Monoclonal antibodies to immune serum antibodies to o foot-and-mouth disease virus and uses thereof

Abstract

The application discloses a monoclonal antibody of immune serum antibody for O-type foot-and-mouth disease virus and application thereof, and relates to the technical field of animal virology and animal infectious disease detection. The application uses vp1 protein as an immunogen, uses polypeptides containing B cell antigen epitopes and inactivated antigens as screening origins respectively, screens out monoclonal antibodies capable of competing with O-type foot-and-mouth disease virus inactivated vaccine and synthetic peptide vaccine immune background serum, and establishes an O-type foot-and-mouth disease virus solid-phase competitive ELISA kit, which greatly shortens the detection time and improves the sensitivity, specificity and stability of the kit.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of animal virology and detection technology of animal infectious diseases, and particularly relates to a monoclonal antibody of immune serum antibody against O-type foot and mouth disease virus. BACKGROUND

[0002] Foot And Mouth Disease (FMD) is an acute, febrile, and contagious disease of cloven-hoofed animals (pigs, cattle, sheep, camels, etc.) caused by Foot And Mouth Disease Virus (FMDV) infection. The disease is listed as the first class A infectious disease by the World Organization for Animal Health (OIE), and has a great harm to the livestock production. Elimination and control of foot and mouth disease is a worldwide problem that is concerned by governments. The main measure for the control and elimination of foot and mouth disease in China is compulsory immunization. At present, the inactivated vaccine and synthetic peptide vaccine are most widely used. Foot and mouth disease vaccine immunization mainly induces the production of neutralizing antibodies, and a safe and effective vaccine is a prerequisite for successful prevention, control and even ultimate elimination of foot and mouth disease.

[0003] Serological detection is a qualitative or quantitative identification of specific antibodies in serum, and is most widely used in foot and mouth disease diagnosis, quarantine and evaluation of animal population foot and mouth disease vaccine efficacy. Virus neutralization test is the most classical serological diagnostic method for foot and mouth disease, but the test has the disadvantages of using live virus, long operation time, and difficult to repeat the test results. Liquid phase blocking ELISA (LPB) developed since 1986 has become a substitute method for virus neutralization test due to its rapidity, good repeatability, and no use of live virus, but it also has the disadvantages of high false positive rate and inability to detect serum antibodies of polypeptide vaccine immunization background. Solid phase competitive ELISA method (SPC) has better specificity, more simple operation, more stable results, and easy to repeat in addition to the advantages of liquid phase blocking ELISA method (LPB), and it was established as one of the designated methods for foot and mouth disease serological detection in international trade by the World Organization for Animal Health (OIE) in 2004.

[0004] Foot-and-mouth disease virus is easy to mutate, and seven virus serotypes have been identified, O, A, C type (i.e. European type), SAT1, SAT2, SAT3 (South Africa 1, 2, 3 type, i.e. African type) and Asia I (Asia I type). There is no cross reaction between the seven virus serotypes, and each serotype includes different variants. The study of the antigenic site of FMDV shows that the structural proteins VP1, VP2, VP3 and VP4 are involved in the formation of the antigenic site. The antigenic site of O type foot-and-mouth disease virus is mainly located on the structural protein VP1, and VP1 is the main component for inducing animals to produce neutralizing antibodies, among which the 141-160, 200-213 amino acid peptide segments can protect animals against virus attack and are important B cell epitopes. They are also important targets for the establishment of O type foot-and-mouth disease virus vaccine immunization and diagnosis method.

[0005] In the prior art, for example, CN114921418A provides a hybridoma cell strain 1D3 of O type foot-and-mouth disease virus-like particle monoclonal antibody, kit and detection method. The method can detect O type foot-and-mouth disease virus-like particle vaccine immune serum antibodies, and is used for distinguishing O type foot-and-mouth disease wild virus, whole virus inactivated vaccine and virus-like particle vaccine immune serum. For example, CN106405092A provides an O type foot-and-mouth disease virus antibody solid-phase competitive ELISA kit based on type-specific monoclonal antibody. The kit contains HRP-labeled O type foot-and-mouth disease virus-specific monoclonal antibody. For example, CN109799342A provides an O type foot-and-mouth disease virus antibody competitive ELISA detection kit. Rabbits are immunized with O type foot-and-mouth disease virus-like particles assembled from structural proteins VP0, VP1 and VP3, and the competitive antibodies obtained by separation are directly labeled with HRP. However, the above prior art all uses the conventional monoclonal antibody screening method, and does not provide the detection effect of the currently marketed various O type foot-and-mouth disease vaccine immune serum, and cannot evaluate the immune effect of the currently marketed vaccine. SUMMARY

[0006] In view of the above deficiencies of the prior art, the present application provides a monoclonal antibody for O type foot-and-mouth disease virus immune serum antibody, and also provides a corresponding solid-phase competitive ELISA kit. The vp1 protein is used as an immunogen, and the polypeptide containing the B cell epitope and the inactivated antigen are used as screening agents, respectively, to screen the monoclonal antibody which can compete with the O type foot-and-mouth disease virus inactivated vaccine and the synthetic peptide vaccine immune background serum. The O type foot-and-mouth disease virus solid-phase competitive ELISA kit is established, the detection site of which has competition effect on the currently marketed inactivated vaccine and synthetic peptide vaccine, thereby improving the sensitivity, specificity and stability of the kit. The immune effect of the O type foot-and-mouth disease vaccine can be accurately evaluated, which has production guiding significance for O type foot-and-mouth disease prevention and control in farms, and the operation is simple, the detection time is greatly shortened, and the cost is saved. The above technical effects are achieved by the following technologies.

