title: Efficacy of protein A-HRP in an immunological study of black rockfish (Sebastes schlegeli Higendorf) humoral immune responses.
abstract: The efficacy of protein A-horse radish peroxidase (HRP), as compared to that of mouse polyclonal antibody raised against purified Ig, in detection of black rockfish (Sebastes schlegeli Higendorf) immunoglobulin (Ig) was examined. Protein A affinity chromatography successfully purified Ig from black rockfish serum; the purified-Ig could be visualised as two protein bands (MW 70 and 25kDa) following resolution with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. In SDS-PAGE immunoblot profiles of the purified-Ig, the mouse polyclonal antibody recognised both the light chain and heavy chains of rockfish Ig, whereas protein A-HRP immunostained only the heavy chain of rockfish Ig. These results suggest that protein A-HRP may be used to detect rockfish antibody-antigen complexes in immunoassays. In a 2-DE immunoblot assay for exploring antigenic profiles of Lactococcus garvieae KG9408, protein A-HRP successfully detected specific antibodies to antigenic proteins of L. garvieae in the rockfish Ig. In addition, enzyme linked immunosorbent assay (ELISA) showed a high correlation between the results obtained for positivity of L. garvieae when protein A-HRP and the mouse polyclonal antibody-was used to analyse samples from 25 diseased rockfish. These results collectively indicate that protein A-HRP has a high affinity for Ig, and may be useful for new investigations into the humoral immune responses of rockfish.
authors.affiliation: Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju, 660-701, South Korea.
authors.forename: Sung-Hyun
authors.initials: SH
authors.lastname: Kang
authors.forename: Gee-Wook
authors.initials: GW
authors.lastname: Shin
authors.forename: K J
authors.initials: KJ
authors.lastname: Palaksha
authors.forename: Yong-Seung
authors.initials: YS
authors.lastname: Shin
authors.forename: Young-Rim
authors.initials: YR
authors.lastname: Kim
authors.forename: Eun-Young
authors.initials: EY
authors.lastname: Lee
authors.forename: Euy-Hoon
authors.initials: EH
authors.lastname: Suh
authors.forename: Nam-Eung
authors.initials: NE
authors.lastname: Huh
authors.forename: Myung-Ju
authors.initials: MJ
authors.lastname: Oh
authors.forename: Tae-Sung
authors.initials: TS
authors.lastname: Jung
mesh_terms: D000818:Animals; D000917:Antibody Formation / Q000276:immunology*; D002846:Chromatography, Affinity / Q000662:veterinary; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D005399:Fishes / Q000276:immunology*; D006735:Horseradish Peroxidase / Q000276:immunology / Q000378:metabolism; D007118:Immunoassay / Q000379:methods / Q000662:veterinary*; D015151:Immunoblotting / Q000662:veterinary; D007136:Immunoglobulins / Q000276:immunology* / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000276:immunology* / Q000378:metabolism
chemical_list: D007136:Immunoglobulins; D013205:Staphylococcal Protein A; D006735:Horseradish Peroxidase