Anti-pyrazinamide specific monoclonal antibody and application thereof in tuberculosis medication monitoring

Abstract

The invention discloses an anti-pyrazinamide specific monoclonal antibody and an application thereof in tuberculosis medication monitoring. 4-(pyrazine-2-formylamino) butyric acid is coupled with carrier protein to prepare a pyrazinamide artificial complete antigen, and an animal is immunized by the pyrazinamide artificial complete antigen to prepare the pyrazinamide monoclonal antibody. The pyrazinamide monoclonal antibody prepared by the invention has high-sensitivity and high-specificity recognition capability, the lowest limit of detection (LOD) of pyrazinamide is 42.522 ng / mL, the IC50 value is 261.8 ng / mL, the detection range is 77.268-1447.072 ng / mL, and the pyrazinamide monoclonal antibody has a wide application prospect in tuberculosis medication monitoring.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology, and in particular to an anti-pyrazinamide specific monoclonal antibody and an application thereof in monitoring the use of tuberculosis drugs. Background Art

[0002] Pyrazinamide (PZA) is a first-line anti-tuberculosis drug with strong bactericidal activity, which can effectively kill retained bacteria in macrophages in the body that cannot be killed by other drugs. Traditionally, PZA is administered according to the weight of children and adults, but the concentration and metabolic rate of PZA in the body are affected by potential pharmacokinetics and pharmacodynamics. Based on this, it is crucial to monitor the blood concentration of patients taking pyrazinamide.

[0003] At present, the determination methods of pyrazinamide at home and abroad mainly rely on conventional instrumental analysis methods, such as high performance liquid chromatography (HPLC), high performance liquid chromatography tandem mass spectrometry (HPLC-MS), amino acid analyzer, fluorescence spectroscopy, and capillary chromatography tandem mass spectrometry (CE-MS). These methods have the disadvantages of complex pre-treatment, expensive equipment, cumbersome operation, and strong professionalism, which are difficult to meet the needs of rapid detection of on-site or large-scale samples.

[0004] Among the existing rapid detection technologies, immunoassay technology has the advantages of high sensitivity, strong specificity, rapidity, and easy operation. It has been widely used in the field of drug residue detection and has many advantages over high-performance liquid chromatography-tandem mass spectrometry. Compared with the mass spectrometry method, it can greatly reduce the threshold for use, facilitate implementation in primary hospitals, and even allow patients to test and guide medication at home. However, there are currently no reports on detection antibodies for pyrazinamide. Therefore, it is of great significance to develop an antibody that can specifically detect pyrazinamide. Summary of the invention

[0005] In order to overcome the above-mentioned defects and deficiencies in the prior art, the present invention provides an anti-pyrazinamide specific monoclonal antibody and its application in monitoring tuberculosis medication.

[0006] The first object of the present invention is to provide the use of 4-(pyrazine-2-carboxamido)butyric acid in the preparation of pyrazinamide artificial antigens and / or pyrazinamide antibodies.

[0007] The second object of the present invention is to provide a pyrazinamide antibody.

[0008] The third object of the present invention is to provide a pyrazinamide monoclonal antibody.

[0009] A fourth object of the present invention is to provide biomaterials.

[0010] The fifth object of the present invention is to provide the use of the above-mentioned pyrazinamide antibody, pyrazinamide monoclonal antibody and / or biological material in the preparation of pyrazinamide detection products.

[0011] The sixth object of the present invention is to provide a kit for detecting pyrazinamide.

[0012] The seventh object of the present invention is to provide a method for detecting pyrazinamide.

[0013] Therefore, the present invention claims the following:

[0014] Application of 4-(pyrazine-2-carboxamido)butyric acid in preparing pyrazinamide artificial antigen and / or pyrazinamide antibody.

[0015] A pyrazinamide antibody is prepared by using 4-(pyrazine-2-carboxamido)butyric acid coupled carrier protein as an immunogen.

