbodiimide method, active ester method, and the like. The
antibody also may be physically adsorbed orl the surface of the carrier.
The antibodies of the invention also can be used to locate and
monitor TAT complex in vivo. For example, one or more antibodies of the
invention can be labeled with a radionuclide such as 11-indium. The
labeled antibody then can be injected intravenously into a human and the
10 human scanned to determine where the labelled antibody accumulates.
The labeled antibody typically will differentially accumulate in areas having
high concentrations of TAT complex, e.g. where blood clotting is occurring
such as in a hemorrhage site. The amount of labeled antibody can be
determined by known scanning methods such as by using a scintigraphic
1 5 camera.
The antibodies of the invention also can be used therapeutically, e.g.
as a carrier for drugs, particularly pharmaceuticals targeted for interaction inthe blood clotting mechanism such as strepo-kinase, TPA and urokinase.
20 The pharmaceutical can be attached to an antibody of the invention by the
same means as attachment of a label as specified above, e.g. by a covalent
linkage that does not inhibit the specificity of the antibody or the desired
pharmacological effect of the drug. The antibody with the attached
pl,ar,l,aceutical can be administered to a human in therapeutically effective
25 amounts by any of a number of known means, e.g. parenterally or orally.
For parenteral adminisl,alion the antibody with attached pharmaceutical is
typically administered in aqueous and non-aqueous sterile iniection
compositions as are known in the art. For oral administration the
therapeutic of the invention may be administered to a human in discrete
WO g41245C6 PCI IUS94/03885
21~7785
units such as capsules or tablets each containing a predeter",ined amount
of the therapeutic, as a solution or a suspension in an aqueous or non-
aqueous liquid, as an oil/water liquid emulsion, in powdered carriers such
as lactose or sucrose, etc.
All documents mentioned herein are incorporated by reference herein
in their entirety.
The following non-limiting examples are illustrative of the invention.
General Comments
Purified samples of TAT complex used in the following Examples
were generally obtained by procedures described in G. Elgue, et al.,
Thrombosis and Hemostasis, 63(3):435 (1990). In brief, thrombin and
antithrombin lll were reacted and the resultant TAT compiex was isolated by
gel filtration. 20% Complete Medium used in the Examples had the
following composition per 100 ml:
1 ml 200 mM 1-glutamine,
1 ml 7.5% Na-bicarbonate,
1 ml 5000 u/ml Penicillin & 5000 mcg/ml Streptomycin,
0.05 ml 0.1M 2 ~-mercaptoethanol,
20 ml Fetal Bovine Serum, and
to 100 ml with RPMI 1640 (Gibco) without L-glutamine.
HAT media used in the Examples had the following composition: to
100 ml of Complete Medium 1 ml 100X Hypoxanthine Thymidine and 2 ml
SOX Aminopterin were added. HATG media used in the Examples had the
following composition: to 100 ml of HAT media 1 ml 100X Glycine was
added. IL-6 used in the Examples was prepared by adding 500 units
WO 94124566 PCT/US94103885
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- 14-
Human Recombinant IL-6 (Collaborative Research Inc.) per ml medium.
STM used in the Examples was prepared by adding 5 ~9 salmonella
typhimurium mitogen (Ribi Immunochemical Research, Inc.) per ml medium.
5 Example 1 - Immunization
The following two groups of BALB/c female mice were immunized
with TAT complex to provide spleen donors for the PEG fusions, disclosed
below, that generated monoclonal antibodies that specifically bind to the
TAT complex.
1. Group 1
BALB/c female mice were sensitized with 7 ~9 each of purified TAT
complex in immunogen emulsion prepared as follows:
0.125 ml TAT complex (325 l~g/ml in 50 mM tris/50 mM NaCI/0.1 M
15 EDTA pH 7.4)
1.500 ml Complete Freund's Adjuvant
1.375 ml Dulbecco's Phosphate Buffered Saline
Each mouse was injected with 0.5 ml of this immunogen emulsion
i.p. The mice were boosted with doses of 5 to 40 ~9 of purified TAT
20 complex i.p. in an immunization protocol that lasted for approximately seven
months.
