AU676907B2 - Immunoassay and test kit for thrombin-antithrombin III complex - Google Patents

Description

WO 94/24566 PCT/US94/03885 -1- IMMUNOASSAY AND TEST KIT FOR THROMBIN-ANTITHROMBIN III COMPLEX BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates an immunoassay for thrombinaniithrombin III complex which comprises antibodies that specifically bind to the complex in a human fluid sample.

2. Background General agreement exists that blood coagulation or clotting includes three essential steps: 1) a complex of substances called prothrombin activator is formed, e.g. in response to rupture of a blood vessel or damage to blood itself; 2) the prothrombin activator catalyzes the conversion of prothrombin to thrombin; and 3) thrombin acts as an enzyme to activate platelets and to convert fibrogen into fibrin threads that enmesh platelets, blood cells and plasma to form the clot itself.

The enzymatic activity of thrombin can be regulated in various ways, including the formation of an inactive complex with antithrombin Ill, known as the thrombin-antithrombin III complex or the "TAT complex".

Antithrombin 11l, or "ATIII", 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 desirable to measure the amount of TAT complex in a human fluid sample. Determination of levels of the TAT complex will provide an WO 94/24566 PCT/US94/03885 -2indication of coagulation activity of a patient and aid in the assessment of thrombotic risk and other disorders such as disseminated intravascular coagulation.

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 XII and XI 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 concentrations 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 concentration of a human thrombin-antithrombin III (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 I -I WO 94/24566 PCT/US94/03885 -3thereof, 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 used herein, "specifically binds to the TAT complex" or other similar phrase indicates that the antibody or antibodies bind to the TAT complex but exhibit substantially or essentially no cross-reactivity with free not complexed) antithrombin III or antithrombin III complexes of other plasma factors, specifically factor Xaantithrombin III complex and factor IXa-antithrombin III complex. Preferably the mixture of antibodies of the assay bind to different epitopes of the TAT 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 antibodies with a human fluid sample, wherein at least one of the antibodies specifically binds to said TAT complex. Preferably the antibodies are 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 Xaantithrombin ill complex and factor IXa-antithrombin III complex. More preferably 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 or even less than about 0.1% cross-reactivity with antithrombin III and

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WO 94/24566 PCT/US94/03885 -4such 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 such lack of cross-reactivity with free (not complexed) thrombin, i.e. the antibodies of the invention preferably exhibit less than about 3% cross-reactivity with free thrombin in reaction with a human plasma sample, more preferably exhibit less than about 1% crossreactivity with thrombin, and still more preferably exhibit less than about 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, 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 III, less than about 0.005% cross-reactivity with factor IXa-antithrombin III complex and less than about 0.02% cross-reactivity with factor Xa-antithrombin III 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 antithrombin III or other complexes of antithrombin III. Thus references herein to an antibody that "binds" to TAT complex, rather than specifically binds thereto, indicates that the antibody binds to the human TAT complex and also may exhibit crossreactivity with antithrombin III, factor Xa-antithrombin III complex and/or factor IXa-antithrombin III complex. Antibodies, including monoclonal antibodies, that bind to the TAT complex and also exhibit cross-reactivity WO 94/24566 PCTUS94/03885 with antithrombin III and complexes thereof have been reported. See J.

Dawes, et al., Thrombosis Research, 36:397 (1984); S. Asakura, et al., Bioch. et Biophy. Acta, 952:37 (1988); Z. Hrkal, Hybridoma, 10(5):633 (1991); and European Published Patent Application No. 0391433A2. In this 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 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 complex that are not exhibited by free ATIII or thrombin.

More particularly, antibodies can be prepared by immunizing a mammal with a purified sample of a TAT complex, or an immunogenic peptide as discussed above, alone or complexed with a carrier. Suitable mammals 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 administered to the mammal by any of a number of suitable routes such as subcutaneous, intraperitoneal, intravenous, intramuscular or intracutaneous injection.

WO 94/24566 PCT/US94/03885 -6- 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 r her 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 (1975). 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 cells 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) lymphoid cell line having a marker such as hypoxanthine-guanine phosphoribosyltransferase deficiency (HGPRT) or thymidine kinase deficiency Preferably a myeloma cell is employed as the lymphoid 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, I I WO 94/24566 PCT/US94/03885 -7et al., Journal of American Medical Association, 199:549 (1967).

