Anti-TAT monoclonal antibody pair or antigen binding fragment thereof, nucleic acid molecule, cell strain, kit and application

Abstract

The invention belongs to the technical field of biology, and discloses an anti-TAT monoclonal antibody pair or an antigen binding fragment thereof, a nucleic acid molecule, a cell strain, a kit and application. The first antibody or the antigen binding fragment thereof and the second antibody or the antigen binding fragment thereof included in the anti-TAT monoclonal antibody pair or the antigen binding fragment thereof provided by the invention have special recognition epitopes, and the first antibody or the antigen binding fragment thereof and the second antibody or the antigen binding fragment thereof jointly form an organic whole; according to the TAT detection kit, the TAT of different structural forms in a to-be-detected sample can be specifically recognized and combined through cooperation, and the TAT detection kit has excellent detection sensitivity and reliability and can be well applied to TAT detection for a non-diagnostic purpose; the TAT detection reagent has important practical significance for research and development of the TAT detection reagent and exploration of thrombus detection value and significance.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an anti-TAT monoclonal antibody pair or an antigen-binding fragment thereof, a nucleic acid molecule, a cell line, a kit and applications thereof. Background Art

[0002] Thrombin-antithrombin III complex (hereinafter referred to as TAT) is a protein complex formed by thrombin and antithrombin molecules in a 1:1 ratio. Among them, thrombin (T) is a type of serine protease, which is transformed by thrombin activated by coagulation factors and has the reactivity to activate fibrinogen to form a reticular structure. There is a large amount of prothrombin in the blood, which has a very high fibrinogen activation rate after activation. Therefore, the blood also contains a large amount of antithrombin (AT) to neutralize thrombin after it has exerted its function, so as to maintain blood homeostasis.

[0003] Antithrombin can actively approach thrombin and neutralize thrombin in an active "sacrificial" way. After the formation of TAT, a stable covalent bond will be formed between thrombin and antithrombin, and the original structures of thrombin and antithrombin will change and rearrange to form a more stable structure. Studies have shown that the formation of TAT is highly correlated with various coagulation diseases such as arterial thrombosis, venous thrombosis and microcirculation thrombosis. The detection of TAT in the blood is of great significance for the diagnosis and treatment of coagulation diseases.

[0004] At present, the methods for detecting TAT in the blood mainly include enzyme-linked immunosorbent assay and chemiluminescent immunoassay. Both of the above methods are to obtain corresponding specific antibodies for the final stable structure of thrombin, antithrombin or TAT, and use the specific recognition ability of specific antibodies to detect TAT in the blood. However, since TAT undergoes structural changes during and after its formation, there are various structural forms of TAT in the blood, and the obtained test results cannot well reflect the actual existence of TAT in the blood, and there are problems such as poor detection sensitivity, detection authenticity and low reliability.

[0005] Therefore, developing an antibody that can specifically recognize and bind to various structural forms of TAT has important practical significance for the research and development of TAT detection reagents and the exploration of their value and significance in thrombosis detection. Summary of the invention

[0006] The first object of the present invention is to provide an anti-TAT monoclonal antibody pair or an antigen-binding fragment thereof. The anti-TAT monoclonal antibody pair or its antigen-binding fragment can well identify TAT of different structural forms, and the obtained test results are highly consistent with the actual presence of TAT in the sample to be tested, with excellent detection sensitivity and reliability, and wide application prospects.

[0007] The second object of the present invention is to provide a nucleic acid molecule.

[0008] The third object of the present invention is to provide a cell line.

[0009] The fourth object of the present invention is to provide a kit.

[0010] A fifth object of the present invention is to provide the use of the above-mentioned anti-TAT monoclonal antibody pair or its antigen-binding fragment, nucleic acid molecule, cell line and / or kit in TAT detection for non-diagnostic purposes.

[0011] Specifically, the anti-TAT monoclonal antibody pair or antigen-binding fragment thereof provided by the present invention specifically comprises: a first antibody or antigen-binding fragment thereof, wherein the first antibody or antigen-binding fragment thereof reacts conformationally with a truncated T antigen, wherein the truncated T antigen comprises an amino acid fragment as shown in SEQ ID NO: 11 or a variant sequence having at least 70% sequence identity with the sequence as shown in SEQ ID NO: 11 and retaining the conformational structure of the corresponding parent sequence; and, a second antibody or antigen-binding fragment thereof, wherein the second antibody or antigen-binding fragment thereof reacts conformationally with a full-length AT antigen and is unreactive with the truncated AT antigen, wherein the full-length AT antigen comprises an amino acid fragment as shown in SEQ ID NO: 2 or a variant sequence having at least 90% sequence identity with the sequence as shown in SEQ ID NO: 2 and retaining the conformational structure of the corresponding parent sequence, wherein the truncated AT antigen is a polypeptide fragment truncated from a partial segment of the full-length AT antigen.

[0012] Furthermore, in the first antibody or antigen-binding fragment thereof, the amino acid sequences of the heavy chain variable region CDR1 to 3 are shown in SEQ ID NOs: 12 to 14, respectively, and the amino acid sequences of the light chain variable region CDR1 to 3 are shown in SEQ ID NOs: 16 to 18, respectively.

[0013] Furthermore, in the first antibody or antigen-binding fragment thereof, the heavy chain variable region amino acid sequence is as shown in SEQ ID NO: 15 or is a variant sequence having at least 70% homology with the sequence shown in SEQ ID NO: 15 and retaining the reactivity of the corresponding parent sequence, and the light chain variable region amino acid sequence is as shown in SEQ ID NO: 19 or is a variant sequence having at least 70% homology with the sequence shown in SEQ ID NO: 19 and retaining the reactivity of the corresponding parent sequence.

[0014] Furthermore, in the second antibody or antigen-binding fragment thereof, the amino acid sequences of the heavy chain variable region CDR1 to 3 are shown as SEQ ID NOs: 20 to 22, respectively, and the amino acid sequences of the light chain variable region CDR1 to 3 are shown as SEQ ID NOs: 24 to 26, respectively.

[0015] Furthermore, in the second antibody or antigen-binding fragment thereof, the heavy chain variable region amino acid sequence is as shown in SEQ ID NO: 23 or is a variant sequence having at least 70% homology with the sequence shown in SEQ ID NO: 23 and retaining the reactivity of the corresponding parent sequence, and the light chain variable region amino acid sequence is as shown in SEQ ID NO: 27 or is a variant sequence having at least 70% homology with the sequence shown in SEQ ID NO: 27 and retaining the reactivity of the corresponding parent sequence.

[0016] The nucleic acid molecule provided by the present invention encodes the above-mentioned anti-TAT monoclonal antibody pair or its antigen-binding fragment.

[0017] The cell line provided by the present invention synthesizes and expresses the above-mentioned anti-TAT monoclonal antibody pair or the antigen-binding fragment thereof.

[0018] The kit provided by the present invention comprises the above-mentioned anti-TAT monoclonal antibody pair or antigen-binding fragments thereof.

[0019] Furthermore, the kit specifically comprises: a solid phase carrier coated with the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof labeled with a marker.

[0020] Furthermore, the solid phase carrier is magnetic inorganic microspheres and / or magnetic polymer microspheres.