[0016] The carrier protein is bovine serum albumin, hemocyanin or ovalbumin. Preferably, the carrier protein is bovine serum albumin.

[0017] 4-(Pyrazine-2-carboxamido)butyric acid uses -CH2-CH2-CH2-COOH as a linker arm, and the carboxyl derivatization site is far away from the characteristic structure of the amide, so that the characteristic structure of the amide is better exposed, thereby achieving specific recognition of pyrazinamide. The pyrazinamide antigen obtained by coupling the protein with -CH2-CH2-CH2-COOH as a coupling arm can accurately obtain antibodies with specific binding ability to pyrazinamide, meeting the requirements of clinical detection specificity, high sensitivity and accuracy of pyrazinamide.

[0018] The CAS number of 4-(pyrazine-2-carboxamido)butyric acid is 209961-11-5, and the structural formula is shown in formula (I):

[0019]

[0020] A pyrazinamide monoclonal antibody, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of CDR1 of the heavy chain variable region is shown in SEQ ID NO: 2, the amino acid sequence of CDR2 of the heavy chain variable region is shown in SEQ ID NO: 3, and the amino acid sequence of CDR3 of the heavy chain variable region is shown in SEQ ID NO: 4;

[0021] The amino acid sequence of CDR1 of the light chain variable region is shown in SEQ ID NO:6, the amino acid sequence of CDR2 of the light chain variable region is KVS, and the amino acid sequence of CDR3 of the light chain variable region is shown in SEQ ID NO:7.

[0022] The amino acid sequence of the heavy chain variable region of the pyrazinamide monoclonal antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5; the pyrazinamide monoclonal antibody has high sensitivity and high specificity recognition ability, and its minimum detection limit LOD for pyrazinamide is 42.522 ng / mL, and IC 50 The value was 261.8 ng / mL, and the detection range was 77.268~1447.072 ng / mL.

[0023] The biomaterial is characterized in that the biomaterial is any one of the following:

[0024] A nucleic acid molecule encoding any of the above-mentioned pyrazinamide monoclonal antibodies, or a completely complementary nucleic acid molecule thereof;

[0025] An expression cassette containing the above nucleic acid molecule;

[0026] A recombinant vector containing the above nucleic acid molecule or expression cassette;

[0027] A recombinant microorganism containing the above nucleic acid molecule, the above expression cassette or the above recombinant vector;

[0028] A recombinant cell line containing the above nucleic acid molecule, the above expression cassette or the above recombinant vector.

[0029] Use of any of the above-mentioned pyrazinamide antibodies, pyrazinamide monoclonal antibodies and / or biological materials in the preparation of a pyrazinamide detection product.

[0030] A kit for detecting pyrazinamide, comprising any of the above-mentioned pyrazinamide antibodies or pyrazinamide monoclonal antibodies, wherein the detection is for the purpose of non-disease treatment diagnosis.

[0031] A method for detecting pyrazinamide, using the above-mentioned kit for detection, wherein the detection is for the purpose of non-disease treatment diagnosis.

[0032] As an practicable manner, the method is an enzyme-linked immunosorbent assay method, which uses 4-(pyrazine-2-carboxamido)butyric acid-coupled carrier protein as a coating source and uses any of the above-mentioned pyrazinamide antibodies or pyrazinamide monoclonal antibodies as a detection antibody for detection.

[0033] Preferably, the carrier protein is chicken ovalbumin.

[0034] Preferably, the pyrazinamide antibody is prepared by using 4-(pyrazine-2-carboxamido)butyric acid coupled to a carrier protein as an immunogen.

[0035] Preferably, the carrier protein is bovine serum albumin.

