CN116284417B

CN116284417B - Thrombin-antithrombin complex antibody, its preparation method and application

Info

Publication number: CN116284417B
Application number: CN202310089381.4A
Authority: CN
Inventors: 王传新; 王醒; 杜鲁涛; 赵志敏; 李娟�; 李培龙
Original assignee: Shandong Aikeda Biotechnology Co ltd; Shandong University
Current assignee: Shandong Aikeda Biotechnology Co ltd; Shandong University
Priority date: 2023-02-09
Filing date: 2023-02-09
Publication date: 2023-10-31
Anticipated expiration: 2043-02-09
Also published as: CN116284417A

Abstract

The application belongs to the field of in-vitro diagnostic medical examination, and particularly relates to a thrombin-antithrombin complex (TAT) antibody, a preparation method and application thereof. The light chain amino acid sequence of the antibody is shown as SEQ ID NO. 7, and the heavy chain amino acid sequence is shown as SEQ ID NO. 9. The result coincidence rate of the kit prepared by the antibody is 94%, and the kit has good result coincidence compared with a standard diagnosis method. Compared with the chemoluminescence detection kit of the Hirschmeikang, the kit of the application has the advantages of equivalent precision and detection limit to the linear verification result.

Description

Thrombin-antithrombin complex antibody, its preparation method and application

Technical Field

The application belongs to the field of in-vitro diagnostic medical examination, and particularly relates to a preparation method and application of a thrombin-antithrombin (TAT) complex antibody.

Background

Thrombosis is the result of the combined actions of multiple factors such as abnormal release of various coagulation factors due to platelet activation and neuroendocrine changes caused by imbalance of blood coagulation and anticoagulation systems, abnormal blood flow and blood components, and vascular endothelial injury.

The marker of coagulation is thrombin formation, but it is rapidly inactivated by thrombomodulin, heparin cofactor, etc. after formation, and thus its concentration cannot be directly measured. After prothrombin is activated, it is rapidly covalently linked to antithrombin by an ester linkage to form TAT, so TAT formation is a marker of thrombin activation in vivo. The half life of TAT is longer (10-30 min), the concentration of thrombin can be reflected by measuring the concentration of TAT, so that the change condition of coagulation-anticoagulation can be monitored, and the TAT has important value for thrombotic diseases such as Disseminated Intravascular Coagulation (DIC), deep Vein Thrombosis (DVT), pulmonary Embolism (PE), cerebrovascular diseases, myocardial infarction, severe hepatitis and the like. Can be combined with other molecular markers such as D-dimer, PAI-1, etc., and can more comprehensively reflect the coagulation and fibrinolysis states of patients.

Currently, quantitative determination methods of plasma TAT are mainly enzyme-linked immunosorbent assay (ELISA) method and chemiluminescent immunoassay method. ELISA method is complex in operation and time-consuming, and is not suitable for single sample measurement. The kit for detecting TAT by chemiluminescent immunoassay used in clinic almost entirely depends on japanese simeconazole (symex), which is expensive. Therefore, development of a domestic, high-quality, low-cost, high-sensitivity and high-specificity TAT antibody and a detection kit is urgent.

Disclosure of Invention

The application provides an antibody capable of being applied to an immunoturbidimetry as well as a preparation method and application thereof aiming at the problems existing in the prior art.

In order to achieve the above purpose, the application is realized by adopting the following technical scheme:

in a first aspect, the application provides a thrombin-antithrombin complex antibody, wherein the light chain amino acid sequence of the antibody is shown as SEQ ID NO. 7, and the heavy chain amino acid sequence of the antibody is shown as SEQ ID NO. 9.

The light chain amino acid sequence of TAT monoclonal antibody is: QIVLTQSPAIMSCAQNLELPTMTCSASSSITYMHWYQQKSENILHHSPKIIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWRSDPPTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

The heavy chain amino acid sequence is: QVHLKESGPGLVASSQSLSITCQKSGTSPKRWGLHWVRQPPGKGGGELPIDWAGGTTNYNSALMSRLSISKDKSKSQVFLKMNSLQTDDTAMYYCARERNGMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

