| Pseudorabies(PR)is a highly infectious disease caused by the pseudorabies virus(PRV).PRV can infect most kinds of mammals,including pigs,sheep,and cattle.The clinical characteristics of PRV are reproductive failure of breeding pigs,dyspnea of adult pigs,and death of piglets.This disease has caused serious economic losses to the Chinese pig industry.In 2011,PRV variants broke out in China.Since then,most of the isolated PRV strains are in the same independent cluster as variants,and they have obvious genetic differences with the Chinese classical strains.Because Bartha-K61(live attenuated vaccine)was derived from classical PRV strain,it can't confer complete protection for pigs who infected PRV variants.However,some vaccines against PRV mutants have been on the market.Anti-g B antibody detection kits are often used to monitor the immune status of vaccinated pigs.In recent years,the mostly used anti-g B antibody detection kits in China were produced by foreign companies.With the application of vaccines derived from Chinese PRV variants,the sensitivity of these kits would not as high as they used to be.Besides,some problems exist in imported kits,such as high prices or some brand's kits haven't acquired national approval number.Therefore,it is necessary to develop domestic kits with better effects to replace these foreign products.To solve the above problems,a blocking ELISA method for anti-g B antibody detection was established by using Chinese PRV variants named TP strain as coating antigen and monoclonal antibody(m Ab)named 312 H as blocking antibody.The results showed that: the best coating conditions were as follows: the antigen protein was diluted to 625 ng/well with carbonate buffer(CBS)and coated at 4?for 12 h;the best blocking conditions were PBST containing 1% BSA as blocking solution and blocked at 37? for 1.5 h;the best dilution of blocking antibody was 1:30;the best reaction conditions for double dilution of serums to be detected were incubation at 37? for 0.5 h;the best substrate reaction conditions were coloration at 37? for 15 minutes.According to the statistical results of 400 serum samples,the ELISA result criteria were obtained: serum samples with S/N value ? 0.6 were judged as positive,and serum samples with S/N value > 0.6 were judged as negative.The results of the specificity test showed that only PRV positive serum could be detected by this method,and there was no cross-reaction with those antibody positive serums against porcine circovirus 2(PCV2),African swine fever virus(ASFV),classical swine fever virus(CSFV)porcine epidemic diarrhea virus(PEDV),porcine reproductive and respiratory syndrome virus 1 strain DV(PRRSV-1),and porcine reproductive and respiratory syndrome virus 2 strain F112(PRRSV-2).The results of the repeatability tests showed that the coefficient of variations of intra-batch tests and inter-batch repeatability tests were less than 3%.The results showed that the sensitivity of anti-g B antibody blocking ELISA was 2-8 times higher than IDEXX detection kit.158 PRV negative and positive sera were detected by this method and IDEXX kit at the same time.The results showed that the coincidence rate between this method and IDEXX kit was99.37%,which indicated that this method had good consistency with IDEXX kit.492 clinical serum samples collected from pig farms were detected by this method and IDEXX kit at the same time.The results showed that the coincidence rate of IDEXX kit and this method was 98.78%,which further proved that this method has a good application prospect as an alternative product of IDEXX kit.In conclusion,this study successfully established a highly specific and sensitive anti-g B antibody blocking ELISA,which can effectively monitor the antibody level before or after vaccination.This method can offer technical support for the PR epidemic monitoring and prevention in China,and lay the foundation for the development of standardized detection kit. |
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