Standardization and validation of a simple, sensitive, second antibody format enzyme immunoassay for growth hormone determination in mithun (Bos frontalis) plasma

Abstract

To facilitate research into the action of growth hormone (GH) in mithun (Bos frontalis), we standardized and validated a simple and highly sensitive enzyme immunoassay (EIA) for GH determination in mithun blood plasma on microtiter plates using biotin-streptavidin amplification system and the second antibody coating technique. Biotin was coupled to GH and used to bridge between streptavidin-peroxidase and immobilized antiserum in competitive assay. The EIA was carried out directly in 25 µl mithun plasma. The GH standards ranging from 0.25 ng/well/25 µl to 128 ng/well/25 µl were prepared in charcoal-treated plasma collected from an aged (>10 years) senile mithun. The sensitivity of EIA procedure was 1.0 ng/ml plasma; the 50% relative binding sensitivity was seen at 36 ng/ml plasma. Plasma volumes for the EIA, namely 12.5 and 25 µl, did not influence the shape of standard curve even though a drop in the optical density (OD)450 observed with higher plasma volumes was due to higher inherent GH content in mithun plasma collected from an aged (>10 years) senile mithun. For the biological validation of assay, two mithuns were administered with synthetic bovine GH-releasing factor (GRF; 10 µg/100 kg body weight; intravenous) and another two were administered sterile normal saline (controls). Jugular blood samples were collected at −60, −45, −30, −15, −10, −5, 0, 5, 10, 15, 30 min and thereafter at 15-min intervals beginning 1 hour before GRF injection up to 8-hr post treatment, and samples were assayed for plasma GH. In two animals, a peak of GH was recorded at 15 min of GRF administration, which confirms the biological validation of the EIA. Plasma GH estimated in blood samples collected for 6 consecutive weeks from two different age groups of mithun (Group I, age 0–3 months; Group II, age 3–4 yr) showed higher (P < 0.0001) mean plasma GH in younger than in adult mithuns and consequently higher growth rates in the younger group. A parallelism test conducted between a buffer standard curve of bovine GH and GH measured from serial dilution of mithun plasma containing high concentration of endogenous GH showed good parallelism with a standard curve. In conclusion, the EIA developed for GH determination in mithun blood plasma is sufficiently reliable, economic, and sensitive enough to estimate mithun GH in all physiologic variations. Zoo Biol 0:1–11, 2005. © 2005 Wiley-Liss, Inc.