THE USE OF N-[BETA-(4-DIAZOPHENYL)ETHYL]MALEIMIDE AS A HETEROBIFUNCTIONAL AGENT IN DEVELOPING ENZYME-IMMUNOASSAY FOR NEUROTENSIN

A heterobifunctional crosslinking agent N-[.beta.-(4-diazophenyl)ethyl]maleimide (DPEM) was newly synthesized and characterized to possess the maleimide group with a stability greater than that previously reported for N-(4-diazophenyl)maleimide. Using the peptide hormone neurotensin (NT) as a model hapten. DPEM was used in the conjugation reaction with bovine serum albumin (BSA) and with .beta.-D-galactosidase (.beta.-Gal) in developing an enzyme immunoassay (EIA) for NT. The NT-DPEM-BSA conjugate elicited anti-NT antibodies in rabbits and the NT-.beta.-Gal conjugate behaved as an enzyme marker of NT in the EIA. The EIA developed double antibody was reproducible and sensitive in detecting NT at concentrations as low as 30 fmol per tube. The specificity of anti-NT serum seems to be primarily toward the carboxy-terminal region of NT, showing cross-reactions with such NT fragments as NT2-13, NT8-13, and NT1-8 for 120, 22, and < 0.1%, respectively. The utility of this assay was also demonstrated by measuring the NT immunoreactivity in several rat organs. DPEM could be useful for developing EIAs for other peptide hormones (even those which contain neither a free amino group nor a free carboxyl group), using the imidazole, phenolic, or indole group(s) of amino acids as a binding site for carrier proteins.