Tat antibodies and uses thereof

Abstract

This invention relates to the field of antibodies, and more particularly to TAT antibodies and their applications. The invention provides coated antibodies comprising a heavy chain variable region and a light chain variable region, wherein: the CDR1, CDR2, and CDR3 regions of the heavy chain variable region have amino acid sequences as shown in SEQ ID No:1 to SEQ ID No:3; and the CDR1, CDR2, and CDR3 regions of the light chain variable region have amino acid sequences as shown in SEQ ID No:4 to SEQ ID No:6. This invention successfully prepared highly specific monoclonal antibodies 7E10 and 2B4 against the thrombin-antithrombin III complex (TAT), wherein 7E10 specifically binds to thrombin (THR), and 2B4 specifically binds to antithrombin III (AT-III). Using these two antibodies in combination enables highly sensitive and specific detection of the TAT complex.

Description

Technical Field

[0001] This invention relates to the field of antibodies, and more particularly to TAT antibodies and their applications. Background Technology

[0002] The thrombin-antithrombin III complex (TAT) is a complex formed by thrombin and antithrombin molecules in a 1:1 ratio. Thrombin is Na+. + Thrombin is an ion-excited protease in the coagulation system and a key enzyme involved in the coagulation cascade reaction. After its formation in vivo, thrombin has a half-life of only a few seconds in the blood, making direct measurement difficult. However, its thrombin portion rapidly binds to antithrombin (AT) to form the TAT complex. Therefore, this indicator is a molecular marker reflecting thrombin formation and can sensitively reflect the degree of activation of the coagulation system, directly reflecting the initiation of the coagulation system. Elevated TAT levels can predict the early risk of thrombosis and recurrence, as well as the early risk of disseminated intravascular coagulation (DIC).

[0003] Currently, Sysmex Medical Electronics (Shanghai) Co., Ltd. is the only imported manufacturer with a domestic registration number for its thrombin-antithrombin III complex assay kit. This kit is used for the quantitative detection of TAT complexes in human plasma and holds a very high market share in western and southeastern China. However, the kit is expensive. Therefore, to better utilize TAT as a disease biomarker, it remains necessary to develop monoclonal antibodies with better detection performance. Summary of the Invention

[0004] In view of this, the present invention provides TAT ​​antibodies and their applications. The present invention successfully prepared highly specific monoclonal antibodies 7E10 and 2B4 against thrombin-antithrombin III complex (TAT), wherein 7E10 specifically binds to thrombin (THR) and 2B4 specifically binds to antithrombin III (AT-III). The pairing of these antibodies enables highly sensitive and specific detection of the TAT complex. Through a double-antibody sandwich screening method, 7E10 was selected as the coating antibody and 2B4 as the enzyme-labeled antibody, establishing a TAT antigen detection kit based on magnetic microparticle chemiluminescence immunoassay. Experiments show that the paired antibodies exhibit excellent signal response and low background interference in detection, and are highly correlated with the detection results of mainstream imported brands (Sysmex). 2 (>0.99), demonstrating excellent detection performance and clinical application potential. Furthermore, this invention provides methods for antibody preparation, sequence information, and labeling processes, offering a crucial technical foundation for the early diagnosis of TAT-related diseases, thrombosis risk assessment, and reagent kit development.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a coated antibody comprising: a heavy chain variable region and a light chain variable region, wherein:

[0007] (1) The CDR1, CDR2, and CDR3 regions of the heavy chain variable region have amino acid sequences as shown in SEQ ID No:1, SEQ ID No:2, and SEQ ID No:3, respectively; and

[0008] (2) The CDR1, CDR2, and CDR3 regions of the light chain variable region have amino acid sequences as shown in SEQ ID No:4, SEQ ID No:5, and SEQ ID No:6, respectively; and / or

[0009] (3) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (1) or (2), and which has the same function as the amino acid sequence described in (1) or (2); or

[0010] (4) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (1), (2) or (3).

[0011] In some embodiments of the present invention, the above-mentioned coated antibody includes:

[0012] (5) The heavy chain variable region has the amino acid sequence shown in SEQ ID No:7; and

[0013] (6) The light chain variable region has an amino acid sequence as shown in SEQ ID No:8; and / or

[0014] (7) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (5) or (6), and which has the same function as the amino acid sequence described in (5) or (6); or

[0015] (8) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (5), (6) or (7).

[0016] In some embodiments of the present invention, the sequence of SEQ ID NO:1 in the above-mentioned coated antibody is: GFTFSDYG.

[0017] In some embodiments of the present invention, the sequence of SEQ ID NO:2 in the above-mentioned coated antibody is: ISNLAYSI.

[0018] In some embodiments of the present invention, the sequence of SEQ ID NO:3 in the above-mentioned coated antibody is: ARGGGYYEYFDV.

[0019] In some embodiments of the present invention, the sequence of SEQ ID NO:4 in the above-mentioned coated antibody is: QNIVHSNGNTY.