Hybridomas, grown after the fusion procedure, are screened such as b, radioimmunoassay or enzyme immunoassay for secretion of antibodies that bind specifically to the TAT complex, e.g. antibodies are selected that bind 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 expanded 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 10 4 L/M, more preferably greater than about 1 x 10 5 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 concentration of TAT complex of about 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 invention: withih assay values for control levels I and II 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 94/24566 PCT/US9403885 -8- Competitive assays have shown that preferred antibodies of the invention are at least substantially or essentially non-competitive, i.e. two or more antibodies of the invention show little or no binding interference among themselves when reacted simultaneously with a sample of TAT complex. By use of such antibodies in an immunoassay, a test sample can be incubated simultaneously 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 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 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 are reported to bind to TAT complex, but will only do so when contacted with a plasma 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 5B46, 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 given Accession No HB 11329 (TAT-4-C44-T-3:ST; expresses C44-T); Accession No HB 11328 (TAT-5-B46-4; expresses 5B46); Accession No HB 11327 (TAT-4-C72E; expresses C72); Accession No HB 11326 (TAT- 1-D2M; expresses D2M).

Thus antibodies of the invention are particularly suitable for use in an immunoassay for detecting and measuring the concentration of TAT complex in a human fluid sample. A particularly preferred immunoassay of the invention is a sandwich assay in which the capture antibody is immobilised using known methods such as physical adsorption onto the 10 surface of a support. Suitable supports include, polymer materials Ssuch 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 microtitre plate is generally preferred.

15 The detectably labelled antibody of the assay is formed by coupling o* e 9 the antibody to a reporter, e.g. a radioactive isotope, an enzyme, a fluorescent or luminescent reagent and the like. Typically used radioactive isotopes 1261, Tc 9 9m and 3H. Known isotope labb;ing methods include lactoperoxidase and Hunter-Bolton methods for 1251 and reduction methylation for 3 H. See A. Bolton, et al., Biochem. 133:529-539 (1972). Enzyme tags may include peroxidases, alkaline phosphatases, I1-Dgalactosidases, 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 labelling of an antibody with such enzymes are known in the art.

Methods suitable for a specific enzyme and antibody can depend on

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WO 94/24566 PCTUS94/03885 available reactive moieties on the enzyme and antibody and can be readily ascertained empirically.

The assay of the invention is illustrated by the following protocol using peroxidase as the label of the conjugate antibody. A test sample, e.g.

a human plasma sample, is added to a TAT complex antibody 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 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 contains 20 mmol Tris, 500 mmol 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 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 g/l imidazole, 17.5 g/l sodium chloride and 4 ml/liter Tween 20. If 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 94/24566 PCT/US94/03885 -11 a quantitative determination of the TAT complex concentration in the plasma test sample can be determined by compar,,,son 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 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 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 minutes. 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 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 using the antibodies of the invention and related apparatus. A suitable purification procedure provides coupling an antibody of the invention on an appropriate 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 94/24566 PCTIUS94/03885 12complex. The antibody can be suitably coupled onto the carrier by known methods, e.g. ite cyanogen bromide method and glutaraldehyde method, aqueous carbodiimide method, active ester method, and the like. The antibody also may be physically adsorbed on 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 lahpled with a radionuclide such as 11-indium. The labeled antibody then can be injected intravenously into a human and the 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 camera.

The antibodies of the invention also can be used therapeutically, e.g, as a carrier for drugs, particularly pharmaceuticals targeted for interaction in the blood clotting mechanism such as strepo-kinase, TPA and urokinase.

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 pharmaceutical can be administered to a human in therapeutically effective amounts by any of a number of known means, e.g. parenterally or orally.

For parenteral administration the antibody with attached pharmaceutical is typically administered in aqueous and non-aqueous sterile injection compositions as are known in the art. For oral administration the therapeutic of the invention may be administered to a human in discrete WO 94/24566 PCT/US94/03885 13units such as capsules or tablets each containing a predetermined amount of the therapeutic, as a solution or a suspension in an aqueous or nonaqueous liquid, as an oil/water liquid emulsion, in powdered carriers such as lactose or suc.)se, etc.

All documents mentioned herein are incorporated by reference herein in their entirse;.

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 III were reacted and the resultant TAT complex 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 6-mercaptoethanol, 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 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 I I WO 94/24566 PCT/US94/03885 14- Human Recombinant IL-6 (Collaborative Research Inc.) per ml medium.

STM used in the Examples was prepared by adding 5 Ag salmonella typhimurium mitogen (Ribi Immunochemical Research, Inc.) per ml medium.

