(1.Luoyang Laipusheng Information Technology Co., Ltd., Luoyang, Henan 471000, China; 2.Anyang Animal Disease Prevention and Control Center, Anyang 455000, Henan, China)

Abstract:In this paper, monoclonal antibodies were prepared for the existing AnSA synthetic peptides of foot-and-mouth disease type O virus, and a competitive ELISA method for the detection of foot-and-mouth disease type O virus antibodies was established. BALB/c mice were immunized with known purified AnSA synthetic peptide conjugates, and then the splenocytes of the immunized mice were fused with SP2/0 myeloma cells in the logarithmic phase of growth, the positive hybridoma cells were screened and purified by iELISA method and limiting dilution method, and the titer of ascites of four hybridoma cells was screened out by ELISA method, and the ascites titer of four hybridoma cells was screened out at more than 1∶51200. A competitive ELISA method was established to detect FMD O virus antibody using a known purified FMD O virus AnSA synthetic peptide conjugate as antigen and its corresponding enzyme-labeled monoclonal antibody, and its specificity, sensitivity and reproducibility were verified. Experimental data show that the specificity of the method is 100%, the sensitivity is 95%, and the coefficient of variation between batches is <10%, which has a promising application. Through clinical testing of a large number of samples, it was concluded that the positive coincidence rate of the method was 87.65%, which could be used to evaluate the level of protective antibodies in the whole animal population after vaccine immunization.

Keywords: Foot-and-mouth disease type O virus, AnSA peptide, competitive ELISA, monoclonal antibody

Abstract

The study aimed to synthetize peptides of existing foot-and-mouth disease virus(FMDV) type O AnSA to generate polyclonal antibodies, and established a competitive ELISA method to detect the antibody of FMDV type O. We mithridatized Balb/c mice with the synthetic peptides conjugate AnSA and fused splenocytes from immunized mice with myeloma cells SP2/0 on logarithmic phase. Then, we evaluated the titer of immunized mice ascites with ELISA and used the ELISA with limiting dilution methods to elect and purify the positive hybridoma cells. Four hybridoma cell lines were elected and the titer were above 1∶51200. The HRP-McAb was prepared by modified NaIO 4 method. The antibody of FMDV type O was detected by mean of competitive ELISA and the specificity, sensibility and repeatability were validated. The results showed that the specificity was 100%, sensibility was 95% and the coefficient variance was less than 10%. A large number of samples were tested by clinical testing and the results showed 87.65% positive detection rate. This method can evaluate the level of protective antibody after mithridatized by vaccine.in the fauna.

Key words：FMDV type O; polypeptide AnSA; competitive ELISA; monoclonal antibody

Foot and mouth disease is an acute, violent, and highly contagious disease caused by the foot-and-mouth disease virus (FMDV), which is classified as a class A animal disease by the World Health Organization (Office International des Epizooties, OIE), and is also listed as the first class of infectious diseases by the Chinese government [1]. There are seven serotypes of the virus: O, A, C, Asia-I, SAT1, SAT2 and SAT3, of which type O is the most prevalent. Timely evaluation of the immune protection effect of FMD vaccine and detection of diseased animals are the key to prevention and control.

Because the clinical symptoms of many diseases are similar, it is difficult to confirm the diagnosis from clinical diagnosis alone, and it must be carried out in conjunction with laboratory diagnosis. Laboratory diagnosis of foot-and-mouth disease can be broadly divided into three categories: first, biological experiments, mainly virus isolation and identification, including animal experiments and cell culture, second, serological diagnosis, including complement fixation test, indirect sandwich ELISA, virus neutralization test, hemagglutination test, immunodiffusion test, liquid phase blocking ELISA, and competitive ELISA, and third, molecular biology diagnostic technology, including ordinary PCR, real-time quantitative PCR technology, and gene chip technology [2].

At present, OIE methods include virus neutralization assay (VNT) and liquid-phase blocking ELISA (LPB-ELISA), which is a more classic method, but it is not suitable for large-scale application because it is time-consuming and laborious. Liquid phase blocking ELISA is relatively accurate, but due to the complexity of the operation steps and the long time, it brings a lot of inconvenience to the user.

The purpose of this study is to establish a convenient, rapid and accurate competitive ELISA method for the detection of protective antibody levels of FMD virus type O in animal serum. Firstly, according to the AnSA site on the VP1 of FMD O virus, a polypeptide was designed and synthesized, and then a monoclonal antibody with high sensitivity and specificity was screened out with polypeptide as antigen, and then a competitive ELISA method for the detection of FMD O virus antibody was established. Through the comparison of virus neutralization experiments, it is concluded that the coincidence rate of positive samples detected by this method can reach 87.65%, the coincidence rate can reach 92% compared with the liquid phase blocking ELISA detection kit of the Lan Research Institute, and the coincidence rate can reach 82% compared with the imported similar kits, which has a good application prospect.

