Development and characterization of a competitive polyclonal antibody enzyme-immunoassay for salmon insulin-like growth factor-II.
This paper describes the development and validation of a competitive, polyclonal antibody enzyme-immunoassay (EIA) for the measurement of salmon and trout insulin-like growth factor-II (IGF-II). A polyclonal antiserum was raised against a synthetic peptide epitope, corresponding to amino acid residues 1-9 of the N-terminus of mature Atlantic salmon (Salmo salar) IGF-II. The antiserum was purified by hydrophobic charge induction chromatography (HCIC). The partially purified immunoglobulins were used in an enzyme-immunoassay system (EIA) resulting in a highly specific assay for salmon IGF-II with cross-reactivity of less than 0.01% for recombinant salmon IGF-I and recombinant salmon growth hormone (GH), and 5.57% for salmon insulin (sIns). The recombinant salmon IGF-II (rsIGF-II) standard curve limit of detection was 1.37 ng/ml with an EC(50) of 44.97+/-0.82 ng/ml. Intra- and interassay coefficients of variation were determined at 7.47% (n=15) and 7.42% (n=15), respectively. Added rsIGF-II was adequately recovered from acid-treated Atlantic salmon and rainbow trout (Oncorhynchus mykiss) plasma samples. Parallel dose-response inhibition curves were demonstrated for the plasma of both fish species tested. Circulating IGF-II levels of 22.26+/-2.66 and 18.24+/-1.43 ng/ml were determined for acid-treated plasma of normal adult Atlantic salmon and rainbow trout, respectively. This EIA should prove to be useful in the study of factors which influence circulating plasma levels of IGF-II in these fish species.
School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia. Ryan.Wilkinson@flinders.edu.au
Ryan J
RJ
Wilkinson
Phillip
P
Elliott
Art
A
Hohmann
Geoffrey
G
Francis
John
J
Carragher
D000818:Animals; D000906:Antibodies; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D007106:Immune Sera; D007335:Insulin-Like Growth Factor II / Q000032:analysis* / Q000276:immunology; D011817:Rabbits; D012474:Salmon; D013045:Species Specificity; D014337:Trout
D000906:Antibodies; D000939:Epitopes; D007106:Immune Sera; D007335:Insulin-Like Growth Factor II