Efficacy of protein A-HRP in an immunological study of black rockfish (Sebastes schlegeli Higendorf) humoral immune responses.
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.
Kang
Sung-Hyun
SH
Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju, 660-701, South Korea.
Shin
Gee-Wook
GW
Palaksha
K J
KJ
Shin
Yong-Seung
YS
Kim
Young-Rim
YR
Lee
Eun-Young
EY
Suh
Euy-Hoon
EH
Huh
Nam-Eung
NE
Oh
Myung-Ju
MJ
Jung
Tae-Sung
TS
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
D007136:Immunoglobulins; D013205:Staphylococcal Protein A; D006735:Horseradish Peroxidase