Example 1 Immunization The following two groups of BALB/c female mice were immunized with TAT complex to provide spleen donoes for the PEG fusions, disclosed below, that generated monoclonal antibodies that specifically bind to the TAT complex.

1. Groupo 1 BALB/c female mice were sensitized with 7 g each of purified TAT complex in immunogen emulsion prepared as follows: 0.125 ml TAT complex (325 ig/ml in 50 mM tris/50 mM NaCI/0.1 M 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 Pg of purified TAT complex i.p. in an immunization protocol that lasted for approximately seven months.

2. Group 2 BALB/c female mice were sensitized with 10 pg each of purified TAT complex in immunogen emulsion prepared as follows: 0.120 ml TAT complex (321 gg/ml) 1.000 ml Complete Freund's Adjuvant 0.880 ml Dulbecco's Phosphate Buffered Saline WO 94/24566 IFCT/IUS94/03885 Each mouse was injected with 0.5 ml of this immunogen emulsion i.p. The mice were boosted with doses of 10 to 11 pg of TAT complex i.p.

in an immunization protocol that lasted approximately nine weeks.

Example 2 ELISA Assay for TAT Antibodies 1. Coating 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 ug/ml. 100 p. of each solution (10 ng TAT complex or ATIII) was placed in each well of the 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 2-8*C until use.

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 incubated on 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 antibody (latron) was used as a positive control in all assays.

Example 3 Preparation of Hybridomas Hybridomas secreting monoclonal antibodies to TAT were generated by two cell fusions. The PEG fusion technique employed based upon the WO 94/24566 PCT/US94/03885 16technique 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 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 Splenocvte 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 g 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 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 Application, 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 counting methods.

Myeloma preparation The myeloma cells were harvested mechanically, pooled, and centrifuged at 200 x g for 10 minutes after which the supernatant was WO 94/24566 PCT/US94/03885 17removed by aspiration, and the cells resuspended in 50 ml of Complete Medium. The viable cell count of the myeloma cells was 2.9 x 106 viable cells per ml at a viability of 76%, Fusion of the sDlenocvtes 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 10 8 The volume was brought up to 50 ml with cold 20% Complete Medium without serum and the cells then centrifuged at 200 x g 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 (37 C) water bath. 1 ml of PEG solution warmed to 37 C was added to the pellet and incubated for 1 minute. The PEG solution was then diluted by addition of ml of warm 20% Complete Medium without serum. The fused cells were incubated at 37 C for 10 minutes and then centrifuged at 200 x g for minutes.

The fused cell pellet was then resuspended in 50 ml of Complete Medium and plated at cell densities of 1.09 x 105 to 4.26 x 10 per well. The cells were plated in a final volume of 200 pl per well of Complete Medium, 20% Complete Medium with 2.5 Ag/ml STM or Complete Medium with 250 units/ml IL-6. After overnight incubation at 37 C and 10%CO 2 one half of the medium in each well was aspirated and the cells feed with 20% HAT, 20% HAT with 5 Mg/ml STM or 20% HAT with 500 units/ml IL-6. The cells were visually scanned and feed periodically WO 94/24566 PCT/US94/03885 18with these media 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.

2. Fusion of Hvbridoma Splenocyte preparation 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 ml of RPMI 1640; the viable cell count of the splenocytes was 6 x 107 at a viability of 95% by trypan blue exclusion technique.

Mveloma 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 splenocvtes and mveloma 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 107. 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 (37*C) water bath. 1 to ml of PEG solution warmed to 37 C was added to the pellet by dropwise addition over 1 to 1.5 minutes. The PEG solution was then diluted by 1 WO 94/24566 PCT/US94/03885 -19addition of warm RPMI 1640 to a volume of 50 ml. The fused cells were then centrifuged at 200 x g for 10 minutes.

After aspirating the supernatant, the fused cell pellet was then resuspended in 50 ml of 20% Complete Medium and plated at 100 ul per well, or a splenocyte density of 1.2 x 106 viable cells per well. After overnight incubation at 37'C and 10% CO,, 100 gl 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 Screening for Antibodies that Bind Specifically to the TAT 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 coated microplates (1 ag/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 tests in the presence of ATIII (up to 300 /g/ml) in solution and by performing sandwich ELISAs on plasma samples spiked with TAT complex (up to 50ng/ml). For each ELISA assay performed, 20 ul of supernatant was added to 80 pl of Sample/conjugate Diluent in wells of the coated microplates. The new antibodies were used as the capture (10pg/ml) and the conjugate used was a TAT complex monoclonal (D2M). Antibodies WO 94/24566 PCT/US94/03885 which showed a low blank, no cross-reactivity with ATIII and which could detect decreasing TAT levels in linearly diluted plasma were selected.

