Preparation and characterization of monoclonal antibodies to pregnenolone 16-alpha-carbonitrile inducible rat liver cytochrome P-450.
Hybridomas were prepared from mouse myeloma cells and spleen cells derived from BALB/c female mice immunized with purified rat hepatic pregnenolone 16-alpha-carbonitrile (PCN) induced cytochrome P-450 2a/PCN-E. The monoclonal antibodies (MAbs) thus obtained were screened for binding to the purified P-450 2a/PCN-E by radioimmunoassay. Eleven independent hybrid clones produced MAbs, each of which was of a single mouse immunoglobulin subclass of the IgG1, IgG2a or IgG2b type. Each of the MAbs produced by the eleven individual hybrid clones bound strongly to P-450 2a/PCN-E as assessed by radioimmunoassay and immunoprecipitation of P-450 2a/PCN-E in Ouchterlony double-immunodiffusion plates. Of the eleven MAbs, three also bound strongly to the phenobarbital-inducible rat liver cytochrome P-450 PB-4. Thus, two classes of MAbs were obtained, one class specific for P-450 2a/PCN-E and a second class that bound to both PCN- and phenobarbital-inducible P-450 forms. The reactivities of one MAb from each class toward eight highly purified rat hepatic cytochromes P-450 were examined using solid phase enzyme-linked immunosorbent analyses. The MAb designated C2 was found to be specific for P-450 2a/PCN-E and did not cross-react with seven other P-450 forms. This MAb was shown to be an effective probe for monitoring, by Western blotting, the induction of microsomal P-450 2a/PCN-E by PCN and phenobarbital. The MAb designated C1 reacted both with P-450 2a/PCN-E and with the two major phenobarbital-inducible P-450 forms, PB-4 and PB-5. None of the MAbs was inhibitory towards P-450 2a/PCN-E-dependent aryl hydrocarbon hydroxylase, benzphetamine N-demethylase, ethoxycoumarin O-deethylase or ethymorphine N-demethylase activity, indicating that the epitopes recognized by these MAbs are not directly associated with catalytic activity. The strong reactivities of three of the MAbs with both P-450 2a/PCN-E and P-450s PB-4 and PB-5 indicate that these two structurally quite different cytochrome P-450 families share at least one common epitope. These new MAbs are additions to our library of MAbs to different cytochromes P-450 and should help further our understanding of the relationship of cytochrome P-450 phenotype and multiplicity to inter-individual differences in drug and carcinogen metabolism and sensitivity.
D015660:7-Alkoxycoumarin O-Dealkylase; D000818:Animals; D000911:Antibodies, Monoclonal / Q000145:classification / Q000276:immunology*; D000918:Antibody Specificity; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology*; D003907:Dexamethasone / Q000494:pharmacology; D004790:Enzyme Induction; D005260:Female; D007114:Immunization; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008862:Microsomes, Liver / Q000201:enzymology; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors; D010105:Oxygenases / Q000037:antagonists & inhibitors; D011285:Pregnenolone Carbonitrile / Q000494:pharmacology*; D051381:Rats; D012737:Sex Factors
D000911:Antibodies, Monoclonal; D011285:Pregnenolone Carbonitrile; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; D015660:7-Alkoxycoumarin O-Dealkylase; D001189:Aryl Hydrocarbon Hydroxylases; C019781:benzphetamine N-demethylase; D010089:Oxidoreductases, N-Demethylating