Targeted antipeptide antibodies to cytochrome P450 2C18 based on epitope mapping of an inhibitory monoclonal antibody to P450 2C51

Arch Biochem Biophys. 1997 Feb 15;338(2):157-64. doi: 10.1006/abbi.1996.9817.


The epitope recognized by the inhibitory monoclonal antibody designated 2F5, which was raised against P450 2C5, was mapped to amino acids 237-260 by immunoblotting using a combination of recombinant antigens and chimeric and partial fusion proteins constructed from rabbit P450s 2C2, 2C4, 2C5, and 2C16, which are recognized by 2F5, and from 2C1 and 2C3, which are not. When the sequence of the epitope for 2F5 (amino acids 237-260) was compared with those of other rabbit 2C P450s, a single lysine residue at position 253 appeared to be a likely determinant of 2F5 immunoreactivity. Substitution of lysine for glutamic acid 253 in P450 2C3 (2C3E253K) conferred immunoreactivity and the ability of 2F5 to inhibit progesterone metabolism catalyzed by P450 2C3E253K. Sequence alignment revealed that this epitope lies in close proximity to the epitope identified for LKM-1 autoantibodies to P450 2D6. Based on these results, an antipeptide antibody was raised to the corresponding region (amino acids 252-263) of human P450 2C18. The resulting antipeptide antiserum recognizes P450 2C18 but not P450 2C8, 2C9, or 2C19. However, the antipeptide 2C18 antiserum did not inhibit 2C18-catalyzed diazepam N-demethylation. Human 2C P450s were also quantitated by immunoblot analysis in a panel of six human liver microsomes using Escherichia coli expressed P450s as standards. Analysis of immunoblots indicated that, if present, P450 2C18 was expressed at very low levels (<2.5 pmol/mg), whereas P450s 2C8, 2C9, and 2C19 were easily detected.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal / immunology
  • Aryl Hydrocarbon Hydroxylases*
  • Blotting, Western
  • Cytochrome P-450 CYP2C19
  • Cytochrome P-450 Enzyme Inhibitors
  • Cytochrome P-450 Enzyme System / analysis
  • Cytochrome P-450 Enzyme System / immunology*
  • Cytochrome P450 Family 2
  • Enzyme-Linked Immunosorbent Assay
  • Epitope Mapping
  • Humans
  • Microsomes, Liver / enzymology
  • Mixed Function Oxygenases / analysis
  • Molecular Sequence Data
  • Peptides / immunology
  • Rabbits
  • Recombinant Fusion Proteins
  • Sequence Alignment
  • Steroid 21-Hydroxylase / antagonists & inhibitors
  • Steroid 21-Hydroxylase / immunology*


  • Antibodies, Monoclonal
  • Cytochrome P-450 Enzyme Inhibitors
  • Peptides
  • Recombinant Fusion Proteins
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • Aryl Hydrocarbon Hydroxylases
  • CYP2C18 protein, human
  • CYP2C19 protein, human
  • CYP2C5 protein, Oryctolagus cuniculus
  • Cytochrome P-450 CYP2C19
  • Cytochrome P450 Family 2
  • Steroid 21-Hydroxylase