A Highly Sensitive Sandwich Enzyme Immunoassay of Human Growth Hormone in Serum Using Affinity-Purified Anti-Human Growth Hormone Fab′-Horseradish Peroxidase Conjugate

Abstract

Abstract A highly sensitive sandwich enzyme immunoassay (EIA) for human growth hormone (hGH) was developed. hGH to be assayed was incubated with an anti-hGH IgG-coated polystyrene ball, and then the polystyrene ball after washing was incubated with anti-hGH Fab1 -peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was correlated to the amount of hGH to be assayed. Polystyrene balls were coated by physical adsorption with rabbit anti-hGH IgG which was not affinity-purified. Rabbit anti-hGH Fab1 which had been affinity-purified was coajugated with horseradish peroxidase using N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate. The sensitivity was 0.06 pg of hGH per tube or 6 pg/ml of serum when 0.01 ml of serum samples was used. No significant interference was observed in the presence of 8 ng/tube of human prolactin, 8 μU/tube of human thyroid-stimulating hormone, 50 mU/tube of human luteinizing hormone, 10 mU/tube of human follicle-stimulating hormone, or 5 U/tube of human chorionic gonadotropin. The recoveries of hGH added to human sera were 96.2-121 %. The coefficients of within-assay and between-assay variations were 3.6-10.0 % and 5.6-9.8 % respectively. The regression equation and coefficient for correlation to radio-immunoassay (RIA) were Y(EIA)=1.04X(RIA)-0.60 and 0.98 (n=137), respectively.