[0020] In some embodiments of the present invention, the sequence of SEQ ID NO:5 in the above-mentioned coated antibody is: KVS.

[0021] In some embodiments of the present invention, the sequence of SEQ ID NO:6 in the above-mentioned coated antibody is: FQGSQIPYT.

[0022] In some embodiments of the present invention, the sequence of SEQ ID NO:7 in the above-mentioned coated antibody is: EVKLVESGGGLVQPGGSRKLSCAASGFTFSDYGMAWFRQAPGKGPEWVAFISNLAYSIYYADTVTGRFTISRENAKNTLYLEMSSLRSEDTAMYYCARGGGYYEYFDVWGAGTTVTVSS.

[0023] In some embodiments of the present invention, the sequence of SEQ ID NO:8 in the above-mentioned coated antibody is: DVLMTQSPLSLPVSLGDQASISCRSSQNIVHSNGNTYLEWYLQKAGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKIRRVEAEDLGVYYCFQGSQIPYTFGGGTKLGIK.

[0024] The present invention also provides a labeled antibody comprising: a heavy chain variable region and a light chain variable region, wherein:

[0025] (9) The CDR1, CDR2, and CDR3 regions of the heavy chain variable region have amino acid sequences as shown in SEQ ID No:9, SEQ ID No:10, and SEQ ID No:11, respectively; and

[0026] (10) The CDR1, CDR2, and CDR3 regions of the light chain variable region have amino acid sequences as shown in SEQ ID No:12, SEQ ID No:13, and SEQ ID No:14, respectively; and / or

[0027] (11) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (9) or (10), and which has the same function as the amino acid sequence described in (9) or (10); or

[0028] (12) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (9), (10) or (11).

[0029] In some embodiments of the present invention, the labeled antibody includes:

[0030] (13) The heavy chain variable region has the amino acid sequence shown in SEQ ID No: 15; and

[0031] (14) The light chain variable region has an amino acid sequence as shown in SEQ ID No:16; and / or

[0032] (15) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (13) or (14), and which has the same function as the amino acid sequence described in (13) or (14); or

[0033] (16) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (13), (14) or (15).

[0034] In some embodiments of the present invention, the sequence of SEQ ID NO:9 in the above-mentioned labeled antibody is: GYSFTDYN.

[0035] In some embodiments of the present invention, the sequence of SEQ ID NO:10 in the above-mentioned labeled antibody is: IEPYNGGT.

[0036] In some embodiments of the present invention, the sequence of SEQ ID NO:11 in the above-mentioned labeled antibody is: ARDNYRYDGFAY.

[0037] In some embodiments of the present invention, the sequence of SEQ ID NO:12 in the above-mentioned labeled antibody is: SVSY.

[0038] In some embodiments of the present invention, the sequence of SEQ ID NO:13 in the above-mentioned labeled antibody is: STS.

[0039] In some embodiments of the present invention, the sequence of SEQ ID NO:14 in the above-mentioned labeled antibody is: QQRSIFPPT.

[0040] In some embodiments of the present invention, the sequence of SEQ ID NO:15 in the above-mentioned labeled antibody is: EIQLQQSGPELVKPGASVKVSCKASGYSFTDYNMYWVKQSHGKSLEWIGYIEPYNGGTSYNQKFKGKATLTVDKSSSTALMHLHSLTSEDSAVYYCARDNYRYDGFAYWGQGTLVTVSA.

[0041] In some embodiments of the present invention, the sequence of SEQ ID NO:16 in the above-mentioned labeled antibody is: QIVLTQSPAIMSASPGEKVAITCSANSSVSYMHWFQQKPGTSPKLWIYSTSNLVSGVPARFSGSGSGTSYSLPISRMEAEDAATYYCQQRSIFPPTFGGGTKLEVK.

[0042] The present invention also provides conjugated antibodies, including: conjugated antibody 1 or conjugated antibody 2;

[0043] The conjugated antibody 1 comprises: the above-described coated antibody and the labeled molecule 1;

[0044] The conjugated antibody 2 comprises: the above-mentioned labeled antibody and labeled molecule 2;

[0045] The marker molecule 1 and the marker molecule 2 may be the same or different.

[0046] The present invention also provides an antibody combination comprising: antibody 1 and antibody 2;

[0047] The antibody 1 is selected from the above-mentioned coated antibody or the above-mentioned conjugated antibody 1;

[0048] The antibody 2 is selected from the above-mentioned labeled antibody or the above-mentioned conjugate antibody 2.

[0049] This invention also provides biomaterials comprising: any of the following:

[0050] (17) A nucleic acid molecule encoding the above-coated antibody or the above-labeled antibody or the above-conjugated antibody or the above-combination of antibodies;

[0051] (18) An expression vector containing nucleic acid molecules as described in (17);

[0052] (19) Transformation and / or transfection of host cells with the expression vector described in (18);

[0053] (20) A mixture obtained by culturing host cells as described in (19).

