CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST N-(γ-MALEIMIDOBUTYRYLOXY) SUCCINIMIDE (GMBS)-CONJUGATED POLYAMINE, SPERMINE
APPLICATION TO IMMUNOCYTOCHEMISTRY
1994 年 27 巻 6 号 p. 515-525
詳細
1994 年 27 巻 6 号 p. 515-525
Anti-spermine monoclonal antibodies (ASPM-1 and ASPM-2 MAbs) against N-(γ-maleimidobutyryloxy) succinimide (GMBS)-conjugated spermine have been demonstrated to be useful for polyamine (PA) immunocytochemistry (ICC) (Fujiwara et al. 1994). Based on the evaluated antibody specificity of the MAbs we studied the immunostaining mechanism in the PA ICC using glutaraldehyde-conjugated Spm (Spm-GA-BSA conjugates) as simulation examples of tissue sections fixed with glutaraldehyde (GA) by enzyme-linked immunosorbent assays (ELISA). It was demonstrated that ASPM-1 bound to the GA conjugates to almost the same degree as to Spm-GMBS-BSA conjugates used for screening the MAbs, but that ASPM-2 did so about ten-times less than to the GMBS conjugates. However, no immunoreaction occurred in either case with paraformaldehyde (PFA)-conjugated Spm. This completely agrees with the results of the present PA ICC, showing an immunoreaction for PAs in a Hela cell line and a variety of cells in the rat adenohypophysis when fixed with GA or a mixture of GA and PFA, but none with PFA alone. The present study also suggests that the Spm-GA-BSA conjugates consist of at least two types of Spm derivatives, one conjugating Spm through only one of the two primary amino groups in the molecule, and another in which Spm has reacted with GA at both the primary amino groups. The former may react primarily with ASPM-1 MAb and the latter with ASPM-2 MAb. This may confirm our previously proposed idea that PAs are converted to a variety of PA derivatives during the fixation process of PA ICC. The model ELISA procedures should prove useful in assessing immunostaining mechanisms in ICC.