[0036] Preferably, the pyrazinamide monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of CDR1 of the heavy chain variable region is shown in SEQ ID NO: 2, the amino acid sequence of CDR2 of the heavy chain variable region is shown in SEQ ID NO: 3, and the amino acid sequence of CDR3 of the heavy chain variable region is shown in SEQ ID NO: 4;

[0037] The amino acid sequence of CDR1 of the light chain variable region is shown in SEQ ID NO:6, the amino acid sequence of CDR2 of the light chain variable region is KVS, and the amino acid sequence of CDR3 of the light chain variable region is shown in SEQ ID NO:7.

[0038] The amino acid sequence of the heavy chain variable region of the pyrazinamide monoclonal antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The present invention discloses an anti-pyrazinamide specific monoclonal antibody and its application in monitoring the use of tuberculosis drugs. The present invention uses 4-(pyrazine-2-carboxamido)butyric acid coupled carrier protein to prepare pyrazinamide artificial complete antigen, and immunizes animals with the pyrazinamide artificial complete antigen to prepare pyrazinamide monoclonal antibody. The pyrazinamide monoclonal antibody prepared by the present invention has high sensitivity and high specificity recognition ability, and its minimum detection limit LOD for pyrazinamide is 42.522ng / mL, and IC 50 The value was 261.8 ng / mL, and the detection range was 77.268-1447.072 ng / mL, which has broad application prospects in monitoring tuberculosis drug use. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 These are the UV scanning identification results of the pyrazinamide artificial complete antigen prepared in Example 1 and the pyrazinamide detection antigen prepared in Example 2.

[0042] Figure 2 This is the SDS-PAGE identification result of the pyrazinamide antibody 10B9 prepared in Example 3, wherein lane 1 is the pyrazinamide antibody 10B9 and lane 2 is the marker.

[0043] Figure 3 The standard curve of pyrazinamide was used for indirect competitive ELISA detection.

[0044] Figure 4 The results of specificity analysis of pyrazinamide monoclonal antibody 10B9 are shown in Figure 2. DETAILED DESCRIPTION

[0045] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0046] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0047] Pyrazinamide hapten derivative: 4-(pyrazine-2-carboxamido)butyric acid, purchased from Leyan, product number 1220408, CAS number 209961-11-5, structural formula as shown in formula (I):

[0048]

[0049] Example 1 Synthesis of pyrazinamide artificial complete antigen

[0050] 1. Experimental Methods

[0051] The pyrazinamide artificial complete antigen was obtained by coupling the pyrazinamide hapten derivative with bovine serum albumin (BSA) through the EDC / NHS two-step method.

[0052] 1. Weigh 2.5 mg of pyrazinamide hapten derivative, 3 mg of NHS, and 20 mg of EDAC, dissolve in 800 μL of DMF, and place in a shaker at 15°C for 12 h;

[0053] 2. After the reaction is completed, the reaction solution was centrifuged at 2000r / min for 3min, 500μL of the supernatant was slowly added dropwise to 2.5mL BSA (4mg / mL, dissolved in 0.1M NaHCO3 buffer) solution, and placed in a shaker at 37°C for reaction for 16h;

[0054] 3. After the reaction, centrifuge at 4°C, 10,000 r / min for 8 min, divide the supernatant into dialysis bags, and dialyze with PBS buffer at 4°C for 3 days. Change the dialysate 3 times a day to remove free pyrazinamide hapten derivatives that are not coupled to BSA;

[0055] 4. After dialysis, the obtained pyrazinamide artificial complete antigen was filtered through a microporous filter membrane and then packaged into centrifuge tubes and stored at -20°C.

[0056] The prepared pyrazinamide artificial complete antigen was identified by ultraviolet scanning.

[0057] 2. Experimental Results

[0058] The results are as follows Figure 1 As shown. Figure 1It can be seen that the pyrazinamide hapten derivative was successfully coupled with the carrier protein BSA, indicating that the pyrazinamide artificial complete antigen was successfully prepared.

[0059] Example 2 Synthesis of pyrazinamide detection antigen

[0060] 1. Experimental Methods

[0061] The pyrazinamide detection antigen is obtained by coupling the pyrazinamide hapten derivative with chicken ovalbumin (OVA) through the EDC / NHS two-step method.

