Development And Application Of Indirect ELISA For Detection Of Antibody Levels Of Feline Parvovirus/feline Calicivirus/feline Herpesvirus Type 1

With the continuous improvement of the material life of the masses,more and more people begin to seek spiritual satisfaction,thus the existence of companion animals is needed in life,which promotes the vigorous development of the pet market.The breeding of pet cats is particularly prominent.It is very important to diagnose and prevent common cat infectious diseases.Feline parvovirus/feline calicivirus/feline herpesvirus type 1 are very easy to infect young cats and are prone to cause cross infection.There’s no specific treatment for any of these viruses,yet vaccine immunization is mainly used to control those disease.It is necessary to assess post-immunization antibody levels,and the antibody level may decrease after immunization.We need to measure antibody levels after vaccination,so that we can accurately assess the immune effect of the vaccine and promote subsequent prevention.Therefore,it is urgent to establish an antibody detection method for FPV/FCV/FHV-1.This study aims to establish an indirect ELISA method for detecting FPV VP2,FCV VP1 and FHV-1g B antibodies.the dominant epitopes of FPV VP2 protein,FCV VP1 protein and FHV-1 g B protein were tandem expressed as recombinant proteins and purified as envelope antigens.The checkerboard method was used to optimize the reaction conditions of each step.FPV indirect ELISA results showed that the amount of antigen coating of FPV was 8 μg/m L,the optimal dilution of serum was 1 :400;the optimum dilution of secondary antibody is 1:10 000;the optimal time of substrate chromogenic solution was 15 min.The results of specificity test showed that there was no cross reaction with FCV and FHV positive serum.The sensitivity of this method to FPV positive serum was1: 5 120;the coefficients of variation within and between groups were less than 10%.FCV indirect ELISA results showed that the amount of antigen coating of FCV was 2 μg/m L,the optimal dilution of serum was 1 :1 000;the optimum dilution of secondary antibody is 1:10 000;the optimal time ofsubstrate chromogenic solution was 15 min.The results of specificity test showed that there was no cross reaction with FPV and FHV positive serum.The sensitivity of this method to FCV positive serum was 1: 40 960;the coefficients of variation within and between groups were less than 5%.FHV-1 indirect ELISA results showed that the amount of antigen coating of FHV-1 was 0.5 μg/m L,the optimal dilution of serum was 1:1 000;the optimum dilution of secondary antibody is 1:10 000;the optimal time of substrate chromogenic solution was 15 min.The results of specificity test showed that there was no cross reaction with FPV and FCV positive serum.The sensitivity of this method to FHV-1 positive serum was 1:20 480;the coefficients of variation within and between groups were less than 5%.In conclusion,FPV VP2 protein,FCV VP1 protein and FHV-1 g B protein were successfully expressed and purified.Indirect ELISA methods have been successfully established for FPV/FCV/FHV-1antibody detection and the bag was full virus methods of parallel comparison,according to the test result has high consistency.