[0021] Furthermore, based on the mass of the solid phase carrier, the coating amount of the first antibody or antigen-binding fragment thereof is 1 to 20 wt %.

[0022] Furthermore, the marker is selected from one or more of acridinium ester, terpyridine ruthenium and isoluminol and their derivatives.

[0023] Furthermore, the mass ratio of the second antibody or antigen-binding fragment thereof to the marker is (1-5):1.

[0024] The present invention provides the use of the above-mentioned anti-TAT monoclonal antibody pair or antigen-binding fragment thereof, nucleic acid molecule, cell line and / or kit in TAT detection for non-diagnostic purposes.

[0025] Beneficial effects:

[0026] The first antibody or its antigen-binding fragment and the second antibody or its antigen-binding fragment included in the anti-TAT monoclonal antibody pair or its antigen-binding fragment provided by the present invention have special recognition epitopes, and the two together constitute an organic whole, and cooperate with each other to achieve specific recognition and binding of TAT with different structural forms in the sample to be tested, have excellent detection sensitivity and reliability, can be well applied to TAT detection for non-diagnostic purposes, and have important practical significance for the research and development of TAT detection reagents and the exploration of their value and significance in thrombus detection.

[0027] It is speculated that the reason why the anti-TAT monoclonal antibody pair or its antigen-binding fragment has the above-mentioned effect may be that: the conformational structure of the truncated T antigen that can react with the first antibody or its antigen-binding fragment is relatively stable, and TAT and its spontaneous structural changes have little effect on the conformational structure of the segment covered by the truncated T antigen in the T antigen, that is, the conformational structure of the segment covered by the truncated T antigen in TAT with different structural forms has a high consistency, so that the first antibody or its antigen-binding fragment can better specifically recognize TAT with different structural forms; at the same time, the second antibody or its antigen-binding fragment and The full-length AT antigen undergoes a conformational reaction and has no reaction with the truncated AT antigen. The epitope is the amino acid sequence of the full-length AT antigen and the conformational structure formed. The recognition information is rich and complex. It is highly tolerant of the structural changes brought about by the "combination of T antigen and AT antigen and the structural changes after the formation of TAT" to the AT antigen, so that the second antibody or its antigen-binding fragment can well recognize TAT of different structural forms. Through the mutual cooperation between the above two antibodies and their antigen-binding fragments for different recognition effects of TAT, a more comprehensive and accurate specific detection of TAT in the sample to be tested can be achieved.

[0028] In some specific embodiments, in the first antibody or antigen-binding fragment thereof, the amino acid sequences of the heavy chain variable region CDR1 to 3 are preferably as shown in SEQ ID NOs: 12 to 14, respectively, and the amino acid sequences of the light chain variable region CDR1 to 3 are preferably as shown in SEQ ID NOs: 16 to 18, respectively; in the second antibody or antigen-binding fragment thereof, the amino acid sequences of the heavy chain variable region CDR1 to 3 are preferably as shown in SEQ ID NOs: 20 to 22, respectively, and the amino acid sequences of the light chain variable region CDR1 to 3 are preferably as shown in SEQ ID NOs: 24 to 26, respectively; in this case, the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof have better detection sensitivity and reliability, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a graph showing the experimental results of gel electrophoresis provided in Example 1 of the present invention;

[0030] Figure 2 This is a graph showing the experimental results of the detection sensitivity test of the TAT chemiluminescent detection kit provided in Example 7 of the present invention;

[0031] Figure 3 This is one of the experimental result graphs of the comparative test between the TAT chemiluminescent detection kit provided in Example 7 of the present invention and the mainstream TAT detection kit on the market (old clinical serum sample);

[0032] Figure 4 FIG2 is the experimental result of the comparative test between the TAT chemiluminescent detection kit provided in Example 7 of the present invention and the mainstream TAT detection kit on the market (fresh clinical serum sample). DETAILED DESCRIPTION

[0033] Table 1.

[0034]

[0035]

[0036]

[0037] In the present invention, based on a deep understanding of the mechanism of action of thrombin and antithrombin and the structure of each protein and its complex, the inventor creatively discovered through extensive and in-depth research that the use of antibodies that "can react conformationally with specific truncated segments of T antigens" and antibodies that "can only react conformationally with full-length AT antigens" can well achieve specific recognition and binding of TAT of different structural forms in the sample to be tested, effectively solving the problems of poor detection sensitivity, detection authenticity and low reliability in existing TAT detection. Based on this, the technical solution of the present invention is obtained.

[0038] The present invention provides an anti-TAT monoclonal antibody pair or an antigen-binding fragment thereof, which specifically includes a first antibody or an antigen-binding fragment thereof and a second antibody or an antigen-binding fragment thereof. Wherein, the first antibody or its antigen-binding fragment can react conformationally with T antigen, truncated T antigen obtained by truncation of T antigen, and the segment covered by the truncated T antigen in TAT. The second antibody or its antigen-binding fragment reacts conformationally with the full-length AT antigen and has no reactivity with the truncated AT antigen, that is, the second antibody or its antigen-binding fragment only reacts conformationally with the full-length AT antigen.

[0039] In the present invention, the conformational reaction is defined as a reaction process in which antibodies need to recognize specific conformational epitopes on antigens to achieve specific recognition and binding of antigens, that is, conformational epitopes on antigens are necessary and sufficient conditions for achieving antibody and antigen reactions.

[0040] In the present invention, the truncated T antigen specifically includes an amino acid fragment as shown in SEQ ID NO: 11 or a variant sequence having at least 70% sequence identity with the sequence as shown in SEQ ID NO: 11 and retaining the conformational structure of the corresponding parent sequence. Wherein, the variant sequence is obtained by substituting, deleting or adding one or more amino acids to the amino acid fragment as shown in SEQ ID NO: 11, and the substitution, deletion or addition does not affect the conformational structure of the truncated T antigen or has a minimal effect on the conformational structure of the truncated T antigen. The homology between the variant sequence and the amino acid fragment as shown in SEQ ID NO: 11 can be 70%, 75%, 80%, 85%, 90%, 93%, 97%, 99%, 99.9999% or any value therebetween.

[0041] In some specific embodiments, in the first antibody or antigen-binding fragment thereof, the amino acid sequences of the heavy chain variable region CDR1 to 3 are preferably shown in SEQ ID NOs: 12 to 14, respectively, and the amino acid sequences of the light chain variable region CDR1 to 3 are preferably shown in SEQ ID NOs: 16 to 18, respectively. In this case, the first antibody or antigen-binding fragment thereof can better react with the region covered by the truncated T antigen in TAT to generate a conformational reaction, and does not have a better detection performance.

[0042] More specifically, in the first antibody or antigen-binding fragment thereof, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 15 or is a variant sequence having at least 70% homology with the sequence shown in SEQ ID NO: 15 and retaining the reactivity of the corresponding parent sequence. The variant sequence is obtained by substituting, deleting or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO: 15, and the substitution, deletion or addition does not affect the reactivity of the first antibody or antigen-binding fragment thereof, and the homology between the variant sequence and the amino acid fragment shown in SEQ ID NO: 15 can be 70%, 73%, 78%, 80%, 85%, 90%, 93%, 97%, 99%, 99.9999% or any value therebetween.