[0062] 1. Weigh 2.5 mg of pyrazinamide hapten derivative, 3 mg of NHS, and 20 mg of EDAC, dissolve in 800 μL of DMF, and place in a shaker at 15°C for 12 h;

[0063] 2. After the reaction is completed, the reaction solution was centrifuged at 2000r / min for 3min, 300μL of the supernatant was slowly added dropwise to 2.5mL of L OVA (6mg / mL, dissolved in 0.1M NaHCO3 buffer) solution, and the mixture was placed in a shaker at 37°C for 16h;

[0064] 3. After the reaction, centrifuge at 4°C, 10,000 r / min for 8 min, divide the supernatant into dialysis bags, and dialyze with PBS buffer at 4°C for 3 days. Change the dialysate 3 times a day to remove free pyrazinamide hapten derivatives that are not coupled to OVA;

[0065] 4. After dialysis, the obtained pyrazinamide detection antigen was filtered through a microporous filter membrane and then dispensed into centrifuge tubes and stored at -20°C.

[0066] The prepared pyrazinamide detection antigen was subjected to ultraviolet scanning identification.

[0067] 2. Experimental Results

[0068] The results are as follows Figure 1 As shown. Figure 1 It can be seen that the pyrazinamide hapten derivative was successfully coupled to the carrier protein OVA, indicating that the pyrazinamide detection antigen was successfully prepared.

[0069] Example 3 Preparation and identification of pyrazinamide monoclonal antibody

[0070] 1. Experimental Methods

[0071] 1. Preparation of specific pyrazinamide-positive hybridoma cells

[0072] Balb / c mice aged 6 to 8 weeks were selected as experimental animals and kept for one week before the experiment. The pyrazinamide artificial complete antigen prepared in Example 1 was diluted to 1 mg / mL with PBS, mixed and emulsified with an equal amount of Freund's adjuvant, and the initial immunization dose was set at 200 μL / mouse per time, and multiple immunizations were performed subcutaneously on the back of the neck; the second immunization was performed two weeks later, and the immunization antigen was emulsified with Freund's incomplete adjuvant, and the injection site and dose were the same as the initial immunization; the third booster immunization was performed two weeks later, and the operating steps were the same as the second; 7 days after the third immunization, the mouse blood was collected by tail cutting, and the serum titer was detected by ELISA.

[0073] Mice with good immunity were screened for hybridoma fusion experiments. Mouse spleen cells were fused with Sp2 / 0 cells to obtain fused cells. After hybridoma cells were screened from the fused cells using HAT screening medium, the pyrazinamide detection antigen prepared in Example 2 was used as the ELISA plate coating antigen, and the cell supernatant of the hybridoma cells was taken for titer and competition determination. The high-quality hybridoma cells identified as pyrazinamide positive were stored in liquid nitrogen and named 10B9.

[0074] 2. Antibody Preparation and Purification

[0075] Hybridoma cells 10B9 were cultured to the logarithmic growth phase and the cell density was adjusted to 2×10 6 / mL. 500 μL of ascites adjuvant was injected into the peritoneum of 8-12 week old Balb / c mice, and 500 μL of hybridoma cells 10B9 were injected into the peritoneum 15 days later. The ascites containing antibodies were collected 7-10 days later, and the antibodies were purified by Protein A affinity chromatography. The purified antibodies were stored in PBS by dialysis to obtain pyrazinamide antibody 10B9, and the purification effect was detected by SDS-PAGE.

[0076] 3. Sequence determination of pyrazinamide antibody 10B9

[0077] The light chain and heavy chain genes of the pyrazinamide antibody 10B9 were amplified, purified and sequenced.

[0078] 2. Experimental Results

[0079] The results of SDS-PAGE were as follows: Figure 2 The molecular weights of the two bands corresponding to the pyrazinamide antibody 10B9 were about 27 kDa and 50 kDa, respectively, which were consistent with the expected protein size, and the purity was greater than 99%.