- 2. Grouo 2
BALB/c female mice were sensitized with 10 ,ug each of purified TAT
25 complex in immunogen emulsion prepared as follows:
0.120 ml TAT complex (321 l~g/ml)
1.000 ml Complete Freund's Adjuvant
0.880 ml Dulbecco's Phosphate Buffered Saline
wo ~nA~ PCT~S94/~5
213778~
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Each mouse was injected with 0.5 ml of this immunogen emulsion
i.p. The mice were boosted with doses of 10 to 11 ,.Lg of TAT complex i.p.
in an immunkalion protocol that lasted approximately nine weeks.
ExamDle2-ELlsA Assay for TAT Antibodies
1. Coatina Microtiter plates with TAT and ATIII
TAT complex and ATIII were each separately diluted in a coating
buffer (carbonate buffer pH 9) at a concentration of 1 ~g/ml. 100 ~l of
each solution (10 ng TAT complex or ATIII) was placed in each well of the
10 microtiter plates, which were sealed and incubated overnight at 2-8 C. The
contents of the wells were then aspirated and the plates were washed once
with wash/storage buffer, the wash aspirated, and the plates again
resealed. The plates were stored at 2-80C until use.
15 2. Mouse Serum Titration
Sera collected from the immunized mice from each of Groups 1 and
2 of Example 1 above were assayed for titers of TAT complex antibodies by
ELISA on TAT complex coated microplates. Serially diluted sera (usually
tested at dilutions of 1:1000 to as high as 1:3,125,000) were incllh~ted on
20 the microplates and probed with enzyme-labeled sheep or goat anti-mouse
conjugates which were subsequently reacted with substrate. Relative titers
were determined according to the strength of the color reaction, which was
read spectrophotometrically at 405 nm. Pre-immune sera was used as the
negative control and a commercially available mouse TAT monoclonal
25 antibody (latron) was used as a positive control in all assays.
Exam~le 3 - Preparation of Hybridomas
Hybridomas secreting monoclonal antibodies to TAT were generated
by two cell fusions. The P~G fusion technique employed based upon the
wo s4n4s66 PcTlus94lo38ss
2~3~ $ 16-
technique disclosed by G. Kohler, et al., Nature 256:495 (1975). The
myeloma cells used were HRPT-minus P3 - X63-Ag8.653 (P3X) (ATCC CRL
1580). Selection for hybrids was accomplished using HAT media
(hypoxanthine, aminopterin and thymidine); unfused P3X myeloma cells will
5 not survive in this medium as they lack the apparatus to use hypoxanthine
to produce purines and the aminopterin present in the medium block
endogenous synthesis of purines and pyrimidines.
1. Fusion to form Hybridoma TAT-4
10 Splenocyte preparation
Spleen cells were obtained from a mouse immunized with TAT
complex as described for Group 1 of Example 1 above. The cells were
teased from the spleen using a forceps and needle, then suspended in 12
ml of cold 20% Complete Medium without serum (RPMI 1640 base, Gibco).
The cells were then centrifuged as 200 x 9 for 10 minutes after which the
supernatant was removed by aspiration, and the cells resuspended again in
the cold medium. This washing process was repeated twice, and the cells
resuspended in a final volume of 10 ml. The viable cell count of the
splenocytes was 1.7 x 108 at a viability of 98% by trypan blue exclusion
20 technique. Counting methods employed were as disclosed by M. Absher,
'Hemacytometer Counting' in: Kruse, P.F. and Patterson, M.K. eds, Tissue
Culture Methods and Ap~lication, Academic Press pp. 395-397: 1973. In
brief, a volume of cell suspension is mixed with an equal volume of 0.02%
Trypan Blue and the cells counted in a hemacytometer by standard
25 counting methods.
Myeloma preparation
The myeloma cells were harvested mechanically, pooled, and
centrifuged at 200 x 9 for 10 minutes after which the supernatant was
wo 94~ 2 1 3 7 7 8 5 ~tUS94/03~
removed by as~i,dlio", and the cells resuspended in 50 ml of 20%
Complete Medium. The viable cell count of the myeloma cells was 2.9 x
106viable cells per ml at a viability of 76%.
