JPH07508103A - Immunoassay and test kit for thrombin-antithrombin 3 complex - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Immunoassay and test kit for thrombin-antithrombin 111 complex Cut Background of the invention 1. Field of invention The invention includes antibodies that specifically bind to such complexes in human fluid samples. Concerning an immunoassay for the thrombin-antithrombin complex. .

2. Background It is generally agreed that blood clotting involves three essential steps. Sunawa l) prothrombin activity, e.g. in response to rupture of a blood vessel or damage to the blood itself; A complex of substances called prothrombin activator is formed.2) This prothrombin activator 3) Thrombin catalyzes the conversion of prothrombin to thrombin activates platelets and fibrinogen (which binds platelets, blood cells and plasma). Acts as an enzyme that converts fibrin into fibrin fibers (which form blood clots themselves).

The enzymatic activity of thrombin is expressed by the thrombin-antithrombin complex or rTAT. Involves the formation of an inactive complex with antithrombin, known as the complex It can be controlled in various ways. Antithrombin ■ or FAT III'' is A plasma protein that forms a complex with several serine proteinases in addition to thrombin. It is one of the teinase inhibitors. Thus, ATIII is a serine protease. It is known that it forms complexes with factor Xna, factor Xa, Xa, and factor Xa. ing.

It is desirable to measure the amount of TAT complex in a human fluid sample. TAT complex Quantification of the level of coalescence provides an indication of a patient's clotting activity and the risk of thrombosis. and other diseases such as disseminated intravascular coagulation.

Assays for TAT complexes in human plasma include, for example, TAT complexes as well as , XIa, Xa, and complexes of factors IXa and ATI, etc. Selecting the concentration of TAT complex rather than measuring the concentration of the total complex of ATIll[in A selective assessment must be provided. Measurement of the “total” complex of such ATIs provides an indication of the equilibrium of several reactions, some of which are associated with specific TAT complexes. It will reflect conditions other than those related to level. For example, factors X and XI Contact activation of primarily results in activation of the complement and kinin systems, thus XII a-ATn [and X I a-ATn [complex is not an indicator of thrombotic risk Will.

Therefore, one means to quantify the level of TAT complex in human fluid samples. It would be desirable to have one. The assay measures the total ATI complex in the sample. It provides a selective assessment of the concentration of TAT complex in a test sample rather than a measurement. has an assay for the concentration of TAT complex in a human fluid sample. That would be especially desirable.

Summary of the invention The present invention provides the ability to detect human thrombin-antithrombin I [ Provides an immunoassay for the detection and concentration measurement of TAT) complexes. This a The assay involves incubating a human fluid sample with at least two antibodies or immunoreactive molecules thereof. contact with a monoclonal antibody, preferably a monoclonal antibody or a reactive fragment thereof. wherein at least one of the antibodies is present in a liquid sample. Binds specifically to the TAT complex. Preferably, each of the antibodies in this assay is T Binds specifically to the AT complex. rTAT used here? specifically binds to 3j coalescence. "combining" or another similar phrase means that the antibody or antibodies are However, free (i.e., uncomplexed) antithrombi antithrombin complexes of Xa and other plasma factors, especially factor Xa-antithrombin complexes. The antithrombin complex and the antithrombin complex are substantially or essentially This shows that there is no cross-reactivity. Preferably, the antibodies in this assay The mixture of antibodies binds to different epitopes of the TAT complex, i.e. The body is not competitive. In addition, the antibodies of the immunoassays of the invention are preferably At the same time, a human liquid supply is used so that only one incubation and washing step is used. sample. Other aspects of the invention are disclosed below.

Detailed disclosure of the invention The immunoassay of the invention involves the reaction of at least two antibodies with a human fluid sample. wherein at least one of the antibodies is specific to the TAT complex. Join. Preferably, the antibodies are monoclonal antibodies and the assay Both of the two antibodies specifically bind to the TAT complex. Specific to TAT complex The antibodies of the assay that bind to or in similar assays on human plasma samples, AT■, factor Xa - antithrombin ■ complex and factor 1 Xa - antithrombin ■ complex, Preferably it exhibits less than about 3% cross-reactivity. More preferably, the anti-inflammatory agent of the present invention The body has less than about 1% cross-reactivity f with ATII [and such ATIII complexes]. 1. Does not indicate L. Even more preferably, the antibodies of the invention are as described in Example 7 below. In the described assay or similar assays using human plasma samples, less than about 0.2% of tithrombin and such antithrombin complexes; Furthermore, it exhibits less than about 0.1% cross-reactivity. The antibody of the present invention may be exhibiting a lack of cross-reactivity with thrombin (unformed), i.e. The antibody of the present invention has approximately 3% free thrombin in reaction with human plasma samples. less than about 1% cross-reactivity, more preferably less than about 1% cross-reactivity with thrombin; Even more preferably, the contact reaction of the antibody with a human plasma sample results in the release of free trophins. It shows less than about 0.2%, even less than about 0.1% cross-reactivity with Both are also preferred. In particular, particularly preferred monoclonal antibodies of the invention are human Upon contact with a liquid sample, it binds to the human TAT complex and binds to thrombin. of about 0. (Cross-reactivity less than 105%, approximately 0.005% with antithrombin■ Less than 0.005% cross-reactivity with the 1st Xa-antithrombin complex Full cross-reactivity and less than about 0.02% with the Xa-antithrombin complex. It was found that it showed only cross-reactivity. "Antibody J, r of the present invention" used herein Antibody J or other similar term refers to whole immunoglobulin as well as )'ab, )'a specifically binds to the TAT complex, including b', )'(ab')z and F(V). containing antigen-binding or immunoreactive fragments that are compatible with each other.

