Determination of urinary acetylpolyamines by a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA).

Abstract

A monoclonal antibody (mAb), ASPM-2, produced against N-(gamma-maleimidobutyryloxy)-succinimide (GMBS)-conjugated polyamine spermine [Spm; Fujiwara et al. (1994) Histochemistry 102, 397-404] was used for the development of an enzyme-linked immunosorbent assay (ELISA) for acetylpolyamines (Ac-PAs) in human urine. The ELISA is based on the principle of competition between an analyte and Spm-glutaraldehyde-bovine serum albumin conjugate-coated polystyrene microtiter wells for the mAb, followed by immunoreaction with biotinylated anti-mouse immunoglobulin and horseradish peroxidase-streptavidin. The ASPM-2 mAb showed strong immunoreaction with N1,N12-diacetylspermine (2Ac-Spm), N-monoacetylspermine (Ac-Spm), and N1-acetylspermidine (N1-Ac-Spd), the EC50 values being 29, 50, and 51 microM, respectively, but no cross-reaction with other PA-related compounds or amino acids. The method was used to measure urinary Ac-PA levels in healthy subjects and cancer patients, without pretreatment of the specimens, mean concentrations of 3.25 and 2.80 mumol per 24-h urine, respectively (as N1-Ac-Spd), being found. The ASPM-2 ELISA for N1-Ac-Spd, which is the PA most relevant to the analysis of human urine among the three Ac-PAs mentioned above, is specific and accurate, and can easily be used to analyze large numbers of specimens in parallel. It should thus have potential for studying the relationship between urinary N1-Ac-Spd levels and cancer.