[0054] In some embodiments of the present invention, in the above-described biological material, the sequence of the nucleic acid molecule encoding the heavy chain variable region of the antibody is as shown in SEQ ID NO:17; the sequence of the nucleic acid molecule encoding the light chain variable region of the antibody is as shown in SEQ ID NO:18; and / or

[0055] The sequence of the nucleic acid molecule encoding the heavy chain variable region of the labeled antibody is shown in SEQ ID NO:19; the sequence of the nucleic acid molecule encoding the light chain variable region of the labeled antibody is shown in SEQ ID NO:20.

[0056] The present invention also provides the use of any of the following in the preparation of products for detecting thrombin-antithrombin complexes:

[0057] (21) The above-mentioned coating antibody; and / or

[0058] (22) The above-mentioned labeled antibodies; and / or

[0059] (23) The above-mentioned conjugated antibodies; and / or

[0060] (24) The above antibody combination; and / or

[0061] (25) The above-mentioned biological materials.

[0062] This invention also provides detection reagents and / or detection kits, including any of the following:

[0063] (26) The above-mentioned coating antibody; and / or

[0064] (27) The above-mentioned labeled antibodies; and / or

[0065] (28) The above-mentioned conjugated antibodies; and / or

[0066] (29) The above antibody combination; and / or

[0067] (30) The above-mentioned biological materials.

[0068] The present invention also provides a method for detecting thrombin-antithrombin complexes for non-diagnostic purposes, comprising detecting a sample with the above-described detection reagents and / or detection kits.

[0069] This invention successfully prepared highly specific monoclonal antibodies 7E10 and 2B4 targeting the thrombin-antithrombin III complex (TAT). 7E10 specifically binds to thrombin (THR), and 2B4 specifically binds to antithrombin III (AT-III). Using these two antibodies in combination enables highly sensitive and specific detection of the TAT complex. Through a double-antibody sandwich screening method, 7E10 was selected as the coating antibody and 2B4 as the enzyme-labeled antibody, establishing a TAT antigen detection kit based on magnetic microparticle chemiluminescence. Experiments showed that the paired antibodies exhibited excellent signal response and low background interference in detection, and were highly correlated with the detection results of a mainstream imported brand (Sysmex). 2 (>0.99), demonstrating good detection performance and clinical application potential. Furthermore, this invention provides methods for antibody preparation, sequence information, and labeling processes, offering a crucial technical foundation for the early diagnosis of TAT-related diseases, thrombosis risk assessment, and reagent kit development. Attached Figure Description

[0070] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0071] Figure 1 The image shows the SDS-PAGE plot of the 7E10 antibody reduction.

[0072] Figure 2 The image shows the SDS-PAGE diagram of antibody reduction with 2B4.

[0073] Figure 3 This invention demonstrates the correlation between the pairing and Sysmex. Detailed Implementation

[0074] This invention discloses TAT ​​antibodies and their applications.

[0075] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0076] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0077] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0078] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0079] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0080] The coated antibody (7E10) provided by this invention:

[0081] Heavy chain variable region amino acid sequence: EVKLVESGGGLVQPGGSRKLSCAASGFTFSDYGMAWFRQAPGKGPEWVAFISNLAYSIYYADTVTGRFTISRENAKNTLYLEMSSLRSEDTAMYYCARGGGYYEYFDVWGAGTTVTVSS (as shown in SEQ ID NO:7).

[0082] (CDR is: GFTFSDYG (as shown in SEQ ID NO:1)..._ISNLAYSI (as shown in SEQ ID NO:2)..._ARGGGYYEYFDV (as shown in SEQ ID NO:3)).

[0083] Heavy chain variable region nucleic acid sequence: GAGGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTACGGAATGGCGTGGTTTCGACAGGCTCCAGGGAAGGGGCCTGAGTGGGTAGCGTTCATTAGTAATTTGGCATATAGTATTTA CTATGCGGACACTGTGACGGGCCGATTCACCATCTCTAGAGAGAATGCCAAGAACACCCTGTACCTGGAAATGAGCAGTCTGAGGTCTGAGGACACAGCCATGTATTACTGTGCAAGGGGGGGGGGTTACTACGAGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA (as SEQ ID NO:17).

[0084] The amino acid sequence of the light chain variable region is: DVLMTQSPLSLPVSLGDQASISCRSSQNIVHSNGNTYLEWYLQKAGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKIRRVEAEDLGVYYCFQGSQIPYTFGGGTKLGIK (as shown in SEQ ID NO:8).

[0085] (CDR is: QNIVHSNGNTY (as shown in SEQ ID NO:4).___KVS (as shown in SEQ ID NO:5).......___FQGSQIPYT (as shown in SEQ ID NO:6)).

[0086] Light chain variable region nucleic acid sequence: GATGTTTTGATGACCCAAAGTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAACATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAAGCAGGCCAGTCTCCAAAGCTCCTGATCTACAAA GTCTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTGAAGATCAGAAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACAAATTCCGTACACGTTCGGAGGGGGGACCAAGCTGGGAATAAAA (as in SEQ ID NO:18).

