Development of a sensitive and accurate enzyme-linked immunosorbent assay (ELISA) system that can replace HPLC analysis for the determination of N1,N12-diacetylspermine in human urine

N-1,N-12-Diacetylspermine (DiAcSpm)-specific antibodies were raised in rabbits, using N-acetylspermine coupled to mercaptosuccinylated BSA via N-(4-maleimidobutyryloxy)succinimide as an antigen. Highly DiAcSpm-specific antibodies were enriched from crude sera through a series of affinity-based fractionations, A competitive ELISA system, intended for measuring DiAcSpm in solution, was constructed using this antibody preparation, with N-acetylspermine coupled to a synthetic peptide via N-(8-maleimidocapryloxy)succinimide as a solid phase antigen, The K-i value for DiAcSpm with this competitive ELISA system was 33 nM, and the cross-reactivity with DiAcSpm, AcSpm, DiAcSpd, N-1-AcSpd, and N-8-AcSpd was 100, 0.29, 0.20, 0.033, and 0.055%, respectively. This procedure can be applied to the determination of DiAcSpm in human urine samples, giving highly reproducible results. The coefficients of variation obtained were 6.7 and 4.2% for within-run and between-run precision, respectively. The correlation coefficient between DiAcSpm concentrations in urine estimated by ELISA and those by HPLC analysis was calculated to be 0.99, and the regression equation was expressed as y = 1.04x + 0.026 mu M.