Development Of Universal Competitive ELISA For Serological Detection Of Capripoxvirus

The Poxvirinae family comprises several viruses of veterinary and medical importance.Poxviruses are the largest and most complex viruses.They are linear double-stranded DNA viruses of 130 to 300kilo base pairs.Poxviruses virion is oval or brick-shaped and can be visualized on light microscopy.Poxvirus diseases occur in most animal species and are of considerable economic importance in some regions of the world.The clinical symptom of every poxvirus shares a similar feature of pox lesions on the skin or the mucosa of infected animals or humans.Most poxviruses are cross-reactive because of this reason a single diagnostic technique can help for the detection of antibodies against a wide range of poxviruses infection in animals.Enzyme-linked immunosorbent(ELISA)assay is one of the diagnostic methods used for the detection of antibodies against poxviruses after coating immunogenic proteins.Since most poxviruses are antigenically related and owning similar immunogenicity,a single diagnostic method can detect different antibodies of poxvirus infection at once.Hence,in this study,a competitive ELISA(c-ELISA)method that can detect Capripoxpoxvirus infection was developed and validated.For the development of the current Competitive ELISA(c-ELISA)method,polyclonal antibodies were produced after rabbits were immunized with formalin-inactivated virion of the Goatpox and Sheeppox viruses subcutaneously.The antibody titer in the serum of rabbits against both virions of Goatpox and Sheeppox was evaluated and No significant difference was observed(p<0.05).After comparison of antibody titration,Immunoglobulin G was purified using ammonium sulfate precipitation method and the concentration of IgG was determined as 2.29 mg/ml and 2.18 mg/ml for virion of Goatpox virus and Sheeppox virus respectively.The immunoglobulin G having 25kDa light and 67kDa heavy chain were analysed by SDS-PAGE.Moreover,additional serum antibodies were produced for comparison of recombinant proteins after immunization of rabbits with Lumpy skin disease virus vaccine,inactivated virion of Goatpox,and Sheeppox viruses as described above.Hereafter,Goatpox virus A27R,Goatpox virus L1R,and Sheeppox virus A33R recombinant proteins having a molecular weight of 35kDa,26kDa,and 19kDa respectively were expressed,purified,and coated.The immunogenicity of each of the proteins was compared through titration of antibodies produced in the rabbits using the Indirect ELISA technique.During the comparison,three of the proteins revealed a significant detection ability with a slightly better antibody titer observed for Goatpox virus A27R protein.As a result,Goatpox virus A27R protein was selected and coated for the development of competitive ELISA(c-ELISA).The concentration of the selected recombinant protein was estimated as 3.54mg/ml.Subsequently,the concentrations of working antigen and serum dilution were optimized as 2 ?g/ml and 1:2 respectively.The currently developed Competitive ELISA method was piloted with a competition of two primary antibodies targeting the coated antigen.The polyclonal antibodies and serum were diluted with various concentrations in PBS and the competitive ELISA method was applied.The competition between polyclonal antibodies versus positive serum and polyclonal antibody versus negative serum were compared.Concerning this ELISA method,the OD value of the competition between polyclonal antibodies and negative serum was higher due to less competition.Whereas less OD values are recognized on the other side.This ELISA method has a miscellaneous advantage while compared with traditional ELISA.It is easy to produce polyclonal antibodies instead of monoclonal antibody and has many epitopes for binding with the coated antigen.The developed c-ELISA has a test sensitivity of 96.07%and diagnostic specificity of 97.0%.No cross-reaction with non-poxvirus antibodies were detected.As described,the principal aim of this study was to produce ELISA that detects different poxvirus antibodies,therefore this method can detect antibodies against Lumpy skin disease virus,Sheeppox virus,and Goatpox virus,and the effects of this ELISA method were highly correlated with diagnoses made by the PCR test.