[0007] The monoclonal antibody of the immune serum antibody of the synthetic peptide vaccine and the inactivated vaccine for O type foot-and-mouth disease virus, including the heavy chain variable region and the light chain variable region, the amino acid sequences of the three complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain are respectively as shown in SEQ ID NO. 1-3; the amino acid sequences of the three complementarity determining regions CDR1, CDR2 and CDR3 of the light chain are respectively as shown in SEQ ID NO. 4-6.

[0008] Preferably, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID NO. 7, and the nucleotide sequence of the light chain variable region is as shown in SEQ ID NO. 8.

[0009] The application further provides a preparation method of the above-mentioned monoclonal antibody, comprising the following steps:

[0010] S1, constructing a recombinant expression plasmid of the vp1 protein according to the gene sequence of the vp1 protein of the O type foot-and-mouth disease virus, and obtaining the vp1 protein by expression and purification;

[0011] Meanwhile, the peptide segments of 141-160 and 200-213 of the vp1 protein of the O type foot-and-mouth disease virus are connected in series to obtain a synthetic polypeptide;

[0012] S2, preparing a cell suspension of immune spleen cells by immunizing animals with the vp1 protein obtained in step S1;

[0013] S3, performing cell fusion of the immune spleen cells prepared in step S2 with myeloma cells, and performing indirect ELISA detection using a commercially available inactivated antigen of foot-and-mouth disease O type, and the vp1 protein and the synthetic polypeptide prepared in step S1 coated plates, respectively, to obtain several selected hybridoma cell strains;

[0014] S4, using the several selected hybridoma cell strains obtained in step S3 to perform competitive ELISA screening with the serum immunized with the commercially available inactivated vaccine and the serum immunized with the commercially available synthetic polypeptide vaccine, respectively, to obtain a positive hybridoma cell strain;

[0015] S5, preparing the monoclonal antibody using the positive hybridoma cell strain prepared in step S4.

[0016] Preferably, in the preparation method of the above-mentioned monoclonal antibody, the optimized gene sequence of the vp1 protein of the O type foot-and-mouth disease virus in step S1 is as shown in SEQ ID NO. 9.

[0017] Preferably, in the preparation method of the above-mentioned monoclonal antibody, after the indirect ELISA detection in step S3, the cells with P / N≥2.1 are the selected hybridoma cell strains.

[0018] Preferably, in the preparation method of the monoclonal antibody, the hybridoma cell strain with PI value of the two sera of step S4 both being greater than or equal to 60% is the positive hybridoma cell strain.

[0019] The application also provides a solid-phase competitive ELISA detection kit for antibodies of a synthetic peptide vaccine and an inactivated vaccine against O-type foot-and-mouth disease virus, comprising the HRP-labeled monoclonal antibody of claim 1.

[0020] Preferably, the solid-phase competitive ELISA detection kit further comprises an enzyme-labeled plate on which O-type foot-and-mouth disease virus vp1 protein is coated, a sample diluent (such as phosphate buffer), a sample washing solution (such as a washing solution containing phosphate buffer and Tween), a color developing solution (single-component TMB), and a termination solution (2M dilute sulfuric acid).

[0021] Compared with the prior art, the application has the following advantages:

[0022] 1. The monoclonal antibody and the corresponding solid-phase competitive ELISA detection kit are suitable for detecting sera immunized by O-type foot-and-mouth disease virus inactivated vaccine and synthetic peptide vaccine. The coincidence rate of the detection of sera immunized by inactivated vaccine is high by using the classical method of the foot-and-mouth disease O-type liquid-phase blocking ELISA antibody detection kit produced by Lanzhou Veterinary Research Institute; the sensitivity of the detection of sera immunized by synthetic peptide vaccine is better, and the serum antibody can be detected 28 days after the immunization of the synthetic peptide vaccine; compared with the Zhengzhou Zhongdao Pig Foot-and-Mouth Disease Virus O-type vp1 structural protein antibody enzyme-linked immunosorbent assay diagnostic kit, the coincidence rate of the detection of sera immunized by synthetic peptide vaccine is high.

[0023] 2. The monoclonal antibody and the solid-phase competitive ELISA detection kit are suitable for detecting antibodies of O-type foot-and-mouth disease virus vaccine, and do not react to other subtypes of foot-and-mouth disease virus, such as A-type and Asial-type viruses, and have good specificity.

[0024] 3. The O-type foot-and-mouth disease virus solid-phase competitive ELISA detection kit has the advantages of simple operation, short time, no need of professional operation, low requirement for operation site, good stability of the kit, long storage period, simultaneous processing of a large number of samples, and large-scale application in clinical and scientific research. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 SDS-PAGE electrophoretogram of O-type foot-and-mouth disease virus vp1 protein;

[0026] Figure 2 Determination results of mouse titers in the screening method determined in Example 1;

[0027] Figure 3 SDS-PAGE electrophoretogram of the caprylic acid-ammonium sulfate purification of the monoclonal antibody 6C12. DETAILED DESCRIPTION

[0028] The technical solutions of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0029] Example 1: Preparation of monoclonal antibody of immune serum antibody against O type foot-and-mouth disease virus

[0030] 1. Test materials

[0031] (1) Experimental animals, antigens and cells

[0032] 8-week-old SPF BALB / c female mice were purchased from Wuhan Experimental Animal Center; SP / 20 cells were purchased from Wuhan Virus Institute;

[0033] O type foot-and-mouth disease virus inactivated antigen and A type foot-and-mouth disease virus inactivated antigen were both purchased from Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences.

