Development Of A Monoclonal Antibody Sandwich ELISA For Antigen Detection Of Porcine Circovirus Type 2

Porcine circovirus(PCV)discovered in 1974 is a non-enveloped,single-stranded circle DNA virus and divided into 3 types.Porcine circovirus type 1(PCV1)has no pathogenicity to pigs,while Porcine circovirus type 2(PCV2)can cause porcine circovirus-associated desease(PCVAD)including porcine multimodal wasting syndrome(PMWS),dermatitis and nephrotic syndrome(PDNS),piglet congenital tremor(CT),necrotizing hypertrophic pneumonia(PNP),porcine respiratory syndrome(PRDC),sow reproductive disturbance and so on.Porcine circovirus type 3(PCV3)is discovered in recent years and its pathogenicity and pathogenesis remain to be studied.Therefore,prevention and control of PCV2 infection has become the top priority in pig industry.Vaccine immunization is considered to be one of effective measure to fight PCVD,so the quality of vaccine has great influence on the prevention and control of PCVD.But there are many kinds of commercialized PCV2 vaccines with different efficacy test and it is difficult to form a unified evaluation standard.There are many factors affecting vaccine quality and the amount of antigen content is the most important one.So it is necessary to establish a general quantitative method of PCV2 antigen for evaluating vaccine quality.Capsid of PCV2 is made up of 60 Cap proteins coded by ORF2,which are the only structural protein with good immunogenicity and antigenicity.Therefore,the absolute quantitative method for PCV2 Cap is undoubtedly a more attractive method.This method is also very necessary for evaluating the quality of vaccines and even for substituting for the test method of immune challenge.In this study,Balb/c mice were immunized with PCV2 Cap protein expressed in E.coli.Two strains hybridoma cell line secreting,named 7D7 and 5C2,were gotten after lymphocyte was prepared from spleen by detecting antibody level,and screened by cell fusion,subcloning and indirect ELISA.Indirect sandwich ELISA showed that both 7D7 and 5C2 could react with PCV2 whole virus and PCV2 Cap protein and recognize different antigen epitopes.Among them,7D7 had a better reactivity.The results of SDS-PAGE and Western Blot showed that 7D7 and 5C2 with linear epitope could not only specifically recognize the PCV2 Cap protein(about 26 kDa)expressed in E.coli,but also specifically react with the PCV2 Cap protein(about 30 kDa)expressed by baculovirus.And an indirect ELISA results showed that the two monoclonal antibodies were IgG2 a subtype.In view of its good reactivity,7D7 was labeled with horseradish peroxidase by sodium iodate method to form the enzyme-labeled monoclonal antibody 7D7-HRP.Using monoclonal antibody 7D7 as capture antibody and 7D7-HRP as detection antibody,a monoclonal antibody sandwich ELISA for PCV2 antigen detection method was successfully established.The linear range of Cap protein,a standard antigen expressed in baculovirus,was 0.1096 ng/mL-14.2222 ng/mL,with a correlation coefficient of 0.99,a coefficient of variation of less than 10%,and a recovery rate of over 90%.This method had no cross reaction with porcine epidemic diarrhea virus(PEDV),porcine reproductive and respiratory syndrome virus(PRRSV),foot and mouth disease virus(FMDV)and other viral antigen.And the detection of PCV2 vaccine from 13 manufacturers showed that the results of different enterprises were significantly different.The antigenic content of 7 vaccines exceeded the upper limit of the method,and the detection values of the 6 vaccines were within the linear range of the standard curve.One of the vaccines had a lower antigen content,which was at least 20 times different from the higher content.In this study,two monoclonal antibodies against PCV2 with different linear epitopes were successfully screened,which provided experimental materials for further study of the distribution and characteristics of PCV2 epitopes.The established monoclonal antibody sandwich ELISA method for PCV2 antigen content has low background value,good specificity,strong sensitivity and high accuracy.It lays a foundation for the research of production technology,quality control of PCV2 vaccine and the study of alternative methods of traditional vaccine efficacy test.It also provides technical support for solving the problems of PCV2 vaccine and manufactures variety,inconsistent quality standards and difficult supervision.