In a second aspect, the present application provides a method for preparing thrombin-antithrombin complex antibodies, comprising the steps of:

s1: preparation of monoclonal antibodies

Immunizing a mouse with complete Freund's adjuvant and natural antigen, separating spleen cells, fusing the spleen cells with SP2/0 cells under the action of a PEG fusion agent, and screening monoclonal antibodies on a HAT selection medium;

s2: acquisition of monoclonal antibody sequences

Extracting RNA of the hybridoma cell strain obtained in the step S1, amplifying sequences of DNA of light chain and heavy chain variable regions of the antibody by RT-PCR, and sequencing the amplified DNA sequences;

s3: construction of monoclonal antibody sequences

Light chain: adding the DNA sequence of the light chain variable region obtained by sequencing in the S2 to the DNA sequence of the IgK conserved region of the mouse antibody;

heavy chain: adding the heavy chain variable region DNA sequence obtained by sequencing in the step S2 to the IgG1 conserved region DNA sequence;

s4: expression of monoclonal antibodies

Constructing antibody light chain and heavy chain expression vectors by utilizing a genetic engineering technology, transfecting the vectors into a CHO cell line to obtain a CHO cell line capable of generating thrombin-antithrombin complex antibodies, and carrying out stable expression of the antibodies;

s5: monoclonal antibodies were purified using protein G sepharose.

Further, in step S2, the 5' primer sequence is: ATTGT CCTTA ATGGG GGG.

Light chain hybrid primer:

5'-ACAGT TGGTG CAGCA TCTGC-3' (IgK conserved 5' -end reverse primer),

5'-GGCGA AGACT TGGGC TGGCC-3' (reverse primer at 5' end of IgL1 conserved region),

5'-GGAGT GGACT TGGGC TGACC-3' (IgL 2 conserved 5' -end reverse primer).

Heavy chain hybrid primer:

5'-CTAGG GGGTG TCGTT TTAGC-3' (reverse primer 5' to the IgG1 conserved region),

5'-GATGG GGCTG TTGTT TTAGC-3' (reverse primer 5' to the IgG2a conserved region),

5'-GATGG GGGTG TTGTT TTAGC-3' (reverse primer 5' to the IgG2b conserved region),

5'-GATGG GGCTG TTGTT GTAGC-3' (reverse primer 5' to IgG3 conserved region).

Further, in step S1, spleen cells fused with myeloma cells were taken as a cell suspension prepared by taking the spleen the third day after the last immunization, and the cell suspension was prepared with myeloma cells according to the following ratio 1:6 to 1:10, and cell fusion under the action of PEG.

Further, the CHO cell line capable of producing thrombin-antithrombin complex antibodies in step S4 has a preservation number of CCTCC NO: C202201, which is named as Chinese hamster ovary cell CHO TAT, and is preserved in China center for type culture Collection, the preservation number of CCTCC NO: C202201, the preservation date of 2022, and 12 days of 01 month, and the preservation address of: eight branches of 299 Wuhan university in Wuhan district of Wuhan, hubei province. .

In a third aspect, the application provides the use of thrombin-antithrombin complex antibodies in an immunonephelometric in vitro diagnostic kit.

Further, the kit comprises an R1 reagent and an R2 reagent, wherein the R2 reagent comprises TAT monoclonal antibody latex, bovine serum albumin and ProClin 300.

The result coincidence rate of the kit prepared by the thrombin-antithrombin complex antibody is 94%, and the kit has good result coincidence compared with a standard diagnosis method. The regression equation of the theoretical and measured values of the linear verification test is y=1.009 x-0.1040, r2=0.9999, and the deviation is less than 5%. Compared with the chemoluminescence detection kit of the Hirschmeikang, the kit of the application has the advantages of equivalent precision and detection limit to the linear verification result.

Drawings

The application is further described below with reference to the accompanying drawings.

FIG. 1 is a schematic diagram of the ELISA of the application for detecting TAT mAb 7 cross-reacts with antithrombin;

FIG. 2 is a schematic diagram showing the cross-reaction of the TAT mAb 7 detected by ELISA of the present application with protease.

Detailed Description

In order that the above objects, features and advantages of the application will be more clearly understood, a further description of the application will be provided with reference to specific examples. It should be noted that, without conflict, the embodiments of the present application and features in the embodiments may be combined with each other.

In the following description, specific details are set forth in order to provide a thorough understanding of the application, however, the application may be practiced otherwise than as described herein, and therefore the application is not limited to the specific embodiments of the disclosure that follow.

Example 1

This example provides a process for the preparation of monoclonal antibodies.

Preparation of tat antibodies.

1.1 immunization of BALB/C mice was performed by conventional methods, the immunogen being a native TAT protein (purchased from Alpha Diagnostic intl., inc.).