[0034] (2) Main reagents and preparation

[0035] Liquid paraffin was purchased from China Pharmaceutical; goat anti-mouse IgG-HRP, HAT and Freund's adjuvant were purchased from Sigma Company; fetal bovine serum and RPMI-1640 culture medium were purchased from Gibico Company; TMB was purchased from BIOFX Company; 96-well enzyme-labeled plate was purchased from Jin Canhua;

[0036] Binding buffer (pH value 7.4): weigh Na2HPO4 2.84 g, NaCl 29.22 g, imidazole 1.36 g, adjust pH value to 7.4, and add injection water to 1000 ml.

[0037] Elution buffer (pH value 7.4): weigh Na2HPO4 2.84 g, NaCl 29.22 g, imidazole 13.60 g, adjust pH value to 7.4, and add injection water to 1000 ml.

[0038] 2. Test method

[0039] (1) Preparation of O type foot-and-mouth disease virus antigen

[0040] The O type foot-and-mouth disease virus antigen includes VP1 protein and synthetic polypeptide.

[0041] ① Synthesis of VP1 protein base sequence and synthetic polypeptide sequence

[0042] The VP1 gene sequence of the O-type foot-and-mouth disease virus MYA / 01 / 1998 strain published on NCBI (Genebank accession number: KR01154) was codon-optimized according to the E. coli expression system, and the sequence is shown as SEQ ID NO. 9. It was synthesized by Wuhan Jin Kai Rui Biological Engineering Co., Ltd. and connected to the pET-42a vector to construct a recombinant expression plasmid of the VP1 protein.

[0043] Meanwhile, the 141-160 and 200-213 amino acid peptide segments of the VP1 protein were connected in series through a linker sequence (amino acid sequence PGS S), and the sequence of the synthetic polypeptide was obtained. The complete amino acid sequence is shown as SEQ ID NO. 10, which was synthesized by Nanjing Jin Sui Biological Engineering Co., Ltd. for standby use.

[0044] ② Expression and purification of the target protein (VP1 protein)

[0045] A. Expression of the target protein

[0046] The recombinant expression plasmid of the VP1 protein was transformed into BL21 (Rossta) competent cells. The expression strain was inoculated into 1000 mL of fresh LB medium containing 100 μg / mL of kanamycin at a ratio of 1:100;

[0047] Then, the bacterial solution was cultured at 37°C and 180 r / min until the OD 600 When the OD reached 0.5-0.6, IPTG was added to a final concentration of 0.6 mmol / L, and the induction expression was performed at 16°C and 180 r / min overnight.

[0048] The induced bacterial solution was centrifuged at 8000 r / min for 10 min, the bacterial cells were resuspended with 20 mL of PBS, and the bacterial cells were placed on ice and broken by ultrasonic waves until the solution was clear and no longer viscous.

[0049] The broken solution was centrifuged at 10000 r / min for 30 min, and the supernatant after centrifugation was filtered with a 0.45 μm filter. The collected filtrate was stored at 2-8°C as a sample for column loading and standby use.

[0050] B. Purification of the target protein

[0051] The specific steps of affinity chromatography purification are: first, equilibrate the chromatography column with 10 column volumes of Binding Buffer, then load the sample, after loading is completed, equilibrate the chromatography column with another 10 column volumes of Binding Buffer, then elute with Elution Buffer, start collecting when a protein peak appears on the instrument screen, stop collecting after the peak ends, and maintain a liquid flow rate of 1.0 mL / min throughout the process;

[0052] The collected protein has a protein concentration of 1.447 g / mL measured by an ultramicro nucleic acid protein concentration detector, and the protein is detected by polyacrylamide gel electrophoresis (SDS-PAGE) with a size of about 25 kDa, which meets the expectation, as shown in Figure 1 , that is, VP1 protein is obtained.

[0053] (2) Animal immunization

[0054] The VP1 protein obtained after purification is used to immunize 8-week-old Balb / c mice for a total of 4 times using a conventional immunization method. The specific method is as follows:

[0055] For the first immunization, mix equal volumes of Freund's complete adjuvant and VP1 protein, emulsify thoroughly, and then immunize the mice at 0.2 mL per mouse (100 μg per mouse) by subcutaneous injection at multiple points on the back. A total of 5 mice are immunized.

[0056] The second and third immunizations are performed on the 14th and 28th days, respectively, using equal volumes of Freund's incomplete adjuvant and VP1 protein, emulsifying thoroughly, and then immunizing the mice at 0.2 mL per mouse (100 μg per mouse) by injection into the axillary fossa and inguinal region.

[0057] On the 36th day, collect serum from the mice's tails to measure the titer, and perform a fusion when the requirements are met. Three days before fusion, the mice are given a booster immunization in the abdominal cavity at 0.2 mL per mouse (200 μg per mouse). Three days later, the mice are euthanized by removing the eyeballs to collect blood, and the cell suspension of immune spleen cells is prepared.

[0058] (3) Determination of screening method (indirect ELISA detection method)

[0059] ① Optimal antigen coating concentration is selected by square array titration test

[0060] Sheep anti-mouse IgG labeled with horseradish peroxidase (HRP) is used as the secondary antibody, and the mouse serum is diluted by a commonly used dilution factor of 50 times. The specific steps are as follows:

[0061] A. Dilute the antigen (VP1 protein or synthetic polypeptide) by 1:50, 1:100, 1:200, 1:400, 1:800, 1:1000, and 1:2000, and coat 100 μL per well at 4°C overnight.