[0087] The labeled antibody (2B4) provided by this invention:

[0088] Heavy chain variable region amino acid sequence: EIQLQQSGPELVKPGASVKVSCKASGYSFTDYNMYWVKQSHGKSLEWIGYIEPYNGGTSYNQKFKGKATLTVDKSSSTALMHLHSLTSEDSAVYYCARDNYRYDGFAYWGQGTLVTVSA (as shown in SEQ ID NO:15).

[0089] (CDR: GYSFTDYN (as shown in SEQ ID NO:9) ....___IEPYNGGT (as shown in SEQ ID NO:10) ....___ARDNYRYDGFAY (as shown in SEQ ID NO:11)).

[0090] Heavy chain variable region nucleic acid sequence: GAGATCCAGCTACAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGGTATCCTGCAAGGCTTCTGGTTACTCATTCACTGACTACAACATGTACTGGGTGAAACAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATATTGAGCCTTACAATGGTGGGACTAG CTACAACCAGAAATTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCCTCATGCATCTCCACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGACAATTATAGGTACGACGGTTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (as SEQ ID NO:19).

[0091] The amino acid sequence of the light chain variable region is: QIVLTQSPAIMSASPGEKVAITCSANSSVSYMHWFQQKPGTSPKLWIYSTSNLVSGVPARFSGSGSGTSYSLPISRMEAEDAATYYCQQRSIFPPTFGGGTKLEVK (as shown in SEQ ID NO:16).

[0092] (CDR: SVSY (as shown in SEQ ID NO:12) ... ___ STS (as shown in SEQ ID NO:13) ... ___ QQRSIFPPT (as shown in SEQ ID NO:14)).

[0093] Light chain variable region nucleic acid sequence: CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCGCCATCACCTGCAGTTCCAACTCAAGTGTTTCTTACATGCACTGGTTCCAGCAGAAGCCAGGCACTTCTCCCAAACTCTGGATTTATAGTACATCCAAC CTGGTTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTCTCTCCCAATCAGCCGAATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAAAGGAGTATTTTCCCACCCACGTTCGGGGGGGGGACCAAGCTGGAAGTAAAA (as SEQ ID NO:20).

[0094] In Examples 1 to 10 of this invention, all raw materials and reagents used can be purchased from the market.

[0095] The luminescent substrate A and luminescent substrate B involved in this invention are manufactured by Antu Biotechnology and have a batch number of 2023100610.

[0096] The present invention will be further illustrated below with reference to the embodiments:

[0097] Example 1: Preparation of thrombin, antithrombin III, and thrombin-antithrombin III complex

[0098] The amino acid sequences of thrombin (P00734) and anti-thrombin III (P01008) were found on the UniProt website. The corresponding gene sequences were derived using software and synthesized. The obtained DNA sequences were cloned into the PCMV02 vector (with Hind III and Not I restriction sites on both sides), then transformed and plated. Clones were picked and plasmids were extracted. The recombinant plasmids were transfected into HEK293 cells under the following conditions: PEI / plasmid mass ratio of 3:1, and transfection ratio of 2 μg plasmid / 1 mL HEK293 cells. Cell supernatant was collected 5-7 days after transfection. The supernatant was purified using a nickel column to obtain high-purity thrombin (THR) protein and antithrombin III (AT-III) protein. The elution buffer was replaced with PBS by dialysis, and the protein concentration was determined using the Bradford method.

[0099] The recombinant THR and AT-III proteins prepared above were adjusted to a concentration of 1.0 mg / mL, mixed in a 1:1 volume ratio, incubated at 37°C for 30 minutes, filtered through Superdex 200 gel filter media, equilibrated with PBS at 1.0 mL / min, and the TAT complex was collected.

[0100] P00734: (as shown in SEQ ID NO:21).

[0101] P01008: MYSNVIGTVTSGKRKVYLLSLLLIGFWDCVTCHGSPVDICTAKPRDIPMNPMCIYRSPEKKATEDEGSEQKIPEATNRRVWELSKANSRFATTFYQHLADSKNDNDNIFLSP LSISTAFAMTKLGACNDTLQQLMEVFKFDTISEKTSDQIHFFFAKLNCRLYRKANKSSKLVSANRLFGDKSLTFNETYQDISELVYGAKLQPLDFKENAEQSRAAINKWVSNKTEGRIT DVIPSEAINELTVLVLVNTIYFKGLWKSKFSPENTRKELFYKADGESCSASMMYQEGKFRYRRVAEGTQVLELPFKGDDITMVLILPKPEKSLAKVEKELTPEVLQEWLDELEEMMLVV HMPRFRIEDGFSLKEQLQDMGLVDLFSPEKSKLPGIVAEGRDDLYVSDAFHKAFLEVNEEGSEAAASTAVVIAGRSLNPNRVTFKANRPFLVFIREVPLNTIIFMGRVANPCVK (as SEQ ID NO:22).

