2 MONOCLONAL-ANTIBODIES RECOGNIZING DIFFERENT EPITOPES ON RAT CYTOCHROME IIB1 REACT WITH HUMAN IIE1

To identify human cytochromes P450 (P450) in the CYP2B subfamily, 14 h uman liver microsomal samples were screened by immunoblots developed w ith monoclonal antibodies that recognized seven distinct epitopes on r at IIB1. Two of these antibodies recognized a protein in all of the sa mples. This protein was termed P450BE. Using video-imaging densitometr y, the levels of P450BE were determined and compared with levels of ot her P450s. An excellent correlation was seen (r = 0.87) between P450BE and human IIE1. However, rat IIE1 did not react in immunoblot and enz yme-linked immunosorbant assays with the two anti-rat IIB1 monoclonal antibodies. As previously observed, the levels of IIE1 in the samples correlated well (r = 0.88) with the ability of these human liver micro somes to N-demethylate N-nitrosodimethylamine. The levels of P450BE al so correlated well (r = 0.91) with the ability of the microsomes to N- demethylate N-nitrosodimethylamine. In addition, excellent correlation s were obtained when the levels of P450BE and IIE1 were compared with the ability of the microsomes to O-deethylate ethoxycoumarin (r = 0.87 and r = 0.85, respectively). To identify the protein recognized by th e anti-rat IIB1 antibodies, P450BE was purified from microsomes prepar ed from human liver D. Amino-terminal amino acid sequence obtained mat ched the corresponding sequence of human IIE1. In addition, purified h uman IIE1 and P450BE migrated with the same apparent molecular weight in polyacrylamide gels. Furthermore, proteolytic maps of P450BE and II E1, generated with two proteases, were found to be identical. Sequence alignments and antigenicity calculations identified three regions of rat IIB1 as likely candidates for the epitopes shared in common with h uman IIE1. In conclusion, this study indicates that caution must be ta ken when interpreting the results of immunochemical assays when specie s lines are crossed.