[0062] B. The next day, the enzyme-labeled plate was dried, and the blocking solution (NaCl 8 g, KCl 0.2 g, Na2HPO4·12H2O 2.9 g, KH2PO4 0.2 g, BSA 5 g, and deionized water to 1000 mL, pH = 7.4) was added at 150 μL / well, and the plate was incubated at 4°C overnight, then dried and stored for use. Before use, the plate was washed 3 times with the washing solution;

[0063] C. The positive serum (collected from mice immunized with VP1 protein) and blank mouse serum were diluted 40 times, respectively, and added to the corresponding antigen-coated plate, and incubated at 37°C for 30 min, and washed 5 times;

[0064] D. The goat anti-mouse IgG was diluted at 1:1000, 1:2000, 1:3000, 1:5000, 1:8000, and 1:0000, and 100 μL / well was added, and incubated at 37°C for 30 min, and washed 5 times;

[0065] E. 100 μL / well of the color developing solution was added, and the plate was incubated at room temperature for 10 min, and the reaction was terminated with the termination solution (50 μL / well). The OD 630 was measured by an enzyme-labeled instrument.

[0066] F. The indirect ELISA detection method was used for screening.

[0067] The ratio of the positive serum to the negative serum was large, and the OD 630 value of the corresponding antigen dilution was close to 1, and the corresponding antigen dilution was selected as the optimal working concentration of the indirect ELISA reaction system.

[0068] Results The dilution multiple of the selected VP1 protein was 500 times, the dilution multiple of the synthetic polypeptide was 1000 times, the dilution multiple of the foot-and-mouth disease virus O antigen was 50 times, and the dilution multiple of the goat anti-mouse IgG was 5000 times. The supernatant of the hybridoma cells was detected, and the OD 630 value was greater than 1.0, which was determined as positive.

[0069] ② Mouse titer determination

[0070] After the establishment of the indirect ELISA method, the optimal coating concentration was used to detect the antibody titer of the immunized mice, and the detection results are shown in Table 1. Figure 3

[0071] The mouse with the highest immune titer detection value was selected for additional immunization once in the abdominal cavity, and the spleen of the mouse was taken for fusion three days later.

[0072] (4) Cell fusion and screening

[0073] ① Preparation of feeder cells

[0074] ​Take one BALB / c mouse to remove the eyeball and blood, according to the conventional method to separate the negative serum.

[0075] The mice were killed and soaked in 75% alcohol for 10 minutes, then moved to a clean bench. The mouse's abdomen was fixed upward, the skin in the middle of the abdomen was lifted with an ophthalmic clamp, and a small incision was made horizontally with an ophthalmic scissors. Do not cut the peritoneum. Use an ophthalmic scissors and an ophthalmic clamp to tear the skin upward, and the spleen can be seen under the peritoneum. Then use the ophthalmic clamp to lift the peritoneum, cut it horizontally, tear it upward, and completely expose the spleen. Sterilely take the spleen and put it into a sterile homogenizer.

[0076] Add 4 mL of incomplete medium and grind, then add another 8 mL. After standing for 2 minutes, 8 mL of the upper suspension was taken and placed in a 50 mL centrifuge tube. Add another 8 mL of incomplete medium, stand for 2 minutes, and as much as possible to take the upper suspension and place it in a 50 mL centrifuge tube. Note that the tissue block should not be sucked up. Add incomplete medium to 40 mL.

[0077] Centrifuge at 1000 r / min for 10 min, discard the supernatant, and add 40 mL of resuspension. Centrifuge at 1000 r / min for 10 min, add 10 mL of HAT complete medium, and store at 37°C.

[0078] ② Preparation of myeloma cells

[0079] Three days before fusion, the myeloma cells were cultured to expand the cells to the logarithmic growth phase. On the day of fusion, the cells were blown off the bottle wall with 15 mL of incomplete medium and collected in a 50 mL centrifuge tube. Centrifuge at 1000 r / min for 10 min. Resuspend the cell pellet in 10 mL of incomplete medium and count.

[0080] ③ Preparation of immune spleen cells

[0081] Prepare the feeder cells as above. After resuspension, count and store at 37°C.

[0082] ④ Fusion

[0083] Take 120 mL of HAT complete medium, 40 mL of incomplete medium, and 1 mL of 50% PEG and preheat them at 37°C. Prepare a 500 mL beaker of 37°C water.

[0084] Mix the SP2 / 0 myeloma cells and immune spleen cells in a 50 mL centrifuge tube at a ratio of 1:10. Centrifuge at 1000 rpm / min for 10 min, discard the supernatant, and use sterile filter paper to absorb it. Gently tap the bottom of the tube to slightly loosen the cell pellet.

[0085] Put the centrifuge tube containing the cell mixture in a 37℃ water bath, then slowly drip in the pre-warmed to 37℃ fusion with 50% PEG1500 0.8mL, gently stirring, 1min, add 1min;

[0086] Then slowly add 37℃ pre-warmed incomplete medium 40mL: 1mL in the first min; 1mL in the second min; 3mL in the third to fourth min; 10mL in the fifth min; finally slowly add the incomplete medium to 40mL, each time when adding, need to slowly add, and gently stirring.