[0102] Example 2: Establishment of TAT hybridoma cell line

[0103] (1) Animal immunization

[0104] This embodiment uses the TAT complex prepared in Example 1 to immunize BALB / c mice and detects the serum titer. The specific steps are as follows: BALB / c mice aged 6-8 weeks were selected and immunized according to the following procedure: For the initial immunization, 25 μg of TAT complex was mixed with an equal volume of Freund's complete adjuvant, emulsified, and injected subcutaneously at multiple sites; 14 days after the first immunization, 12.5 μg of TAT complex was mixed with Freund's incomplete adjuvant, emulsified, and used as a booster immunization. 14 days after the second immunization, 12.5 μg of TAT complex was mixed with Freund's incomplete adjuvant, emulsified, and used as a booster immunization. 14 days after the third immunization, blood was collected and serum was separated. ELISA plates were coated with 1 μg / mL TAT complex, and an indirect ELISA test was performed to determine the serum titer. Mice with a serum titer of 1 / 10000 or higher were preferred for fusion immunization.

[0105] (2) Cell fusion

[0106] Feeder cell preparation: Mice were euthanized by dislocation and placed in a bottle of 75% alcohol for 2 minutes. Then, the mice were fixed to a foam board in a biosafety cabinet, the abdominal skin was opened, and PBS was gently injected subperitoneally with a syringe. The liquid containing feeder cells was washed out from the other side. The cells were then centrifuged at 200g for 10 minutes at room temperature. The supernatant was discarded and the cells were used for later use.

[0107] Cell fusion: Cell fusion was performed 3 days after the mice were immunized in step (1). After removing the eyeballs of the mice and collecting blood, the mice were euthanized by dislocation and placed in a bottle of 75% alcohol for 2 minutes. Then, the mice were fixed on a foam board in a biosafety cabinet, the abdominal skin was opened to locate the spleen, which was removed with forceps and placed in a 200-mesh stainless steel filter membrane to gently crush the cells. The cells were then gently washed with DMEM culture medium and centrifuged at 200g for 10 minutes at room temperature. The supernatant was discarded and the cells were used for further processing. 2.0×10 7 NS1 myeloma cells and 2.0 × 10 8 Mix the spleen cells thoroughly, centrifuge at 200g for 10 minutes, discard the supernatant, gently vortex to mix, and incubate at 37°C. Add 1 mL of 50% PEG1500 aqueous solution over 90 seconds, then add 20 mL of DMEM culture medium. Centrifuge at 200g for 10 minutes, discard the supernatant, repeat the washing process once, centrifuge at 200g for 10 minutes, discard the supernatant, and obtain hybridoma cells. Seed the cells into 10 96-well plates, 150 μL per well. Add 10 feeder cells... 4 Add 100 μL of cells / well to each of the 10 96-well cell culture plates described above. After labeling the culture plates, incubate them in a cell culture incubator at 37°C with 5% CO2. Maintain the culture in HAT selection medium for 7-10 days, then replace with HT culture medium. During the selection culture period, when the hybridoma cells cover 1 / 10 of the bottom area of ​​each well, you can begin detecting specific antibodies and screening for the desired hybridoma cell lines.

[0108] (3) Screening and subcloning of positive hybridoma cell lines

[0109] The purified TAT complex was diluted to 1 μg / mL with PBS and coated onto a 96-well ELISA plate, incubated overnight at 4°C. The plate was washed twice with PBST. 200 μL of 1% BSA in PBS was added to each well, and the plate was blocked at room temperature for 2 hours, then patted dry on folded paper. Sample loading: 0.1 mL of the test sample was added to each well, incubated at 37°C for 1 hour, and then washed. Blank wells (without sample), negative control wells (blank culture medium), and positive control wells (immunized mouse serum) were also prepared. 0.1 mL of the sample was added to each well, incubated at 37°C for 1 hour, and then washed three times. Secondary antibody addition: 0.1 mL of 1 / 5000 diluted HRP-goat anti-mouse IgG antibody was added to each well. The plate was incubated at 37°C for 1 hour and washed three times. Substrate solution addition and color development: 0.1 mL of TMB substrate solution was added to each well, and the plate was incubated at room temperature for 10 minutes. 0.1 mL of 0.2 M H₂SO₄ was added to each well. The results were determined by measuring the OD value using a microplate reader at 450 nm. A positive result was defined as an OD value greater than 2.1 times that of the negative control (calculated after zeroing the blank control well). Single clones of hybridoma cells resistant to the TAT complex were selected.

[0110] Following the above method, the selected positive hybridoma cells were subcloned. The original wells were diluted with HAT selective medium using a limiting dilution method and re-distributed into 96-well plates. The cell density was adjusted to 5-10 cells / mL based on observed cell morphology and quantity. 100 μL of diluted cells was added to each well of a cell culture plate containing a feeder cell layer prepared the previous day. The plates were incubated statically at 37°C with 5% CO2. On days 7-10 (when hybridoma cells covered 1 / 10 of the well bottom area), the medium was changed, and the reactivity of the cell supernatant was tested using the same screening method. This cloning process was repeated at least three times. Cells from the positive wells were then sequentially transferred to 24-well and 6-well plates for further culture and cryopreservation.