1.2 primary immunization after full emulsification of 100. Mu.g antigen with Freund's complete adjuvant, intraperitoneal or subcutaneous multipoint injections were made. Every two weeks thereafter, 50 μg of antigen was mixed with incomplete adjuvant and injected for four total immunizations.

1.3 boost once 3 days prior to fusion, then remove spleen to prepare spleen cell suspension.

1.4 fusion with SP2/0 cells under the action of PEG fusion agent and screening of monoclonal on HAT selection medium.

1.5, obtaining 17 anti-TAT specific monoclonal antibody strains respectively named TAT mAb 1 to TAT mAb 17 according to sequence numbers.

2. Quality control of antibodies.

Myeloma cells: SP2/0, which does not synthesize or secrete immunoglobulin per se, meets the requirements of preservation.

Cell fusion: spleen was taken for the third day after last immunization to prepare cell suspension, and the cell suspension was prepared with myeloma cells according to a ratio of 1:6 to 1:10, and cell fusion under the action of PEG.

Cloning: the hybridoma secreting the target antibody is screened by ELISA method, and cloned by limiting dilution method to obtain hybridoma cell line. After cloning and continuous passage inspection, the positive rate of the monoclonal antibody is 100 percent after continuous cloning.

3. Identification of monoclonal antibodies.

Specificity analysis: ELISA reaction is carried out on the obtained monoclonal antibody and a series of irrelevant antigens, and whether the obtained antibody generates cross reaction with the irrelevant immunogen or not is detected. As a result, TAT mAb 7 prepared in this example showed substantially no cross-reaction with other antigens.

Example 2

Obtaining monoclonal antibody sequences.

1. Obtaining antibody DNA sequence.

1.1 Total RNA was extracted from monoclonal cells according to the protocol of Invitrogen ™ PureLink ™ RNA minikit.

1.2 RNA from the antibody light and heavy chain variable regions was then reverse transcribed into cDNA according to the protocol of the Thermo Scientific ™ ReverteAid RT reverse transcription kit, as described in Table 1 below.

Table 1 reaction system (42 ℃,30 min)

1.3 the addition of a polyC tail at the rear end of the reverse transcription product according to Invitrogen ™ terminal deoxynucleotidyl transferase (TdT) instructions.

The specific reaction system is shown in Table 2 below:

table 2 reaction system (37 ℃,15 min)

1.4 amplification of antibody light and heavy chain DNA by PCR techniques according to the instructions of Thermo Scientific ™ PCR Master Mix (2X), respectively. The PCR reaction conditions were: preheating at 95 ℃ for 5min, circulating (denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30 s) and long-time extension (at 72 ℃ for 10 min). The reaction system is shown in Table 3.

TABLE 3 reaction system

The 5' end primer sequence is as follows: ATTGT CCTTA ATGGG GGG.

Light chain hybrid primer:

5'-ACAGT TGGTG CAGCA TCTGC-3' (IgK conserved 5' -end reverse primer),

5'-GGAGT GGACT TGGGC TGACC-3' (IgL 2 conserved 5' -end reverse primer).

Heavy chain hybrid primer:

5'-CTAGG GGGTG TCGTT TTAGC-3' (reverse primer 5' to the IgG1 conserved region),

5'-GATGG GGCTG TTGTT GTAGC-3' (reverse primer 5' to IgG3 conserved region).

1.5 PCR purification kit instructions for the purification of PCR products according to the Charge switch ™ protocol.

DNA sequencing

And (3) carrying out DNA sequencing on the purified PCR product by using a Sanger sequencing method, wherein a sequencing result is shown in a sequence table.

The amino acid sequence of the TAT mAb 7 light chain variable region is shown as SEQ ID NO. 1, and the DNA sequence is shown as SEQ ID NO. 2.

The amino acid sequence of the heavy chain variable region of TAT mAb 7 is shown as SEQ ID NO. 3, and the DNA sequence is shown as SEQ ID NO. 4.

Example 3

This example provides the construction of monoclonal antibody sequences.

And adding a mouse antibody IgK light chain conserved region DNA sequence SEQ ID NO. 5 after the antibody light chain variable region DNA sequence to obtain the modified antibody light chain sequence.

And adding a mouse heavy chain IgG1 conserved region DNA sequence SEQ ID NO. 6 after the Fab end DNA sequence of the antibody heavy chain variable region to obtain the modified antibody heavy chain sequence.

