An attempt of the development of solid-phase enzyme immunoassay of gonadotropin releasing hormone.

GESHI Masaya, KASUGA Naoki, IMAGAWA Kenichi, MORI Junichi

doi:10.1262/jrd1977.32.172

A solid-phase competitive enzyme immunoassay of gonadotropin releasing hormone (GnRH) was examined. Enzyme-labeled antigens were prepared by coupling synthetic GnRH with peroxidase using bis-diazotized benzidine and purified by Sephadex G-25 column chromatography. Antiserum raised against [Glu¹]-GnRH-rabbit serum albumin conjugate in a male horse was used. Immunoglobulin G (IgG) fraction of the antiserum used for a solid-phase was prepared by an (NH₄)₂SO₄ precipitation method, purified by Sephacryl S-300 column chromatography, and adsorbed to a polystyrene ball.<BR>Three-tenths ml of 0.05 M phosphate buffer (pH 7.5), 0.1 ml of standard or sample solution, 0.1 ml of peroxidase-labeled GnRH solution and a solid-phase polystyrene ball were added in a test tube. After incubation at 4°C for 24 hours, a polystyrene ball was washed three times with 2 ml of saline, and transferred to a new tube. The enzyme activity was measured with a spectrophotometer (absorbance at 492 nm).<BR>A dose-response curve was obtained between 1 to 1000 ng/ml of GnRH when a solid-phase coated with 50 μg/ml IgG solution and peroxidase-labeled GnRH solution (×100) were used. Acid extract of bovine pituitary stalk produced dose-response curve indistinguishable from synthetic GnRH, but those of anterior lobe, liver and muscle didn't produced.<BR>These results show that GnRH is assayed by this method.

An attempt of the development of solid-phase enzyme immunoassay of gonadotropin releasing hormone.

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