DEVELOPMENT OF A SENSITIVE AND ACCURATE ENZYME-LINKED-IMMUNOSORBENT-ASSAY (ELISA) SYSTEM THAT CAN REPLACE HPLC ANALYSIS FOR THE DETERMINATION OF N-1,N-12-DIACETYLSPERMINE IN HUMAN URINE

N-1,N-12-Diacetylspermine (DiAcSpm)-specific antibodies were raised in rabbits, using N-acetylspermine coupled to mercaptosuccinylated BSA v ia 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 fo r measuring DiAcSpm in solution, was constructed using this antibody p reparation, with N-acetylspermine coupled to a synthetic peptide via N -(8-maleimidocapryloxy)succinimide as a solid phase antigen, The K-i v alue for DiAcSpm with this competitive ELISA system was 33 nM, and the cross-reactivity with DiAcSpm, AcSpm, DiAcSpd, N-1-AcSpd, and N-8-AcS pd was 100, 0.29, 0.20, 0.033, and 0.055%, respectively. This procedur e can be applied to the determination of DiAcSpm in human urine sample s, giving highly reproducible results. The coefficients of variation o btained were 6.7 and 4.2% for within-run and between-run precision, re spectively. 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.