Preparation of Monoclonal Antibodies against N-(γ-Maleimidobutyryloxy)succinimide (GMBS)-Conjugated Acetylspermine, and Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for N1,N12-Diacetylspermine

We have developed three mouse monoclonal antibodies (mAb) of types IgG1 and IgG2h, i.e. anti-acetylspermine (Ac-Spm)-1 and 2 (ACSPM-1 and 2), and anti-acetylspermine (Ac-Spm)-3 (ACSPM-3), respectively, against Ac-Spm conjugated to bovine serum albumin via a heterobifunctional cross-linker, N-(γ-maleimidobutyryloxy)succinimide (GMBS). Among these mAbs, ACSPM-2 was the most useful for the development of an enzyme-linked immunosorbent assay (ELISA) for acetylpolyamines (Ac-PAs) with glutaraldehyde (GA)-conjugated N1,N12-diacetylspermine (2Ac-Spm) or acetylspermine (Ac-Spm) as the solid phase antigen. However, GMBS-conjugated Ac-Spm did not behave as a solid phase antigen in the competitive ELISA. The ELISA is based on the principle of competition between an analyte and the conjugated antigen for the mAb, followed by immunoreaction with biotinylated anti-mouse immunoglobulin and horseradish peroxidase-streptavidin. The ACSPM-2 mAb reacted with 2Ac-Spm to the highest degree, followed by Ac-Spm, N1-acetylspermidine (N1-Ac-Spd), N1,N8-diacetylspermidine (2Ac-Spd), and spermine (Spm), the EC50 values being 0.06, 0.25, 7.0, 10, and 60 μM, respectively, but exhibited almost no cross-reaction with other polyamine-related compounds or amino acids. The method was used to determine the urinary Ac-PA levels in healthy subjects, the average value of 0.36 μg of 2Ac-Spm/g creatinine (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 correlation of urinary 2Ac-Spm levels in cancers.