Incubations: TAT in diluent or plasma/ATIII in diluent 2 hours at room temperature; conjugate 1 hour room temperature; substrate 20 min. room 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'104 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, .7:305 (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 co;npetitive assay with HRP-conjugated and unconjugated antibody on antigen coated plates (1/g/ml TAT), Example 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 10pg/mi of C44-T or C72 F (ab') 2 Conjugates were diluted to 1.g/ml and competing antibodies were added at concentrations of 110, 11, and 0.11 gg/ml. TAT incubation 1 hour at room temperature, Conjugate IgG incubation 30 minutes at room temperature and substrate incubation 15 minutes at room temperature. Results showed that a number of different combinations of the four TAT antibodiu. the invention of D2M,

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WO 94/24566 PCT/US94/03885 -21 C72, C44-T and 5B46 did not interfere, or interfered very little with each others binding.

Example 7 Determination of Antibody Specificity Microtiter plates were coated with C44-T F (ab') 2 (10.g/ml). Other complexes of ATIII (factor XaATIII or factor IXaATIII) were incubated at for 60 minutes at room temperature. The plates were then washed three times and conjugate (5B46 labeled with HRP) was added at After incubation for 30 minutes the plates were washed three times and substrate 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 microtiter wells. A sample of patient plasma containing TAT complex and a measured amount of monoclonal 5B46, covalently labeled with an enzyme, specifically horseradish peroxidase, were then added to the immobilized C44-T for minutes at 37*C. The unreacted conjugate (5B46) 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.

Claims (26)

1. An immunoassay for detection of a human thrombin-antithrombin III complex in a human fluid sample, comprising: contacting a first antibody with thb fluid sample; and contacting a second antibody with the fluid sample, wherein each of the first and second antibodies binds to the human thrombin-antithrombin III complex, wherein at least one of the first and second antibodies specifically binds to the human thrombin-antithrombin III complex such that said antibody binds to the thrombin-antithrombin III complex but exhibits Ssubstantially or essentially no cross-reaction with free antithrombin III, or antithrombin III complexes of other plasma factors and wherein the amount of binding of at least one of the first and second antibodies to the thrombin-antithrombin III complex is detected directly or indirectly. .1

2. The immunoassay of claim 1 wherein both of said antibodies specifically bind to the human thrombin-antithrombin III complex. o•9

3. The immunoassay of claim 1 wherein the first and second antibodies are essentially non-competitive with respect to binding to the complex.

4. The immunoassay of claim 1 wherein the first and second antibodies are contacted substantially simultaneously with the fluid sample.

The immunoassay of claim 1 wherein the first antibody is contacted with the fluid sample and then the second antibody is contacted with the fluid sample. (Y i T. a\

6. The immunoassay of claim 1 wherein the first antibody is adsorbed onto a solid matrix and the second antibody is conjugated to a detectable label.

7. The immunoassay of claim 1 wherein the fluid sample includes a quantity of TAT complex and the quantity of TAT complex in the fluid sample is determined.

8. The immunoassay of claim 1 wherein the first and second antibodies are monoclonal antibodies. o l* *4*

9. The immunoassay of claim 1 wherein the fluid sample is plasma.

10. The immunoassay of claim 1 wherein the at least one antibody that specifically binds to the thrombin-antithrombin III complex exhibits less than about 1% cross-reactivity with each of thrombin, antithrombin, serine protease factor Xa-antithrombin III complex, and serine protease factor IXa- antithrombin 1II complex.

11. The immunoassay of claim 1 wherein the at least one antibody that specifically binds to the thrombin-antithrombin III complex exhibits less than about 0.2% cross-reactivity with each of thrombin, antithrombin, serine protease factor Xa-antithrombin III complex, and serine protease factor IX,-antithrombin III complex. -i 1

12. The immunoassay of claim 1 wherein the first antibody has at least the following characteristic of D2M, C72, C44-T or 5B46: an affinity constant with the human thrombin-antithrombin III complex of greater than about 1 x 104 I/m; and wherein said antibody exhibits less than about 1 cross-reactivity with each of thrombin- antithrombin, serine protease factor X-antithrombin III complex, and serine protease factor IX-antithrombin III complex.