If only one of the antibodies in the immunoassay specifically binds to the TAT complex, If so, the other antibodies in the assay must bind to the TAT complex, but only if For example, cross-reactivity with antithrombin or other antithrombin complexes. may also be shown. Here, [binds] to the TAT complex (specifically binds to it) When referring to an antibody, the antibody binds to the human TAT complex. Also, antithrombin ■, factor Xa-antithrombin ■ complex and/or It may also show cross-reactivity with the factor 1 Xa-antithrombin complex. show. It binds to the TAT complex and cross-reacts with antithrombin and its complex. antibody (monochrome-(199+)) and European Patent Application Publication No. 03 See 911133A2. In this embodiment of the invention, preferably TAT Antibodies that specifically bind to the complex are used as carrier antibodies and sandwich-tied. immobilized on a substrate in a sample assay, and cross-reactive antibodies are labeled. However, it is possible to use cross-reactive antibodies as carrier antibodies. using an antibody that specifically binds to the TAT complex as a labeled conjugate. Can also be done.

Human fluid samples used in the assays of the invention include, for example, blood or urine. etc., any sample containing a TAT complex. Typically, blood A plasma sample is used.

Antibodies of the invention can be prepared using techniques generally known in the art. and typically for purified samples of human plasma TAT complexes. Created. The antibody is not expressed by free ATRI or thrombin. from an immunogenic peptide comprising one or more epitopes of the TAT complex. It may be extracted.

More specifically, the antibody is a purified sample of TAT complex or an immunogen as described above. Immunizing mammals with sex peptides (alone or complexed with carriers) It can be prepared by Suitable mammals include sheep, goats, rabbits, guinea pigs, Typical laboratory animals such as rats and mice are included. rats and mice, In particular, mice are preferred for obtaining monoclonal antibodies. antigen to said mammal by any of a number of suitable routes, such as subcutaneous, intraperitoneal, intravenous, intramuscular or subcutaneous injection, etc. It may also be administered by this method.

Preferably, immunization is by subcutaneous, intraperitoneal, or intravenous injection. Optimal immunization interval, Dosages etc. can vary over a relatively wide range and may be determined experimentally based on this disclosure. can be done. The typical procedure involves injecting the antigen several times over many weeks. Accompany. Antibodies are collected from the immunized animal's serum by standard techniques and TAT replicated. Screened to find antibodies specific for the conjugation. monoclonal antibody can be produced in cells that produce antibodies, and those cells can be hybridized. monochrome cells by using standard fusion techniques to form doma cells. can be used to generate null antibodies. G.Kohler et. al., Nature, 256: 1156 (1975). and. Typically, this involves fusion of antibody-producing cells with immortal cell lines such as myeloma cells. It involves producing hybrid cells. As an alternative, monoclonal Husa et al., 5science, 256:1275. One suitable prototype that can be produced from cells by the method of (+989) Cole has a composition comprising purified TAT complex for about 2 to 7 months. Provide intraperitoneal immunization of mice, which is performed over a period of time. Then from the immunized mice Pancreatic cells can be removed. Prior to removal of pancreatic cells, the immunized mouse sera from the school are assayed for titers of antibodies specific for the TAT complex. . The isolated mouse pancreatic cells were then treated with hypoxanthine-guanine phosphoribo siltransferase deficiency (HGPRT-) or thymidine kinase deficiency (TK - a suitable homologous or heterologous (preferably homologous) phosphorus with a marker such as fused to the Pacytic cell line. Preferably, bone uNm cells are used as the lymphoid cell line. It will be done. Myeloma cells and pancreatic cells, for example, have a myeloma cell:spleen cell ratio of about 1:4. and mixed. Cells were fused by polyethylene glycol (PEG) method. You can do that. G, Kohler at al, Nature (above) checking ... The hybridoma thus cloned is, for example, RPMI-1 Grow in a medium such as 6110. G, E, More et al.

