Tubular magnetic particle chemiluminiscence detection kit for detecting foot and mouth disease virus type A neutralizing antibody and application of tubular magnetic particle chemiluminiscence detection kit

Abstract

The invention provides a tubular magnetic particle chemiluminiscence detection kit for detecting a foot and mouth disease virus type A neutralizing antibody. The tubular magnetic particle chemiluminiscence detection kit is characterized by comprising a magnetic particle coupling monoclonal antibody W125 working solution and an enzyme-labeled antibody working solution, an antibody in the enzyme-labeled antibody working solution is a monoclonal antibody W145. Magnetic particles are used as a solid phase carrier to be coupled with an FMDV type A bovine monoclonal antibody W125 (MPs-W125), alkaline phosphatase is used for marking an FMDV type A bovine monoclonal antibody W145 (AP-W145), FMDVA / WH / CHA / 09 146S is used as a detection antigen, 3-(2-spiral adamantane)-4-methoxy-4-(3-phosphoryloxy)-phenyl-1, 2-dioxane (AMPPD) is used as a luminous substrate, and the rapid detection kit is assembled. Meanwhile, a full-automatic chemiluminescence immunoassay analyzer is matched, so that quantitative, rapid and automatic detection of the FMDV A type neutralizing antibody is realized.

Description

Technical Field

[0001] The invention provides a tubular magnetic particle chemiluminescence detection kit for detecting foot-and-mouth disease virus type A neutralizing antibody and application thereof, belonging to the technical field of in vitro detection. Background Art

[0002] Foot and mouth disease (FMD) is an acute, febrile, highly contagious disease caused by foot and mouth disease virus (FMDV). It mainly infects even-toed ungulates such as pigs, cattle, and sheep, causing huge economic losses to animal husbandry and seriously affecting international trade. The World Organization for Animal Health (WOAH) lists it as a notifiable animal infectious disease, and my country lists it as the first of the first category of animal infectious diseases. FMDV is a small RNA virus with high variability. There are seven serotypes: O, A, C, Asia 1, SAT1, SAT2, and SAT3. There is no cross-immune protection between the serotypes. There are also differences in antigenic structure between different lineages of the same serotype, which cannot provide good cross-immune protection against heterologous strains. The main FMDV serotypes prevalent in my country are type O and type A. FMDV type A is the serotype with the largest antigenic variation. The antigenic variation of different lineages of viruses is large, and the cross-immune protection effect is poor, which has brought great trouble to the development of vaccines and diagnostic reagents. In countries where foot and mouth disease is prevalent, inactivated vaccines are mainly used for immunization and prevention of FMD. The detection of antibody levels after vaccination is an important basis for evaluating the immune effect and formulating the immune program. Neutralizing antibodies are the main protective immune components of the host against FMDV infection. The virus neutralization test (VNT) is the "gold standard" for detecting neutralizing antibodies, but this method requires the use of live viruses, which has the risk of spreading the virus. It needs to be operated in a high-level biosafety laboratory, the experimental cycle is long, the cell state has a great influence on the experimental results, the results are poorly reproducible, and it is not suitable for large-scale serum sample detection. Liquid blocking ELISA and solid competitive ELISA for detecting FMDV structural protein antibodies are widely used because of their safe operation and good reproducibility. However, the traditional ELISA method based on rabbit and guinea pig polyclonal antisera detects both neutralizing antibodies and non-neutralizing antibodies, and has a low correlation with attack protection. Therefore, establishing a safe, efficient, rapid, and fully automatic neutralizing antibody detection method can provide strong technical support for foot-and-mouth disease prevention and control.