[0111] Example 3 Antibody Subtype Identification

[0112] Dilute the TAT antigen with 0.05M pH 9.5 carbonate buffer to a final concentration of 1 μg / mL. Add 0.1 mL to each well of a 96-well polystyrene plate and incubate at 37°C for 2 hours or at 4°C overnight. Then block with 0.2 mL of 0.01M pH 7.4 PBS containing 1% BSA per well and incubate at 37°C for 2 hours for detection. Add 0.1 mL of hybridoma supernatant to each well, incubate at 37°C for 1 h, wash six times with water, then add 1000-fold diluted goat anti-mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibody, incubate at 37°C for 0.5 h, wash six times with water, then add 5000-fold diluted HRP-rabbit anti-goat IgG, incubate at 37°C for 30 min, wash as above, then add 100 μL of an equal volume of substrate and chromogenic solution to each well, incubate at 37°C for 10 min, stop the reaction with dilute sulfuric acid solution, add 50 μL to each well, and measure the absorbance at 450 nm. The results show that the 7E10 antibody of this invention is IgG1 subtype, and the 2B4 antibody is IgG2b subtype.

[0113] Table 1. Results of TAT antibody subtype identification

[0114]

[0115] Example 4: Large-scale preparation of monoclonal antibodies

[0116] Eight-week-old BALB / c mice were injected intraperitoneally with 0.5 mL of Freund's incomplete adjuvant, followed by an intraperitoneal injection of 1 × 10⁻⁶ mg / mL two weeks later. 6Hybridoma cells will produce ascites 7-10 days after inoculation. Closely observe the animal's health and signs of ascites. When ascites is abundant but the mouse is close to death, euthanize it and aspirate the ascites into a test tube using a dropper. 5-10 mL of ascites can be obtained from one mouse. Alternatively, ascites can be extracted using a syringe, and can be collected repeatedly. Centrifuge the obtained ascites at 3000g for 10 minutes, discarding the upper layer of oil and the bottom precipitate. Collect the supernatant and aliquot it at -20℃. After thawing and equilibrating the ascites supernatant to room temperature, add 1 / 10 volume of 1M Tris-HCl pH 8.0 to adjust the sample pH to 8.0. Equilibrate the protein G affinity column with 20 column volumes of 100mM pH 8.0 Tris-HCl. Load the pH-adjusted ascites supernatant onto the column, then wash with 20 column volumes of 100mM pH 8.0 Tris-HCl. Finally, elute the antibody with 100mM Glycine-HCl pH 2.5. Add the antibody eluent to a concentration tube and centrifuge at 3000×g for 20 minutes at room temperature. Centrifuge in batches to a final volume of 1 mL / concentration tube (2 tubes). Add 4 mL of 10 mM PBS pH 7.4 buffer and centrifuge again at 3000×g for 20 minutes at room temperature. Repeat centrifugation three times to adjust the antibody buffer to 10 mM PBS pH 7.4. Add 10 mM PBS pH 7.4 to a final antibody concentration of 5 mg / mL. Finally, aliquot the concentrated antibody solution into centrifuge tubes (2 mL / tube) and store at -20°C.

[0117] Example 5 Sequence Analysis of Monoclonal Antibodies

[0118] Hybridoma cell lines were cultured in DMEM medium supplemented with 10% serum in 10 cm diameter cell culture dishes (37℃, 5% CO2). After 7 days of culture, the cells were transferred to 15 mL centrifuge tubes, counted using a hemocytometer, and 4 × 10⁴ cells were collected. 6 Centrifuge the cells at 200g for 5 minutes, discard the supernatant, and invert the centrifuge tube to drain the liquid. Use a QIAGEN reverse transcription kit to synthesize cDNA from the cells.

[0119] The synthesized cDNA was used as a template for PCR amplification using specific primers. 10 μL of each PCR product was loaded onto a 1% agarose gel for electrophoresis. The target fragment was cut from the agarose gel and extracted using a DNA extraction kit. The extracted DNA fragment was ligated into the pEASY-T1 cloning vector and transformed into Trans1-T1 competent cells. The transformed bacterial colonies were picked and cultured overnight in LB medium for DNA sequencing.

[0120] Example 6: Identification of TAT antibody specificity

[0121] In Example 1, the THR antigen, AT-III antigen, and TAT complex prepared were diluted with PBS and coated onto 96-well microplates at equimolar concentrations of 10 nmol / mL (100 μL / well). After overnight coating at 4°C, the microplates were washed twice with PBST solution (300 μL / well each time). After washing and drying, the microplates were blocked with PBS containing 1% BSA (200 μL / well) at room temperature for 2 hours. After drying, 7E10 and 2B4 antibodies were diluted to 0.5 μg / mL with PBS containing 1% BSA and added to the blocked microplates (100 μL / well), and incubated at 37°C for 1 hour. The microplates were washed three times with PBST (300 μL / well each time). After drying, HRP-labeled goat anti-mouse IgG antibody diluted 5000-fold with PBS containing 1% BSA was added to the microplates, and incubated at 37°C for 1 hour. Wash the plate three times with PBST, 300 μL / well. Pat dry, add 100 μL of TMB substrate solution to each well, incubate at room temperature for 10 minutes, then add 100 μL / well of 1M H2SO4 to terminate the reaction. Measure the absorbance at 450 nm using a microplate reader. The experimental results are shown in Table 2, indicating that 7E10 is a thrombin antibody and 2B4 is an antithrombin antibody, and their pairing can achieve specific detection of the TAT complex.