Journal of American Medical As5ociat ion, +99: See 549 (+967). Proliferated after fusion operation The developed hybridomas are radioactive for secreting antibodies that specifically bind to the TAT complex. Screened by immunoassay or enzyme immunoassay, etc. For example, an antibody is selected that binds to the TAT complex but not to ATII.

This screening preferably uses an enzyme immunoassay. Like that Hybridomas that show positive results in a comprehensive screening are developed and used in the limiting dilution method. Therefore, it can be cloned.

Further screening will explore TAT replication in solution as well as in human fluid samples. A procedure is preferably performed to select antibodies capable of binding the conjugate.

The antibodies of the invention preferably have a preferably greater than about IX+O'L/M, more preferably greater than about 1×+O''L/M shows a large affinity constant for the TAT complex, and such a constant is similar to that of Frigue t et al, J, Immunol, Methods, 77: 3 05 (1985).

Preferred antibodies of the present invention are highly effective against TAT complexes in human fluid labs. High sensitivity. For example, a preferred antibody is about 0.55 ml of plasma sample per ml of plasma sample. Additive material with a concentration of TA Tra aggregates of n g or more that is essentially 100% It was detected with accuracy with a recovery rate of .

The immunoassays of the invention generally exhibit excellent accuracy. For example, preferred embodiments of the present invention The following results were obtained for the assay. That is, the control level I at n=80 Coefficients of variation within the same-lot assay for and ■ were 3.9% and 6, respectively. 8% and coefficient of variation of 9.3% for lot-to-lot assay precision values for n=80, respectively. and 12.2%.

Competitive assays are those in which the preferred antibodies of the invention are at least substantially or essentially non- Showed to be competitive. That is, the two or more antibodies of the present invention Samples exhibit little or no binding interference between themselves when reacted simultaneously. I don't. By using such antibodies in immunoassays, test samples can be samples simultaneously (i.e., in a single incubation step) in the assay. can be incubated with both combined capture antibody and labeled conjugate antibody. Wear. This applies to each of the capture and conjugate antibodies (e.g. Utilize separate incubation and washing steps (to avoid binding interference) It is quite different from previous assays that

Preferred antibodies of the invention contain undiluted or TAT-conjugated antibodies. Human liquid samples, especially human plasma samples, only diluted with solutions It was also found that it could bind to the TAT complex within. In particular, if the liquid sample is When diluted with only a smaller volume of diluent, the preferred antiseptic of the present invention It was found that the body can bind to TAT complexes in human fluid samples. this is, binds to the TAT complex, but to a significant extent (e.g. plasma or other test sample It binds only when in contact with plasma samples diluted (more than 0 times diluted). This antibody is completely different from at least some of the previous antibodies that have been reported. the Such dilutions increase the complexity of TAT complex assays utilizing such antibodies. It is undesirable to obtain.

Particularly preferred monoclonal antibodies include 02M, C72, CIJ4-T and 5 B46, a mouse monoclonal that specifically binds to the TAT complex. antibody. Hybridomas expressing such monoclonal antibodies are + 2301 Parklawn Drive, Rockville, Mary land 20852 American Type Cu1ture Co1 1ection (AT CC) (expressing D2M).

The antibodies of the invention are thus useful for the detection and concentration of TAT complexes in human fluid samples. Particularly suitable for use in immunoassays for measurements. Features of the present invention One of the preferred immunoassays for This is a sandwich assay in which the capture antibody is immobilized using known methods. suitable Supports include polymeric materials such as polypropylene or polystyrene; Glass, metal, cross-linked dextran, agarose, etc. The support is a tray , can be of various shapes such as balls, rods, test tubes, etc. micro tie tarp Trays with wells, such as trays, are generally preferred.

The detectably labeled antibodies of this assay can be linked to a reporter such as a radioisotope. Formed by binding to a topical element, an enzyme, a fluorescent or luminescent reagent, etc. be done. Typically used radioactive isotopes are +251,! 1 wa They are Tc and 1H. Known isotope labeling methods include Toperoxidase and Hunter-Porton method, reductive methylation for 3H law is included. A. Bolton et al., Biocham, J. , 133: 529-539 (+972). For enzyme installation , peroxidase, alkaline phosphatase, β-D-galactosidase, Includes glucose oxidase and others. Peroxidases are particularly preferred.

Peroxidases are found in various sources, such as horseradish, pineapple, and yam. Selected from those obtained from sugarcane, java beans, corn, etc. Ru. Horseradish peroxidase is a particularly preferred material. By covalent bond Means for such enzymatic antibody labeling are known in the art. . Suitable methods for particular enzymes and antibodies include It depends on the reactive moiety and can be readily confirmed experimentally.

