title: Development and validation of an enzyme-linked immunosorbent assay for the quantification of cytochrome 3A4 in human liver microsomes.
abstract: Little is known about the influence of hepatic pathologies on cytochrome P450 (CYP) mediated drug metabolism in children. The determination of the abundance of the different isoforms in pediatric microsomes may provide valuable information on the mechanisms of possible changes in activity. Until now, western blotting was mostly used for abundance measurements, but this technique only provides semi-quantitative data. Therefore, this study aimed to develop and validate an indirect ELISA for the quantification of the most important CYP isoform, CYP3A4, in human liver microsomes, using commercially available reagents. Samples, calibrators and validation samples were diluted to a final concentration of 10 μg microsomal protein/ml. A polyclonal antibody raised against the full length human protein was used as primary antibody; horseradish peroxidase conjugated secondary antibodies for detection. The assay was validated for sensitivity, working range and calibration, accuracy and precision. Amounts of CYP3A4 between 2 and 300 pmol/mg microsomal protein could be quantified with a 5-parameter logistics function with 1/x weighting factor. Coefficients of variation of intra and inter assay variability were between 9.54 and 13.98% (16.34% at LLOQ), and between 10.51 and 14.55% (19.44% at LLOQ), respectively. The relative error (%RE) varied between -5.96 and 6.68% (11.53% at LLOQ), and the total error between 11.93 and 21.23% (30.97% at LLOQ). The cross-reactivity of the method with human CYP2E1 showed to have no significant effect on the accuracy of the results. Successful analysis of five samples from an ongoing study demonstrated the usefulness of the method.
authors.affiliation: Laboratory of Medical Biochemistry and Clinical Analysis, Ghent University, Harelbekestraat 72, 9000 Gent, Belgium. 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: D000906:Antibodies / Q000276:immunology; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology*; D015203:Reproducibility of Results
chemical_list: D000906:Antibodies; D003577:Cytochrome P-450 Enzyme System; C408695:Cyp3a41a protein, mouse