Establishment And Application Of ELISpot Assay For Detection Of PCV2-Specific Memory B Cells

Porcine circovirus type-2(PCV2)is the main pathogen of various swine diseases such as post-weaning multisystem wasting syndrome(PMWS)and porcine dermatitis and nephropathy syndrome(PDNS)in weaned piglets,and PCV2 has a wide epidemic range in China,serious infection,and is harmful to the pig industry.At this stage,vaccine immunization is the main means of preventing porcine circovirus disease(PCVD),so the assessment of vaccine-induced humoral immune response is particularly important.However,conventional antibody detection methods cannot distinguish maternal antibodies,vaccine antibodies and wild virus infection antibodies,plus the characteristics of PCV2 vaccine and other factors.Through conventional antibody detection methods,it is difficult for us to objectively determine whether the vaccine has produced significant effect.In this study,we tried to evaluate the immune effect of PCV2 vaccine at the cellular level.The ELISpot method for the detection of PCV2-specific memory B cells was successfully established by isolating primary lymphocytes from porcine peripheral blood and stimulating and differentiating memory B cells in lymphocytes into antibody-secreting cells(ASCs),adding them into ELISpot plate wells coated with Cap protein,capturing specific B cells using Cap protein,followed by adding biotinylated secondary antibody and HRP-labeled streptavidin,etc.,and optimizing various reaction conditions,determining that the coating concentration of Cap protein was 1.25 μg/m L,the concentration of biotinylated secondary antibody was 5 μg/m L,and the concentration of HRP-labeled streptavidin was0.25 μg/m L.Forty piglets with high maternal antibody were randomly divided into 4 groups and injected with three different brands of PCV2 vaccine and normal saline,respectively.The dynamic changes of humoral antibody level,humoral immune memory level and PCV2 viremia at different stages before and after immunization were studied by ELISpot combined ELISA and fluorescence quantitative PCR.The results of ELISA showed that the growth and decline of PCV2 antibodies in each immunized group were very similar to those in the group injected with normal saline,and all of them had been in a downward trend;while the results of ELISpot showed that the levels of specific memory B cells after immunization with the three vaccines were significantly increased compared with those in the normal saline group,and the levels of specific memory B cells induced by vaccine A were the highest,followed by vaccine B and vaccine C,respectively;the results of quantitative PCR showed that the PCV2 infection rates in vaccine A,vaccine B,vaccine C and the control group were 11.1%,10%,44.4%,and 44.4%,respectively.The above studies showed that in the presence of maternal antibodies,the use of ELISpot to detect the level of PCV2-specific memory B cells can better reflect the humoral immune level of the vaccine and the effect of resisting PCV2 infection.This method can be used to evaluate the immune effect of PCV2 vaccine and is of great significance for the prevention and control of PCV2.