title: Development of monoclonal antibody based sandwich ELISA for the rapid detection of pathogenic Vibrio parahaemolyticus in seafood.
abstract: Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are considered important virulence factors of Vibrio parahaemolyticus and strains producing either of these or both are considered pathogenic. In this study, we generated monoclonal antibodies (mAbs) against purified TRH recombinant protein of pathogenic V. parahaemolyticus. Sandwich enzyme-linked immunosorbent assays (ELISA) using the hybridoma clone 4B10 showed higher sensitivity of detection compared to other clones. Using mAb 4B10 based sandwich ELISA, we could detect pathogenic V. parahaemolyticus in 41.18% (14 out of 34) of the seafood samples analyzed. PCR targeting the toxR gene showed the presence of V. parahaemolyticus in 64.7% (22 out of 34) seafood samples. Further, PCR targeting the virulence genes showed that 6 seafood samples harboured the tdh gene while 9 harboured the trh gene indicating the presence of pathogenic V. parahaemolyticus. Our results show that mAb 4B10 sandwich ELISA developed in this study could be used as a rapid method for screening seafood samples for the presence of pathogenic V. parahaemolyticus.
authors.affiliation: Department of Fishery Microbiology, Karnataka Veterinary, Animal and Fisheries Sciences University, College of Fisheries, Mangalore 575 002, India.
authors.forename: Ballamoole Krishna
authors.initials: BK
authors.lastname: Kumar
authors.forename: Pendru
authors.initials: P
authors.lastname: Raghunath
authors.forename: Devananda
authors.initials: D
authors.lastname: Devegowda
authors.forename: Vijay Kumar
authors.initials: VK
authors.lastname: Deekshit
authors.forename: Moleyur Nagarajappa
authors.initials: MN
authors.lastname: Venugopal
authors.forename: Iddya
authors.initials: I
authors.lastname: Karunasagar
authors.forename: Indrani
authors.initials: I
authors.lastname: Karunasagar
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001427:Bacterial Toxins / Q000737:chemistry / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005516:Food Microbiology / Q000379:methods*; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D016133:Polymerase Chain Reaction; D017747:Seafood / Q000382:microbiology*; D012680:Sensitivity and Specificity; D014736:Vibrio parahaemolyticus / Q000302:isolation & purification*
chemical_list: D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D006460:Hemolysin Proteins; C068489:thermostable direct hemolysin