Production of polyclonal antibody against the recombinant PirBvp protein of Vibrio parahaemolyticus

Acute hepatopancreatic necrosis disease (AHPND) is caused by toxin-producing strains of Vibrio parahaemolyticus which contain deadly binary toxins PirA vp and PirB vp encoded in pVA1 plasmid. The polyclonal antibodies against PirB vp protein could be used to develop immunochromatographic test strip for in-field diagnosis of AHPND. In this study, PirB vp gene was amplified, cloned, and expressed in E. coli . The expressed protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot probed with 6xHis antibodies. Then, the recombinant PirB vp (rPirB vp ) was purified using Ni-Sepharose column. Rabbits were immunized with the purified rPirB vp , and produced antibodies were analyzed using Ouchterlony double immunodiffusion. The antibody titration and antibody purification were performed by ELISA and affinity chromatography, respectively. Finally, antibody specificity and sensitivity were evaluated by dot blotting. The present study showed a high titer of polyclonal antibodies in rabbit serum after immunization and the titer increased steadily during the immunization schedule. The highest titer of antibody reached up to 2,560,000 with LOD of 0.1 ng/mL. The purified antibodies showed no cross-reactivity with proteins from other Vibrio species, and the detection threshold ranged from 6.25 to 12.5 ng toxin/dot. This study highlights the production of high titer and specific polyclonal antibodies as an initial material towards the development of lateral-flow strip test.