[0003] The sensitivity and specificity of diagnostic test kits depend largely on the selection of monoclonal antibodies. Whether antibodies that recognize a single epitope can represent the overall antibody response of different epitopes on the FMDV capsid. Therefore, the epitope recognized by the monoclonal antibody should be immunodominant and overlap with the epitopes recognized by most FMDV antibodies produced by the host animal. In the early stage of this study, bovine monoclonal antibodies W125 and W145 were successfully developed through single B cell antibody technology. Both are broad-spectrum neutralizing antibodies within the FMDV A type, which can neutralize all representative strains prevalent in China, including viruses from the two branches of G1 (representative strain A / WH / CHA / 09FMDV) and G2 (representative strain A / GDMM / CHA / 2013 FMDV) in the SEA97 lineage, and can also neutralize the AF72 vaccine strain (A22 subtype strain). Antibody W145 recognizes the key amino acid at position RGD+2 of VP1 of type A virus, binds to the receptor binding site of the virus, and exerts a neutralizing effect that blocks the virus from invading cells (Li et al, 2023, PLoS Pathogens, 2023,19(11):e1011811). At the same time, the binding of antibodies to the neutralizing sites of VP3 and VP2 adjacent to this site will also produce a steric hindrance effect on the binding of W145 antibodies, thereby accurately reflecting the response level of neutralizing antibodies. The receptor binding site is the most important neutralizing site and the neutralizing site with the strongest immunodominance. By accurately detecting the antibody response at this site, the response level of neutralizing antibodies can be truly reflected. The antigenic sites recognized by W125 cover the BC, EF and HI loops on VP2 and the BB node and HI loop on VP3. The recognition sites of W145 combined with W125 antibodies cover almost all the dominant neutralizing antigenic epitopes on the structural proteins of FMDV (Li et al, 2023, Plos Pathogens).

[0004] Chemiluminescence immunoassay (CLIA) technology combines enzyme-linked immunosorbent assay and chemiluminescence technology, and has the advantages of high sensitivity, simple operation, and easy automation. It is widely used in clinical diagnosis, drug analysis, animal disease detection, etc. Magnetic particles (MP) are new solid phase carrier materials for CLIA. Compared with traditional polystyrene microplates, magnetic particles (MP) have a large surface area and are more firmly connected to antigens / antibodies through covalent bonds. The immune reaction is carried out in an almost liquid state, and it has the characteristics of rapidity, high throughput, full automation, and convenient operation. Summary of the invention

[0005] In view of this, the present invention aims at the deficiencies of existing VNT and ELISA detection technologies and provides a magnetic particle CLIA kit and its application for detecting FMDVA type neutralizing antibodies in a safe, rapid and automated manner. The kit can quantitatively detect FMDVA type neutralizing antibodies in animal serum for evaluating the immune efficacy of vaccines.

[0006] The tubular magnetic microparticle chemiluminescence detection kit for detecting neutralizing antibodies of type A of foot-and-mouth disease virus of the present invention comprises a magnetic microparticle-coupled monoclonal antibody W125 working solution and an enzyme-labeled antibody working solution. The amino acid sequence of the heavy chain variable region of the monoclonal antibody W125 is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2. The enzyme in the enzyme-labeled antibody working solution is alkaline phosphatase; the antibody in the enzyme-labeled antibody working solution is monoclonal antibody W145, the amino acid sequence of the heavy chain variable region of the monoclonal antibody W145 is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4.

[0007] The amino acid sequence of the heavy chain variable region of monoclonal antibody W125: QVQLRESGPSLVKPSQTLSLTCTVSGFSLSTYAVYWVRQAPGKALECLGSVSSGDYLTYNPALKSRLTITKDNSKSEVSLSVSTVTPEDTATYYCAKSHSSGYNGWIDFGCYEFTGYGPRYVDAWGQGVQVTVSS; The amino acid sequence of the light chain variable region of monoclonal antibody W125: WAQAVLTQPSSVSGSLGQRVSITCSGSSSNVGLGNYVSWFQQIPGSAPRTLIYGATNQASGVPDRFSGSRSGNTATLTISSLQAEDEANYFCASPDSSQTIFGSGTTLTVLG; The amino acid sequence of the heavy chain variable region of monoclonal antibody W145: QAQLRESGPSLVKPSQTLSLTCTVSGFSLSTCAVVWVRQTPGKALEWIGRLARDGTTYYNPALKSRLSITKDNSKTRVSLSLSSVTPEDTATYYCGKDVPDYIGYAEEIDVDVWGQGLLVTVSS; The amino acid sequence of the light chain variable region of monoclonal antibody W145: WAQAVLTQPSSVSGSLGQRVSITCSGSSSNVGQGDYVSWFQQIPGSAPRTLIYAATSRASGVPDRFSGSRSGNTATLTIRSFQAEDEADYFCSSPDNSGNAYFGSGTTLTVLG; In the magnetic particle-coupled monoclonal antibody W125 working solution, the mass ratio of the magnetic particles to the monoclonal antibody W125 is 100:(1-10).