[0043] More specifically, in the first antibody or antigen-binding fragment thereof, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 19 or a variant sequence having at least 70% homology with the sequence shown in SEQ ID NO: 19 and retaining the reactivity of the corresponding parent sequence. The variant sequence is obtained by substituting, deleting or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO: 19, and the substitution, deletion or addition does not affect the reactivity of the first antibody or antigen-binding fragment thereof; the homology of the variant sequence to the amino acid fragment shown in SEQ ID NO: 15 can be 70%, 75%, 80%, 85%, 90%, 93%, 97%, 99%, 99.9999% or any value therebetween.

[0044] More specifically, the type of the first antibody or antigen-binding fragment thereof may be, but is not limited to, one or more of: Fab, F(ab')2, Fd, Fv, dAb, single-chain antibody, chimeric antibody, monoclonal antibody and polyclonal antibody.

[0045] It should be noted that the reactivity of the first antibody or its antigen-binding fragment to the truncated T antigen is determined by the variable region, and those skilled in the art select a suitable constant region from the prior art to assemble with the above-mentioned variable region according to the actual usage scenario and its application requirements to obtain the first antibody or its antigen-binding fragment, that is, the present invention does not particularly limit the specific amino acid sequence of the constant region of the first antibody or its antigen-binding fragment.

[0046] In the present invention, the full-length AT antigen specifically includes an amino acid fragment as shown in SEQ ID NO: 2 or a variant sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 2 and retaining the conformational structure of the corresponding parent sequence. Wherein, the variant sequence is obtained by replacing, deleting or adding one or more amino acids to the amino acid fragment as shown in SEQ ID NO: 2, and the substitution, deletion or addition does not affect the conformational structure of the full-length AT antigen or has a minimal effect on the conformational structure of the full-length AT antigen. The homology between the variant sequence and the amino acid fragment as shown in SEQ ID NO: 2 can be 90%, 93%, 95%, 97%, 99%, 99.9999% or any value therebetween.

[0047] In the present invention, the truncated AT antigen is defined as a polypeptide fragment truncated from a partial segment of the full-length AT antigen, and the truncated AT antigen does not retain the conformational structure of the full-length AT antigen.

[0048] In some specific embodiments, in the second antibody or antigen-binding fragment thereof, the amino acid sequences of the heavy chain variable region CDR1 to 3 are preferably as shown in SEQ ID NOs: 20 to 22, respectively, and the amino acid sequences of the light chain variable region CDR1 to 3 are preferably as shown in SEQ ID NOs: 24 to 26, respectively. In this case, the second antibody or antigen-binding fragment thereof can better react with TAT in a conformational manner and has better detection performance.

[0049] More specifically, in the second antibody or antigen-binding fragment thereof, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 23 or is a variant sequence having at least 70% homology with the sequence shown in SEQ ID NO: 15 and retaining the reactivity of the corresponding parent sequence. Wherein, the variant sequence is obtained by substituting, deleting or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO: 23, and the substitution, deletion or addition does not affect the reactivity of the second antibody or antigen-binding fragment thereof, and the homology between the variant sequence and the amino acid fragment shown in SEQ ID NO: 23 can be 70%, 80%, 85%, 90%, 93%, 97%, 99%, 99.9999% or any value therebetween.

[0050] More specifically, in the second antibody or antigen-binding fragment thereof, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 27 or is a variant sequence having at least 70% homology with the sequence shown in SEQ ID NO: 27 and retaining the reactivity of the corresponding parent sequence. The variant sequence is obtained by substituting, deleting or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO: 27, and the substitution, deletion or addition does not affect the reactivity of the second antibody or antigen-binding fragment thereof; the homology between the variant sequence and the amino acid fragment shown in SEQ ID NO: 27 can be 70%, 75%, 80%, 85%, 90%, 93%, 97%, 99%, 99.9999% or any value therebetween.

[0051] More specifically, the type of the second antibody or its antigen-binding fragment may be, but is not limited to, one or more of: Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, ScFv fragment, humanized antibody, chimeric antibody, monoclonal antibody and polyclonal antibody.

[0052] It should be noted that the reactivity of the second antibody or its antigen-binding fragment to the full-length AT antigen is determined by the variable region, and those skilled in the art select a suitable constant region from the prior art to assemble with the above variable region according to the actual use scenario and application requirements to obtain the second antibody or its antigen-binding fragment. The present invention does not particularly limit the specific amino acid sequence of the constant region of the second antibody or its antigen-binding fragment.

[0053] Based on the purpose of obtaining the above-mentioned anti-TAT monoclonal antibody pair or antigen-binding fragment thereof, the present invention also provides a nucleic acid molecule. The nucleic acid molecule encodes the above-mentioned anti-TAT monoclonal antibody pair or antigen-binding fragment thereof.

[0054] In the present invention, the nucleic acid molecule is limited to carrying the genetic information encoding the above-mentioned first antibody or its antigen-binding fragment and / or the second antibody or its antigen-binding fragment. Those skilled in the art can design the nucleotide sequence and structure of the nucleic acid molecule according to actual needs, and the present invention does not impose any particular limitation on its specific nucleotide sequence and structure.

[0055] Based on the purpose of obtaining the above anti-TAT monoclonal antibody pair or antigen-binding fragment thereof, the present invention also provides a cell strain. The cell strain synthesizes and expresses the above anti-TAT monoclonal antibody pair or antigen-binding fragment thereof.

[0056] In the present invention, the cell line is limited to the ability to synthesize the above-mentioned first antibody or its antigen-binding fragment and / or the second antibody or its antigen-binding fragment. Those skilled in the art can select a corresponding preparation method from the prior art to obtain the cell line according to actual needs, and the present invention does not impose any special limitation on it.

[0057] Based on the excellent specific recognition and binding ability of the above-mentioned TAT monoclonal antibody pair or its antigen-binding fragment for TAT of different structural forms, the present invention also provides a kit. The kit specifically includes the above-mentioned anti-TAT monoclonal antibody pair or its antigen-binding fragment.

[0058] In the present invention, the first antibody or its antigen-binding fragment is preferably a capture antibody, and the second antibody or its antigen-binding fragment is preferably a detection antibody, that is, when the kit is used for TAT detection, the first antibody or its antigen-binding fragment first reacts with TAT to achieve capture and fixation of TAT, and the second antibody or its antigen-binding fragment is used to detect TAT that has been fixed by the first antibody or its antigen-binding fragment. At this time, the reactivity of the second antibody or its antigen-binding fragment with TAT is less interfered by other proteins and drugs in the complex sample to be tested, and it can still react well with the first antibody or its antigen-binding fragment-TAT, showing great advantages in the detection of complex samples to be tested.

[0059] In the present invention, specific examples of methods for detecting TAT using the anti-TAT monoclonal antibody pair or antigen-binding fragments thereof in the kit include but are not limited to: one or more of enzyme-linked immunosorbent assay, chemiluminescent immunoassay, immunochromatography and colloidal gold method.

[0060] In some specific embodiments, the kit specifically uses chemiluminescent immunoassay to detect TAT; in this case, the kit includes: a solid phase carrier coated with the first antibody or its antigen-binding fragment and the second antibody or its antigen-binding fragment labeled with a marker.

