Enzyme Immunoassay for the Determination of Des-Gly¹⁰-NH₂-LH-RH-ethylamide (Fertirelin) in Bovine Plasma

Abstract

A double-antibody solid-phase enzyme immunoassay for determining des-Gly¹⁰-NH₂-LH-RH-ethylamide (fertirelin) in bovine plasma was developed. Antiserum was raised against fertirelin-BSA conjugate, and enzyme-labeled antigens were prepared by coupling fertirelin analogues bearing an N-terminal amino group with β-D-galactosidase using N-(m-maleimidobenzoyloxy)-succinimide (MBS). The antiserum cross-reacted hardly at all with LH-RH and only slightly with peptides bearing a different C-terminal alkyl substituent, but it was less specific for the N-terminus. Plasma specimens were extracted with antibody-immobilized cellulose in order to minimize substances interfering with the assay. This assay system could detect as little as 0.2 ng/ml of fertirelin in the absence of plasma extract and 1.0 ng/ml in the presence of the extract. The mean recovery of fertirelin added to plasma was 80.9% and the coefficients of variation were 14.4% (within assay) and 17.5% (between assay).