[0008] The magnetic particle concentration in the magnetic particle-coupled monoclonal antibody W125 working solution is 0.5 mg / mL; the concentration of the enzyme-labeled antibody working solution is 1:1000.

[0009] The kit also includes a detection antigen, a concentrated washing solution, a luminescent substrate, a quality control product, a diluent, and a standard product.

[0010] The detection antigen is 146S antigen, and the detection antigen concentration is 0.25 μg / mL. The concentrated washing solution is a PBS buffer with a pH value of 7.4 containing Tween-20 with a volume concentration of 0.1% to 0.5%. The concentrated washing solution contains 2000.0g of sodium chloride, 50.0g of potassium chloride, 725.0g of disodium hydrogen phosphate dodecahydrate, 50.0g of potassium dihydrogen phosphate, and 125mL of Tween 20 per 10000mL of water, and the pH value is 7.4. The luminescent substrate is 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoacyl)-phenyl-1,2-dioxetane. The quality control products include quality control product 1 and quality control product 2, quality control product 1 is 0~10U negative serum, and quality control product 2 is a standard product with a FMDV type A antibody content of 100U.

[0011] The diluent is a diluent used for the magnetic particle-coupled monoclonal antibody W125 working solution and the enzyme-labeled antibody working solution, and the diluent is a 0.1M MOPS buffer with a pH of 7; the MOPS buffer contains 0.1-2% BSA, 0.01-0.5% Tween-20, 0.01-0.5% ProClin300, 0.01-0.5M MgCl 2 , 0.01~0.5M NaCl, 0.05~3% hydrolyzed milk protein and 0.1~1% PEG 6000.

[0012] The standard substance is antiserum with FMDVA type neutralizing antibody content of 3200U, 1600U, 800U, 400U, 200U, 100U, 50U, 25U, 12.5U, 6.25U, 3.125U (1:4), 1.56U (1:2), and 0U.

[0013] The monoclonal antibodies W125 and W145 used in the present invention are both FMDV host-derived antibodies developed using single B cell antibody technology, and have unique antibody gene sequence information, which realizes the digital permanent preservation of antibody information and will not be lost; the expression plasmid of the antibody can be synthesized according to the sequence information, and the neutralizing antibody can be obtained by transfecting CHO cells for expression. The detection target of the kit is the neutralizing antibody produced after virus infection or vaccine immunization. The antibody W145 recognizes the key amino acid RGD+2 of type A virus VP1, binds to the receptor binding site of the virus, and plays a neutralizing role in blocking the virus from invading cells. The receptor binding site is the most important neutralizing site and the neutralizing site with the strongest immunodominance. The response level of the neutralizing antibody can be truly reflected by accurately detecting the antibody response at this site. The W125 recognition antigen site covers BC, EF and HI loops on VP2 and BB nodes and HI loops on VP3. The recognition site of W145 combined with the W125 antibody covers almost all the dominant neutralizing antigen epitopes on the FMDV structural protein. Both W145 and W125 are FMDVA type intratype broad-spectrum neutralizing antibodies, which can neutralize all representative strains prevalent in my country, such as FMDVA / WH / CHA / 09 (SEA97 lineage G1 branch), FMDV A / GDMM / CHA / 2013 (SEA97 lineage G2 branch) and AF72 vaccine strain (A22 subtype strain), forming a universal detection model for detecting neutralizing antibodies after immunization with different type A foot-and-mouth disease vaccines.

[0014] During the test, the sample to be tested, FMDVA type antigen, AP-W145 and appropriate amount of MPs-W125 were first mixed and incubated at 37 ℃ for 15 minutes; the magnetic beads were adsorbed on a magnetic plate for 1 minute, the reaction solution was discarded, and the magnetic beads were resuspended in washing solution and then adsorbed and washed. This was repeated 4 times, and the washing solution was discarded. 100μL of chemiluminescent substrate AMPPD was added, the magnetic beads were mixed, and incubated at 37 ℃ for 3 minutes. The relative luminescence intensity (RLU) was detected and the antibody content was calculated. This method was used to detect the standard, fit the standard curve, and detect 343 serum samples with clear background. The optimal Cut-off value of the CLIA method was determined to be 41.395U through ROC curve analysis. At this time, the sensitivity of the method was 95.93% and the specificity was 100%.