[0122] Table 2. Antibody Specificity of the Invention

[0123]

[0124] Example 7 Horseradish peroxidase (HRP) labeling of TAT antibody

[0125] Take 3 mg of the TAT antibody prepared in Example 4 and add it to a dialysis bag (10 kDa, 1 cm wide). Dialyze overnight at 4°C in 2 L of 10 mM PBS solution (pH 7.4). The next day, place the dialysis bag containing the antibody solution into 1 L of 10 mM carbonate buffer (pH 9.5) and dialyze at room temperature with stirring for 2 hours, in preparation for coupling with activated HRP.

[0126] Meanwhile, accurately weigh 1 mg of HRP using an analytical balance and dissolve it in 0.2 mL of ultrapure water to achieve an HRP concentration of 5 mg / mL. Add 40 μL of 0.1 M NaIO4 to the above HRP solution and incubate on a horizontal shaker at room temperature in the dark for 20 minutes. Add the activated HRP solution to a dialysis bag (10 kDa, 1 cm wide) and dialyze overnight at 4°C in 2 L of 1 mM sodium acetate buffer (pH 4.4). Carefully aspirate the dialyzed HRP solution and transfer it to a new 1.5 mL centrifuge tube. Add 1 / 10 volume of 0.2 M carbonate buffer to raise the pH of the activated HRP solution to 9.0-9.5. Mix the above antibody and HRP and incubate on a horizontal shaker at room temperature in the dark for 4 hours. After the reaction is complete, add 10 μL of freshly prepared NaBH4 (using pre-cooled ultrapure water) and incubate overnight at 4°C in the dark to terminate the reaction. After termination, transfer the antibody solution to a dialysis bag (10 kDa, 1 cm wide) and dialyze at room temperature with stirring for 2 hours. Finally, transfer the solution to a brown centrifuge tube and store at 4°C.

[0127] Example 8: Screening of TAT antigen-paired antibodies using a double-antibody sandwich assay

[0128] The TAT monoclonal antibody prepared in Example 4 was diluted to a final concentration of 2 μg / mL with 0.05 M pH 9.5 carbonate buffer. 0.1 mL was added to each well of a 96-well Costar luminescent plate and incubated at 37°C for 2 hours or overnight at 4°C. The plate was then blocked with 150 μL of 0.01 M pH 7.4 PBS containing 1% BSA and incubated at 37°C for 2 hours for detection. Add TAT antigen prepared in Example 1 (Ag-1: 100 ng / mL, Ag-2: 5 ng / mL, Ag-3: 0 ng / mL) serially diluted with 2% BSA to each well, incubate at 37°C for 1 hour, wash six times with water, then add HRP-labeled TAT antibody prepared in Example 7 diluted 2000 times, incubate at 37°C for 30 minutes, wash as above, then add 100 μL of equal volumes of substrate solution A and substrate solution B to each well, incubate at room temperature in the dark for 5 minutes, and measure the luminescence value with a luminometer. The results are shown in Table 3. The titers and gradients of 7E10 coating + 2B4 labeling and 9E3 coating + 2B4 labeling are comparable and good. 7E10 coating + 2B4 has better background and is preferred for subsequent magnetic microparticle reagent establishment.

[0129] Table 3. Results of antibody pairing screening using the plate sandwich method

[0130]

[0131]

[0132]

[0133] Example 9: Establishment of a TAT antigen detection kit based on magnetic microparticle chemiluminescence method

[0134] (1) Preparation of magnetic microparticle suspension components

[0135] Take 20 mg of carboxyl-modified microparticle solution. Magnetic microspheres with superparamagnetic properties, uniform particle size, and carboxyl (COOH-) active groups on their surface are separated by sedimentation under the action of a magnet for 10 minutes. The supernatant is discarded. The sedimented magnetic microspheres are washed three times with 0.05 M MES pH 6.0 activation buffer, 2 mL each time. Then, the microspheres are thoroughly resuspended in 1 mL of 0.05 M MES pH 6.0 buffer and EDC is added. The mixture is incubated at room temperature for 30 minutes. Mix 1.0 mg of 7E10 antibody with 20 mg of the activated magnetic microsphere solution, gently shake to mix, and react at 4 °C for 6 hours. Separate by magnetic sedimentation for 10 minutes, discard the supernatant, and resuspend in PBS containing 1% BSA. After washing three times, the microspheres are resuspended in PBS containing 1% BSA. This is component R1 of the reagent magnetic microparticle suspension.