[0087] 1000rpm / min centrifugation for 10min, discard the supernatant, the precipitate was resuspended with HAT complete medium, evenly added to 96 well culture plate 6 blocks, placed in 5% CO2, 37℃ constant temperature incubator.

[0088] Half the amount of liquid after 5d, full liquid after 7d, when the clone hole to the hole bottom area 1 / 4~1 / 3, take the supernatant for detection.

[0089] ⑤Hybridoma cell screening

[0090] First, respectively, using the foot and mouth disease O inactivated antigen (Chinese Academy of Agricultural Sciences, Lanzhou Institute of Veterinary Medicine), VP1 protein and synthetic polypeptide antigen coated plate for indirect ELISA detection (with the same as the foregoing indirect ELISA screening detection method), select P / N≥2.1 cells are selected hybridoma cell lines;

[0091] Second, the supernatant of the selected hybridoma cell lines, respectively, using inactivated vaccine immunized serum and serum with synthetic peptide vaccine immunization for competitive ELISA screening, two kinds of serum PI (i.e. 1-S / N) ≥60% of the cells are positive hybridoma cell lines; The screening results are shown in Table 1.

[0092] Table 1 selected hybridoma cell lines and competitive ELISA screening results

[0093]

[0094]

[0095] ⑥Hybridoma cell cloning culture

[0096] The positive hybridoma cells of ELISA detection were promptly cloned and cultured, using the limited dilution method. The steps are as follows:

[0097] A. feeder cells: as described above; 0.1mL per well in 96 well plate;

[0098] B. ELISA positive wells with a pipette, gently blow mixed, dilution in 96-well plate, under a microscope to count, the total number of cells in the hole 100-200 or so cells are pumped out to 10 mL complete medium. After mixing, 0.1 mL per well into the 96-well plate has been coated with feeder cells. Incubate at 37°C, 5% CO2 and saturated humidity conditions;

[0099] C. Observation and record the number of cells per well clone, when the cell to the bottom of the hole 1 / 4 ~ 1 / 3 half of the liquid, and the supernatant ELISA indirect detection;

[0100] D. Clone number, OD 630 high value positive wells, it is cloned again.

[0101] 3-4 times cloning operation, until all the cloning cell hole detection positive rate of 100%, that is, the anti-O FMDV monoclonal antibody hybridoma cell line 6C12, should be promptly expanded and frozen.

[0102] (5) preparation of monoclonal antibodies and labeled verification

[0103] ① Preparation of monoclonal antibodies

[0104] Select 8 weeks of age BALB / c female mice, under sterile conditions, each intraperitoneal injection of 0.5 mL sterile liquid paraffin.

[0105] Preparation of cells: select the growth of hybridoma cells 6C12 using cell bottle subculture, sterile conditions, with a pipette gently blow bottle wall, so that the cells fall off, into a 15 mL centrifuge tube, 1000r / min centrifugation 10 min, add the right amount of incomplete medium resuspended, trypan blue count adjustment of cell number to 1 x 10 6 -6 x 10 6

[0106] About two weeks later, the hybridoma cells 6C12 resuspended using RPMI-1640 medium into the abdominal cavity of mice. Each mouse intraperitoneal injection of 0.5 mL of the cells, daily observation of mice, the abdominal slowly swell, about 7-10 d, the mouse spirit, do not move, when the ascites, room temperature 1000r / min centrifugation 10 min, the supernatant, which contains 6C12 monoclonal antibody ascites.

[0107] ② ascites titer detection

[0108] ​The ascites of 6C12 monoclonal antibody was serially diluted, and the antibody titer was determined by indirect ELISA. The maximum dilution with a P / N value greater than 2.1 was the ELISA titer, which was 1:102400.

[0109] ③ Ascites-specific detection

[0110] The inactivated antigens of foot-and-mouth disease type A and type O (purchased from the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences) and BHK-21 cell suspension were coated on an enzyme-labeled plate at 100 μL per well and 4°C overnight. The specificity of the ascites was determined by indirect ELISA. 100 μL of the ascites of 6C12 monoclonal antibody diluted with PBS (1:1000) was added to each well, and incubation was performed at 37°C for 30 min.

[0111] The plate was washed, and 100 μL of goat anti-mouse IgG-HRP diluted with PBS (1:5000) was added to each well, and incubation was performed at 37°C for 30 min.

[0112] The plate was washed, 100 μL of color developing solution was added to each well, and reaction was performed at room temperature for 10 min. The reaction was terminated by adding a termination solution, and the OD value was read by an enzyme-labeled instrument. 630 The results showed that the ascites of monoclonal antibody 6C12 reacted with the inactivated antigen of type O but not with the inactivated antigen of type A, and had good specificity.

[0113] ④ Purification of ascites antibody (caprylic acid-ammonium sulfate method)

[0114] A. 2 mL of ascites was centrifuged at 1000 r / min for 10 min, and the supernatant was taken. While stirring, 4 times the volume of 0.06 mol / L sodium acetate buffer (pH=4.5) was added.

[0115] B. While stirring, n-octanoic acid was added dropwise to a final concentration of 25 μL / mL, and stirring was performed at room temperature for 30 min. Centrifugation was performed at 4°C and 10000 r / min for 30 min, and the precipitate was discarded, and the supernatant was collected.

[0116] C. The supernatant in step (2) was filtered with filter paper, and the pH was adjusted to 7.4.