[0015] In summary, the present invention uses magnetic particles as solid phase carriers to couple FMDV A type bovine monoclonal antibody W125 (MPs-W125), uses alkaline phosphatase (AP) to label FMDV A type bovine monoclonal antibody W145 (AP-W145), FMDVA / WH / CHA / 09146S as detection antigen, and 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoacyl)-phenyl-1,2-dioxetane (AMPPD) as luminescent substrate, establishes a magnetic particle CLIA method for detecting FMDV A type neutralizing antibodies, and assembles it into a rapid detection kit. At the same time, it is equipped with a fully automatic chemiluminescence immunoassay to achieve quantitative, rapid and automated detection of FMDV A type neutralizing antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 .Four reference standard curves for CLIA method; Figure 2 .ROC curve analysis was used to determine the CLIA cut-off value; Figure 3 .Cross-reactivity of CLIA with FMDV type O and Asia type 1; Figure 4 .CLIA cross-reactivity with other viral antibodies. DETAILED DESCRIPTION

[0017] The present invention is described in detail below in conjunction with specific embodiments.

[0018] Example 1 CLIA method development 1. Materials Magnetic particles (magnetic particles are magnetic Fe 3 O 4 Microparticles) were purchased from MBL, JSR Group, and AP and AMPPD were products of Sigma. FMDV type A bovine broad-spectrum neutralizing antibodies W125 and W145 were prepared and preserved by our laboratory using single B cell antibody technology. The antibody preparation method is described in patent document ZL201810929067.1.

[0019] LYC100T fully automatic chemiluminescent immunoassay analyzer was provided by Lanzhou Zouyan Biotechnology Co., Ltd.; SEPMAG®A200 mL biomagnetic separator was purchased from sepmag.

[0020] The control serum and FMDV A / WH / CHA / 09146S antigen were prepared and stored by the National Foot-and-Mouth Disease Reference Laboratory.

[0021] The amino acid sequence of the heavy chain variable region (HV1) of W145 is shown in GenBank (Accession Number OR540351.1), and the amino acid sequence of the light chain variable region (LV1) is shown in GenBank (Accession Number OR540395.1). The amino acid sequence of the heavy chain variable region of antibody W125 is shown in GenBank (Accession Number OR540349.1), and the amino acid sequence of the light chain variable region is shown in GenBank (Accession Number OR540393.1). The HV1 and HV2 coding sequences of the antibody are respectively connected to the pcDNA3.4 eukaryotic expression vector containing the constant region sequence of the heavy chain and light chain of bovine IgG2, and the heavy chain and light chain expression plasmids are mixed and transfected into CHO suspension culture cells in a mass ratio of 2:3, and the complete IgG2 subtype antibody is expressed, and the antibody is purified by affinity chromatography. The preparation method of the antibodies W125 and W145 is described in the patent document patent ZL201810929067.1.

[0022] 2. Preparation of Magnetic Microparticles (MPs)-coupled Monoclonal Antibody W125 Working Solution (1) Take 20 mg of magnetic microparticles (MPs), adsorb them using a biomagnetic separator for 1 min, discard the supernatant, resuspend them with 2 mL of 0.1 mol / L 2-morpholineethanesulfonic acid (MES, pH 5.0), and repeat this process for 4 times. 100:1 20000 (2) Add 200 μL each of N-hydroxysuccinimide (NHS, 10 mg / mL) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 10 mg / mL) to activate the MPs, and mix and react at room temperature for 30 min. Then, wash the MPs 4 times with 2 mL of 0.1 mol / L MES (pH 5.0).

[0023] (3) Add 200 μg of highly purified monoclonal antibody W125, shake at room temperature for 3 h, then add 200 μL 10% BSA and shake at room temperature for 3 h to prepare magnetic particle-monoclonal antibody conjugate (MPs-W125). Finally, wash with PBST 3 times, disperse with 40 mL preservation solution to make magnetic particle working solution, and store at 4°C for later use.