[0080] The sequencing results showed that the amino acid sequence of the heavy chain variable region of the pyrazinamide antibody 10B9 is as shown in SEQ ID NO: 1:

[0081] QVQLQQSGPELVRPGVSVKISCKGSGYTFTDYAMHWVKQSHARSLVWI GVISAYYTFTNYNQFKGTATLTVDKSSSTAYMELARLTSEDSALYYCAKGA TGTYFDYWGQGTSVTVSS (SEQ ID NO: 1);

[0082] The amino acid sequence of the heavy chain variable region CDR1 of the pyrazinamide antibody 10B9 is shown in SEQ ID NO: 2:

[0083] GYTFTDYA (SEQ ID NO: 2);

[0084] The amino acid sequence of the heavy chain variable region CDR2 of the pyrazinamide antibody 10B9 is shown in SEQ ID NO: 3:

[0085] VISAYYTFT (SEQ ID NO: 3);

[0086] The amino acid sequence of the heavy chain variable region CDR3 of the pyrazinamide antibody 10B9 is shown in SEQ ID NO: 4:

[0087] AKGATGTYFDY (SEQ ID NO: 4);

[0088] The amino acid sequence of the light chain variable region of the pyrazinamide antibody 10B9 is shown in SEQ ID NO: 5:

[0089] DIMLTQTPLSLPVSLGDQASISCRSIQSLIHRNGITYLHWYLQKPGQSPKL LIYKVSNRFSGVPDRFSGSRSGTDFTLKITRVEAEDLGVYFCFQGTHVPYTFG GGTKLEIK (SEQ ID NO: 5);

[0090] The amino acid sequence of the light chain variable region CDR1 of the pyrazinamide antibody 10B9 is shown in SEQ ID NO: 6:

[0091] QSLIHRNGITY (SEQ ID NO: 6);

[0092] The amino acid sequence of the light chain variable region CDR2 of the pyrazinamide antibody 10B9 is KVS;

[0093] The amino acid sequence of the light chain variable region CDR3 of the pyrazinamide antibody 10B9 is shown in SEQ ID NO: 7:

[0094] FQGTHVPYT (SEQ ID NO: 7).

[0095] Example 4 Establishment of pyrazinamide indirect competitive ELISA detection method

[0096] 1. Experimental Methods

[0097] The pyrazinamide detection antigen prepared in Example 2 was selected as the coating source, the coating source concentration was 0.03125 μg / mL, and the pyrazinamide antibody 10B9 concentration was 0.03125 μg / mL.

[0098] 1. Coating: dilute the pyrazinamide detection antigen to 0.03125 μg / mL with 0.01M pH 9.6 carbonate buffer, add the diluted coating antigen to the ELISA plate, 100 μL per well, and incubate in a 4°C refrigerator overnight;

[0099] 2. Blocking: Wash the plate twice, pat dry, add 150 μL / well of blocking solution, incubate in a 37°C constant temperature water bath for 1 hour, pat dry, and dry in a 37°C drying oven for 1 hour;

[0100] 3. Competitive binding with primary antibody: add 50 μL of pyrazinamide standard solution of serial concentrations and 50 μL of pyrazinamide antibody 10B9 to the standard wells, add 50 μL of sample solution containing pyrazinamide and 50 μL of pyrazinamide antibody 10B9 to the sample wells, shake and mix, incubate in a 37°C water bath for 30 min, pour off the liquid in the wells, wash the plate 3 times, and pat dry;

[0101] 4. Binding with secondary antibody: dilute the enzyme-labeled secondary antibody IgG-HRP 5000 times with PBS, add 100 μL to each well, bind at 37°C for 30 min, wash with washing buffer PBST, and pat dry;

[0102] 5. Color development: Add 100 μL of color development solution to each well and place in a 37°C constant temperature water bath for 10 min;