TAT monoclonal antibody (modified TAT mAb 7 antibody) has light chain amino acid sequence shown in SEQ ID NO. 7 and DNA sequence shown in SEQ ID NO. 8.

The heavy chain amino acid sequence of TAT monoclonal antibody (modified TAT mAb 7 antibody) is shown as SEQ ID NO. 9, and the DNA sequence is shown as SEQ ID NO. 10.

Example 4

This example provides for expression of monoclonal antibodies.

Expression of tat monoclonal antibodies.

The synthetic DNA sequence SEQ ID NO. 8 was ligated into the pcDNA3.1 plasmid.

The synthetic DNA sequence SEQ ID NO. 10 was ligated into the pcDNA3.1 plasmid.

The two plasmids were 1:1 mixed and the plasmids were transfected into a CHO cell line for expression according to the instructions of the Gibco ™ ExpiFectamine ™ CHO transfection kit. The cell line is named as Chinese hamster ovary cell CHO TAT, and is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C202201 and the preservation date of 2022, 01 and 12 days.

2. And (5) purifying the antibody.

Monoclonal antibodies were purified using protein G sepharose.

2.1 Protean G agarose gel was packed in a column with a bed volume of 1mL at 0.75X2 cm.

2.2 wash 5mL with 20mM phosphate buffer, PBS solution at pH 7.4 (buffer A).

2.3 1mL of cell lysate was diluted to 5mL with buffer A, filtered through a 0.45 μm filter and loaded, and 5mL was rewashed with buffer A.

2.4 elution with 100mM glycine-HCl buffer (buffer B, pH 2.7) at a flow rate of 1mL/min, collection of eluted antibody, immediate neutralization of antibody with 1M Tris-HCl buffer (buffer C, pH 9.0) and collection.

2.5 washing with purified water 10mL and then with 20% ethanol 10mL, regenerating the column and storing in an environment of 4-8deg.C.

Example 5

This example provides for the preparation of an immunoturbidimetric reaction reagent.

Preparation of R1 reagent

The concentration of 3-bis (2-hydroxyethyl) amino-2-hydroxypropanesulfonic acid (DIPSO) buffer was 0.05mol/L and the pH was adjusted to 7.5 with 0.1mol/L HCl solution. Then 0.15mol/L sodium chloride and 0.1% triton X-100,0.02%ProClin 300 are added. Wherein, the DIPSO buffer solution plays a role in stabilizing the pH value of the reaction system, sodium chloride provides an ionic environment for the reaction system, triton X-100 is a surfactant, and ProClin 300 plays a role in the reaction system as a preservative.

2. R2 reagent preparation

2.1 Preparation of 0.05mol/L (pH 7.2) phosphate buffer

First separately prepare NaH of 0.05mol/L ₂ PO ₄ Solution and 0.05mol/L Na ₂ HPO ₄ Is a solution of (a) and (b). Wherein 0.05mol/L NaH ₂ PO ₄ The preparation method comprises the following steps: 7.8015g of NaH was weighed out ₂ PO ₄ -2H ₂ O, added to 1L of water. Similarly, 17.911g of Na ₂ HPO ₄ -12H ₂ O is dissolved in 1L of water to prepare 0.05mol/L Na ₂ HPO ₄ Is a solution of (a) and (b). Then 28ml NaH was taken ₂ PO ₄ Solution and 72ml Na ₂ HPO ₄ The solution was stirred and mixed.

2.2 Preparation of 0.5mol/L MES buffer (pH 6.0)

97.6g of 2-morpholinoethanesulfonic acid was weighed, added to 1L of purified water, dissolved sufficiently at room temperature, and pH was adjusted to 6.0 (0.5 mol/L) with sodium hydroxide.

2.3 weighing of other materials

2.3.1 NHS: NHS 2g was weighed out, dissolved in 5mL of purified water with stirring, and stirred for 5min to allow complete dissolution.

2.3.2 EDC: 1.4g of EDC is weighed, 3mL of purified water is added, and stirring and dissolution are carried out; stirring for 5min to dissolve thoroughly.

2.3.3 polystyrene latex beads: 2.5mL of polystyrene latex beads were measured.

2.3.4 bovine serum albumin: 10g of bovine serum albumin was weighed.

2.3.5 ProClin 300: proClin 300.2 mL was measured.

2.4 activation of polystyrene latex microspheres

2.4.1 the dissolved EDC solution was added to the NHS solution and mixed well.