Fusion of the sDlenocytes and myeloma cells
The splenocytes and 15 ml of the myeloma cell suspension were
combined at spleen cell to myeloma cell ratio of approximately 4:1 with a
total viable cell count of 2.13 x 108. The volume was brought up to 50 ml
with cold 20% Complete Medium without serum and the cells then
centrifuged a,t 200 x 9 for 10 minutes. The cell pellet was then washed
twice with this medium at a volume of 50 ml. After the final wash, the
supernatant was removed by aspiration and the pellet centrifuged at 200 x
g for 3 minutes and the remaining supernatant aspirated. The cells were
then fused with 40% PEG (molecular weight 7,000 to 9,000) buffered in
RPMI 1640; fusion was performed in a tube held in a warm (370C) water
bath. 1 ml of PEG solution warmed to 37 o C was added to the pellet and
incubated for 1 minute. The PEG solution was then diluted by addition of
20 ml of warm 20% Complete Medium without serum. The fused cells were
incllb~ted at 370C for 10 minutes and then centrifuged at 200 x 9 for 10
minutes.
The fused cell pellet was then resuspended in 50 ml of 20%
Complete Medium and plated at cell densities of 1.09 x 105 to 4.26 x 105
per well. The cells were plated in a final volume of 200 ~11 per well of 20%
Complete Medium, 20% Complete Medium with 2.5 ~Lg/ml STM or 20%
Complete Medium with 250 units/ml IL-6. After overnight incubation at
37OC and 10%CO2, one half of the medium in each well was aspira~ed and
the cells feed with 20% HAT, 20% HAT with 5 ~g/ml STM or 20% HAT with
500 units/ml IL-6. The cells were visually scanned and feed periodically
wo s4n4s6c Pcr/uss4/0388s
2~3~1185 - ~8-
with these media for several weeks while the growth of the hybridomas was
",o"ilored and growing wells screened for the presence of anti-TAT
complex antibodies beginning at day 12 to 14 post-fusion.
2. Fusion of Hybridoma TAT-5
SDlenocyte Dreparation
Spleen cells were obtained from a mouse immunized with TAT
complex as described for Group 2 of Example 1 above. The cells were
teased from the spleen using forceps and scissors then washed three
times in cold RPMI 1640. The cells were resuspended in a final volume of
40 ml of RPiAI 1640; the viable cell count of the splenocytes was 6 x 107 at
a viability of 95% by trypan blue exclusion technique.
Myeloma preparation
The myeloma cells were harvested mechanically pooled and
washed three times in cold RPMI 1640. The cells were resuspended in a
final volume of 10 ml; the viable cell count of the myeloma cells was 3.6 x
106 viable cells per ml at a viability of 71%.
Fusion of the splenocytes and myeloma cells
The splenocytes and 4.2 ml of the myeloma cell suspension were
combined at a spleen cell to myeloma cell ratio of approximately 4:1 with a
total viable cell count of 7.5 x 10'. The cell mixture was centrifuged at 200 x
g for 8 minutes and the supernatant aspirated. The cells were then fused
with 40% PEG (molecular weight 7 000 to 9 000) buffered in RPMI 1640;
fusion was performed in a tube held in a warm (370C) water bath. 1 to 1.5
ml of PEG solution warmed to 37 o C was added to the pellet by dropwise
addition over 1 to 1.5 minutes. The PEG solution was then diluted by
wo 94~ 21 3 7785 PCT/us94lo388s
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addition of warm RPMI 1640 to a volume of 50 ml. The fused cells were
then centrifuged at 200 x 9 for 10 minutes.
After aspila~ing the supernatant, the fused cell pellet was then
resuspended in 50 ml of 20% Complete Medium and plated at 100 ~l per
well, or a splenocyte density of 1.2 x 106viable cells per well. After
overnight incubation at 37OC and 10% CO2, 100 1.l of 20% HATG (HAT
medium supplemented with glycine) was added to each well. The cells
were visually scanned and feed periodically with 20% HATG for several
weeks, while the growth of the hybridomas was monitored and growing
wells screened for the presence of anti-TAT complex antibodies beginning
at day 12 to 14 post-fusion.
