Establishment Of ELISA Methods And Production Of Monoclonal Antibody Of VP1 Proteins Of Coxsackieviruses A6 And B4

As members of the enterovirus genus,Picornaviridaefamily,coxsackie virus was first isolated during the polio epidemic in Coxsackie in New York in 1948.Most of the children under 6 years of age are susceptible to coxsachie virus infections,with the major clinical symptom of herpes in hands,feet and mouth,which is known as Hand,Foot,and Mouse Diseases(HFMD).A few patients can develop neurological diseases such as aseptic meningitis,neurogenic edema and other complications.The major pathogens causing HFMD include EV71 and CVA16,however,numerous studies have shown that the pathogens causing HFMD have been constantly changing.In this study,the VP1protein of CVA6 and CVB4 was successfullyobtained using a prokaryotic expression system,and they were subsequently used to establish an ELISA detection method targeting the VP1 protein.In addition,circulating CVA6and CVB4 strains isolated by our laboratory were selected to produce monoclonal antibodies against the VP1 proteins,which would provide a rapid and accurate detection technology for monitoring and diagnosis of HFMD epidemics.1.Construction and identification of the VP1 protein prokaryotic expressionof CVA6 and CVB4TheVP1 gene of CVA6 and CVB4 were cloned into the expression vector pET-32a,and was then transformed into the E.coli DH5α.The positive recombinant plasmids were identified and transformed into the plasmid E.coli BL21(DE3).Via the IPTG induction,the VP1 genes are efficiently expressed in the form of fusion protein.SDS-PAGE analysis showed that the molecular weight of the recombinant protein expressed was approximately 50k Da,which was also confirmed by protein mass spectrum.With the purified recombinant protein being used as antigen,an indirect ELISA method for detecting CVA6 and CVB4 serum antibodies was established and optimized.The optimal coating concentration of CVB4 VP1 antigen was 8 ug/well;the optimal dilution of serum was 200 times;the optimal incubation time was 60 min;the best working concentration is 1:1600.The optimized method was used to detect 15serum samples includingCVA6 negative serums,CVB4 positive serum,EV71 and other types of Coxsackie virus positive serums.The results showed that the purified CVA6 capsid protein VP1 had good antigenicity and the established indirect ELISA method showed high specificity,sensitivity and reproducibility.The same method was used to establish an indirect ELISA method for CVB4.The optimal coating concentration of CVB4 VP1 antigen was determined to be 7.5 ug/well;the optimal dilution of serum was 100 times;the optimal incubation time was 60 min;the optimal working concentration of the secondary antibody is 1:1600.The established method was used to detect 18 samples,including CVB4 negative serums,CVB4 positive serums,EV71,CVB3 and other types of Coxsackie virus positive serums.The results showed that the purified CVB4 capsid protein VP1 had certain cross reaction with CVB3,however,no cross-reaction with other virus-infected sera was observed.In particular,the purified CVB4 capsid protein VP1 had good antigenicity and the established indirect ELISA method showed high specificity,sensitivity and reproducibility.In this step,two indirect ELISA methods were established,which can be applied to the detection of serum antibodies after infection with CVA6and CVB4,laying a foundation for further development of diagnostic reagents and mouse-derived monoclonal antibodies.2.Production and Identification of CVA6and CVB4 Monoclonal AntibodiesAfter the purified CVA6 and CVB4 viruseswere mixed with adjuvant,respectively,6-week-old BALB/c mice were immunized three times and serum antibodies reached1:5000.The spleen cells of the mice were fused with the SP20 myeloma cells in an exponential growth phase.Hybridoma cells were screened using the ELISA method established in the previous step.A total of2 CVA6and 4 CVB4 hybridoma cell lines were obtained.These hybridoma cells were injected into the abdominal cavity of the mice to produce ascites.The antibody titer of the ascites ranged from 10~4 to10~6,respectively.Six primary monoclonal antibodies were identified and analyzed.The subtypes of CVB4-1,CVB4-2,CVB4-3,and CVB4-4 were IgM,IgA,IgM,and IgA,respectively.The subtypes of both CVA6-1 and CVA6-2 were IgM.Indirect immunofluorescence showed that CVA6 virus-infected cells had specific bright green fluorescence with the two monoclonal antibodies against CVA6,and CVB4 virus-infected cells also showed specific bright green fluorescence with the four specific monoclonal antibodies against CVB4,suggesting the high specificity of the monoclonal antibodies obtained in the present study.