13. The immunoassay of claim 1 or 12 wherein the second antibody has at least the following characteristic of D2M, C72, C44-T, or 5B46: l* an affinity constant with the human thrombin-antithrombin III complex of greater than about 1 x 104 I/m; and wherein said antibody exhibits less than about 1% cross-reactivity with each of thrombin- antithrombin, serine protease factor X,-antithrombin III complex, and serine protease factor IXa-antithrombin III complex.

14. The immunoassay of claim 6 wherein the first antibody has at least the following characteristic of C44-T and the second antibody has at least the following characteristic of 5B46: an affinity constant with the human thrombin-antithrombin III complex of greater than about 1 x 104 I/m; and wherein each of said antibodies exhibits less than about 1% cross-reactivity with each of thrombin-antithrombin, serine protease factor X-antithrombin III complex, and serine protease factor IX,-antithrombin III complex.

The immunoassay of claim 1 wherein the second antibody is D2M, C72, C44-T or 5B46. r

16. The immunoassay of claim 6 wherein the first antibody has at least the following characteristic of C44-T and the second antibody has at least the following characteristic of 5B46: an affinity constant with the human thrombin-antithrombin III complex of greater than about 1 x 104 I/m; and wherein each of said antibodies exhibits less than about 1% cross-reactivity with each of thrombin-antithrombin, serine protease factor Xa-antithrombin III complex, and serine protease factor IXa-antithrombin III complex.

17. The immunoassay of claim 6 wherein the first antibody is C44-T and the second antibody is 5B46.

18. A test kit when used in the immunoassay of any one of claims 1 to 17 for the detection of a human thrombin-antithrombin III complex in a test :sample, comprising a first and second antibody, wherein each of the first and second antibodies binds to the human thrombin-antithrombin III complox, wherein at least one of said antibodies can specifically bind to the human thrombin-antithrombin III complex, such that the at least one of said antibodies binds to the thrombin-antithrombin III complex but exhibits S** substantially or essentially no cross-reaction with free antithrombin III, or antithrombin III complexes of other plasma factors and wherein the binding of at least one of the first and second antibodies to the thrombin- antithrombin III complex is detected directly or indirectly.

19. The test kit of claim 18 wherein both of said antibodies specifically bind to the human thrombin-antithrombin III complex.

I The test kit of claim 18 wherein one of said antibodies specifically binds to the human thrombin-antithrombin III complex, and the other of said antibodies binds to the human thrombin-antithrombin III complex.

21. The test kit of claim 18 wherein the first antibody has at least the following characteristic of D2M, C72, C44-T or 5B46: an affinity constant with the human throrbin-antithrombin III complex of greater than about 1 x 104 I/m; and whet in said antibody exhibits less than about 1% cross-reactivity with each of thrombin- S' antithrombin, serine protease factor Xa-antithrombin III complex, and serine 4 4 .4 protease factor IX-antithrombin III complex. o 4

22. The test kit of claim 18 wherein the second antibody has at least the foilowing characteristic of D2M, C72, C44-T or 5B46: an affinity constant with the human thrombin-antithrombin III complex of greater than about 1 x 104 I/m; and wherein said antibody exhibits less than about 1 cross-reactivity with each of thrombin- antithrombin, serine protease factor Xa-antithrombin III complex, and serine protease factor IXa-antithrombin III complex.

23. A monoclonal antibody that binds to a human thrombin-antithrombin III complex, with an affinity constant with the human thrombin- antithrombin III complex of greater than about 1 x 104 I/m; and wherein said antibody binds to the thrombin-antithrombin III complex and exhibits less than about 1% cross-reactivity with each of thrombin-antithrombin, serine protease factor X,-antithrombin III complex, and serine protease Sfactor IXa-antithrombin III complex. M 27

24. The monoctonal antibody of claim 23 that has at least the following characteristic of D2M, C72, C44-T or 5846: an affinity constant with the human thrombin-antithrombin III complex of greater than about 1 x 104 I/m; and wherein said antibody exhibits less than about 1% cross-reactivity with each of thrombin- antithrombin, serine protease factor Xa-antithrombin III complex, and serine protease factor IX-antithrombin III

