ENZYME-LINKED-IMMUNOSORBENT-ASSAY (ELISA) FOR LUTEINIZING-HORMONE-RELEASING HORMONE (LH-RH) USING A HETEROBIFUNCTIONAL CROSS-LINKING AGENT,N-[BETA-(4-DIAZOPHENYL)ETHYL]MALEIMIDE

Luteinizing hormone-releasing hormone (LH-RH) was first labeled with a n enzyme, beta-D-galactosidase (beta-Gal; EC 3.2.1.23), using N-[beta- (4-diazophenyl)ethyl]maleimide (DPEM) as a heterobifunctional cross-li nking agent. An antigen was similarly prepared by coupling LH-RH to me rcaptosuccinylated bovine serum albumin with DPEM and was used for the immunization of rabbits for antibodies against LH-RH. A new, simple e nzyme-linked immunosorbent assay (ELISA) for LH-RH was developed by us ing the principle of direct competition between LH-RH and beta-Gal-lab eled LH-RH for anti-LH-RH IgG which had been adsorbed to the plastic s urface of microtiter plates. LH-RH concentrations lower than 50 pg/ass ay 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 specifici ty of this ELISA seems to be primarily toward the C-terminal region of LH-RH, showing a cross-reaction with the LH-RH6-10 fragment to the sa me extent as with LH-RH, but no cross-reaction with the LH-RH1-3 and L H-RH4-6 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 a nonradioactive, inexpensive and rapid method, and might be useful for elucidating experimental hypothalamic-pituitary-gonad interaction s.