Enzyme immunometric assay of thyroliberin (TRH).

ABSTRACT

An enzyme immunometric assay of thyroliberin (TRH) using monoclonal antibodies and a derivatization procedure is described. This assay, named SPIE-IA, involves a four step procedure after chemical derivatization of TRH and biological samples by diazotized APEA. Step 1 derivatized TRH was immunocaptured by a monoclonal anti-TRH antibody coated on a 96-well microtiter plate. Step 2 after washing, derivatized TRH was cross-linked via its amino group to the wells using glutaraldehyde. Step 3 washing and treatment with NaOH. Step 4 measurement of bound TRH using a monoclonal anti-TRH antibody labeled with acetylcholinesterase. The minimal detectable concentration was 0.1 pmol/ml with a coefficient of variation less than 10% in the 0.156-10 pmol/ml range. This assay is 26-fold more sensitive and more specific than the competitive enzyme immunoassay using the same monoclonal capture antibody, derivatized TRH and TRH-acetylcholinesterase conjugate as tracer. Good correlation was observed between SPIE-IA and a sensitive competitive enzyme immunoassay using polyclonal antibodies.