Development of a polyclonal antibody-based sandwich ELISpot assay for interleukin-4/13B1 in rainbow trout Oncorhynchus mykiss

Abstract In mammals, interleukin-4 (IL-4) and IL-13 are important Th2 cytokines that enhance antibody production by B cells and proliferation of Th2 cells. Salmonid fish have three homologs to both of these cytokines as a result of four rounds of whole genome duplication events, designated as IL-4/13A , IL-4/13B1 , and IL-4/13B2 . In this study, we aimed to develop a sandwich ELISpot assay for the evaluation of Th2 immune responses in rainbow trout Oncorhynchus mykiss after immersion vaccination. We first performed gene expression analyses of the three homologs after immersion vaccination with inactivated Yersinia ruckeri . IL-4/13B1 increased significantly its levels of transcription in the gills at 7 days postvaccination. Thus, we obtained two polyclonal antibodies (pAbs) in rabbit (detection antibody) and guinea pig (capture antibody) against rainbow trout IL-4/13B1 and used them for a sandwich-ELISpot assay. The ELISpot assay clearly detected IL-4/13B1 production and revealed a significant increase in the number of IL-4/13B1-producing cells in the gills at 7 days postimmersion vaccination compared with the nonvaccinated group. This new tool has allowed us to evaluate a Th2 response mediated by IL-4/13B1 in rainbow trout, and revealed that IL-4/13B1-producing cells increased mainly in the gills after immersion vaccination.