25. The monoclonal antibody of claim 23 that is D2M, C72, C44-T or 5B46. 1

26. The immunoassay of claim 14 wherein the second antibody is D2M, *Dated this 19 day of September 1996 DADE INTERNATIONAL INC C72, C44-T or 5B46 and wherein the second bntibody is not the same as Pa the first orneys for the Applicanttibody. PETER MAXWELL ASSOCIATES 9.o Dated this 19 day of September 1996 DADE INTERNATIONAL INC Patent Attorneys for the Applicant PETER MAXWELL ASSOCIATES II I JN1'ERNATIONAL SJ? ARCH I I ,PORTI'jnterrmtional application No. PCT/US 94/03885 A. CI.ASSIFICATION OF SUBJE-CI MATTER IPC 5 G01N33/86 C12P21/08 According to International Patent Classification (ll'C or to both national classification and ll'C Ui. FIELDS SEA RC1ED Minimum documnentation searched (classification syslcn followed by classification symbols) IPC 5 G01N C07K Documentation searchcd other than minimum documentation to thc extent that such documents arc included in the fields searched Electronic data base consulted during thc international search (name of data base and, where practical, search tenns used) C. DOCUMENTS CONSIDERED TO BE RELEVANT Category Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No, Y THROMBOSIS RESEARCH, 1-23 vol.36, no.5, 1984, US. pages 397 409 J. DAWES ET AL. 'Monoclonal antibodies directed against human alpha-thrombin and the thrombin-antithrombin III complex' see the whole document Y EP,A,0 431 169 (TEIJIN LIMITED) 12 June 1-23 1991 see the whole document Y EP,A,O 479 721 (CIBA-GEIGY AG) 8 April 1-23 1992 see abstract; claims j;1Further document are listed in the continuation of box C. Patent family members are listed in annex. *Special categories of cited documents. T* later document published after the internatinal filing date A doumet dfinng te gnerl sate f te at wich s nt o pronty date and not in conflict wvith the application but 'A ouetdfiigtegnrlaeofncecitedih t o o, understand the principle or theory underlying the considered to be of particular rlvneinvention HE earlier document but published on or after the international W7( document of particular relevance; the claimed inv~ention filing date cannot be considered novel or cannot be considered to 'I1' document which may throw doubts on pnionity claim(s) or involve an inventive step when the document is taken alone which is cited to establish the publication date of another document of particular relevance;, the claimed invention citation or other special reason (as spcitfied) cannot be considered to involve an inventive step when the document referring to an oral disclosure, uce, exhibition or document is combined with one or more other such docu. other meant ments, rich combination beng obvious to a person skilled document published pnior to the international filing date hut in the art. later than the prioity date claimed W& document member of the same patent family Date of the actual completion oF the international search Date of mailing of the international search report 29 July 1994 I0.D. Y Name and mailing address of the ISA Authorized officer rEuropean Patent Office, $818 Patentlaan 2 Nt.- 2280 (IV Rilswilk Tel. 31.70) 340.2040, Tx. 31 651 cplo rdrifth. Fax: t 31.70) 340.3016Grf ih G Fotrq PCT!ISAJ210 (itcond theet) Ouly 190) page 1 of 2 INTIERNATIONAL SEARCH REPOWI' Internationl application No. PCT/US 94/03885 C.(Conunuation) DOCUMENTS CONSIDERED TO DRE RELEVANT Category Citation of docurnent. with indication, wh~ere appropriate, of thc relevant passages RelovAnt to claim No. A EP,A,O 391 433 (TEIJIN LIMITED) 10 October 1990 cited in the application A JOURNAL OF IMMUNOLOGY vol.127, no.1, July 1981, BALTIMORE US pages 239 244 F. C. MCOUFFIE ET AL. 'Antigenic changes produced by complex formation between thrombin and antithrombin-III' rotn PCTIISN210 (conUituation of smoid thet) (July 1992) page 2 of 2 *rINTERNATIONAL SEARtCH REPORT' inentoa-plcto o inofainon patent f~1tly mrcathcrx PnTerntina 9 pplc0 Patent document Publication Patent family Publication cited in search report date member(s) -1date EP-A-0431169 12-06-91 AU-B- 628921 24-09-92 AU-A- 4948390 13-08-90 CA-A- 2025563 20-07-90 WO-A- 9008320 26-07-90 EP-A-0479721 08-04-92 AU-A- 8471391 20-05-93 EP-A-0391433 10-10-90 AU-B- 628960 24-09-92 AU-A- 5297390 11-10-90 CA-A- 2014016 07-10-90 JP-A- 3048158 01-03-91 romn pCTIISA'210 (patent family annetx) (July 1992)