Example 4 - Screeninq for Antibodies that Bind S~ecifically to the TAT
1 5 ComPlex
Samples of supernatant collected from the growing hybridomas
generated in fusions TAT-4 and TAT-5 as described in Example 3 above
were screened for activity against TAT complex and ATIII using the antigen
20 coated microplates (1 ~g/ml) described in Example 2 above. Each of the
twenty-one antibodies which tested positive for binding to TAT complex and
negative for binding to ATIII was selected for the following further screening.
Further screening was carried out by performing sandwich ELISA
25 tests in the presence of ATIII (up to 300 ~g/ml) in solution and by
performing sandwich ELlSAs on plasma samples spiked with TAT complex
- (up to 50ng/ml). For each ELISA assay performed, 20 ~l of supernatant
was added to 80 ~LI of Sample/conjugate Diluent in wells of the coated
microplates. The new antibodies were used as the capture (10~Lg/ml) and
30 the conjugate used was a TAT complex monoclonal (D2M). Antibodies
WO s4~As66 PCT/US94103885
2,~3~ S - 20 -
which showed a low blank, no cross-reactivity with ATIII and which could
detect decreasiny TAT levels in linearly diluted plasma were selected.
Incut-~tions: TAT in diluent or plasma/ATIII in diluent 2 hours at room
temperature; conjugate 1 hour room temperature; substrate 20 min. room
5 temperature. On the basis of such successive screens, four antibodies
were selected for further consideration D2M, C72, C44-T, and 5B46.
Example 5 - Affinity Data
The affinity constant for the C44-T capture antibody was determined
to be 3 x 10~ L/M. This constant was obtained using the method described
in Friguet, et al., Measurements of True Affinity Constant in Solution of
Antigen-Antibody complexes by Enzyme-Linked Immunoadsorbent Assay, J.
Immunol. Methods, 77:305 (1985).
The apparent affinity constant for the 5B46 conjugate antibody was
determined to be 3.8 x 107. This constant was obtained by performing a
competitive assay with HRP-conjugated and unconjugated antibody on
antigen coated plates (1~g/ml TAT).
ExamDle 6 - Competition assay
Competition assays using different combinations of conjugate and
cold antibody were performed on C72 and C44-T plates. Plates were
coated with 10~g/ml of C44-T or C72 F (ab')2. Conj~g~tes were diluted to
1.1~g/ml and competing antibodies were added at concenL,alions of 110,
11, and 0.11 ~g!ml. TAT incubation 1 hour at room temperature,
Conjugate + IgG incubation 30 minutes at room temperature and slJbsLraLe
incub~tion 15 minutes at room temperature. Results showed that a number
of different combinations of the four TAT antibodies of the invention of D2M,
WO 94124566 PCIIUS94103885
2l37785
C72, C44-T and 5B46 did not interfere, or interfered very little with each
others binding.
Exam~le 7 - Determination of Antibodv sDecificity
Microtiter plates were coated with C44-T F (ab')2 (10,l~g/ml). Other
complexes of ATIII (factor XaATIII or factor IXaATIII) were incubated at
50ng/ml for 60 minutes at room temperature. The plates were then washed
three times and conjugate (5B46 labeled with HRP) was added at 5.5~g/ml.
After incubation for 30 minutes the plates were washed three times and
sub:,lrate was added. The reaction was stopped after 15 minutes for
treatment with 1M sulfuric acid. This antibody pair demonstrated no
significant cross-reactivity with either complex, specifically with factor Xa-
ATIII complex less than 0.004% cross-reactivity was observed; and with
factor IXa-ATIII complex less than 0.02% cross-reactivity was observed.
ExamPle 8 - Sandwich Assay Using Monoclonal Antibodies
Monoclonal C44-T was immobilized on a solid carrier of ",icroliLer
wells. A sample of patient plasma containing TAT complex and a measured
amount of monoclonal 5B46, covalently labeled with an enzyme, specifically
hor~eradish peroxidase, were then added to the immobilized C44-T for 30
minutes at 37 ~C. The unreacted conjugate (~B46) and any unreacted TAT
are then removed by washing. The amount of bound labeled antibody is
directly proportional to the concentration of TAT complex in the test sample.
This invention has been described in detail with reference to
preferred embodiments thereof. However, it will be appreciated that those
skilled in the art, upon consideration of the disclosure, may make
modification and improvements within the spirit and scope of the invention.