1. Main materials and methods

1.1 Main instruments and reagents

Microplate reader (BIORAD, ImarkTM), microplate washer (Beijing Tuopu Analytical Instrument Co., Ltd., DEM-3 type), electronic balance (OHAUS Instrument Co., Ltd., AR224CN), thermostatic magnetic stirrer (Shanghai Sealer Instrument Co., Ltd., 81-2 type), vortex instrument (Thermo Fisher Technology Co., Ltd.), microplate plate is Costar 96-well high-adsorption microplate plate in the United States.

Foot-and-mouth disease virus type O AnSA peptide was synthesized and identified by Sangon Bioengineering (Shanghai) Co., Ltd.; SP2/0 multiple myeloma cells were preserved by our company's laboratory; 6-8-week-old female Balb/c mice were purchased from Henan Provincial Laboratory Animal Center; complete Freund's adjuvant, incomplete Freund's adjuvant, horseradish peroxidase (HRP), sodium periodate and 8-aza-guanine were purchased from Sigma; Foot-and-mouth disease type O inactivated vaccine was purchased from Xinjiang Tiankang;AnSA peptide-OVA, AnSA peptide-KLH is prepared in our laboratory.

Foot-and-mouth disease standard yin, The positive serum was purchased from the Lanzhou Veterinary Research Institute, 500 copies of foot-and-mouth disease negative serum were collected from cattle without FMD history, and 500 samples were collected from fattening pigs in Henan African epidemic areas without FMD history and without FMD vaccine, and no outbreak of foot-and-mouth disease was determined by liquid phase blocking ELISA detection, 10 negative pig serum swine vesicular disease was positive, but foot-and-mouth disease was confirmed negative, 10 negative pig serum vesicular stomatitis was positive, but foot-and-mouth disease was confirmed negative, 10 negative pig serum vesicular stomatitis was positive, but foot-and-mouth disease was confirmed negative, 10 cattle negative serum Asia-I positive, type O was confirmed negative, 10 bovine negative serotype A was confirmed as positive, and type O was negative。 200 samples of FMD type O positive reference serum were obtained from 21-28 days of recovered pig serum of confirmed single FMD O virus strain, 200 samples of pig serum 15-28 days after three immunization of FMD O vaccine, and 20 samples of artificially infected FMD O pig serum.

1.2 Preparation of monoclonal antibodies

The foot-and-mouth disease type O inactivated vaccine was demulsified to prepare the foot-and-mouth disease type O inactivated virus, and the 146S virus antigen was obtained by sucrose gradient centrifugation. Eight mice are immunized with foot-and-mouth disease type O inactivated virus and conjugated antigen AnSA peptide-KLH according to the Immunization method of A, Sanyal et al.[3], and shock immunization with a double dose of AnSA polypeptide-KLH (without adjuvant) on the first 3 days of fusion. Then, indirect ELISA was used to determine the serum titer, and after the titer was reached, the spleen could be taken and the cells were fused according to the conventional method.

SP2/0 cells in logarithmic growth phase were fused with immune spleen cells at a ratio of 1:10 under PEG1500 action, and 10 days later, AnSA peptide-OVA was coated, and positive hybridoma cells were screened by iELISA. The positive well cells were cloned and purified for 4 times by limiting dilution, and the screened hybridoma cells were intraperitoneally injected with BALB/c mice to prepare ascites. Monoclonal antibodies to ascites fluid are purified by the caprylic acid-ammonium sulfate method.

1.3 Preparation of enzyme-labeled monoclonal antibody and determination of antigen-coated concentration and enzyme-labeled antibody concentration

The modified sodium periodate method was used to prepare enzyme-labeled monoclonal antibodies. The working concentration of the enzyme-labeled monoclonal antibody and the coating concentration of the AnSA peptide-OVA were determined by checkerboard titration.

1.4 Reaction systems for competing ELISA methods

AnSA peptide-OVA was diluted to an appropriate coating concentration with carbonate buffer (pH 9.6, 0.01 M), added to the microplate at an amount of 100ul/well, and left to zero the wells overnight at 2-8 °C (16-24 h). Wash twice with PBST, pat dry, add 200 ul of blocking solution per well, block at 37 °C for 1 h, wash, and dry. 50 μL of the 20-fold dilution of the serum to be tested was added to the wells, 2 wells of each of the positive and negative controls were set, 50 μL of enzyme conjugate was added immediately, and incubated at 37°C for 30 min (± 1 min). After washing for 5 times, 100 μL of chromogenic solution (mixed in the proportion of chromogenic solution A and B), incubated at 37 °C for 15min, 50 μL of stop solution was added, and the OD450 value was measured with a microplate reader and the serum inhibition rate was calculated.

