Preparation of Antibodies Highly Specific to N¹, N⁸-Diacetylspermidine, and Development of an Enzyme-Linked Immunosorbent Assay (ELISA) System for Its Sensitive and Specific Detection

Abstract

N⁸-Acetylspermidine was coupled to mercaptosuccinylated BSA using a bifunctionalcross-linker, N-(4-maleimidobutyryloxy)succinimide, and the resulting conjugate was used to raise N¹, N⁸-diacetylspermidine (DiAcSpd)-specific antibodies in rabbits. DiAc-Spd-specific antibodies were enriched from crude sera through a series of affinity-based fractionations using ligands with structures mimicking thoseof DiAcSpd and monoacetyl-spermidines. With the N⁸-acetylspermidine-BSA conjugate as a solid phase antigen in a competitive ELISA system, the selectivity for DiAcSpd over other polyamine species was high, but competition by DiAcSpd added to the fluid phase was too weak for the system to be applicable to measurement of the concentration of DiAcSpd in human urine. In contrast, with the N1-acetylspermidine BSA conjugate adsorbed on the ELISA plate, DiAcSpd efficiently competed for the same antibody, thus yielding a sensitive competitive ELISA system for measuring DiAcSpd. The K¹ value for DiAcSpd with the latter competitive ELISA system was 54 nM, and the cross-reactivity with DiAcSpd, N¹, N¹²-diacetylsper-mine, N⁸-acetylspermidine, N¹-acetylspermidine, and acetylputrescine was 100, 1. 2, 0.74, 0. 12, and 0. 08%, respectively. The DiAcSpd-specific antibodies and the competitive ELISA system developed in this study will prove to be useful for analyzing the urinary level of DiAcSpd, that was recently shown to be a promising diagnostic and prognostic indicator of malignant disorders.