Development of non-radioisotopic immunoassay systems for measuring flounder IGF-I.
A time-resolved fluoroimmunoassay system (TR-FIA) for measuring flounder insulin-like growth factor-I (IGF-I) was developed using biotinylated flounder IGF-I, anti-fish IGF-I antiserum and europium-avidin conjugate. The detection limit per well was <5 pg/well corresponding to <0.5 ng/ml in a basic procedure for sample of 10 microl/well and to <0.08 ng/ml in a procedure modified for high volume samples (up to 70 microl/well). Specificity of the assay was validated using various IGF-Is and insulins. All IGFs except seabream IGF-I showed very low or no crossreactivity. Binding inhibition curves for flounder and seabream IGF-Is were completely identical to each other. Intra- and interassay variations ranged from coefficients of variations of 3.9% to 7.2%. Recovery tests using barfin flounder plasma varied from 82.7 to 101.6% in the added range from 20 to 160 ng/ml. This assay system was applied for measuring total plasma IGF-I in barfin flounder injected porcine growth hormone (GH). A group injected with GH at the dose of 0.05 IU/gBW showed a significant increase of total plasma IGF-I compared with those of albumin-injected (control) and initial groups. In addition, I was able to substitute time-resolved fluorometric detection in this assay system with enzymatic fluorometric detection (FIA). Binding inhibition curve for flounder IGF-I in this substituted assay system showed equal performance with that of the TR-FIA system. Correlation of IGF-I levels between TR-FIA and FIA was high (r(2)=0.957) in plasma samples from barfin flounders in various physiological conditions. Thus, the present study shows precision and efficiency of two non-radioisotopic immunoassay systems for measuring flounder IGF-I.
Andoh
Tadashi
T
Hokkaido National Fisheries Research Institute, Fisheries Research Agency, Kushiro, Japan. andoh@fra.affrc.go.jp
D000818:Animals; D001360:Avidin; D005063:Europium; D005432:Flounder / Q000378:metabolism*; D015200:Fluoroimmunoassay / Q000379:methods*; D005470:Fluorometry; D013006:Growth Hormone / Q000008:administration & dosage; D007106:Immune Sera / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D011485:Protein Binding; D015203:Reproducibility of Results; D013045:Species Specificity
D007106:Immune Sera; D001360:Avidin; D005063:Europium; D007334:Insulin-Like Growth Factor I; D013006:Growth Hormone