[0136] (2) Preparation of enzyme conjugate components

[0137] Take the horseradish peroxidase-labeled 2B4 antibody prepared in Example 7, dilute it 1 / 5000 times with PBS containing 1% BSA and mix well to obtain enzyme conjugate component R2.

[0138] (3) Preparation of dilute components

[0139] PBS with 1% BSA was used as sample dilution component R3.

[0140] Example 10 Application of the Magnetic Microparticle TAT Antigen Detection Kit

[0141] Using the kit prepared with the preferred paired antibodies of the present invention (Example 9), calibrated with an A2000 plus fully automated chemiluminescence immunoassay analyzer, 18 Sysmex clinical samples were tested. The correlation R between the paired antibodies of the present invention and Sysmex was [missing information]. 2 >0.99.

[0142] Table 4. Correlation between the TAT-paired antibodies of this invention and Sysmex.

[0143]

[0144] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A coating antibody characterized in that, include: Heavy chain variable region and light chain variable region, wherein: (1) The CDR1, CDR2, and CDR3 regions of the heavy chain variable region have amino acid sequences as shown in SEQ ID No:1, SEQ ID No:2, and SEQ ID No:3, respectively; and (2) The CDR1, CDR2, and CDR3 regions of the light chain variable region have amino acid sequences as shown in SEQ ID No:4, SEQ ID No:5, and SEQ ID No:6, respectively; and / or (3) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (1) or (2), and which has the same function as the amino acid sequence described in (1) or (2); or (4) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (1), (2) or (3).

2. The coating antibody of claim 1, wherein include: (5) The heavy chain variable region has an amino acid sequence as shown in SEQ ID No:7; and (6) The light chain variable region has an amino acid sequence as shown in SEQ ID No:8; and / or (7) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (5) or (6), and which has the same function as the amino acid sequence described in (5) or (6); or (8) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (5), (6) or (7).

3. A labeled antibody characterized in that, include: Heavy chain variable region and light chain variable region, wherein: (9) The CDR1, CDR2, and CDR3 regions of the heavy chain variable region have amino acid sequences as shown in SEQ ID No:9, SEQ ID No:10, and SEQ ID No:11, respectively; and (10) The CDR1, CDR2, and CDR3 regions of the light chain variable region have amino acid sequences as shown in SEQ ID No:12, SEQ ID No:13, and SEQ ID No:14, respectively; and / or (11) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (9) or (10), and which has the same function as the amino acid sequence described in (9) or (10); or (12) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (9), (10) or (11).

4. The labeled antibody of claim 3, wherein the antibody is a monoclonal antibody. include: (13) The heavy chain variable region has an amino acid sequence as shown in SEQ ID No:15; and (14) The light chain variable region has an amino acid sequence as shown in SEQ ID No:16; and / or (15) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (13) or (14), and which has the same function as the amino acid sequence described in (13) or (14); or (16) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (13), (14) or (15).

5. A conjugated antibody, characterized in that, include: Conjugated antibody 1 or conjugated antibody 2; The conjugated antibody 1 comprises: the coated antibody and the labeling molecule 1 as described in claim 1 or 2; The conjugated antibody 2 comprises: the labeled antibody and the labeled molecule 2 as described in claim 3 or 4; The marker molecule 1 and the marker molecule 2 may be the same or different.

6. Antibody combination, characterized in that include: Antibody 1 and Antibody 2; The antibody 1 is selected from the coated antibody as described in claim 1 or 2 or the conjugated antibody as described in claim 5; The antibody 2 is selected from the labeled antibody as described in claim 3 or 4 or the conjugate antibody as described in claim 5.

7. Biomaterials characterized in that, include: Any of the following items: (17) A nucleic acid molecule encoding the coated antibody as described in claim 1 or 2, the labeled antibody as described in claim 3 or 4, the conjugated antibody as described in claim 5, or the antibody combination as described in claim 6; (18) An expression vector containing nucleic acid molecules as described in (17); (19) Transformation and / or transfection of host cells with the expression vector described in (18); (20) A mixture obtained by culturing host cells as described in (19).

8. Any of the following may be used in the preparation of products for detecting thrombin-antithrombin complexes: (21) The coated antibody as described in claim 1 or 2; and / or (22) The labeled antibody as described in claim 3 or 4; and / or (23) The conjugated antibody as described in claim 5; and / or (24) The antibody combination as described in claim 6; and / or (25) The biomaterial as described in claim 7.

9. A test reagent and / or test kit, characterized in that, Includes any of the following: (26) The coated antibody as described in claim 1 or 2; and / or (27) The labeled antibody as described in claim 3 or 4; and / or (28) The conjugated antibody as described in claim 5; and / or (29) The antibody combination as described in claim 6; and / or (30) The biomaterial as described in claim 7.

10. A method for detecting thrombin-antithrombin complexes for non-diagnostic purposes, characterized in that, This includes testing the sample with the detection reagent and / or detection kit as described in claim 9.

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