1.5 Determination of the critical value of yin and yang

The OD450nm value was measured in 150 O-negative bovine serum samples and 150 O-negative pig serum samples of FMD by competitive ELISA, and the PI value of serum was calculated, and the critical value of yin and yang was determined according to the distribution of inhibition rate of yin and yang serum.

1.6 Specificity test

Competitive ELISA was used to detect 10 strains of pigs positive for vesicular disease, 10 samples of pigs positive for vesicular disease, 10 samples of pigs positive for Asia-I positive and 10 samples of positive pigs for type A, 500 samples of pigs with type O-negative for foot-and-mouth disease, and 500 samples of bovine serum for type O-negative of foot-and-mouth disease, respectively, and the PI value of serum was determined.

1.7 Sensitivity test

The PI value of 200 swine sera immunized with inactivated vaccine against FMD type O, 200 recovered pig serums and 10 samples of pig sera infected with FMD type O virus were determined by competitive ELISA, and 10 samples of strongly positive pig serum of FMD O virus and 10 samples of weakly positive pig serum of FMD O virus were determined.

1.8 Repeatability test

3 batches of enzyme labels were coated, 10 negative and positive pig serums were repeatedly detected to calculate the inter-batch variation, and 1 negative serum was repeated 20 times to calculate the intra-batch variation.

1.9 Clinical Trials

A total of 2000 serum samples from pig farms in Xinjiang, Inner Mongolia, Heilongjiang, Shandong, Sichuan, Henan and other provinces were detected by competitive ELISA, and the positive detection rate was calculated.

2 Results and Analysis

2.1 Determination of monoclonal antibody titer and reactivity

The results showed that the antibody titers of all mice were between 1:6400~1:51200, and the serum antibody titers of No. 2 and No. 3 mice were detected 7 days after four immunizations.

After cell fusion, indirect ELISA was used to screen four hybridoma cell lines that could stably secrete anti-FMD O-type AnSA polypeptide monoclonal antibody, named #O3A2, #O4C7, and #O6E5和#O8D4, respectively.

The collected cell culture supernatant, ascites supernatant, and purified antibodies were detected using the established indirect ELISA method. The results showed (Table 1) that the ascites titer of the four monoclonal antibodies was the second after purification, followed by the antibody titer after purification, and the titer of cell culture supernatant.

Table 1 Titer determination results of monoclonal antibodies

	Cell culture supernatant	ascites	Purified antibodies
#O3A2	0.597222222	1:51200	1:25600
#O4C7	1.152777778	1:102400	1:51200
#O6E5	1.152777778	1:102400	1:102400
#O8D4	2.263888889	1:150000	1:100000

  2.2 Determination of suitable working concentration
  The results (Table 2) showed that the coating concentration of AnSA peptide-OVA and monoclonal antibody-HRP was 1:1600, and the dilution concentration of monoclonal antibody-HRP was 1:1600.
  Table 2 Determination of antibody titer in immunized mice

Enzyme-labeled antibodies	Antigen dilution
Dilution factor	100	200	400	800	1600	3200	6400	12800
0.736111111	3.489	3.457	3.186	2.874	2.476	2.088	1.787	1.478
1.430555556	3.477	3.359	2.784	2.541	2.306	1.972	1.608	1.209
2.819444444	3.419	3.268	2.501	2.274	2.198	1.704	1.476	1.072
5.597222222	3.357	2.909	2.247	2.045	1.748	1.465	1.184	0.897
1:16000	3.104	2.373	2.079	1.793	1.564	1.309	1.027	0.791
1:32000	2.944	2.373	1.844	1.651	1.386	1.042	0.853	0.504
1:64000	2.685	2.091	1.588	1.408	1.173	0.891	0.642	0.422
1:128000	2.507	1.747	1.325	1.087	0.713	0.502	0.324	0.324
Negative control	0.142	0.177	0.184	0.196	0.165	0.172	0.188	0.167
Blank control	0.058	0.047	0.065	0.077	0.082	0.056	0.043	0.051

  2.3 Determination of the critical value of yin and yang
  300 bovine serum and porcine serum samples that had been identified as negative specimens were diluted 40 times for testing, and the PI value of each serum was counted, and the PI value of positive and negative serum was finally determined to be 40% according to the formula (+3SD). If the PI value < 0.4, the sample should be judged negative for antibodies. If the PI value ≥ 0.4, the sample should be judged to be antibody positive. The PI value of 40% was set as the critical value, and the positive and negative results were clearly distinguished, and the difference was greater than 10%.
  2.4 Specificity test
  10 samples of swine vesicular disease-positive serum, swine vesicular stomatitis-positive serum, Asia-I positive serum, and type A serum,
  500 samples of foot-and-mouth disease virus type O negative pig serum and 500 samples of foot-and-mouth disease type O negative bovine serum were detected with competitive ELISA and the PI values were all below 0.4, and the test results were negative.
  Table 4 Serum-specific test results of different properties

