Enzyme-Linked Immunosorbent Assay (ELISA) for Luteinizing Hormone Releasing Hormone (LH-RH) Using a Heterobifunctional Cross-Linking Agent, N-[β-(4-Diazophenyl)ethyl]maleimide

Abstract

Luteinizing hormone-releasing hormone (LH-RH) was first labeled with an enzyme, β-D-galactosidase (β-Gal; EC 3.2.1.23), using N-[β-(4-diazophenyl)ethyl]maleimide (DPEM) as a heterobifunctional cross-linking agent. An antigen was similarly prepared by coupling LH-RH to mercaptosuccinylated bovine serum albumin with DPEM and was used for the immunization of rabbits for antibodies against LH-RH. A new, simple enzyme-linked immunosorbent assay (ELISA) for LH-RH was developed by using the principle of direct competition between LH-RH and β-Gal-labeled LH-RH for anti-LH-RH IgG which had been adsorbed to the plastic surface of microtiter plates. LH-RH concentrations lower than 50 pg/assay well were measurable reproducibly by the ELISA, the sensitivity of which was found to be about 6250 times greater than the corresponding high performance liquid chromatography (HPLC) procedure. The specificity of this ELISA seems to be primarily toward the C-terminal region of LH-RH, showing a cross-reaction with the LH-RH_6-10 fragment to the same extent as with LH-RH, but no cross-reaction with the LH-RH_1-3 and LH-RH_4-6 fragments. Using this assay, LH-RH levels were easily measured in the blood and urine of rats following the administration of LH-RH in a single dose of 0.5 mg/kg i.v. The present, newly developed ELISA is nonradioactive, inexpensive and rapid method, and might be useful for elucidating experimental hypothalamic-pituitary-gonad interactions.