[0117] D. While stirring, saturated ammonium sulfate solution (ammonium sulfate volume / total volume≤45%) was added to the filtrate obtained in step (3). When the filtrate became a white turbid liquid, stirring was continued for 30 min. Centrifugation was performed at 4°C and 10000 r / min for 30 min.

[0118] E. The supernatant was discarded, and the precipitate was resuspended with 2 mL of 10 mM Tris-HCl (pH=9.0). In 100 times the volume of 10 mM Tris-HCl (pH=9.0), dialysis was performed at 4°C overnight. After dialysis, the volume became 3.2 mL, and the sample was aliquoted and stored at low temperature.

[0119] F. SDS-PAGE electrophoresis analysis of monoclonal antibody 6C12 purified by octanoic acid-ammonium sulfate was performed, and the results are shown in Figure 2. Figure 2 Two bands were observed, which were the heavy chain and light chain of IgG, with sizes of 55 kDa and 25 kDa, respectively. The concentration of monoclonal antibody 6C12 after dialysis was 7.1 mg / mL, as determined by a protein nucleic acid concentration detector.

[0120] (5) Subtype identification of monoclonal antibody

[0121] The Ig subtype of monoclonal antibody 6C12 was identified according to the instructions of the mouse monoclonal antibody subtype identification kit (purchased from Beijing Yiqiao Godz). The identification result was IgG3 subtype, as shown in Table 2 below.

[0122] Table 2 Subtype identification results of monoclonal antibody 6C12

[0123]

[0124] (6) Determination of variable region sequence of monoclonal antibody

[0125] The mRNA of hybridoma cell 6C12 was extracted, and after reverse transcription into cDNA, PCR amplification was performed using universal primers for variable regions. The amino acid sequences of the three complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region were respectively shown in SEQ ID NO. 1-3; the amino acid sequences of the three complementarity determining regions CDR1, CDR2 and CDR3 of the light chain were respectively shown in SEQ ID NO. 4-6.

[0126] The nucleotide sequence of the heavy chain variable region was shown in SEQ ID NO. 7, and the nucleotide sequence of the light chain variable region was shown in SEQ ID NO. 8.

[0127] (7) Preparation of HRP-labeled purified monoclonal antibody (sodium periodate method)

[0128] Take 5.0 mg HRP dissolved in 1 mL ddH2O, add 0.5 mL NaIO4, grass green; 4°C for 30 min, add 0.16 mol / L ethylene glycol 0.5 mL to stop the reaction, room temperature for 30 min, the solution turns brown yellow; add 5 mg monoclonal antibody 6C12, dialysis at 4°C overnight, the dialysate is 0.05M pH9.5 CB buffer; the next day, add 5 mg / mL sodium borohydride 0.2 mL, 4°C for 2 h, add an equal volume of saturated ammonium sulfate, 4°C for 30 min, 3000 r / min centrifugation for 15 min, discard the supernatant; use 20 mM pH7.4 PB buffer to dialyze overnight; the next day, 3000 r / min centrifugation for 15 min, the supernatant is diluted to a constant volume, which is the HRP-labeled monoclonal antibody.

[0129] Example 2: Antibody solid-phase competitive ELISA detection kit and corresponding method for immune serum against O-type foot-and-mouth disease virus synthetic peptide vaccine and inactivated vaccine

[0130] 1. Antigen and serum

[0131] Foot-and-mouth disease virus O-type VP1 protein, monoclonal antibody 6C12-HRP, foot-and-mouth disease virus O-type antibody positive serum and foot-and-mouth disease virus O-type antibody negative serum were all prepared by Wuhan Keqian Biological Co., Ltd. The foot-and-mouth disease virus O-type antibody positive serum and the foot-and-mouth disease virus O-type antibody negative serum were detected by the foot-and-mouth disease virus O-type antibody liquid-phase blocking ELISA kit from Lanzhou Veterinary Research Institute, with titers of 1:256 and less than 1:8, respectively.

[0132] 2. Related reagents

[0133] Coating buffer: Na2CO3 1.59 g, NaHCO3 2.93 g, constant volume to 1000 mL with deionized water;

[0134] Blocking solution: NaCl 8 g, KCl 0.2 g, Na2HPO4·12H2O 2.9 g, KH2PO4 0.2 g, BSA 5 g, constant volume to 1000 mL with deionized water (pH=7.4), same as in Example 1;

[0135] Sample diluent: NaCl 8 g, KCl 0.2 g, Na2HPO4·12H2O 2.9 g, KH2PO4 0.2 g, Tween-20 0.5 mL, constant volume to 1000 mL with deionized water (pH=7.4);

[0136] Stop solution: 27.2 mL concentrated sulfuric acid (H2SO4) added to 900 mL deionized water, constant volume to 1000 mL, aliquot, 10 mL / bottle;

[0137] 20-fold concentrated washing solution: NaCl 160 g, KCl 4 g, Na2HPO4·12H2O 58 g, KH2PO4 4 g, Tween-20 10 mL, deionized water to 1000 mL (pH = 7.4);

[0138] Protective agent: bovine serum albumin (BSA) 5.00 g, Tween-20 0.5 mL, sodium chloride (NaCl) 8.00 g, sodium phosphate dibasic (Na2HPO4·12H2O) 2.90 g, potassium dihydrogen phosphate (KH2PO4) 0.20 g, potassium chloride (KCl) 0.20 g, sodium thiomersal 0.20 g, sucrose 2 g, dissolved with water for injection and constant volume to 1000 mL, stored at 2-8℃ for standby.