The assay of the present invention is based on the following protocol using peroxidase as a label for the conjugate antibody. Illustrated by rotor. If the test sample, e.g. human plasma sample, TAT-conjugated antibodies (i.e. , an antibody that binds to the TAT complex), an antibody-antigen reaction is performed, followed by , peroxidase-labeled TAT-conjugated antibodies obtained as outlined above. The conjugate is added and then further antibody-antigen reactions are performed. Applicable. Appropriate Diluents include those known in the art for use in immunoassays. Contains diluents. Especially for dissolving antibodies for contact with test samples One preferred solution is 20mM Tris, UOOmM sodium chloride, 0. 05 mg/ml mouse IgG and 5% BSA.

Preferably, both the carrier antibody and the conjugate antibody of the assay of the invention are TAT Antibodies that specifically bind to the complex and are bound to the support, human fluid samples and and the labeled antibody are incubated together, followed by the reaction of the TAT complex. A single wash stage to remove all unreacted labeled antibodies and human plasma samples Floor is done. Suitable cleaning agents include: Cleaning agents known in the art are included. One of the particularly preferred wash buffers is 27.2g/j7 imidazole, +7.5g/47 sodium chloride and 4　ml　　/　I! Contains Tween 20. Perioki if necessary Add the substrate for the sidase to the assay and measure the absorbance or fluorescence of the resulting material. The reaction products are assayed for enzymatic activity. Detected The combined amount of labeled antibody is the amount of TAT complex in the human plasma sample subjected to the assay. is directly proportional to the concentration of In this way, the absorbance or fluorescence of the test sample is determined by a known amount of T. By comparing the absorbance values obtained from standard solutions containing the AT complex, blood Quantitative measurements of TAT complex concentration in plasma test samples can be achieved. trial To facilitate the interpretation of values obtained from test samples, a number of standard solutions are available. It is desirable to create a calibration curve from the absorbance values.

One particularly preferred immunoassay of the invention was performed as follows. Acquisition mode Labeled with noclonal antibody and horseradish peroxidase (HRP) The conjugated monoclonal antibody was incubated with human plasma samples for 30 minutes at 37°C. Incubate for a while. The plate was then washed with HRP substrate for 15 minutes at room temperature. Incubate for minutes and quantify bound conjugate.

One of the preferred immunoassays of the present invention is for individuals with disseminated intravascular coagulation. (n = 15) were used to measure plasma samples. The assay results are -TAT complex significantly elevated in plasma samples of all patients except humans showed the level. 12 of the individuals tested were higher than 160 ng/mi' had TAT complex levels.

The TAT conjugate is prepared using a carrier conjugate conjugated to one or more antibodies of the invention. can be purified from human plasma samples, and the invention therefore provides the antibodies and Methods for obtaining purified TAT complexes using associated equipment are included. suitable In one purification procedure, the antibodies of the invention are transferred to a gel or resin such as in the art. binding to a known suitable carrier, then loading the carrier into a column, and then loading the carrier into a column. Flow the sample solution containing the TAT complex through the column to selectively release the TAT complex. Adsorb the union. The antibody can be prepared using, for example, the cyanogen bromide method and the glutaraldehyde method, onto the carrier by the aqueous carbodiimide method, active ester method, or other known methods. Can be combined appropriately. The antibody may also be physically adsorbed onto the surface of the carrier. You may let them.

Antibodies of the invention can also be used to localize and monitor the TAT complex in vivo. Can be used to monitor. For example, one or more anti- The body can be labeled with a radionuclide such as indium-11. labeled Antibodies are then injected intravenously into humans and where the labeled antibodies accumulate. The person is scanned to measure. Labeled antibodies are typically High concentrations of TAT complexes, such as areas where blood clotting is occurring, such as bleeding sites. differentially accumulates in different areas. The amount of labeled antibody is measured using a scintigraphy camera. can be quantified by known scanning methods, such as using

Antibodies of the invention may also be directed to interactions with, for example, drugs, particularly in the blood coagulation mechanism. drugs such as streptokinase, TPA and urokinase. It can also be used therapeutically as a carrier for cancer. The drug is labeled as specified above. the specificity of the antibody or the desired pharmacological effect of the drug. can be attached to the antibodies of the invention by linkage through non-detrimental coexisting bonds. . The drug-attached antibody can be administered in a therapeutically effective amount, e.g., parenterally or orally. It can be administered to humans by any number of known means, including orally. Non For oral administration, drug-attached antibodies are typically It is administered as aqueous and non-aqueous sterile injectable compositions known in the art. oral administration For example, the therapeutic agents of the invention can be packaged in capsules, each containing a predetermined amount of the therapeutic agent. solutions in aqueous or non-aqueous liquids or as discrete units such as cells or tablets; as a suspension, as an oil/water liquid emulsion, as a powder carrier such as lactose or sucrose; It can be administered to humans in the body.

