[Measurement of urine human growth hormone levels by ultra-highly sensitive enzyme immunoassay].

A highly sensitive enzyme immunoassay (EIA) for measurement of urine hGH was set up by a modification of the method of Hashida and Ishikawa, which was a sandwich enzyme immunoassay using anti-hGH antibody coated polystyrene balls and anti-hGH antibody-peroxidase conjugate. Anti-hGH serum was obtained in rabbits by subcutaneous injections of hGH emulsified in complete Freund's adjuvant. In order to reduce non-specific binding to the solid phase, anti-hGH IgG was precipitated from rabbit serum followed by digestion to F(ab')2 and affinity purification. Fab'-peroxidase conjugate was produced by maleimide method. The assay procedure was as follows. 1. 100 microliters of urine samples or hGH standard were incubated with anti-hGH IgG coated polystyrene balls. 2. Polystyrene balls were then incubated with Fab'-peroxidase conjugate. Polystyrene balls were carefully washed three times in saline after incubation with Fab'-peroxidase conjugate, which reduced contamination and non-specific binding. 3. Peroxidase activity bound to the balls was assayed by enzyme reaction using 3(p-hydroxyphenyl) propionic acid as a substrate, and fluorescence intensity was measured by a spectrofluorophotometer (Shimadzu RF-540). Reducing the energy of excitation by setting the slit width of the spectrofluorophotometer at 2nm made it possible to gain stable fluorescence. The minimum detectable quantity of hGH was 30fg/tube in the assay, so that the detection limit was 0.3 pg/ml when 100 microliters of urine samples were used. Coefficient of intra- and inter-assay variation was 6.0% and 8.6%, respectively. The recovery was 98.8 +/- 2.8 (+/- SE) on average. Multiple dilution of acromegalic urine and urine after insulin injection produced dose-response curves parallel to those of the standards. Urine hGH levels in acromegalic patients were significantly greater than those in normal subjects. These findings indicate that sensitive EIA of urine hGH is potentially useful for evaluating the pituitary function.