[0024] 3. Preparation of Alkaline Phosphatase (AP) Labeled Monoclonal Antibody W145 Working Solution (1) AP enzyme activation: sodium periodate (NaIO 4) 15 mg / mL and AP 10 mg / mL were shaken and mixed at a mass ratio of 1.5:1, and reacted at 4°C for 1 h. Then, an equal volume of ethylene glycol (1%) was added to the mixture to reduce the excess NaIO 4 , 4°C, 2h, to complete the activation of AP.

[0025] (2) AP-labeled monoclonal antibody: Mix monoclonal antibody W145 with activated AP at a mass ratio of 1:2, dialyze in carbonate buffer at pH 9.6 and concentration of 0.5 mol / L, and store at 4°C overnight. Then add sodium borohydride (NaBH4), react at 4°C for 1 hour, and dialyze in PBS overnight to prepare alkaline phosphatase-labeled antibody (AP-W145). Finally, add PBS containing 50% glycerol to the dialyzed product and store at -20°C or below for future use. Dilute to working concentration with enzyme-labeled antibody diluent when using and store at 4°C.

[0026] 4. Definition of antibody content in FMDVA type and preparation of standard A serum sample with an FMDVA antibody titer of 1:128 was taken for testing by virus neutralization test (VNT), and the antibody content was defined as 100 active units, i.e. 100 U. The antibody content of the standard (serum with a VNT titer of 1:4096) was 3200 U. Use newborn calf serum to dilute the standard sample in multiples to prepare antibody contents of 3200U (1:4096), 1600U (1﹕2 048), 800U (1﹕1024), 400U (1﹕512), 200U (1﹕256), 100U (1﹕128), 50 (1﹕64), 25 (1﹕32), 12.5U (1﹕16), 6.25U (1﹕8), 3.125U (1﹕4), 1.56U (1﹕2), and 0U, and store at -20℃ or below for future use. The corresponding relationship between the antibody titer (1﹕x) and the active content U is U=0.78125x.

[0027] 5. CLIA detection process of FMDVA type neutralizing antibody magnetic particles (1) Add 40 μL of the sample to be tested, standard, and positive and negative serum into the reaction cups respectively.

[0028] (2) Add 25 μL MPs-W125, 100 μL AP-W145, and 100 μL 146S antigen, mix well, and incubate at 37 °C for 15 min. Use a magnetic plate to adsorb the magnetic beads for 1 min, discard the reaction solution, add washing solution to resuspend the magnetic beads, and then adsorb and wash again. Repeat this process 4 times and discard the washing solution.

[0029] (3) Add 100 μL of chemiluminescent substrate AMPPD, mix the magnetic beads, and incubate at 37 °C for 3 min.

[0030] (4) Detect the relative luminescence intensity (RLU) and calculate the antibody content.

[0031] 6. Preparation of Standard Curve Use CLIA to test standard serum, with antibody concentration (U) as the horizontal axis and luminescence value (RLU) as the vertical axis, to draw a four-parameter standard curve y=A 2 +(A 1 -A 2 ) / (1+(x / x 0 ) p ), the standard curve is as follows Figure 1 shown.

[0032] 7. Optimization of Magnetic Microparticle CLIA Method 7.1. Optimization of Antibody-Conjugated Magnetic Particle Concentration MPs-W125 was diluted with diluent (0.1M pH7 MOPS buffer) at MPs concentrations of 0.1, 0.25, 0.5, and 1 mg / mL, respectively, for the detection of standard products. The standard curve was fitted with antibody content as X value and RLU as Y value. The positive and negative controls were used as the serum to be tested, and the luminescence intensity ratio (LIR) N / P value of the positive and negative sera was calculated after the test. By comparing the standard curve R 2 The results showed that when the MPs concentration was 0.5 mg / mL, the standard curve R 2 The LIRN / P value of positive and negative samples was 0.9997, and the LIRN / P value of positive and negative samples was 1131, which was the best test result.

[0033] The MPs-W125 diluent is 0.1M pH7 MOPS buffer, which contains 0.1~2% BSA, 0.01~0.5% Tween-20, 0.01~0.5% ProClin300, 0.01~0.5M MgCl 2 , 0.01~0.5M NaCl, 0.05~3% hydrolyzed milk protein and 0.1~1% PEG 6000.