[0061] More specifically, the solid phase carrier is a material conventionally used in existing chemiluminescent immunoassays. Those skilled in the art can make adaptive choices from the existing technology according to actual needs, and the present invention does not impose any particular limitation on it. Specific examples of the solid phase carrier include but are not limited to: magnetic inorganic microspheres and / or magnetic polymer microspheres.

[0062] In some preferred embodiments, based on the mass of the solid phase carrier, the coating amount of the first antibody or its antigen binding fragment is preferably 1-20wt%, such as 1wt%, 3wt%, 5wt%, 10wt%, 12wt%, 15wt%, 20wt% or any value therebetween. At this time, the solid phase carrier coated with the first antibody or its antigen binding fragment has a better capture and fixation ability for TAT in the sample to be tested, and the solid phase carrier bound to TAT is easily separated from the reaction mixture and unreacted antibodies.

[0063] More specifically, the marker is a material conventionally used in existing chemiluminescent immunoassays. Those skilled in the art can make adaptive choices from the existing technology according to actual needs, and the present invention does not impose any particular limitation on it. Specific examples of the solid phase carrier include but are not limited to: one or more of acridinium esters, terpyridine ruthenium and isoluminol and their derivatives.

[0064] In some preferred embodiments, the mass ratio of the second antibody or antigen-binding fragment thereof to the marker is preferably (1-5):1, such as 1:1, 1.51:, 2:1, 3:1, 4:1, 5:1 or any value therebetween. At this time, the second antibody or antigen-binding fragment thereof labeled with the marker has better recognition and binding ability for TAT fixed on the solid phase carrier, and can well achieve chemiluminescence effect to generate an ideal detection signal.

[0065] More specifically, the kit may also include other auxiliary materials for immune response, dilution and / or improving antibody stability. Those skilled in the art may make adaptive choices from the prior art according to actual needs, and the present invention does not impose any particular limitation thereto.

[0066] Based on the excellent specific recognition and binding ability of the above-mentioned anti-TAT monoclonal antibody pairs or their antigen-binding fragments for TAT with different structural forms, the present invention also provides the use of the above-mentioned anti-TAT monoclonal antibody pairs or their antigen-binding fragments, nucleic acid molecules, cell lines and / or kits in TAT detection for non-diagnostic purposes.

[0067] Embodiments of the present invention are described in detail below, and the examples of the embodiments are intended to be used to explain the present invention, but should not be construed as limiting the present invention. In the embodiments, if specific techniques or conditions are not specified, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not specified for reagents or instruments used, they are all conventional products that can be obtained commercially.

[0068] Example 1

[0069] This example is used to illustrate the preparation of a TAT antigen complex, which specifically includes:

[0070] (1) Solution preparation: Dissolve human thrombin (Medix Biochemica, catalog number 528-70-1000) in PB buffer (20 mM, pH = 6.5) to obtain a thrombin solution with a concentration of 1 kU / mL;

[0071] Take PB buffer (20 mM, pH = 6.5) to dissolve human antithrombin (Medix Biochemica, product number 123-10-1) to obtain an antithrombin solution with a concentration of 1 mg / mL;

[0072] PBS buffer (10 mM, pH=7.4) was used to dissolve heparin sodium (Sigma, catalog number H3149) to obtain a heparin sodium solution with a concentration of 10 μg / mL.

[0073] (2) Preparation of TAT antigen complex: Thrombin solution and antithrombin solution were mixed at a volume ratio of 1:1, and 1 / 5T volume of heparin sodium solution was added, and the mixture was reacted at 37° C. for 30 min to obtain a TAT antigen complex solution.

[0074] The thrombin solution, antithrombin solution and TAT antigen complex solution were subjected to 12% SDS-PAGE gel electrophoresis. Figure 1 As shown. Figure 1 The results show that the TAT antigen complex solution includes thrombin-antithrombin complex and antithrombin.

[0075] Example 2

[0076] This example is used to illustrate the preparation of TAT monoclonal antibody, which specifically includes:

[0077] 1. Mouse immunization

[0078] (1) Basic immunization of mice: (i) The TAT antigen complex solution provided in Example 1 was titrated using a TAT detection reagent (Sysmex), and the TAT antigen complex solution was diluted to 1 mg / mL using a PB buffer (20 mM, pH = 6.5), and then mixed with an equal volume of Freund's adjuvant (Sigma, catalog number F5881) to obtain an emulsion;

[0079] (ii) 6-8 week old BALB / c female mice were immunized by intramuscular injection of 250 μL of the solid-liquid suspension per time, and boosted every 2 weeks. Before each immunization, 200 μL of ocular venous blood was collected and the serum titer was measured by indirect ELISA. When the serum titer of mice reached the plateau, the mice were stopped from being immunized and rested for 2 months before cell fusion.

[0080] (2) Booster immunization of mice: (i) diluting the TAT antigen complex solution to 0.5 mg / mL using PB buffer (20 mM, pH = 6.5);

[0081] (ii) 72 h before cell fusion, mice were anesthetized with ether, the abdominal skin was opened to remove the spleen, and 100 μL of 0.5 mg / mL TAT antigen complex solution was injected longitudinally along the spleen for booster immunization. After the injection, the abdominal skin incision was surgically sutured quickly.

[0082] 2. Cell fusion and hybridoma cell screening

[0083] (1) Preparation of feeder cells: (i) Preparation of macrophage feeder cell fluid: A BALB / c mouse of about 6 weeks old was killed by cervical dislocation, rinsed with tap water, and immersed in 75% ethanol solution for 5 min; the mouse was placed on a clean bench with its abdomen facing upward; the mouse abdominal skin was lifted with forceps, a small cut was made, and the skin was pulled up and down with large forceps to fully expose the abdomen; the peritoneum was lifted with sterile ophthalmic forceps, and then an appropriate amount of culture medium was injected into the abdominal cavity with a 5 mL syringe; the mouse limbs were gently lifted with another sterile ophthalmic forceps, and finally the culture medium was aspirated into a centrifuge tube with a syringe to obtain peritoneal cell fluid; the cell density of the peritoneal cell fluid was adjusted to 3×10 using RPMI-1640 culture medium (Gibco, catalog number 31800105). 5 cell / mL, to obtain macrophage feeder cell solution;

[0084] (ii) Preparation of thymic feeder cell solution: A 13-day-old BALB / c mouse was killed by cervical dislocation, rinsed with tap water, and immersed in 75% ethanol solution for 5 min. The mouse was placed on a clean bench with its abdomen facing upward. The abdominal skin of the mouse was lifted with forceps, and the skin of the abdomen and chest was cut open. The chest cavity was cut open with another pair of clean scissors, and the milky white thymus was taken out with forceps. After grinding, the thymus was passed through a 200-mesh cell sieve to obtain thymic feeder cell solution with a cell density of 3×10 5 cell / mL.