血清类型	血清数/份	阴性数/份	特异性（%）
水泡病毒阳性猪血清	10	10	100
水泡型口炎阳性猪血清	10	10	100
Asia-I型阳性猪血清	10	10	100
A型阳性猪血清	10	10	100
阴性猪血清	500	500	100
阴性牛血清	500	500	100

  2.5 Sensitivity test
  200 samples of pig serum were immunized with FMD O vaccine, 200 samples of recovered pig serum after infection, 10 samples of pig serum infected with FMD O virus, 10 samples of strongly positive pig serum of FMD O virus, and 10 samples of weakly positive pig serum of FMD O virus, and the PI value of serum was determined by competitive ELISA, and the total positive coincidence rate was 95%. The results are shown in Table 5 below.
  Table 5 Results of serum susceptibility tests for different properties

Serum type	Sera/copy	Number of negatives/copy	Number of positives/copies	sensitivity（%）
Vaccine immunized porcine serum	200	2	198	99
Recovered porcine serum	200	6	194	97
Poisoned pig serum	10	1	9	90
Strongly positive porcine serum	10	0	10	100
Weakly positive porcine serum	10	2	8	80

  2.6 Repeatability test
  The coefficient of variation between 10 negative and 10 positive serums was 9.6% for 10 negative and 10 positive sera independently repeated three times by competitive ELISA, and the coefficient of variation between batches was 6.2% for 1 negative serum 20 times.
  2.7 Clinical Trials
  A total of 2000 serum samples were tested from pig farms in Xinjiang, Inner Mongolia, Heilongjiang, Shandong, Sichuan, Henan and other provinces by competitive ELISA, and 247 negative samples and 1753 positive samples were measured by calculating their PI values, with a negative rate of 12.35% and a positive rate of 87.65%.
  3 Discussion and conclusions
  In recent years, solid-phase blocking ELISA detection kits have developed rapidly and are popular because of their ease of operation and short time consumption. At present, solid-phase blocking ELISA detection kits are mainly imported, and few are domestically produced [5]. In this study, a direct competition ELISA detection method was established, which was short, convenient and fast, and only needed 1 hour to complete the experiment. Its sensitivity and specificity are highly correlated with VNT and LPB-ELISA, and the coincidence rate can reach about 90%, and the coincidence rate is >85% compared with similar imported products.
  The key to this method is to select the appropriate epitope to prepare monoclonal antibodies with high specificity and high affinity. FMDV antigen mutation is frequent, and antigen variability between subtypes is large, so it is necessary to select highly conserved antigenic sites. VP1 protein is exposed to the surface of FMD virions and is the main component that induces the production of neutralizing antibodies, among which peptides 140~160 (G-H loop) and 200~213 are key epitopes for the production of neutralizing antibodies[6][7]. Sensitivity, specificity, and precision are important indicators for testing ELISA kits, which are directly related to the accuracy of the test. In this study, a competitive ELISA method with strong specificity, high sensitivity, convenience, reliability, practicality, and good stability was established. After the large-scale application of this method, it will greatly reduce the workload of testing personnel, and it is easy to operate, which is conducive to the promotion to the grassroots level, and will play an important role in the detection and diagnosis of foot-and-mouth disease.
  References
  [1] Xie Qingge, Foot-and-mouth disease[M], Beijing: China Agriculture Press, 2004.
  [2] Ning Yibao, Wang Mingjun.Development history and prospect of veterinary biological products technology in China[J], Chinese Journal of Veterinary Medicine, 2002(08):16-19.
  [3] Sanger D V，The replication of picornavirus，J Gen Virol，1979，45:1～13.
  [4] Sanyal A, Venkataramanan R, Pattnaik B. Antigenic features of foot and nouth disease viruser O type Asial as revealed by monoclonal antibodies and neutralization escape mutants. Virus Res, 1997, 4: 168-173.
  [5] Gao Yanchun, Zhao Deming.Analysis of the current situation and development suggestions of China's veterinary biological products industry[J].Industry perspective，2011,47（2）：55-59.
  [6] P.Zamorano，A  10-Amino-Acid Linear Sequence of VP1 of Foot and Mouth Disease Virus Containing B-andT-Cell Epitopes Induces Protection inMice Virology，1995,212:614～621.
  [7] Ruben Marrero，A computational study of the interaction of the foot and mouth disease virus VP1 with monoclonal antibodies，Journal of Immunological Methods，2015,12052:1～7.