title: Quantification of cytochrome 2E1 in human liver microsomes using a validated indirect ELISA.
abstract: CYP2E1 is an important cytochrome P450 isoform in many endogenous processes and in the metabolism of organic solvents, a number of drugs and pre-carcinogens. Information on the abundance of the enzyme may be valuable in various types of research in the field of toxicology and pharmacology. An indirect ELISA for the quantification of CYP2E1 in human liver microsomes was developed and successfully validated. All samples, including validation samples and calibrators, were diluted to a final concentration of microsomal protein of 10μg/ml. Detection of the antigen was obtained through binding of a polyclonal antibody raised against the full length protein, followed by the addition of horseradish peroxidase conjugated secondary antibodies and enzymatic detection. A five-parameter logistics function with 1/x weighting was used for quantification within the concentration range of 4-256pmol CYP2E1/mg microsomal protein. The method showed acceptable intra- and inter-assay precision, with calculated coefficients of variation of 6.3-15.2% and 11.3-21.0%, respectively. The relative error varied between -2.3 and 8.9%, and the total error between 16.0 and 27.2%. No significant cross reactivity with other abundant CYP isoforms was observed. The method was evaluated through the analysis of samples from a pharmacokinetic study, and the comparison with the CYP2E1 activity in those samples.
authors.affiliation: Laboratory of Medical Biochemistry and Clinical Analysis, Ghent University, Harelbekestraat 72, 9000 Gent, Belgium. Electronic address: Lies.DeBock@UGent.be.
authors.forename: Lies
authors.initials: L
authors.lastname: De Bock
authors.forename: Pieter
authors.initials: P
authors.lastname: Colin
authors.forename: Koen
authors.initials: K
authors.lastname: Boussery
authors.forename: Jan
authors.initials: J
authors.lastname: Van Bocxlaer
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry; D002138:Calibration; D019392:Cytochrome P-450 CYP2E1 / Q000032:analysis* / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007313:Insecta; D008862:Microsomes, Liver / Q000737:chemistry / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry; D015203:Reproducibility of Results
chemical_list: D000906:Antibodies; D011994:Recombinant Proteins; D019392:Cytochrome P-450 CYP2E1; D001189:Aryl Hydrocarbon Hydroxylases
keywords: Cytochrome P450; Drug metabolism; Enzyme linked immunosorbent assay; Human liver microsomes; Validation