Production of monoclonal antibodies for detection of Vibrio harvey

Abstract

Monoclonal antibodies (MAbs) against Vibrio harveyi were produced from mice immunized with heat-killed and SDS-mercaptoethanol-treated highly virulent V. harveyi 639. Fifteen MAbs were selected and sorted into 6 groups according to their specificity to various proteins of apparent molecular weight ranging from 8 to 49 kDa. Some antibodies were used for detection of V. harveyi at concentrations as low as 10 4 CFU ml -1 using immunodot blots. Most of the selected MAbs did not show cross-reactivity to other Vibrio species and other Gram-negative bacteria tested. Only 1 MAb (VH39-4E) showed slight cross-reactivity to Aeromonas hydrophila. Another MAb (VH24-8H) bound lightly to V. harveyi 1526 but strongly to V. harveyi 639, allowing rapid differentiation. Two of the MAb groups were used to localize V. harveyi in tissues of infected black tiger shrimp Penaeus monodon by immunohistochemistry. This study demonstrates the versatility of a highly specific immunological tool for the detection of V. harveyi in aquaculture and opens the way for further development of convenient test kits.

Evaluations of lactic acid bacteria as probiotics for juvenile seabass Lates calcarifer

“…After challenges with A. hydrophila , haemorrhagic ulcerations occurred on dead seabass abdomens. These ulcerated tissues reacted with monoclonal antibodies against A. hydrophila prepared previously using the ELISA technique (Phianphak et al 2005; Prahkarnkaeo 2005). Tissues surrounding the liver and gut were also haemorrhagic in all infected seabass.…”

“…Dead seabass from each experimental group were immediately cut open with a sterile scalpel and samples of each organ were collected. Samples were fixed for at least 24 h in Davidson's fixation, immersed in running tap water for 3 h and dehydrated through a graded ethanol series and treated as described previously by Phianphak, Rengpipat, Rukpratanporn, Longyant, Chaivisuthangkura, Sithigorngul and Sithigorngul (2005). A monoclonal antibody against A. hydrophila was placed on tissue and incubated for 5 h, and then washed.…”

Sensitivity improvement of rapidVibrio harveyidetection with an enhanced chemiluminescent-based dot blot

“…Because some Mabs bound loosely to V. harveyi , it might recognize a hidden epitope (Phianphak et al . ). We then tested different dilutions of Mabs to obtain the optimal dilution.…”

“…The traditional dot blot assay required at least 8 h after applying the sample to the nitrocellulose membranes (Phianphak et al . ).…”

Identification of Vibrio spp. in vibriosis Penaeus vannamei using developed monoclonal antibodies

“…However, there is currently no simple, rapid, and accurate technique suitable for routine use by microbiologists and shrimp farmers due to the lack of high quality antibody for the assay. Recently, monoclonal antibodies specific to V. harveyi (Phianphak et al, 2005), V. alginolyticus , and other Vibrio spp. have been developed.…”