[0085] (2) Preparation of myeloma cell fluid: Mouse myeloma cell line Sp2 / 0-Ag14 (Sp2 / 0) was resuscitated and inoculated into RPMI-1640 culture medium (Gibco, catalog number 31800105), and cultured at 37°C and 5% CO2 until the logarithmic growth phase. Before fusion, myeloma cells were removed from the culture flask into a centrifuge tube, washed three times with RPMI-1640 culture medium, and centrifuged at 1000 rpm for 5 min after each wash, and the supernatant was removed; the cells were resuspended in RPMI-1640 culture medium to obtain myeloma cell fluid, and the cell density was 2×10 7 cell / mL, volume 30mL.

[0086] (3) Preparation of immune spleen cell fluid: (i) Eyeballs of BALB / C mice that had undergone primary and booster immunizations were enucleated and bled to death, and the blood was collected and antiserum was prepared as a positive control for antibody detection;

[0087] (ii) Rinse the mouse with tap water, soak it in 75% ethanol solution for 5 minutes, and then place it on the mouse dissection board in the clean bench in the right side lying position; open the abdominal cavity and remove the spleen with aseptic surgery, cut it into small pieces with scissors and place it on a 200-mesh cell sieve, and then squeeze and grind it with a grinding rod (inner core of the syringe), and at the same time add RPMI-1640 culture medium with a blowpipe to obtain broken spleen tissue;

[0088] (iii) Take an appropriate amount of RPMI-1640 culture medium and add it to the crushed spleen tissue, let it stand for 5 minutes, take the upper 2 / 3 of the suspension and transfer it to a 50 mL plastic centrifuge tube, repeat the above operation 3 times, and collect the suspension obtained in each operation; take the collected suspension and centrifuge it at 1000 rpm for 10 minutes, remove the supernatant, and wash the cells with RPMI-1640 culture medium, and resuspend the cells with 30 mL RPMI-1640 culture medium to obtain immune spleen cell fluid, with a cell density of 2×10 7 cell / mL.

[0089] (4) Cell fusion: (i) Prepare 1 mL of PEG-1500 (Sigma, Catalog No. P7181), 30 mL of RPMI-1640 culture medium, and 200 mL of RPMI-1640 complete culture medium containing 20% ​​sterile fetal bovine serum (Rongye, Catalog No. RYS-GF12-05), and preheat them to 37°C;

[0090] (ii) Mix the myeloma cell suspension and immune spleen cell suspension in a 6:1 volume ratio in a 50 mL centrifuge tube, centrifuge at 1500 rpm for 8 min, remove the supernatant, and gently tap the bottom of the tube to loosen the cells into a paste;

[0091] (iii) 1 mL of PEG was added to a centrifuge tube while gently stirring. The PEG was added within 60 s, and then 30 mL of RPMI-1640 complete culture medium was added. After gently stirring to mix, a mixed cell solution was obtained;

[0092] (iv) The mixed cell solution was centrifuged at 1000 rpm for 5 min, the supernatant was removed, a small amount of RPMI-1640 culture medium was taken to carefully disperse the cells and then transferred into 200 mL of RPMI-1640 complete culture medium, and added to a 96-well cell culture plate at a rate of 0.1 mL / well, and cultured at 37° C. and 5% CO2 for 12 h. Then, 0.1 mL / well of RPMI-1640 complete culture medium was taken to add to a 96-well cell culture plate, and cultured at 37° C. and 5% CO2 for 5 d; the cell supernatant in the culture well was replaced by 50-100% of the RPMI-1640 complete culture medium, and cultured at 37° C. and 5% CO2 for 12 d to obtain hybridoma cell solution.

[0093] (5) Screening of hybridoma cells: (i) adding the homemade TAT antigen complex to an ELISA plate at a volume of 0.1 mL / well, incubating overnight at 4°C, washing and blocking, and obtaining an ELISA plate coated with the TAT antigen complex;

[0094] (ii) 50 μL / well of hybridoma cell solution was added to the ELISA plate, incubated at 37°C for 1 h, and then washed 5 times; then GAM-HRP (donated by Beijing Wanyu Meilan Technology Co., Ltd.) was added, incubated at 37°C for 0.5 h, and then washed 5 times; then 50 μL / well of TMB colorimetric reagent (donated by Beijing Wantai Bio-Pharmaceutical Co., Ltd.) was added to the ELISA plate, and after 15 min of reaction in the dark, 50 μL / well of stop solution (donated by Beijing Wantai Bio-Pharmaceutical Co., Ltd.) was added to the ELISA plate, and the 450 / 620 nm value of each well was detected by an ELISA instrument to screen for positive clone wells.

[0095] (6) Cloning of hybridoma cells: The cells in the positive clone wells were diluted in a gradient manner according to a certain concentration using the limiting dilution method, and then inoculated into a 96-well cell culture plate so that only one cell grows in the well. The cloning was repeated 2 to 3 times until 100% positive was confirmed as a stable clone.

[0096] 3. Preparation of monoclonal antibody ascites

[0097] (1) The hybridoma cells obtained after cloning were centrifuged at 1000 rpm for 5 min and the supernatant was removed. The cells were resuspended in RPMI-1640 culture medium and the cell density was adjusted to 1×10 6 cell / mL to obtain the cell solution to be injected.

[0098] (2) Three BALB / c mice were intraperitoneally injected with 0.5 mL of liquid paraffin oil, followed by feeding with a normal diet for 1 week. Each mouse was then intraperitoneally injected with 0.5 mL of the cell solution to be injected, followed by feeding with a normal diet for 7 to 10 days until the mouse abdomen was significantly swollen; the mice were killed by cervical dissection, rinsed with tap water, and immersed in 75% ethanol for 5 minutes. The limbs of the mice were fixed on the mouse dissection table with the abdomen facing up using an injection needle; the mouse abdominal skin was lifted with tweezers, a small cut was made, and then it was cut from both sides toward the back of the mouse. The skin was torn up and down with large tweezers to fully expose the abdomen, and the peritoneum was extracted with sterile ophthalmic tweezers. A small cut was made in the center of the peritoneum to collect the ascites in the mouse abdominal cavity. The ascites was centrifuged at 3000 rpm for 20 minutes, and the supernatant was collected to obtain the monoclonal antibody ascites.

[0099] 4. Purification of monoclonal antibody ascites

[0100] The monoclonal antibody ascites was purified by ammonium sulfate precipitation and Protein A affinity column chromatography (purchased from GE, USA) to obtain monoclonal antibodies 9E1-2, E10C7, E3E12, E3B12, E6E9, E9B3, 8D11 and D14F6.

[0101] Example 3

[0102] This example is used to illustrate the reactivity of the monoclonal antibody provided in Example 2. The test specifically includes:

[0103] 1. Reactivity of monoclonal antibodies with full-length antigens

[0104] (1) The protein concentration of the thrombin solution, antithrombin solution and TAT antigen complex solution provided in Example 1 was adjusted to 300 ng / mL, and each adjusted solution was added to an ELISA plate at an addition amount of 100 μL / well. After incubation at 4°C overnight, the plates were washed and blocked to obtain ELISA plates coated with T antigen, AT antigen or TAT antigen.

