Development Of Colloidal Gold Immunochromatographic Test Strip For PEDV Specific SIgA

Porcine epidemic diarrhea virus(PEDV)is one of the major pathogens causing diarrhea in piglets and is mainly infected by adhesion to intestinal epithelial cells.Pigs can be infected at all ages,but with age the fatality rate declines.PEDV causes the most serious damage to suckling piglets.Piglets infected PEDV showed the clinical symptoms of watery diarrhea,vomiting,and dehydration.PEDV infection can stimulate the humoral immune system to produce corresponding antibodies,of which secretory Ig A(SIg A)is the main effector molecule and plays an important role in mucosal immunity.The SIg A molecule consists of a secretory component(SC),two Ig A molecules and a joining chain(J).Compared with monomeric Ig A and dimer Ig A(d Ig A),SC is a unique component of SIg A,so detection of SC can better reflect the level of SIg A in the body.At present,the methods for detecting PEDV include virus isolation,enzyme linked immunosorbent assay(ELISA),indirect immunofluorescence(IFA),virus neutralization assay,polymerase chain reaction(PCR)and so on.The above detection methods need a long detection time,require expensive equipment,as well as professional personnel to operate.The colloidal gold test strips just make up for the inadequacies of the above detection methods.Therefore,we developed colloidal gold immunochromatographic test strip based on PEDV specific SIg A.In this experiment,SIg A purified from colostrum was used as immunogen to immunize6-week-old BALB/c mice.The antibody titers of the serum were detected by indirect ELISA.Then we fused immune spleen cells with higher titer and SP2/0 myeloma cells using 50% PEG and selected monoclonal positive hybridoma cells.Finally,4 hybridoma cells stably secreting against SIg A monoclonal antibodies were obtained successfully(one of them was a hybridoma cell secreting anti-SIg A SC monoclonal antibody and the monoclonal antibody named 2F9).Both IFA and western blot showed that 2F9 can react with SC specifically.The mice were inoculated with the hybridoma cells secreting 2F9.Then collected and purified 2F9 ascites fluid,and the titer of the purified 2F9 ascitic fluid(concentration was 1.0 mg/ml)was 1:16000,the titer of the cell supernatant was 1:200 by indirect ELISA.In the experiment,colloidal gold solution with particle diameter of 20 nm was prepared by the sodium citrate reduction method.Results showed that the optimal p H for binding the colloidalgold particles to the monoclonal antibody 2F9 was 8.2,and the optimal binding protein amount of the monoclonal antibody was 9 ?g/ml.The monoclonal antibody 2F9 was combined with colloidal gold,purified PEDV was used as the test line(2 mg/ml),and SPA was used as the control line(1.5 mg/ml),finally we prepared the colloidal gold immunochromatographic test strip based on PEDV specific SIg A.The strips showed good specificity.When testing colostrum infected with PEDV,TGEV and PRo V,milk,water as well as phosphate buffered saline using the colloidal gold immunochromatographic test strip,only colostrum infected with PEDV came up positive.The sensitivity results showed that the lowest detection dose was 50-fold dilution of colostrum samples.In addition,the test strips have good repeatability and stability.In this study,we prepared the monoclonal antibody of anti-PEDV specific SIg A,and the colloidal gold immunochromatographic test strip for the testing of swine milk was developed based on the antibody,providing a tool for PEDV-mediated mucosal immune responses.