[0103] 6. Termination: Add 1M sulfuric acid solution to the ELISA plate to terminate the reaction, 50 μL per well;

[0104] 7. Reading: Use a microplate reader to measure the absorbance OD value at a wavelength of 450nm;

[0105] 8. Calculation: Use the logarithm of the standard concentration as the horizontal axis and B / B0 (B0: the absorbance value corresponding to the pyrazinamide standard solution concentration of 0 μg / mL; B: the absorbance value corresponding to other standard concentrations) as the vertical axis to draw a standard curve, use Graphpad software to fit the competition standard curve, and calculate the content of pyrazinamide in the sample solution.

[0106] 2. Experimental Results

[0107] The standard curve of pyrazinamide detected by indirect competitive ELISA is as follows Figure 3 As shown, its linearity is reliable and can be used as a standard curve. 50 The detection range is 77.268-1447.072 ng / mL, and the LOD is 42.522 ng / mL. The blood concentration of pyrazinamide in clinical use is generally 5-20 mg / L (i.e., 5000-20000 ng / mL). Therefore, the detection sensitivity of the pyrazinamide monoclonal antibody 10B9 prepared by the present invention can fully meet the needs of clinical pyrazinamide blood concentration detection.

[0108] Example 5 Specificity Analysis of Pyrazinamide Monoclonal Antibody 10B9

[0109] 1. Experimental Methods

[0110] The indirect competitive ELISA detection method was used to determine the binding ability of the pyrazinamide antibody 10B9 obtained in Example 3 to common clinical first-line anti-tuberculosis drugs (ethambutol, isoniazid, rifampicin and streptomycin).

[0111] The cross-reaction rate (CR) was calculated according to the following formula: CR (%) = IC 50 (Pyrazinamide) / IC 50 (common first-line clinical anti-tuberculosis drugs) × 100%. The smaller the cross-reaction rate, the stronger the specificity.

[0112] 2. Experimental Results

[0113] The specificity analysis results are as follows Figure 4 As shown in Table 1 , except for pyrazinamide, there is no obvious competition for the other first-line anti-TB drugs.

[0114] Table 1 Cross-reaction results of pyrazinamide antibody 10B9 with pyrazinamide and common first-line anti-tuberculosis drugs in clinical practice

[0115] Drug Name <![CDATA[IC 50 (ng / mL)]]> CR(%) Pyrazinamide 261.8 100 Ethambutol ＞10000 ＜2.6％ Isoniazid ＞10000 ＜2.6％ Rifampin ＞10000 ＜2.6％ Streptomycin ＞10000 ＜2.6％

[0116] Example 6 A kit for detecting pyrazinamide

[0117] 1. Composition

[0118] (1) pyrazinamide antibody 10B9 prepared in Example 3;

[0119] The amino acid sequence of the heavy chain variable region of the pyrazinamide antibody 10B9 is shown in SEQ ID NO: 1:

[0120] QVQLQQSGPELVRPGVSVKISCKGSGYTFTDYAMHWVKQSHARSLVWI GVISAYYTFTNYNQFKGTATLTVDKSSSTAYMELARLTSEDSALYYCAKGA TGTYFDYWGQGTSVTVSS (SEQ ID NO: 1);

[0121] The amino acid sequence of the heavy chain variable region CDR1 of the pyrazinamide antibody 10B9 is shown in SEQ ID NO: 2:

[0122] GYTFTDYA (SEQ ID NO: 2);

[0123] The amino acid sequence of the heavy chain variable region CDR2 of the pyrazinamide antibody 10B9 is shown in SEQ ID NO: 3:

[0124] VISAYYTFT (SEQ ID NO: 3);

[0125] The amino acid sequence of the heavy chain variable region CDR3 of the pyrazinamide antibody 10B9 is shown in SEQ ID NO: 4:

[0126] AKGATGTYFDY (SEQ ID NO: 4);

