An evaluation of heterologous antibodies to lactate dehydrogenase-C in the detection of mutations

We report that we are unable to repeat consistently the results published by Ansari et al. (1980) using antibodies to detect mutations in lactate dehydrogenase-C (LDH-C, previously called LDH-X) directly in sperm of mice exposed to the mutagen procarbazine. The approach made use of the interspecies differences in the antigenic sites between the LDH-C of the rat and mouse in sperm. The visualization of mutations in mouse LDH-C was based on the detection of alterations in antigenic sites of mouse LDH-C such that mouse sperm would bind the antibody that was specific for rat LDH-C (presumptive mutants); the antibody was termed specific when it immunofluorescently labeled rat sperm but not mouse sperm. The original work reported increases in the frequency of occurrence of mouse sperm that would bind rat-specific antibody from mice treated with procarbazine as compared control mice; a single absorbed antiserum was used throughout the experiments. In this study, we found that there is too much variation in the frequency of mouse sperm that react with rabbit antibodies to purified rat LDH-C for the system to be useful in mutagenesis studies. The fundamental criterion of antibody specificity was maintained as in the original work. The frequency of labeled mouse sperm depended on the absorption of the antibody on mouse proteins, indicating that the factors denoting a presumptive mutant were associated with the mouse proteins. In some experiments, the frequency of labeled mouse sperm was higher among sperm from procarbazine-treated mice than among sperm from control mice. This increase, however, was not consistently reproducible. After extensive absorption of the antibody on mouse proteins, no presumptive mutants were observed in sperm from treated and control animals; these antibodies continued to immunofluorescently label rat sperm. The absence of presumptive mutants with highly absorbed antibody suggests that natural variation between species may not be appropriate as markers for the detection of mutations without a thorough knowledge of the number of independent events at the DNA level required to produce a change in antigenic recognition.