[0034] 7.2. Optimization of 146S antigen and AP-W145 dilution The 146S antigen was diluted with pH7 PBS buffer at 0.1, 0.25, 0.5, 1, and 2 μg / mL; AP-W145 was diluted with diluent (0.1M pH7 MOPS buffer) at 1﹕500, 1﹕1000, 1﹕2000, 1﹕5000, and 1﹕10000. 100μL of the detection standard was used for all tests. The standard curve was fitted with the antibody content as the X value and RLU as the Y value. The positive and negative controls were used as the serum to be tested, and the positive and negative serum LIRN / P values ​​were calculated after the test. The results showed that when the 146S antigen was diluted to 0.25μg / mL, R 2 The value of R was 0.9994, and the N / P value was 686, which was the best result. When the enzyme-labeled antibody was diluted 1:1000, 2 The value is 0.9989, and the N / P is 1372, which is the best detection result.

[0035] The AP-W145 diluent is 0.1M pH7 MOPS buffer, which contains 0.1~2% BSA, 0.01~0.5% Tween-20, 0.01~0.5% ProClin300, 0.01~0.5M MgCl 2 , 0.01~0.5M NaCl, 0.05~3% hydrolyzed milk protein and 0.1~1% PEG 6000.

[0036] The 146S antigen diluent is 0.1M pH7 PBS buffer, which contains 0.1~2% BSA, 0.1~1% PEG6 000, 0.5~5% trehalose, 0.1~0.5% gelatin and 0.01~0.5% ProClin300 by weight.

[0037] 7.3. Optimization of reaction time and sample volume The sample to be tested, MPs-W125, AP-W145 and 146S antigens were mixed and incubated at 37°C for 5, 10, 15 and 20 min respectively; the sample volume was selected to be 10, 20, 30, 40 and 50 μL respectively, the standard and positive and negative control serum were tested, the standard curve was fitted, the N / P value was calculated, and the standard curve R was compared. 2 The results showed that the best detection result was obtained when 40 μL of sample was added, at which time N / P was 1198.8 and the standard curve R 2 When the incubation time was 15 min, R 2 The value is 0.9985, and the N / P value is 530.9, which is the best effect.

[0038] Example 2 CLIA method cut-off value, sensitivity and specificity determination 1. Serum Samples Positive sera: A total of 221 samples, from cattle, sheep and pigs immunized with FMDV A type inactivated vaccine, confirmed by VNT as positive for FMDV A type neutralizing antibodies.

[0039] Negative sera: 122 in total, from healthy non-immunized cattle and pigs.

[0040] FMDV other serotype positive sera: 30 FMDV O type antibody positive sera; 18 FMDV Asia type 1 antibody positive sera.

[0041] Other virus antibody positive sera: 8 samples of porcine reproductive and respiratory syndrome virus (PRRSV) antibody positive sera; 4 samples of peste des petits ruminants virus (PPRV) antibody positive sera; 3 samples of Seneca virus (SVV), mycoplasma, bovine viral diarrhea virus (BVDV), and swine fever virus (CSFV) antibody positive sera. The serum samples were collected and preserved by the National Key Laboratory of Animal Disease Prevention and Control of Lanzhou Veterinary Research Institute.

[0042] 2. Determination of Cut-off Value The optimized CLIA method was used to detect the standard, fit the standard curve, and detect 221 positive sera and 122 negative sera. The ROC curve was used for analysis, and the Youden index value was used as the indicator. The antibody concentration value when the Youden index was the largest was used as the optimal Cut-off value of the CLIA method and as the judgment standard for the detection of FMDVA type neutralizing antibodies by this method. The results showed that the optimal Cut-off value of this method was 41.395U (titer was 1:52.9856), at which time the AUC value was 0.994, P < 0.001, and the Youden index was 0.959 ( Figure 2 ). The judgment standard is: when the test result is ≥41.395U, it is judged as FMDVA type neutralizing antibody positive, and when it is <41.395U, it is judged as FMDVA type neutralizing antibody negative.