[0105] (2) After the monoclonal antibody was diluted to 0.1 μg / mL using PBS buffer (10 mM, pH = 7.4), the diluted monoclonal antibody was added to the ELISA plate at a volume of 100 μL / well, incubated at 37°C for 0.5 h, and then the plate was washed 5 times; GAM-HRP was added at a volume of 100 μL / well, incubated at 37°C for 0.5 h, and then the plate was washed 5 times, then TMB color developer was added to the ELISA plate at a volume of 100 μL / well, and after reacting in the dark for 15 min, the stop solution was added to the ELISA plate at a volume of 50 μL / well, and the 450 / 620 nm value of each well was detected using an ELISA reader. The results are shown in Table 2.

[0106] Table 2.

[0107]

[0108] From the test results shown in Table 2, it can be seen that the monoclonal antibodies provided in Example 2 can be divided into two types. Monoclonal antibodies E6E9, E3B12, E10C7, 9E1-2 and E3E12 are reactive with both the full-length T antigen and the full-length TAT antigen; monoclonal antibodies E9B3, 8D11 and D14F6 are reactive with the full-length AT antigen and the TAT antigen complex.

[0109] 2. Reactivity of monoclonal antibodies with truncated antigens

[0110] (1) Preparation of truncated antigens: Using the T antigen sequence shown in SEQ ID NO: 1 and the AT antigen sequence shown in SEQ ID NO: 2 as reference sequences, polypeptide fragments as shown in Table 3 were designed and synthesized by Shanghai Biotechnology Co., Ltd. to obtain truncated antigens.

[0111] Table 3.

[0112] name sequence Truncated antigen T-1 SEQ ID NO:3 Truncated antigen T-2 SEQ ID NO:4 Truncated antigen T-3 SEQ ID NO:5 Truncated antigen T-4 SEQ ID NO:6 Truncated antigen AT-1 SEQ ID NO:7 Truncated antigen AT-2 SEQ ID NO:8 Truncated antigen AT-3 SEQ ID NO:9 Truncated antigen AT-4 SEQ ID NO:10

[0113] (2) Determination of reactivity: (i) Preparation of truncated antigen solutions: CB buffer (50 mM, pH = 9.6) was mixed with the truncated antigens shown in Table 3 to obtain truncated antigen solutions, and the concentration of each truncated antigen solution was 5 μg / mL.

[0114] (ii) ELISA plate coating: 100 μL / well of each truncated antigen solution was added to an ELISA plate, incubated at 4°C overnight, and then incubated at 37°C for 2 h, followed by washing and blocking to obtain an ELISA plate coated with the truncated antigen.

[0115] (iii) After the monoclonal antibody was diluted to 0.1 μg / mL using PBS buffer (10 mM, pH = 7.4), the diluted monoclonal antibody was added to the ELISA plate at an amount of 100 μL / well, incubated at 37°C for 0.5 h, and then the plate was washed 5 times; GAM-HRP (1 / 5000 dilution) was added at an amount of 100 μL / well, incubated at 37°C for 0.5 h, and then the plate was washed 5 times, then TMB color developer was added to the ELISA plate at an amount of 100 μL / well, reacted in the dark for 15 minutes, and then the stop solution was added to the ELISA plate at an amount of 50 μL / well, and the 450 / 620 nm value of each well was detected using an ELISA reader. The results are shown in Table 4.

[0116] Table 4.

[0117]

[0118] From the test results shown in Table 4, it can be seen that the monoclonal antibody provided in Example 2 exhibits different reactivity to different truncated antigens, the monoclonal antibody 9E1-2 epitope is the overlapping segment of the truncated antigens T-2 and T-3; the monoclonal antibody E10C7 epitope is the overlapping segment of the truncated antigens T-1 and T-2; the monoclonal antibody E3E12 epitope is the truncated antigen T-4; the monoclonal antibody E3B12 epitope is the overlapping segment of the truncated antigens T-3 and T-4; the monoclonal antibody E6E9 epitope is the overlapping segment of the truncated antigens T-3 and T-4; the monoclonal antibody E9B3 epitope is the truncated antigen AT-3; the monoclonal antibody 8D11 epitope is the overlapping segment of the truncated antigens AT-2 and AT-3; the monoclonal antibody D14F6 has no reactivity to the truncated antigens AT-1 to AT-4 and is reactive to both the full-length AT antigen and the TAT antigen complex, and the epitope is conformational.

[0119] Example 4

[0120] This example is used to illustrate the screening of magnetic bead-coated monoclonal antibodies and acridinium ester-labeled monoclonal antibodies in the antibody sandwich method, taking the monoclonal antibodies provided in Example 2 as the screening objects, and specifically includes:

[0121] 1. Preparation of monoclonal antibodies coated with magnetic beads

[0122] (1) Magnetic microparticles (ThermoFisher Scientific, catalog number 34310D, particle size 1.5-3 μm), EDC and NHS were mixed in a mass ratio of 1:1:1, and then MES solution (50 mM, pH = 5.0) was added until the concentration of magnetic microparticles in the solution was 4 mg / mL. The mixture was activated in a vertical rotating apparatus at 25°C for 20 min to obtain activated magnetic microparticles.

[0123] (2) Activated magnetic microparticles and monoclonal antibodies were mixed at a mass ratio of 1:0.02 and reacted in a vertical rotating apparatus at 25°C for 3 h. After the reaction, the magnetic microparticles were washed three times with PBS buffer (10 mM, pH = 7.4), and then PBS buffer (10 mM, pH = 7.4) containing 142 mM glycine, 0.5% (W / V) bovine serum albumin and 0.05% TritonX-100 was added to make the concentration of the magnetic microparticles in the solution 4 mg / mL, and the reaction was continued in a vertical rotating apparatus at 25°C for 2 h. After the reaction, the magnetic microparticles were washed three times with PBS buffer (10 mM, pH = 7.4), and then PBS buffer (10 mM, pH = 7.4) containing 0.5% (W / V) bovine serum albumin, 0.5% (W / V) casein, 0.05% (W / V) TritonX-100 and 1 g / L Proclin 300 preservatives (Sigma, catalog number 48914-U) in PBS buffer (10 mM, pH = 7.4) to obtain magnetic beads coated with monoclonal antibodies with a magnetic bead particle concentration of 4 mg / mL, which were stored at 4°C for future use.

[0124] 2. Preparation of acridinium ester-labeled monoclonal antibodies

[0125] (1) 50 μg of monoclonal antibody was mixed with PBS buffer (100 mM, pH = 8.0) and the final volume of the solution was 300 μL. Then, 5 μL of 10 μg / mL acridinium ester solution was added, the mixture was shaken and mixed, and the mixture was reacted at room temperature in the dark for 30 min. After the reaction, 200 μL of PBS buffer (100 mM, pH = 8.0) containing 150 mM NaCl and 133 mM glycine was added, the mixture was inverted and mixed, and the mixture was reacted at room temperature in the dark for 30 min. After the reaction, the product was transferred to a dialysis bag, and PBS buffer (10 mM, pH = 7.4) was used as the dialysate. The product was dialyzed at 4°C in the dark, and the PBS buffer was changed every 2 h for a total of 3 times to remove unlabeled acridinium ester to obtain a protein solution.

