PREPARATION OF MONOCLONAL-ANTIBODIES AGAINST N-(GAMMA-MALEIMIDOBUTYRYLOXY)SUCCINIMIDE (GMBS)-CONJUGATED ACETYLSPERMINE, AND DEVELOPMENT OF AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY (ELISA) FOR N-1,N-12-DIACETYLSPERMINE

We have developed three mouse monoclonal antibodies (mAb) of types IgG (1) and IgG(2b), i.e. anti-acetylspermine (Ac-Spm)-1 and 2 (ACSPM-1 an d 2), and anti-acetylspermine (AcSpm)-3 (ACSPM-S), respectively, again st Ac-Spm conjugated to bovine serum albumin ria a heterobifunctional cross-linker, N-(gamma-maleimidobutyryloxy)succinimide (GMBS). Among t hese mAbs, ACSPM-2 was the most useful for the development of an enzym e-linked immunosorbent assay (ELISA) for acetylpolyamines (Ac-PAs) wit h glutaraldehyde (GA)conjugated N-1,N-12-diacetylspermine (2Ac-Spm) or acetylspermine (Ac-Spm) as the solid phase antigen. However, GMBS-con jugated Ac-Spm did not behave as a solid phase antigen in the competit ive ELISA. The ELISA is based on the principle of competition between an analyte and the conjugated antigen for the mAb, followed by immunor eaction with biotinylated anti-mouse immunoglobulin and horseradish pe roxidase-streptavidin. The ACSPM-2 mAb reacted with 2Ac-Spm to the hig hest degree, followed by Ac-Spm, N-1-acetylspermidine (N-1-Ac-Spd), N- 1,N-8-diacetylspermidine (2Ac-Spd), and spermine (Spm), the EC50 value s being 0.06, 0.25, 7.0, 10, and 60 mu M, respectively, but exhibited almost no cross-reaction with other polyamine-related compounds or ami no acids. The method was used to determine the urinary Ac-PA levels in healthy subjects, the average value of 0.36 mu g of 2Ac-Spm/g creatin ine (n = 16) being obtained. The ACSPM-2 ELISA for 2Ac-Spm, which was the PA most relevant to the analysis of human urine among the five PA analogs mentioned above, might have potential for elucidation of the c orrelation of urinary 2Ac-Spm levels in cancers.