Development And Primary Application Of ELISA Method For Detecting The Mucosal Specific SIgA Against Foot-and-mouth Disease Virus Type A

Foot-and-mouth disease virus?FMDV?enters the body mainly through the respiratory tract and digestive tract,mucosal system is the first line of defensing against pathogen invasion.Secretory IgA antibody?sIgA?is a specific marker and main effect factor of mucosal immunity,which plays an important role in resisting the invasion of FMDV.In this study,we expressed the structure protein VP1gene of FMDV type A in Escherichia coli.The puried protein was used as the coating antigen of indirect ELISA to detect the mucosal specific sIgA antibody against FMDV.This ELISA will provide a basic method for monitoring the change of FMDV specific sIgA antibody level and evaluating the mucosal immune effect,provide a new method for early diagnosis of foot-and-mouth disease as well.The specific research contents are as follows:1.Preparation of FMDV mucosal specific sIgA antibody ELISA antigenThis study constructed a prokaryotic expression vector pET30a-VP1,the recombinant plasmid was expression in E.coli BL21?DE3?.The expressed protein was purified by affinity chromatography,using western blotting to verify its good immunogenicity.The molecular weight of purified protein was 24.3ku by SDS-PAGE gel scanning analysis.The protein concentration was 0.7 mg/mL by BCA ^TM assay.2.Establishment of FMDV mucosal specific sIgA antibody ELISAThe purifed VP1 was used as coating antigen,mouse anti-pig IgA monoclonal antibody as second antibody and HRP-conjugated goat anti-mouse IgG as third antibody.Optimized the conditions for FMDV mucosal specific sIgA antibody ELISA,the concentration of coating antigen was 3.5 g/mL,the best sealing liquid is 5%skim milk,the dilution of mouse anti-pig IgA monoclonal antibody and HRP-conjugated goat anti-mouse IgG antibody respectively were 1:5000 and 1:2500.The reaction time of samples,the second antibody and the third antibody were all 37?for30 min.The reaction time of substrate was room temperature for 10min.There is no cross reaction with classical swine fever?CSF?,porcine reproductive and respiratory syndrome?PRRS?,porcine circovirus?PCV?and porcine epidemic diarrhea?PED?specific IgA antibody.The sensitivity was above 95%and the specificity was above87%.The coefficient of variation was between 3.16%and 9.76%,which indicated that the method had good repeatability.3.The clinical application of FMDV mucosal specific sIgA antibody ELISAWe used the ELISA method to detecte nasal swab samples from different time period of pigs infected with FMDV type A.The trend of sIgA was compared with the results of IgG and 3ABC antibodies in serum.Statistical results showed that FMDV specific sIgA antibody can be detected at 1d after cohabitation infection,which earilier than IgG and 3ABC antibodies in serum.SIgA antibody of some samples was positive till 60d.Although the cross reaction between type A and O in this study can not be avoided completely,it can be greatly reduced by adjusting the appropriate cut-off,which is to ensure that the method have good sensitivity and specificity.This ELISA provides a basic method for monitoring the change of FMDV specific sIgA antibody level and evaluating the mucosal immune effect.Besides,it provides a new method for early clinical diagnosis of Foot-and-mouth disease?FMD?.