All documents mentioned herein are incorporated herein by reference in their entirety.

The following non-limiting examples illustrate the invention.

General comments Purified samples of TAT complexes used in the following examples were G, Elgue et al, Thrombosis and Hemosta sis, 63(3):'435 (+990) obtained. In short, by reacting thrombin and antithrombin ■, the obtained The TAT complex was isolated by gel filtration. 20 used in the examples % complete medium had the following composition per 100 mJ':

200mM 1-glutamine, 1m! , 7.5% sodium bicarbonate, 1 mA’ , 5000 units/ml penicillin + 5000μ2ζ/ml streptomycin N, 1mA? 0.1M 2-mercaptoethanol, '-, 05mA', fetal bovine serum, 2 0 mJ, and +OOmI with L-glutamine-free RPMI 1640 (Gibco)! To.

The HΔT medium used in the Examples had the following composition. That is, In 100ml of complete medium, 1mf of 100x hyboxanthin thymidine and 2mj 7 portions of 50x aminopterin were added. The HATG medium used in the examples was It had the following composition. 100mI of HAT medium! Then, 1rnj+ 100x group Added ricin. The IL-6 used in Example 1) was used at a medium concentration of 1 mA? Hit 50 0 units of human recombinant IL-6 (Collaborative Re5search Inc.). The STM used in the example is Salmonellatyphimurium phytodiae per rrl of medium (Ribi Immunochemical Re5earch, Inc.

) was prepared by adding 5 μg.

Example 1 - Immunization method Monoclonal antibodies that specifically bind to the TAT complex were prepared as described below. To provide pancreatic donors for the production of PEG fusions, the following two groups of BALB/c Female mice were immunized with the TAT complex.

1. 1st group 7 μg of each purified TAT replicate in an immunodiene emulsion prepared as follows. In combination, BALB/c female mice were sensitized.

0.125ml TAT complex (50mM) 150mM sodium chloride/ 0. IM EDTA + p 325μg/m in H7-'4 Iri 1.500 ml Complete Freund's Adjuvant L375 ml Dulbacco Lin Acid Buffered Saline Each mouse received 0.5 m of this Immunodien emulsion! ! belly Intracavitary injection. In an immunization protocol that lasted approximately 7 months, mice were A booster immunization was given by intraperitoneal administration of 108 g of purified TAT conjugate.

2. 2nd group 10 μg of each purified TAT in an immunodiene emulsion prepared as follows. BALB/C female mice were sensitized with the complex.

0, 120 ml of TAT complex (321 μg/mj2) 1.000 ml of Complete Freund's adjuvant 0.880 ml Dulbecco phosphate buffer Saline Each mouse was injected intraperitoneally with 0.5 ml of this immunodiene emulsion. I shot it. In an immunization protocol that lasted approximately 9 weeks, mice were exposed to 10 to 11μ Example 2 - TAT antibody boosted by intraperitoneal administration of TAT conjugate of ELISA assay 1, microtiter with TAT and AT II+ Coat the plate with 1 μg of TAT complex and ATII [each separately] diluted with coating buffer (carbonate buffer p) (9) to a concentration of ml. 100μ of each solution β (+Ong of TAT complex or ATI[) was added to each volume of the microtiter plate. The cells were placed in a well, sealed and incubated overnight at 2-8°C. Then the well Aspirate the contents and wash the plate once with wash/storage buffer, aspirate the wash solution. , and the plate was resealed. Store plates at 2-8°C until use. did.

2. Titer measurement of mouse serum The serum collected from each of the immunized mice of Group 1 and Group 2 of Example 1 above was , by ELISA on TAT complex-coated microplates. The titer of the combined antibody was determined. Serially diluted serum (usually 1 volume 00 (tested at dilutions from 0 to 1:3125000) on a microplate. incubated and probed with enzyme-labeled sheep or goat anti-mouse conjugates. , followed by reaction with substrate. The relative titer was determined according to the intensity of the color reaction and was 80 Read spectrophotometrically at 5 nm. For all assays, preimmune serum was used as a negative control, and a commercially available mouse TAT monoclonal antibody (1atron ) was used as a positive control.

Example 3 - Preparation of hybridomas Hybridomas that secrete monoclonal antibodies against TAT are produced by two types of cell lysis. created by the combination. The PEG fusion method used was that of G. Kohler et al. al, , Nature 256: 495 (+975) The myeloma cells used were HRPT-minus P3-X. 63-Ag3.653 (P3X) (ATCC CRL+580) Tea Tsuta. C Selection of hybrids was performed using HAT medium (hypoxanthine, aminopterin and thymidine). This was achieved using P3X myeloma cells that do not fuse produce purines. lacks the equipment to use hyboxanthin and the aminopropylene present in the medium. Because telin blocks endogenous synthesis of purines and pyrimidines, this medium Does not exist.