[0126] (2) The protein solution was taken out, and 10% (W / V) bovine serum albumin was added to the protein solution to make the final concentration of bovine serum albumin in the solution 0.1% (V / V). Then, an equal volume of glycerol was added and mixed by inversion to obtain an acridinium ester-labeled monoclonal antibody with an antibody concentration of 50 ug / mL, which was stored at -15°C in the dark for future use.

[0127] 3. Detection performance of monoclonal antibody combination for TAT antigens with different structural forms

[0128] (1) Preparation of test specimens: Two 5 mL tubes of the TAT antigen complex solution provided in Example 1 were placed in the TAT antigen and stored at 4°C and -20°C for 8 days, respectively, wherein the antigen stored at 4°C was used as specimen one, and the antigen stored at -20°C was used as specimen two (pre-frozen and thawed before testing), and a 20% (W / V) bovine serum albumin solution was used as a negative control specimen.

[0129] (2) Detection of the sample to be tested: 20 μL of the sample to be tested was mixed with 50 μL of the monoclonal antibody coated with magnetic beads and incubated at 37°C for 10 min; the sample was washed with PBS buffer (10 mM, pH = 7.4) containing 0.05% (W / V) Tween-20, and then 50 μL of acridinium ester-labeled monoclonal antibody was added, and the sample was mixed by oscillation and incubated at 37°C for 10 min; the sample was washed with PBS buffer (10 mM, pH = 7.4) containing 0.05% (W / V) Tween-20, and 100 μL of 1% (w / v) hydrogen peroxide solution was added. After pre-excitation at 37°C, 100 μL of 1 mol / L sodium hydroxide solution was added for excitation and detection, and the ratio of sample 1 to sample 2 (sample 1 / sample 2) was calculated. The test results are shown in Table 5.

[0130] Table 5.

[0131]

[0132]

[0133] From the results shown in Table 5, it can be seen that during the thawing process of the TAT antigen complex solution stored at -20°C, the TAT antigen complex undergoes structural changes, so that the sample 2 contains TAT ​​antigen complexes of various structural forms. If the value of sample 1 / sample 2 is closer to 1, it means that the monoclonal antibody composition has better specific recognition and detection capabilities for TAT antigen complexes of different structural forms; and compared with other monoclonal antibody combinations, the combination of monoclonal antibody E6E9 as the coating antibody and monoclonal antibody D14F6 as the labeling antibody has a lower loss rate in the detection, and this combination can detect TAT antigen complexes of more structural forms.

[0134] 4. Detection performance of monoclonal antibody combination on clinical serum samples

[0135] (1) Preparation of test specimens: TAT-positive clinical serum samples (TAT concentration > 50 ng / mL) were taken as specimen 1; TAT-positive clinical serum samples (TAT concentration < 20 ng / mL and > 4 ng / mL) were taken as specimen 2; TAT-negative clinical serum samples (TAT concentration < 4 ng / mL and > 0 ng / mL) were taken as specimen 3; 20% (W / V) bovine serum albumin was taken as a negative control specimen.

[0136] (2) Detection of the sample to be tested: 20 μL of the sample to be tested was mixed with 50 μL of magnetic bead-coated monoclonal antibody and incubated at 37°C for 10 min; the sample was washed with PBS buffer (10 mM, pH = 7.4) containing 0.05% (W / V) Tween-20, and then 50 μL of acridinium ester-labeled monoclonal antibody was added, and the sample was mixed by oscillation and incubated at 37°C for 10 min; the sample was washed with PBS buffer (10 mM, pH = 7.4) containing 0.05% (W / V) Tween-20, and 100 μL of 1% (w / v) hydrogen peroxide solution was added. After pre-excitation at 37°C, 100 μL of 1 mol / L sodium hydroxide solution was added for excitation and detection, and the ratio P / N of the detection mean of sample 1, 2 or 3 to the detection mean of the negative control was calculated. The results are shown in Table 6.

[0137] Table 6.

[0138]

[0139]

[0140] From the test results shown in Table 6, it can be seen that the use of monoclonal antibodies reactive to T as coating antibodies has better detection performance for clinical serum samples, and the detection effects of monoclonal antibodies E6E9 and E3B12 whose epitopes are the overlapping segments of truncated antigens T-3 and T-4 (sequence as shown in SEQ ID NO: 11) are better than those of other monoclonal antibodies reactive to T, and the combination of monoclonal antibody E6E9 and monoclonal antibody D14F6 whose epitope is conformational shows excellent detection effects.

[0141] 5. Structural identification of monoclonal antibodies E6E9 and D14F6

[0142] The monoclonal antibodies E6E9 and D14F6 were sent out for sequencing (sequencing company: General Biotechnology (Anhui) Co., Ltd.) to obtain specific amino acid sequence information. The results are shown in Table 7.

[0143] Table 7.

[0144]

[0145]

[0146] Example 5

[0147] This example is used to illustrate the reactivity of the monoclonal antibody E6E9 provided in Example 2. The test specifically includes:

[0148] 1. Preparation of truncated antigens

[0149] Using the overlapping region of the truncated antigens T-3 and T-4 as the reference sequence, the polypeptide fragments shown in Table 8 were designed, and these polypeptide fragments together covered 50 amino acids of the overlapping region of the truncated antigens T-3 and T-4, and were synthesized by Shanghai Biotechnology Co., Ltd. to obtain truncated antigens.

[0150] Table 8.

[0151] name Amino acid sequence Truncated antigen T-5 NVGKGQPSVLQVVNL Truncated antigen T-6 QVVNLPIVERPVCKD Truncated antigen T-7 PVCKDSTRIRITDNM Truncated antigen T-8 ITDNMFCAGYKPDEG Truncated antigen T-9 FCAGYKPDEGKRGDA

[0152] 2. Reactivity of monoclonal antibodies with truncated antigens

[0153] The method provided in "2. Reactivity of monoclonal antibodies with truncated antigens" of Example 3 was used for detection, and the results are shown in Table 9.

[0154] Table 9.

[0155] Truncated antigen T-5 T-6 T-7 T-8 T-9 Epitope range 514-528 524-538 534-548 544-558 549-563 OD value 0.0000 0.0030 0.0020 0.0050 0.0040

[0156] From the test results shown in Table 9, it can be seen that monoclonal antibody E6E9 is not reactive to truncated antigens T-5 to T9, that is, there is a conformation within the epitope range T 514-563, while the truncated antigen does not have a related conformational structure, and the conformation of this epitope range is not affected in the relevant process of TAT formation.

[0157] Example 6

[0158] This example is used to illustrate a TAT chemiluminescent detection kit and a preparation method thereof. The kit specifically includes M reagent and R reagent, and its preparation specifically includes:

[0159] 1. Preparation of reagent M

[0160] The monoclonal antibody E6E9 coated with magnetic beads was prepared according to the method provided in Example 4, namely, reagent M, and the concentration of magnetic particles in reagent M was 1 mg / mL, and the coating amount of the monoclonal antibody was 80 μg / 1 mg of magnetic beads.

[0161] 2. Preparation of R reagent

[0162] The acridinium ester-labeled monoclonal antibody D14F6 was prepared by referring to the method provided in Example 4, namely, the R reagent, and the labeled amount of acridinium ester in the R reagent was 0.33 μg / 1 μg of the monoclonal antibody D14F6.

