Technical Report Standardization and Validation of a Simple, Sensitive, Second Antibody Format Enzyme Immunoassay for Growth Hormone Determination in Mithun (Bos frontalis) Plasma Mohan Mondal, 1 * Chandan Rajkhowa, 1 and B.S. Prakash 2 1 National Research Centre on Mithun (ICAR), Jharnapani, Medziphema, Nagaland, India 2 Division of Dairy Cattle Physiology, National Dairy Research Institute, Karnal, Haryana, India 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 ml mithun plasma. The GH standards ranging from 0.25 ng/well/25 ml to 128 ng/well/25 ml were prepared in charcoal-treated plasma collected from an aged (410 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 ml, 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 (410 years) senile mithun. For the biological validation of assay, two mithuns were administered with synthetic bovine GH-releasing factor (GRF; 10 mg/100 kg body weight; intravenous) and another two were administered sterile normal saline (controls). *Correspondence to: Dr. Mohan Mondal, Scientist (Animal Physiology), National Research Centre on Mithun (ICAR), Jharnapani, Medziphema, Nagaland-797 106, India. E-mail: mohan_mondal@rediffmail.com Received 09 March 2005; Accepted 10 May 2005 DOI 10.1002/zoo.20069 Published online 22 July 2005 in Wiley InterScience (www.interscience.wiley.com). Zoo Biology 24:483–493 (2005)  c 2005 Wiley-Liss, Inc.