1. Preparation of fused tile cells to form hybridoma TAT-4 Pancreatic cells were prepared as described for the first group in Example 1 above. Obtained from mice immunized with TAT complex. using tweezers and needles Cells were harvested from the spleen and then placed in 12 ml of cold 20% complete medium (RPM) without serum. I161JO basis (Gibco). Cells were then incubated at 200 x g for 10 After centrifugation for a minute, the supernatant was aspirated and the cells were resuspended in cold medium.

This washing step was repeated twice and the cells were resuspended in a final volume of Oml. the tile cell Viable cell counts were determined with a 98% viability test using the Trivan blue exclusion method. It was 1.7 xlO@ pieces. The counting method used was Kruse, P.F., a. ndPatterson, M, ni, ads, Ti5sue Cultur e Methods and Applications.

M in Academic Press pp, 395-397: 1973. By Abshar, “Hemacytometer Counting” The procedure was as disclosed. In short, if a certain volume of cell suspension is equal to 0.02 % trypan blue and in a Hela cytometer by standard counting methods. Cells are counted.

myeloma preparation Myeloma cells were mechanically harvested, pooled, centrifuged at 200Xg for 10 minutes, and then Afterwards, the supernatant was aspirated and the cells were resuspended in 50 ml of 20% complete medium. . The myeloma cell viable cell count was 76% viable per 1 m47. The total number of viable cells was 2.9 x 1O6.

Fusion of tile cells and myeloma cells Add the tile cells and +5 ml of myeloma cell suspension to Il’l! Cell/Myeloma cell suspension Solution 4 = 1 combined for total viable cell count 2-+3x+O'. serum free The volume was brought up to 50+ nl with cold 20% complete medium, then the cells were Centrifugation was performed at xg for 10 minutes. Next, the cell pellet was placed in 50 m of this medium! ! So 2 Washed twice. After the final wash, the supernatant was aspirated and the pellet was incubated at 200×g for 3 Centrifugation was performed for a minute, and the remaining supernatant was aspirated. Then buffered in RPMI 16110 Cells were then fused with 40% PE(-; (molecular weight 7000-9000)). I let it happen. Fusion was performed in tubes kept in a warm (37°C) water bath. 37°C Add 1 ml of warmed PEG solution to the pellet and incubate for 1 minute. . Then 20 mI of warm 20% complete medium without serum! The P The EG solution was diluted. Incubate the fused cells for 10 minutes at 37°C. , and then centrifuged at 200×g for 10 minutes.

The fused cell pellet was then resuspended in 50ml of 20% complete medium. , plate at a concentration of +, 09xlO5 to L26x105 cells per well. It was sown in Cells were grown in 20% complete medium, 2°5μg/mj! STM content of 20% per well of complete medium or 20% complete medium containing 250 units/ml IL-6. The cells were seeded in a final volume of 200 μl. Incubate overnight at 37°C and 10% CO2. After incubation, aspirate half of the medium from each well and add 20% HAT to the cells. $20% HAT with STM at 5 u g/m i or 500 units/ml IL-6 Containing 20% HAT was supplied. Inspect the cells with the naked eye and see if they are 12 to 14 hours after fusion. Monitor hybridoma growth and test growing wells for the presence of anti-TAT complex antibodies. These media were supplied regularly for several weeks while screening for .

2. Fusion of hybridoma TAT-5 Preparation of tile cells Mice immunized with TAT complex as described for Group 2 in Example above. Pancreatic cells were obtained from mice. Cells were removed from the pancreas using forceps and scissors and cooled. Washed three times in PMI 16110. Transfer the cells to the final solution of RPMI 16JIO. It was resuspended in a volume of 140ml. Viable cell counts of tile cells were determined using trypanb. The Roux exclusion method gave a 95% viability of 5xlO'.

myeloma preparation Myeloma cells were mechanically harvested, pooled, and incubated in cold RPMI +61JO. Washed 3 times. Cells were resuspended in a final volume of 10 m7. Myeloma cell survival The viable cell count is 3.3 viable cells per rrl with a 71% viability. There were 6xlO' pieces.

Fusion of tile cells and myeloma cells The tile cells and the myeloma cell suspension of 4.2mj7 were mixed into tile cells:myeloma cells, cell ratio of approx. 4 = 1, resulting in a total viable cell count of 7.5 x lO'. cell mixture The mixture was centrifuged at 200xg for 8 minutes and the supernatant was aspirated.

Cells were then buffered in RPMI 1640 and 10% PEG (molecular weight 7000 to 9000). Fusion was kept in a warm (37 °C) water bath. It was carried out in a tube. 1 to 1.5m heated to 37℃! ! Pellet the PEG solution of The mixture was added dropwise over 1 to 1.5 minutes. The PEG solution was then added to hot RPMI Diluted to 50ml by addition of 16110. The fused cells were then incubated for 20 Centrifugation was performed at 0×g for 10 minutes.