[0163] Example 7

[0164] This example is used to illustrate the detection performance of the TAT chemiluminescent detection kit provided in Example 6. The test specifically includes:

[0165] 1. Detection sensitivity of TAT chemiluminescence detection kit

[0166] (1) Preparation of test samples: The TAT antigen complex solution provided in Example 1 was graded diluted with a 20 wt % NBS solution to obtain a series of graded concentrations of test samples, and a 20 wt % NBS solution was used as a blank control.

[0167] (2) Detection of the sample to be tested: 20 μL of M reagent and 50 μL of the sample to be tested were mixed and incubated at 37°C for 10 min; washed with PBS buffer (10 mM, pH = 7.4) containing 0.05% (W / V) Tween-20, then added with 50 μL of acridinium ester-labeled monoclonal antibody, oscillated and mixed, and incubated at 37°C for 10 min; washed with PBS buffer (10 mM, pH = 7.4) containing 0.05% (W / V) Tween-20, added with 100 μL of 1% (w / v) hydrogen peroxide solution, pre-excited at 37°C, and then added with 100 μL of 1 mol / L sodium hydroxide solution for excitation and detection. The results are shown in Tables 10 and 10. Figure 2 shown.

[0168] Table 10.

[0169] TAT concentration (ng / mL) RLU 120.00 4959768 48.00 1999648 19.20 813309 7.68 322181 3.07 132500 1.23 57060 0.49 26408 0.20 12325 blank 4059

[0170] From Table 10 and Figure 2 The test results shown in the figure show that the detection sensitivity of the TAT chemiluminescence detection kit is 0.20 ng / mL, which can well meet the clinical detection needs, and the detected luminescence signal has a good linear relationship with the TAT concentration in the sample to be tested, that is, Figure 2 The results shown serve as a standard curve.

[0171] 2. Comparison of TAT chemiluminescence detection kit and mainstream TAT detection kits on the market

[0172] (1) Preparation of test specimens: 30 old clinical serum samples from June 2024 and 47 fresh clinical serum samples from July 25 were collected as test specimens, and all test specimens were refrigerated at 4°C.

[0173] (2) Detection of test specimens: Detect each test specimen according to the method provided in "1. Detection sensitivity of TAT chemiluminescence detection kit", and Figure 2 The TAT concentration of each test sample was converted, and the commercially available mainstream TAT detection kit (Sysmex) was used as a control. The detection time and results of each test sample are shown in Table 11 and Figure 3 and 4 shown.

[0174] Table 11.

[0175]

[0176]

[0177] From Table 11 and Figure 3 and 4 The test results shown in FIG. 6 show that the detection results obtained by the TAT chemiluminescent detection kit provided in Example 6 of the present invention are highly consistent with the results obtained by the mainstream TAT detection kits on the market. 2 All are above 0.99.

[0178] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. An anti-TAT monoclonal antibody pair or an antigen-binding fragment thereof, characterized in that: The anti-TAT monoclonal antibody pair or antigen-binding fragment thereof comprises: A first antibody or an antigen-binding fragment thereof, wherein the first antibody or the antigen-binding fragment thereof reacts conformationally with a truncated T antigen, wherein the truncated T antigen comprises an amino acid fragment of a sequence as shown in SEQ ID NO: 11 or a variant sequence having at least 70% sequence identity with the sequence as shown in SEQ ID NO: 11 and retaining the conformational structure of the corresponding parent sequence; and, a second antibody or an antigen-binding fragment thereof, wherein the second antibody or the antigen-binding fragment thereof conformationally reacts with the full-length AT antigen and has no reactivity with the truncated AT antigen, wherein the full-length AT antigen comprises an amino acid fragment as shown in SEQ ID NO: 2 or a variant sequence having at least 90% sequence identity with the sequence as shown in SEQ ID NO: 2 and retaining the conformational structure of the corresponding parent sequence, and the truncated AT antigen is a polypeptide fragment truncated from a partial segment of the full-length AT antigen.

2. The anti-TAT monoclonal antibody pair or antigen-binding fragment thereof according to claim 1, characterized in that: In the first antibody or antigen-binding fragment thereof, the amino acid sequences of the heavy chain variable region CDR1 to 3 are shown in SEQ ID NOs: 12 to 14, respectively, and the amino acid sequences of the light chain variable region CDR1 to 3 are shown in SEQ ID NOs: 16 to 18, respectively.

3. The anti-TAT monoclonal antibody pair or antigen-binding fragment thereof according to claim 2, characterized in that: In the first antibody or antigen-binding fragment thereof, the heavy chain variable region amino acid sequence is as shown in SEQ ID NO: 15 or is a variant sequence having at least 70% homology to the sequence shown in SEQ ID NO: 15 and retaining the reactivity of the corresponding parent sequence, and the light chain variable region amino acid sequence is as shown in SEQ ID NO: 19 or is a variant sequence having at least 70% homology to the sequence shown in SEQ ID NO: 19 and retaining the reactivity of the corresponding parent sequence.

4. The anti-TAT monoclonal antibody pair or antigen-binding fragment thereof according to claim 1, characterized in that: In the second antibody or antigen-binding fragment thereof, the amino acid sequences of the heavy chain variable region CDR1 to 3 are shown in SEQ ID NOs: 20 to 22, respectively, and the amino acid sequences of the light chain variable region CDR1 to 3 are shown in SEQ ID NOs: 24 to 26, respectively.

5. The anti-TAT monoclonal antibody pair or antigen-binding fragment thereof according to claim 1, characterized in that: In the second antibody or antigen-binding fragment thereof, the heavy chain variable region amino acid sequence is as shown in SEQ ID NO: 23 or a variant sequence having at least 70% homology to the sequence shown in SEQ ID NO: 23 and retaining the reactivity of the corresponding parent sequence, and the light chain variable region amino acid sequence is as shown in SEQ ID NO: 27 or a variant sequence having at least 70% homology to the sequence shown in SEQ ID NO: 27 and retaining the reactivity of the corresponding parent sequence.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-TAT monoclonal antibody pair or antigen-binding fragment thereof according to any one of claims 1 to 5.

7. A cell line, characterized in that The cell line synthesizes and expresses the anti-TAT monoclonal antibody pair or antigen-binding fragment thereof according to any one of claims 1 to 5.

8. A kit, characterized in that: The kit comprises the anti-TAT monoclonal antibody pair or the antigen-binding fragment thereof according to any one of claims 1 to 5.

9. The kit according to claim 8, characterized in that The kit comprises: a solid phase carrier coated with the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof labeled with a marker; Optionally, the solid phase carrier is a magnetic inorganic microsphere and / or a magnetic polymer microsphere; Optionally, based on the mass of the solid phase carrier, the coating amount of the first antibody or antigen-binding fragment thereof is 1 to 20 wt %; Optionally, the label is selected from one or more of acridinium ester, terpyridine ruthenium and isoluminol and their derivatives; Optionally, the mass ratio of the second antibody or antigen-binding fragment thereof to the marker is (1-5):

1.

10. Use of the anti-TAT monoclonal antibody pair or antigen-binding fragment thereof according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the cell line according to claim 7 and / or the kit according to claim 8 or 9 in TAT detection for non-diagnostic purposes.

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