2.4.2 adding the measured polystyrene latex beads into the well dissolved EDC and NHS mixed solution, adding 2.5mL of 0.5mol/L MES buffer (pH 6.0), adding 5 times volume (polystyrene latex beads) of purified water for full dissolution, reacting for 30min at room temperature, and continuously stirring.

2.4.3 transfer the prepared polystyrene latex solution into a centrifuge bottle, centrifuge after balancing, 15000rpm,8℃for 50min.

2.4.4 discarding the supernatant, and collecting the centrifugal precipitate; adding 40mL of phosphate buffer, fully suspending polystyrene latex beads by using a stirrer, balancing, centrifuging at 15000rpm and 8 ℃ for 40min; twice in succession.

2.4.5 discarding the supernatant, and collecting the centrifugal precipitate; the polystyrene latex beads were resuspended in 40mL of phosphate buffer and dispersed by sonication for 20min (150W, 3 seconds, 10 seconds apart, 30 times).

2.5 Coupling of TAT antibodies

2.5.1 TAT antibody was added to the above latex solution, stirred at room temperature for 30min, and then transferred to 2-8deg.C for overnight stirring.

2.5.2 Centrifugation at 15000rmp for 50min, and discarding the supernatant. Washing twice with phosphate buffer, centrifuging to precipitate, and discarding supernatant; the monoclonal antibody latex was resuspended in 40mL of phosphate buffer and sonicated for 20 minutes (150 w,3 seconds, 10 seconds apart, 30 times) to disperse.

2.5.3 adding bovine serum albumin and ProClin 300 into monoclonal antibody emulsion, fully dissolving, and adding 460mL of phosphate buffer solution to obtain the R2 reagent.

Laboratory test results:

A. accuracy of detection reagents

50 specimens were clinically taken, and the kit of the application was compared with the Hizimeric kit for clinical negative and positive comparison, and the results are shown in Table 4 below.

TABLE 4 accuracy test results

B. Linear range of detection reagents

Blood samples with detection values of 117.64ng/mL of the Hizimelkang kit were prepared with TAT standard substances with concentrations of 100ng/mL,50ng/mL,25ng/mL,10ng/mL,5ng/mL,1ng/mL and 0ng/mL using the kit of the application as a diluent, and detection results were shown in Table 5 below.

TABLE 5 Linear test results

C. Precision of detection reagent

Blood samples with detection values of 4.15ng/mL and 94.78ng/mL of the Hizimel kit were tested 10 times with the kit of the present application, and the precision of the kit of the present application was measured, and the results are shown in Table 6 below.

TABLE 6 precision measurement results

As can be seen from the above table, the coincidence rate of the results of the kit prepared by using the antibody of the present application is 94%,has good consistency of results compared with standard diagnostic methods. The regression equation of the theoretical and measured values of the linear verification test is y=1.009 x-0.1040, r ² =0.9999, deviation less than 5%. The samples with the same concentration are continuously monitored for 10 times, and the results show that the variation coefficient CV of the kit is less than 5%. The existing method for detecting TAT is less, mainly comprises an ELISA method and a few tubular chemiluminescence methods, and the ELISA method has obvious defects such as poor reproducibility, inconvenient operation and the like.

And (3) a sequence table:

SEQ ID NO:1

QIVLTQSPAIMSCAQNLELPTMTCSASSSITYMHWYQQKSENILHHSPKIIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWRSDPPTFGGGTKLEIK

SEQ ID NO:2:

CAAATTGTTTTCACCCAATCTCCTGCTATTATGTCTTGTGCTCAAAATTTCGAATTCCCTACCATGACCTGTTCTGCTTCTTCTTCTATTACCTATATGCATTGGTATCAACAAAAATCTGAAAATATTTTCCATCATTCTCCTAAAATTATTTATGATACCTCTAAATTCGCTTCTGGTGTTCCTGCTCGTTTTTCTGGTTCTGGTTCTGGTACCTCTTATTCTTTCACCATTTCTTCTATGGAAGCTGAAGATGCTGCTACCTATTATTGTCAACAATGGCGTTCTGATCCTCCTACCTTTGGTGGTGGTACCAAATTCGAAATTAAA

SEQ ID NO:3:

