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 residu...

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Bibliographic Details
Published in:Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology
Main Authors: Wilkinson, Ryan, Elliott, P, Hohmann, R, Francis, G, Carragher, J
Format: Article in Journal/Newspaper
Language:English
Published: Pergamon-Elsevier Science Ltd 2004
Subjects:
Biological Sciences
Biochemistry and Cell Biology
Biochemistry and Cell Biology not elsewhere classified
Eia
Atlantic salmon
Salmo salar
Online Access:https://doi.org/10.1016/j.cbpc.2004.06.016
http://www.ncbi.nlm.nih.gov/pubmed/15465665
http://ecite.utas.edu.au/63067
Description
Summary: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 EC50 of 44.970.82 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 22.262.66 and 18.241.43 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.

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