Validation of immunoassays for bioanalysis: a pharmaceutical industry perspective

Immunoassays are bioanalytical methods in which quantitation of the analyte depends on the reaction of an antigen (analyte) and an antibody. Although applicable to the analysis of both low molecular weight xenobiotic and macr omolecular drugs, these procedures currently find most consistent applicati on in the pharmaceutical industry to the quantitation of protein molecules. Immunoassays are also frequently applied in such important areas as the qu antitation of biomarker molecules which indicate disease progression or reg ression, and antibodies elicited in response to treatment with macromolecul ar therapeutic drug candidates. Currently available guidance documents deal ing with the validation of bioanalytical methods address immunoassays in on ly a limited way. This review highlights some of the differences between im munoassays and chromatographic assays, and presents some recommendations fo r specific aspects of immunoassay validation. Immunoassay calibration curve s are inherently nonlinear, and require nonlinear curve fitting algorithms for best description of experimental data. Demonstration of specificity of the immunoassay for the analyte of interest is critical because most immuno assays are not preceded by extraction of the analyte from the matrix of int erest. Since the core of the assay is an antigen-antibody reaction, immunoa ssays may be less precise than chromatographic assays; thus, criteria for a ccuracy (mean bias) and precision both in pre-study validation experiments and in the analysis of in-study quality control samples, should be more len ient than for chromatographic assays. Application of the SFSTP (Societe Fra ncaise Sciences et Techniques Pharmaceutiques) confidence interval approach for evaluating the total error (including both accuracy and precision) of results from validation samples is recommended in considering the acceptanc e/rejection of an immunoassay procedure resulting from validation experimen ts. These recommendations for immunoassay validation are presented in the h ope that theirconsideration may result in the production of consistently hi gher quality data from the application of these methods. (C) 2000 Elsevier Science B.V. All rights reserved.