Development of monoclonal antibodies specific to ToxA and ToxB of Vibrio parahaemolyticus that cause acute hepatopancreatic necrosis disease (AHPND)

Toxin A (ToxA) and toxin B (ToxB) of Vibrio parahaemolyticus, which cause acute hepatopancreatic necrosis disease (AHPND), were prepared in a bacterial supernatant from Chinese isolates (CN-VPAHPND) by washing bacterial colonies off of TSA cultures. The supernatant was subsequently used in mouse immunization to produce monoclonal antibodies (MAbs). Three groups of MAbs were selected: one MAb specific to ToxA, two MAbs specific to ToxB and one MAb specific to V. parahaemolyticus (CN-VPAHPND). The MAbs specific to ToxA and ToxB recognized all 10 VPAHPND isolates from China, Vietnam, Malaysia and Thailand, but did not bind to the 20 non-VPAHPND isolates from various other sources, including Vibrio spp. and other bacteria. The MAbs specific to toxins were used to detect the recombinant proteins of His-tagged ToxA and GST-ToxB with sensitivities of 200 fmol spot(-1) and 10 fmol spot(-1), respectively, as determined by dot-ELISA. The MAbs were used to detect the toxins produced by bacteria or shrimp tissue lysate spiked with bacteria in a complex tissue sample at concentrations as low as 1 CFU ml(-1) after pre-enrichment of the bacteria for 6 h. The third group of MAb was specific to CN-VPAHPND isolate but did not recognize the other 9 out of 10 VPAHPND isolates from Vietnam, Malaysia and Thailand. However, this MAb demonstrated cross-reactivity to 1 out of 20 of the non-VPAHPND isolates and 3 out of 9 of the V. alginolyticus isolates. These findings indicate that the AHPND epidemic in Southeast Asia was not caused by the CN-VPAHPND isolate. The MAbs specific to ToxA and ToxB produced in this study could be used to detect both toxins directly by dot blotting.