[0043] 3. Sensitivity and specificity of CLIA method Of the 221 positive samples, 212 were detected as positive by the CLIA method, with a sensitivity of 95.93%; of the 122 negative samples, 122 were detected as negative, with a specificity of 100%.

[0044] 4. Cross-reactivity with FMDVO and Asia 1 The CLIA method was used to test 30 serum samples with positive FMDVO antibodies, and one sample was positive, while the other 29 samples were all negative. The results of testing 18 serum samples with positive FMDVAsia1 antibodies were all negative ( Figure 3 ). This indicates that this method has no cross-reaction with other serotypes and is a FMDVA type-specific detection method.

[0045] 5. Cross-reactivity with antibodies against other related viruses The CLIA method was used to test 8 PRRS-positive sera, 4 PPRV-positive sera, and 3 SVA, mycoplasma, BVDV, and CSFV antibody-positive sera. All the results were negative ( Figure 4 ). This indicates that there is no cross-reaction with antibodies against other viruses.

[0046] Example 3 1. Correlation between CLIA methods and VNT The established CLIA method and VNT method were used to test 296 field serum samples, and the results were compared and analyzed for correlation. The positive / negative results are shown in Table 1. The established CLIA method detected 223 positive samples and 73 negative samples; VNT detected 232 positive samples and 64 negative samples, and the consistency rate of the two methods was 92.2%. The Kappa coefficient between the CLIA titer and the VNT titer was calculated, and the Kappa value was 78.19%. This shows that the detection results of the two methods are highly consistent.

[0047] Example 4 Composition and assembly of tubular magnetic microparticle chemiluminescent detection kit for foot-and-mouth disease virus type A neutralizing antibody 1. Kit components: (A) Magnetic microparticle-conjugated monoclonal antibody W125 (MPs-W125) working solution, concentration 0.5 mg / mL; (B) Enzyme-labeled antibody (AP-W145) working solution, concentration 1:1000; (C) Detection antigen (FMDV A / WH / CHA / 09 146S), working concentration 0.25ug / mL; (D) concentrated washing buffer (25×PBST), working concentration 1×PBST; (E) Luminescent substrate (AMPPD); (F) Quality control products: negative serum and standard product with an antibody content of 100 U were packaged as quality control product 1 and quality control product 2 respectively; (G) Dilution solution (0.1 M MOPS buffer, pH 7); (H) Standard The above components are assembled into a tubular magnetic microparticle chemiluminescence detection kit for foot-and-mouth disease virus type A neutralizing antibody according to corresponding specification requirements.

[0048] 2. Kit detection steps: 2.1. Preparation before testing 2.1.1. Sample requirements Serum samples can be stored at 2°C to 8°C if tested within one week. If tested for more than one week, they should be frozen and stored below -20°C.

[0049] 2.1.2. Before using, take the reagent out of the refrigerator and equilibrate it to room temperature.

[0050] 2.1.3. Resuspension of magnetic particle reagent Before loading for the first time, the magnetic particle reagent needs to be mixed to resuspend the magnetic particles that have settled during transportation. After the first loading of the magnetic particle reagent, no further mixing is required.

[0051] 2.1.4. Solution preparation Washing solution (1× washing solution): Dilute the 25× concentrated washing solution provided in this kit with deionized water or distilled water at a ratio of 1:25.

[0052] 2.1.5. Instrument preparation Read the instrument manual, set the instrument parameters according to the determined conditions, place the relevant reagents, samples, and required consumables, and start the test. (For specific settings, refer to the instrument manual).

[0053] 2.2. Standard curve calibration Take the standard samples with antibody contents of 0U, 25U, 100U, 800U, 1600U, and 3200U as calibration samples, and calibrate the standard curve according to the instrument manual. 2 When ≥0.99, quality control product testing can be carried out.

[0054] 2.3. Quality Control When necessary, test the quality control product. The test value must be within the reference range before continuing the sample test. The quality control product needs to be tested in the following situations: a. Each time the kit batch number is changed or calibrated; b. Testing is required at least once within 24 hours.

[0055] 2.4. Sample testing (A) Add 40 μL of the sample to be tested into the reaction cup.

