Hybridomas were prepared from mouse myeloma cells and spleen cells derived from female BALB/c mice that had been immunized with a partially purified ethanol-induced rat liver cytochrome P-450 (P-450et). Monoclonal antibodies (MAbs) produced by the hybridomas were screened for binding to P-450et with a radioimmunoassay. Thirty-one independent hybrid clones produced MAbs that had a high affinity for P-450et. Each clone produced MAbs of a single subclass of the mouse immunoglobulins IgG1, IgG2a, IgM, or IgA. Ten of the 31 MAbs also immunoprecipitated P-450et as determined by Ouchterlony double-immunodiffusion analyses. One of the MAbs was tested for cross-reactivity with other rabbit and rat liver cytochromes P-450 and was found not to cross-react with rat liver P-450 induced by either phenobarbital, β-naphthoflavone, or rabbit liver P-450LM2 or P-450LM4. Nine of the MAbs were tested for cross-reactivity with rat liver clofibrate-induced P-450, rat liver pregnenolone-16-α-carbonitrile-induced P-450, and a human liver P-450. All the MAbs showed no cross-reactivity except for one MAb which cross-reacted with both pregnenolone-16-α-carbonitrile and human P-450 and three MAbs which cross-reacted with human P-450. Three antigen-precipitating MAbs and four nonprecipitating MAbs were tested for their effects on the aniline p-hydroxylase activity of liver microsomes of untreated rats and from rats treated with acetone, pyrazole, methylpyrazole, or imidazole. One of the seven MAbs tested, 1-91-3, inhibited enzyme activity of acetone-, pyrazole-, or methylpyrazole-induced microsomes by 54, 47, and 48%, respectively. This indicates that at least 50% of microsomal cytochrome P-450 aniline p-hydroxylase activity in the latter is a function of a P-450 enzyme that contained the epitope to which the MAb 1-91-3 is directed. With untreated and imidazole-induced microsomes, 32 and 21% inhibition of the enzyme activity was observed. In reconstituted systems containing phospholipid and NADPH-cytochrome P-450 reductase, MAb 1-91-3 inhibited aniline p-hydroxylase activity of purified ethanol-induced P-450et and acetone-induced P-450 by more than 90%. Nitrosodimethylamine demethylase activity of acetone-induced rat microsomes was inhibited by the various MAbs up to 77% and the activity of the purified acetone-induced P-450 was inhibited up to 92%. Western blot analysis indicated that the ethanol-inducible form of cytochrome P-450 recognized by the MAbs was also detected in microsomes of rats treated with acetone, pyrazole, methylpyrazole, and imidazole and in untreated rats in the latter order of decreasing quantity. The MAbs reported here should prove useful for biochemical and epidemiological studies concerning nitrosamines and ethanol.
An abstract form of this paper was presented at the Fifth Annual Congress for Hybridoma Research, 1/26–29, 1986, Baltimore, MD.