Novel enzyme immunoassay for thyrotropin-releasing hormone using N-(4-diazophenyl)maleimide as a coupling agent.
A novel enzyme immunoassay (EIA) for thyrotropin-releasing hormone (TRH) was developed which used N-(4-diazophenyl)maleimide (DPM) as a new heterobifunctional agent capable of cross-linking TRH to mercaptosuccinyl bovine serum albumin and to beta-D-galactosidase. The resulting conjugates act as the immunogen producing anti-TRH serum in rabbits and the enzyme marker of TRH in the EIA, respectively. This EIA with a double-antibody technique was sensitive and reproducible in measuring TRH at concentrations as low as 50 pg per tube, and monospecific to the hormone showing no cross-reactivity with the hormone analogue L-pGlu-L-His-L-Pro and TRH constituents. Using this assay, the distribution of immunoreactive TRH in the brain was determined easily in rats. The use of DPM should provide a valuable new method for developing EIA hitherto possible for other peptide hormones containing neither a free carboxy nor a free amino group, using imidazole, phenolic, and indole group(s) of the amino acid as a reaction site.
D000818:Animals; D000917:Antibody Formation; D001923:Brain Chemistry; D055598:Chemical Phenomena; D002621:Chemistry; D003429:Cross Reactions; D003432:Cross-Linking Reagents / Q000378:metabolism*; D003979:Diazonium Compounds / Q000378:metabolism*; D005696:Galactosidases / Q000378:metabolism; D007124:Immunoenzyme Techniques; D008301:Maleimides / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013870:Thiomalates / Q000378:metabolism; D013973:Thyrotropin-Releasing Hormone / Q000032:analysis*; D014018:Tissue Distribution
D003432:Cross-Linking Reagents; D003979:Diazonium Compounds; D008301:Maleimides; D013870:Thiomalates; C049605:N-(4-diazophenyl)maleimide; D012710:Serum Albumin, Bovine; D013973:Thyrotropin-Releasing Hormone; C046062:2-thiomalic acid; D005696:Galactosidases