After aspirating the supernatant, resuspend the fused cells in 50 mIl of 20% complete medium. , 100 μl per well, i.e. 1.2xl viable cells.

Plate cells were seeded at a density of 6 cells/well. Incubated overnight at 37°C and 10% COz. After incubation, 100% of 20% HATG (HAT medium supplemented with glycine) μl was added to each well. Visually inspect the cells to see if they are higher than 12 to Ill after fusion. Monitor the growth of bridomas and test growing wells for the presence of anti-TA'1 conjugated antibodies. 20% HATG was fed regularly for several weeks while screening.

Example 4 - Screening for antibodies that specifically bind to the TAT complex Example 3 Proliferating hybrids produced by fusion as described in Samples of the supernatant collected from the market TAT-4 and TAT-5 were prepared in Example 2 above. TAT using antigen-coated microplates (lμg/ml) as described in The complex was screened for activity against AT■. Connection to TAT complex Each of the 21 antibodies that tested positive for binding to ATI and negative for binding to ATI Selected for further testing.

Further tests were carried out in the presence of ATII in solution (up to 300 μg/ml). By conducting sandwich EL engineering SA test and spacing TAT composite Sandwich ELISA was performed on the isolated (50 ng/ml) plasma sample. This was done by giving. For each ELISA assay performed, 20 μl of The supernatant was added to 80 μl of sample/conjugate in the wells of the coated microplate. added to the diluent. These new antibodies were used as capture antibodies (10 μg/ The conjugate used was a TAT-conjugated monoclonal antibody (02M). It was. Low blank, ATI [which shows lack of cross-reactivity and diluted linearly] Antibodies were selected that were able to detect reduced TAT levels in plasma.

Incubation: TAT in diluent or plasma/ATII in diluent [, chamber 2 hours at warm temperature. Bonding, 1 hour at room temperature. Substrate, 20 minutes at room temperature. This kind of continuous screen Based on training, four antibodies, 02M, C72, C114-T and 5B46 were selected for further consideration.

Example 5 - Affinity data The affinity constant for the capture antibody CIJil-T was determined to be 3 x lO' L/M. It was done. This constant is based on Friguet et al., Measureme nts of’ True Affinity Con5tant in 5o Solution of Antigen-Antibody complexes byEnzyme-Linked Immunoadsorbent As5 ays, J. Immunol, Methods.

77:305 (1985).

The apparent affinity constant of conjugate antibody 5B46 was determined to be 3.8 xlO'.

This constant was calculated using antibodies conjugated and not conjugated to HRP on antigen-coated plates. were also obtained by performing a competitive assay.

Example 6 - Competition assay For C72 and Cll4-T plates, different sets of conjugates and unlabeled antibodies were added. A competition assay was performed using the combination. The plate was washed with a CI of 10 μg/mj7. J4-T or C72F(ab')2 was coated. zygote to 1.1μg/ml Diluted and competitive antibodies were added to concentrations of 110.11, and 00-11a/ml. T AT incubation at room temperature 1 hour, conjugate + IgG incubation at room temperature 3 0 min, and substrate incubation at room temperature 15 min. The results show that the D2M 1 of the present invention Many different sets of four TAT antibodies: C72, CIJIJ-T, and 5B46. It has been shown that the combination does not or hardly interferes with mutual coupling.

Example 7 - Determination of antibody specificity Microtiter plate, C114-TF(ab')z (Ioμg /ml). Other complexes of ATI[I (factor 1a ATIII or Factor 1 Xa ATII [) was incubated at 50 ng/mj7 for 60 min at room temperature. I bet. The plates were then washed three times and the conjugate (HRP-labeled 5B11 6) at 5.5 μg/mI! was added to a concentration of 30 minutes of incubation Afterwards, plates were washed three times and substrate was added. 1M sulfuric acid after 15 minutes The reaction was stopped by treatment. This pair of antibodies showed no significant interaction with either complex. No differential reactivity was shown, especially when the factor Xa-ATI complex was Only cross-reactivity was observed, with factor 1 Xa-ATI [complex being less than 0.02% Only cross-reactivity was observed.

Example 8 - Sandwich assay using monoclonal antibodies Monoclonal C glI was immobilized on a solid support in a microtiter well. Then, the TAT A sample of patient plasma containing conjugate and a measured amount of monoclonal 5Bl1 6 (labeled by covalently bonding an enzyme, especially horseradish peroxidase), The immobilized C88-T was added for 30 minutes at 37°C. Then, if there is no reaction The conjugate (5BIIJ6) and any unreacted TAT are removed by washing. removed.

