Blood sampling routine of farmed fish being popular to investigate fish health status to forecast and prevent disease outbreak. Enzyme linked immunosorbent assay (ELISA) is the typical assay to monitor fish health status by serum titer elevation after exposure to pathogen. Secondary antibody against fish IgM is the key component of the ELISA. Existing secondary antibodies for fish IgM in market are however, provided for each fish species, and low titer in general as such 40-100 times dilution to use while anti mammal IgM antibodies work with 5,000 – 10,000 times dilution.
In this study, we generated several secondary antibodies against fish IgM. Firstly, seabass IgM and grouper IgM were purified by chromatography (Superdex® 200 / ÄKTA avant). Subsequently, mice were immunized with purified grouper IgM to generate over 10^3 hybridoma clones, and 83 of positive hybridoma cells were chosen by ELISA with purified grouper IgM. Supernatant of the 83 positive cells were tested further against un-purified serum from seabass, grouper, tilapia and bovine serum albumin (BSA: negative control), to evaluate versatility to different fish species and specificity to fish IgM. Eight (8) clones gave high titer with fish serum but minimum titer with BSA. Two (2) clones out of the 8 were specific to grouper serum only. Another 2 clones react with both seabass and grouper serum but not react with tilapia serum. Rest 4 clones showed broad spectre by giving signal to seabass, grouper and tilapia serum. Western blot of grouper serum revealed that our monoclonal antibodies bind IgM. Selected secondary antibodies evaluated in ELISA with iridovirus as an antigen. Significant difference of serums from iridovirus challenged fish (represents disease) and iridovirus naïve fish (as healthy fish). Results above indicate that, we managed to raise monoclonal antibody against fish IgM. Further specifications and preparations towards product launch are undergoing.