Generation and evaluation of polyclonal antibodies specific for ToxA from Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (AHPND) in shrimp.

Abstract

Acute Hepatopancreatic Necrosis Disease (AHPND) is a newly emerging shrimp disease with mortality up to 100 percent caused by Vibrio parahaemolyticus which carries a plasmid encoding for two toxins, ToxA and ToxB. In 2013, the Global Aquaculture Alliance (GAA) estimated shrimp farming decline in Asia accounted for 1-billion US dollar lost. Currently, diagnosis using PCR method does not meet the demand of in situ detection, which is based on antigen-antibody interaction, has not been developed yet. In this present study, we proceeded to create the toxin and its antibody for lateral flow development. First, recombinant toxin ToxA was generated by gene manipulation. After that, purified ToxA was used to immunize rabbits. Finally, antisera from rabbits and protein-A purified antibodies were evaluated for titer, specificity, and detection threshold. Results showed that recombinant ToxA was overexpressed in soluble fraction at 37^oC with 1mM IPTG. Purification by affinity chromatography was able to isolate recombinant ToxA with the purity up to 94.49%. In ELISA experiment, the immunized antisera reached a titer of up to 1/5,210,000 with 1µg/ml of antigen, and detection threshold was 100ng recombinant toxin. After purification, the detection threshold of purified polyclonal antibodies was 25ng toxin per dot. These results laid a groundwork for the development of AHPND detection kit based on antigen - antibody interactions.