[0127] The amino acid sequence of the light chain variable region of the pyrazinamide antibody 10B9 is shown in SEQ ID NO: 5:

[0128] DIMLTQTPLSLPVSLGDQASISCRSIQSLIHRNGITYLHWYLQKPGQSPKL LIYKVSNRFSGVPDRFSGSRSGTDFTLKITRVEAEDLGVYFCFQGTHVPYTFG GGTKLEIK (SEQ ID NO: 5);

[0129] The amino acid sequence of the light chain variable region CDR1 of the pyrazinamide antibody 10B9 is shown in SEQ ID NO: 6:

[0130] QSLIHRNGITY (SEQ ID NO: 6);

[0131] The amino acid sequence of the light chain variable region CDR2 of the pyrazinamide antibody 10B9 is KVS;

[0132] The amino acid sequence of the light chain variable region CDR3 of the pyrazinamide antibody 10B9 is shown in SEQ ID NO: 7:

[0133] FQGTHVPYT (SEQ ID NO: 7).

[0134] (2) Standards, blocking agents, colorimetric solution, antibody diluent, washing solution, stop solution, ELISA plate, and sealing film.

[0135] 2. Usage

[0136] The detection was performed according to the method of Example 4.

[0137] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of 4-(pyrazine-2-carboxamido)butyric acid in the preparation of pyrazinamide artificial antigens and / or pyrazinamide antibodies.

2. A pyrazinamide antibody, characterized in that: The pyrazinamide antibody is prepared by using 4-(pyrazine-2-carboxamido)butyric acid coupled carrier protein as an immunogen.

3. The pyrazinamide antibody according to claim 2, characterized in that The carrier protein is bovine serum albumin.

4. A pyrazinamide monoclonal antibody, characterized in that: The pyrazinamide monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of CDR1 of the heavy chain variable region is shown in SEQ ID NO: 2, the amino acid sequence of CDR2 of the heavy chain variable region is shown in SEQ ID NO: 3, and the amino acid sequence of CDR3 of the heavy chain variable region is shown in SEQ ID NO: 4; The amino acid sequence of CDR1 of the light chain variable region is shown in SEQ ID NO:6, the amino acid sequence of CDR2 of the light chain variable region is KVS, and the amino acid sequence of CDR3 of the light chain variable region is shown in SEQ ID NO:

7.

5. The pyrazinamide monoclonal antibody according to claim 4, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

5.

6. Biomaterial, characterized in that The biological material is any one of the following: A nucleic acid molecule encoding the pyrazinamide monoclonal antibody according to claim 4 or 5, or a completely complementary nucleic acid molecule thereof; An expression cassette containing the above nucleic acid molecule; A recombinant vector containing the above nucleic acid molecule or expression cassette; A recombinant microorganism containing the above nucleic acid molecule, the above expression cassette or the above recombinant vector; A recombinant cell line containing the above nucleic acid molecule, the above expression cassette or the above recombinant vector.

7. Use of the pyrazinamide antibody according to any one of claims 2 to 3, the pyrazinamide monoclonal antibody according to any one of claims 4 to 5, and / or the biomaterial according to claim 6 in the preparation of a pyrazinamide detection product.

8. A kit for detecting pyrazinamide, characterized in that: The method comprises the pyrazinamide antibody according to any one of claims 2 to 3 or the pyrazinamide monoclonal antibody according to any one of claims 4 to 5, wherein the detection is for the purpose of non-disease treatment diagnosis.

9. A method for detecting pyrazinamide, characterized in that: The test kit according to claim 8 is used for the purpose of non-disease treatment diagnosis.

10. The method according to claim 9, characterized in that The method is an enzyme-linked immunosorbent assay method, which uses 4-(pyrazine-2-carboxamido)butyric acid-coupled carrier protein as a coating source, and uses the pyrazinamide antibody described in any one of claims 2 to 3 or the pyrazinamide monoclonal antibody described in any one of claims 4 to 5 as a detection antibody for detection.

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