[0139] O-type foot-and-mouth disease virus solid-phase competitive ELISA test kit negative and positive controls: immunize goats with O-type foot-and-mouth disease inactivated vaccine to prepare positive serum, 32-fold dilution as positive control; sample diluent as negative control.

[0140] 3. Determination of working concentration of each reagent

[0141] (1) Dilute the foot-and-mouth disease virus O-type VP1 protein to a final concentration of 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL, one row for each concentration.

[0142] Add 100 μL to each well and incubate at 2-8℃ for 15 h; discard the coating solution, add 200 μL of blocking solution to each well, and incubate at 37℃ for 2 h; discard the blocking solution and dry at 37℃ for 2 h.

[0143] (2) Dilute the negative and positive sera 2-fold with sample diluent, add 50 μL of the diluted negative and positive sera to columns 1-6 and 7-12 respectively, and add 50 μL of the appropriate concentration of monoclonal antibody 6C12-HRP (enzyme label) to columns 1-6 and 7-12 respectively, incubate at 37℃ for 30 min.

[0144] (3) Discard the liquid in the wells, add 300 μL of washing solution to each well, repeat the washing 5 times, and finally pat dry on absorbent paper. Add 100 μL of substrate color developing solution to each well, develop color at 20-25℃ for 15 min. Add 50 μL of stop solution to each well, and read the OD value within 10 min. 450

[0145] Calculate the OD of the corresponding negative serum wells​450 The ratio of the OD value (N) of the negative serum and the OD value (P) of the positive serum 450nm The ratio of the OD value (N) of the negative serum and the OD value (P) of the positive serum 450 The ratio of the OD value (N) of the negative serum and the OD value (P) of the positive serum

[0146] 4. Optimization of blocking solution

[0147] The phosphate buffer solution containing 0.1%, 0.2%, 0.5% BSA and 5% skim milk was used as the blocking solution, and the operation procedure was the same as above; the OD value (N) of the negative serum and the OD value (P) of the positive serum were compared 450 The ratio of the OD value (N) of the negative serum and the OD value (P) of the positive serum 450 The ratio of the OD value (N) of the negative serum and the OD value (P) of the positive serum

[0148] 5. Optimization of diluent

[0149] Diluent I: phosphate buffer solution;

[0150] Diluent II: phosphate buffer solution containing 0.5% BSA and 0.05% Tween-20;

[0151] Diluent III: phosphate buffer solution containing 0.05% Tween-20; the three diluents were used to dilute the serum to be tested, and the operation procedure was the same as above; the OD value (N) of the negative serum and the OD value (P) of the positive serum were compared 450 The ratio of the OD value (N) of the negative serum and the OD value (P) of the positive serum 450 The ratio of the OD value (N) of the negative serum and the OD value (P) of the positive serum

[0152] 6. Optimization of the optimal dilution multiple of the serum to be tested

[0153] The positive serum and the negative serum with known background were diluted by 2 times, 5 times, 10 times and 20 times, respectively, and then 50 μL of each was added; the other operation procedures were the same as above; the OD value (N) of the negative serum and the OD value (P) of the positive serum were compared 450 The ratio of the OD value (N) of the negative serum and the OD value (P) of the positive serum 450 The ratio of the OD value (N) of the negative serum and the OD value (P) of the positive serum

[0154] 7. Optimization of the optimal reaction time for the competitive binding of the serum, enzyme label and antigen

[0155] After the serum was added, incubation was carried out at 37°C for 15 min, 30 min, 45 min and 60 min, respectively; the other operation procedures were the same as above; the OD value (N) of the negative serum and the OD value (P) of the positive serum were compared 450 The ratio of the OD value (N) of the negative serum and the OD value (P) of the positive serum 450The ratio of the values ​​(P) was used to select the reaction time corresponding to the maximum value of the ratio, and the optimal reaction time was finally determined to be 30 min.

[0156] 8. Optimization of the optimal development time for the substrate

[0157] After adding the substrate, react at room temperature in the dark for 5 min, 10 min, 15 min, and 30 min, respectively. Other procedures are the same as above. Compare the OD of negative serum. 450 Value (N) and positive serum OD 450 The ratio of the values ​​(P) is used to select the color development time corresponding to the maximum value of the ratio, and the optimal color development time for the substrate is determined to be 15 minutes.

[0158] 9. Determination of criteria for solid-phase competitive ELISA antibody detection methods

[0159] The established ELISA method was used to test 150 clinical serum samples with known background (50 samples each from pigs, cattle, and sheep). The results were imported into SPSS 17.0 software, and ROC curve analysis was performed. The cut-off point corresponding to the maximum Youden index was selected as the cutoff point. Based on comprehensive judgment, since the cutoff points for the three species were relatively close, and also for ease of result interpretation, a two-digit integer of 0.50 was ultimately chosen as the cutoff value for the kit. The S / N value of 0.50 was used as the cutoff value for the kit; that is, the kit's judgment criteria are: an S / N value greater than 0.5 indicates antibody negativity, and an S / N value less than 0.5 indicates antibody positivity.

[0160] 10. Sensitivity of solid-phase competitive ELISA antibody detection method

[0161] The established ELISA method was used to detect porcine serum immunized with inactivated vaccine, porcine serum immunized with synthetic peptide vaccine, sheep serum immunized with inactivated vaccine, and bovine serum immunized with inactivated vaccine. The method showed high sensitivity, and the results are shown in Table 3 below.