QVHLKESGPGLVASSQSLSITCQKSGTSPKRWGLHWVRQPPGKGGGELPIDWAGGTTNYNSALMSRLSISKDKSKSQVFLKMNSLQTDDTAMYYCARERNGMDYWGQGTSVTVSS

SEQ ID NO:4:

CAAGTTCATTTCAAAGAATCTGGTCCTGGTTTCGTTGCTTCTTCTCAATCTTTCTCTATTACCTGTCAAAAATCTGGTACCTCTCCTAAACGTWGGTTTCCATWGTTCGTCAACCTCCTGGTAAAGGTGGTGGTGAATTCCCTATTGATWGCTGGTGGTACCACCAATTATAATTCTGCTTTCATGTCTCGTTTCTCTATTTCTAAAGATAAATCTAAATCTCAAGTTTTTTTCAAAATGAATTCTTTCCAAACCGATGATACCGCTATGTATTATTGTGCTCGTGAACGTAATGGTATGGATTATWGGTCAAGGTACCTCTGTTACCGTTTCTTCT

SEQ ID NO:5:

CGTGCAGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG

SEQ ID NO:6:

GCTAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCGCAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGA

SEQ ID NO:7:

QIVLTQSPAIMSCAQNLELPTMTCSASSSITYMHWYQQKSENILHHSPKIIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWRSDPPTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

SEQ ID NO:8:

CAAATTGTTTTCACCCAATCTCCTGCTATTATGTCTTGTGCTCAAAATTTCGAATTCCCTACCATGACCTGTTCTGCTTCTTCTTCTATTACCTATATGCATTGGTATCAACAAAAATCTGAAAATATTTTCCATCATTCTCCTAAAATTATTTATGATACCTCTAAATTCGCTTCTGGTGTTCCTGCTCGTTTTTCTGGTTCTGGTTCTGGTACCTCTTATTCTTTCACCATTTCTTCTATGGAAGCTGAAGATGCTGCTACCTATTATTGTCAACAATGGCGTTCTGATCCTCCTACCTTTGGTGGTGGTACCAAATTCGAAATTAAACGTGCAGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG

SEQ ID NO:9:

QVHLKESGPGLVASSQSLSITCQKSGTSPKRWGLHWVRQPPGKGGGELPIDWAGGTTNYNSALMSRLSISKDKSKSQVFLKMNSLQTDDTAMYYCARERNGMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

SEQ ID NO:10:

CAAGTTCATTTCAAAGAATCTGGTCCTGGTTTCGTTGCTTCTTCTCAATCTTTCTCTATTACCTGTCAAAAATCTGGTACCTCTCCTAAACGTWGGTTTCCATWGTTCGTCAACCTCCTGGTAAAGGTGGTGGTGAATTCCCTATTGATWGCTGGTGGTACCACCAATTATAATTCTGCTTTCATGTCTCGTTTCTCTATTTCTAAAGATAAATCTAAATCTCAAGTTTTTTTCAAAATGAATTCTTTCCAAACCGATGATACCGCTATGTATTATTGTGCTCGTGAACGTAATGGTATGGATTATWGGTCAAGGTACCTCTGTTACCGTTTCTTCTGCTAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCGCAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGA

the present application is not limited to the above-mentioned embodiments, and any equivalent embodiments which can be changed or modified to equivalent changes by those skilled in the art can be applied to other fields by using the above-mentioned technical matters without departing from the technical matters of the present application, and any simple modification, equivalent changes and modification to the above-mentioned embodiments according to the technical matters of the present application still belong to the protection scope of the technical matters of the present application.

Claims

1. The thrombin-antithrombin complex antibody is characterized in that the light chain amino acid sequence of the antibody is shown as SEQ ID NO. 7, and the heavy chain amino acid sequence is shown as SEQ ID NO. 9.

2. A CHO cell line secreting the thrombin-antithrombin complex antibody of claim 1, wherein the CHO cell line has a preservation number of CCTCC NO: C202201.

3. Use of the thrombin-antithrombin complex antibody according to claim 1 for the preparation of a TAT-immunonephelometric in vitro diagnostic kit.

4. A use according to claim 3, characterized in that: the kit comprises an R1 reagent and an R2 reagent, wherein the R1 reagent is 3-bis (2-hydroxyethyl) amino-2-hydroxypropanesulfonic acid buffer solution with the pH of 7.5, sodium chloride, triton X-100 and ProClin 300, and the R2 reagent contains TAT monoclonal antibody latex, bovine serum albumin and ProClin 300.