[0056] (B) Add 25 μL MPs-W125, 100 μL AP-W145, and 100 μL 146S antigen, mix well, and incubate at 37 °C for 15 min. Use a magnetic plate to adsorb the magnetic beads for 1 min, discard the reaction solution, add washing solution to resuspend the magnetic beads, and then adsorb and wash again. Repeat this process 4 times and discard the washing solution.

[0057] (C) Add 100 μL of chemiluminescent substrate AMPPD, mix the magnetic beads, and incubate at 37 °C for 3 min.

[0058] (D) Detect the relative luminescence intensity (RLU) and calculate the antibody content.

[0059] 3. Test kit result determination: When the test result is ≥41.395U (titer is 1﹕52.9856), it is judged as FMDV type A neutralizing antibody positive; when it is <41.395U, it is judged as FMDV type A neutralizing antibody negative.

Claims

1. A tubular magnetic microparticle chemiluminescence detection kit for detecting neutralizing antibodies to foot-and-mouth disease virus type A, characterized in that: It comprises a magnetic particle-coupled monoclonal antibody W125 working solution and an enzyme-labeled antibody working solution; the antibody in the enzyme-labeled antibody working solution is monoclonal antibody W145.

2. The tubular magnetic microparticle chemiluminescence detection kit for detecting neutralizing antibodies to foot-and-mouth disease virus type A according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody W125 is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

2.

3. The tubular magnetic microparticle chemiluminescence detection kit for detecting neutralizing antibodies to foot-and-mouth disease virus type A according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody W145 is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

4.

4. The tubular magnetic microparticle chemiluminescence detection kit for detecting neutralizing antibodies to foot-and-mouth disease virus type A according to claim 1, characterized in that: The enzyme in the enzyme-labeled antibody working solution is alkaline phosphatase.

5. The tubular magnetic microparticle chemiluminescence detection kit for detecting neutralizing antibodies to foot-and-mouth disease virus type A according to claim 1, characterized in that: In the magnetic particle-coupled monoclonal antibody W125 working solution, the mass ratio of the magnetic particles to the monoclonal antibody W125 is 100:(1-10).

6. The tubular magnetic microparticle chemiluminescence detection kit for detecting neutralizing antibodies to foot-and-mouth disease virus type A according to claim 1, characterized in that: The kit also includes a detection antigen, a concentrated washing solution, a luminescent substrate, a quality control product, a diluent, and a standard product.

7. The tubular magnetic microparticle chemiluminescence detection kit for detecting neutralizing antibodies to foot-and-mouth disease virus type A according to claim 6, characterized in that: The detection antigen is FMDVA / WH / CHA / 09 146S.

8. The tubular magnetic microparticle chemiluminescence detection kit for detecting neutralizing antibodies to foot-and-mouth disease virus type A according to claim 6, characterized in that: The concentrated washing solution is a PBS buffer solution with a pH value of 7.4 and containing Tween-20 with a volume concentration of 0.1% to 0.5%; the luminescent substrate is 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoacyl)-phenyl-1,2-dioxetane.

9. The tubular magnetic microparticle chemiluminescence detection kit for detecting neutralizing antibodies to foot-and-mouth disease virus type A according to claim 6, characterized in that: The quality control products include quality control product 1 and quality control product 2, quality control product 1 is 0-10U negative serum, and quality control product 2 is a standard product with a foot-and-mouth disease virus type A antibody content of 100U; The diluent is a diluent used for the magnetic particle-coupled monoclonal antibody W125 working solution and the enzyme-labeled antibody working solution, and the diluent is a 0.1M MOPS buffer with a pH of 7; the MOPS buffer contains 0.1-2% BSA, 0.01-0.5% Tween-20, 0.01-0.5% ProClin300, 0.01-0.5M MgCl2, 0.01-0.5M NaCl, 0.05-3% hydrolyzed milk protein and 0.1-1% PEG 6000 in a mass percentage; The standard is an antiserum with a FMDVA type neutralizing antibody content of 3200U, 1600U, 800U, 400U, 200U, 100U, 50U, 25U, 12.5U, 6.25U, 3.125U, 1.56U, and 0U.

10. Use of the detection kit according to any one of claims 1 to 9 in detecting neutralizing antibodies against foot-and-mouth disease virus type A.

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