. The amount of bound labeled antibody is directly proportional to the concentration of TAT complex in the test sample. Give an example.

The invention has been described in detail with reference to preferred embodiments thereof. However, Modifications within the spirit and scope of the invention may be made by those skilled in the art in light of this disclosure. and that improvements may be made are the continuation of the authorized front page. (51) Int, C1,6 identification symbol Internal office reference number // C12N 151 02 (C12P 21108 C12R1:91) (72) Inventor: Miller, Stape 31st Street Northwest, Sunrise, Florida 33351 United States 11741 (72) Inventor Perez Tejidor, Liliana United States 148 Avenue Court I, Southwest, Miami, Florida 33185 (72) Inventor: Arbuthnot, Kathryn, nee United States 33129 18 Terrance, Southwest, Miami, Florida 459 (72) Inventor Mastin, Erik, El Johnson City, Tennessee 37614 USA Tee, Earl Circle 1

Claims (25)

[Claims] 1. Human thrombin-antithrombin III complex in human fluid samples an immunoassay for the detection of a liquid sample, the method comprising: contacting a first antibody with the liquid sample and contacting a second antibody with the liquid sample, wherein At least one of the first and second antibodies is human thrombin-antithrone. An immunoassay that specifically binds to the Bin III complex. 2. Both of the antibodies are specific for the human thrombin-antithrombin III complex. 2. The immunoassay of claim 1, which binds to. 3. the first and second antibodies are essentially non-competitive for binding to the complex; , the immunoassay of claim 1. 4. contacting the first and second antibodies with the liquid sample substantially simultaneously; The immunoassay of claim 1. 5. contacting the first antibody with the liquid sample and then contacting the second antibody with the liquid sample. 2. The immunoassay of claim 1, wherein the immunoassay is brought into contact with a body sample. 6. the first antibody is adsorbed onto a solid matrix and the second antibody is 2. The immunoassay of claim 1, wherein the immunoassay is labeled with a reporter group. 7. The item of claim 1, wherein the amount of TAT complex in the liquid sample is regulated. Munoassay. 8. The immunoassay of claim 1, wherein the first and second antibodies are monoclonal antibodies. Surname. 9. 2. The immunoassay of claim 1, wherein said liquid sample is plasma. 10. An antibody that specifically binds to the thrombin-antithrombin III complex is , thrombin, antithrombin, serine protease Xa-antithrombin the serine protease factor IXa-antithrombin II complex, and the serine protease factor IXa-antithrombin II complex. 2. The compound of claim 1 exhibits less than about 1% cross-reactivity with each of the I complexes. Immunoassay. 11. An antibody that specifically binds to the thrombin-antithrombin III complex is , thrombin, antithrombin, serine protease Xa-antithrombin the serine protease factor IXa-antithrombin II complex, and the serine protease factor IXa-antithrombin II complex. 1 complex exhibiting less than about 0.2% cross-reactivity with each of the I complexes. 1 immunoassay. 12. the first antibody has the characteristics of D2M, C72, C44-T or 5B46 The immunoassay according to claim 1, which is an immunoassay. 13. the second antibody has characteristics of D2M, C72, C44-T or 5B46 The immunoassay according to claim 1 or 12, which is 14. Claim wherein the first antibody is D2M, C72, C44-T or 5B46. 1 immunoassay. 15. Claim wherein the second antibody is D2M, C72, C44-T or 5B46. 1 or 14 immunoassays. 16. The first antibody has the characteristics of C44-T and the second antibody has the characteristics of 5B46. 7. The immunoassay according to claim 6, wherein the immunoassay has the following characteristics. 17. Claim wherein the first antibody is C44-T and the second antibody is 5B46. 6 immunoassay. 18. Detection of human thrombin-antithrombin III complex in test samples 1. A test kit for testing, comprising a first and a second antibody; at least one of which is specific for human thrombin-antithrombin III complex. A kit that can be combined with 19. Both antibodies are specific for the human thrombin-antithrombin III complex. 19. The test kit according to claim 18, wherein the test kit binds to 20. One of the antibodies is directed against the human thrombin-antithrombin III complex. specifically binds, and the other of the antibodies is human thrombin-antithrombin. 19. The test kit of claim 18, wherein the test kit binds to the protein III complex. 21. the first antibody has the characteristics of D2M, C72, C44-T or 5B46 19. The test kit of claim 18. 22. the second antibody has characteristics of D2M, C72, C44-T or 5B46 19. The test kit of claim 18. 23. A module that specifically binds to the human thrombin-antithrombin III complex Noclonal antibodies. 24. The claimant has the characteristics of D2M, C72, C44-T or 5B46. Monoclonal antibody according to claim 23. 25. 24. The monochrome of claim 23, which is D2M, C72, C44-T or 5B46. null antibody.