[0162] Table 3. Sensitivity test results of solid-phase competitive ELISA antibody detection method

[0163]

[0164]

[0165] 11. Specificity of solid-phase competitive ELISA antibody detection method

[0166] The established ELISA method was used to detect positive serum samples for foot-and-mouth disease type A, Asial, classical swine fever, porcine reproductive and respiratory syndrome (PRRS), and pseudorabies. The S / N values ​​were all greater than 0.5, and the samples were judged as negative. This indicates that the present invention has good specificity for the above-mentioned viruses. The results are shown in Table 4.

[0167] Table 4 Specificity detection results of the solid-phase competitive ELISA antibody detection method

[0168]

[0169] 12. Coincidence rate of the solid-phase competitive ELISA antibody detection method

[0170] Using the established solid-phase competitive ELISA method and the O-type competitive ELISA and O-type antibody liquid-phase blocking ELISA detection kit of Lan Shuenyan, different sampling time pig serum immunized by Shanghai Shenlian synthetic peptide vaccine, Jinyubao Ling A-O bivalent inactivated vaccine, Tiankang Bio A-O bivalent inactivated vaccine, and Zhongnong Weite O-type inactivated vaccine were detected, respectively, wherein the coincidence rate at 14 days after the second immunization was 100%; but at 28 days after the first immunization, the detection sensitivity of the ELISA method established in the application was higher than that of the Lan Shuenyan kit on the synthetic peptide vaccine immunized serum sample, and neither the Lan Shuenyan O-type liquid-phase blocking nor the O-type solid-phase competition could detect the serum antibody of the synthetic peptide vaccine at 28 days after the first immunization. Using the solid-phase competitive ELISA method established in the application, 60 sheep serum immunized by Tiankang Bio A-O bivalent inactivated vaccine were detected by the imported kit of Saide, and the coincidence rate was 93.3%; and 88 pig serum immunized by synthetic peptide vaccine were detected by the Zhengzhou Zhongdao pig foot-and-mouth disease virus O-type vp1 structural protein antibody enzyme-linked immunosorbent assay diagnostic kit, and the coincidence rate was 97.7%. The related detection results are shown in Tables 5-8.

[0171] Table 5 Detection results of the ELISA method of the application and the Lan Shuenyan kit

[0172]

[0173] Table 6 Detection results of the ELISA method of the application and the Saide imported kit

[0174]

[0175] Table 7 Detection results of the ELISA method of the application and the Zhengzhou Zhongdao kit

[0176]

[0177] Table 8 Coincidence rate of the solid-phase competitive ELISA antibody detection method

[0178]

[0179]

[0180] 13. Reproducibility of the solid-phase competitive ELISA antibody detection method

[0181] The 15 known background sera were respectively detected by using 3 O-type foot-and-mouth disease virus competitive ELISA antibody detection kits of the application and 3 kits of the application of different batches, the intra-batch variation coefficient was within 10%, indicating that the kits had good intra-batch repeatability; the inter-batch variation coefficient of the 3 kits was within 15%, indicating that the kits had good inter-batch repeatability.

[0182] 14. Operation steps and result determination of O-type foot-and-mouth disease virus synthetic peptide vaccine and inactivated vaccine immune serum antibody solid-phase competitive ELISA detection kit

[0183] Before use, the enzyme-labeled plate was washed twice, the serum to be detected was diluted with the sample diluent at a ratio of 1:1, and the negative and positive controls were not diluted; 50 μL of the diluted serum to be detected, negative control and positive control were added to the antigen-coated plate wells, with 1 well for the serum to be detected, 2 wells for the positive control and 2 wells for the negative control, then 50 μL of enzyme label was added to each reaction well, incubated at 37℃ for 30 minutes, the solution in the plate well was discarded, 300 μL of washing solution was added to each well, and the plate was washed for 5 times, the last time was dried on the water absorption material, 100 μL of substrate color developing liquid was added to each well, and the color was developed at room temperature (20-25℃) for 15 minutes, 50 μL of termination liquid was added to each well, and finally the OD 450 value of each well was determined on the enzyme-labeled instrument.

[0184] Result determination: if the serum to be detected S / N≤0.5, it is determined to be positive; if the serum to be detected≥0.5, it is determined to be negative.

[0185] The above specific embodiments describe the implementation of the application in detail, but the application is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concepts of the application, the technical solutions of the application can be modified and changed in many simple ways, and these simple changes all belong to the protection scope of the application.

Claims

1. Monoclonal antibodies to the immune serum antibodies of synthetic peptide vaccines and inactivated vaccines against O foot-and-mouth disease virus, characterized in that, The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of three complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain are respectively shown as SEQ ID NO. 1-3; the amino acid sequences of three complementarity determining regions CDR1, CDR2 and CDR3 of the light chain are respectively shown as SEQ ID NO. 4-6.

2. The monoclonal antibody according to claim 1, characterized in that, The nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 7, and the nucleotide sequence of the light chain variable region is shown as SEQ ID NO.

8.

3. A solid phase competitive ELISA test kit for the detection of antibodies against synthetic peptide vaccine and inactivated vaccine of O foot and mouth disease virus characterized by, The monoclonal antibody of claim 1 or 2 is labeled with HRP.

4. The solid phase competitive ELISA test kit according to claim 3, characterized in that, The kit further comprises an enzyme-labeled plate, sample diluent, sample washing solution, color developing solution and termination solution, and the enzyme-labeled plate is coated with O-type foot-and-mouth disease virus vp1 protein.

Images