Journal of Pharmaceutical and Biomedical Analysis 21 (2000) 1249–1273 Validation of immunoassays for bioanalysis: a pharmaceutical industry perspective J.W.A. Findlay a , W.C. Smith b , J.W. Lee c , G.D. Nordblom d , I. Das a, *, B.S. DeSilva e , M.N. Khan f , R.R. Bowsher g a Metabolism and Safety Ealuation, Searle, 4901 Searle Parkway, Skokie, IL 60077, USA b Statistical and Mathematical Sciences, Lilly Research Laboratories, Eli Lilly and Company, Greenfield, IN 46140, USA c MDS Harris, 621 Rose Street, Lincoln, NE 68502, USA d Department of Pharmacokinetics, Dynamics and Metabolism, Parke -Dais Pharmaceutical Research, Diision of Warner -Lambert Co., 2800 Plymouth Road, Ann Arbor, MI 48105, USA e Bioanalytical, Procter and Gamble Pharmaceuticals, Route 320 Woods Corners, Norwich, NY 13815, USA f Clinical Testing Laboratory, Quality Control Department, MedImmune, 636 Research Drie, Frederick, MD 21703, USA g Drug Disposition, Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA Received 10 September 1999; received in revised form 12 October 1999; accepted 13 October 1999 Abstract 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 macromolecular drugs, these procedures currently find most consistent application in the pharmaceutical industry to the quantitation of protein molecules. Immunoassays are also frequently applied in such important areas as the quantitation of biomarker molecules which indicate disease progression or regression, and antibodies elicited in response to treatment with macromolecular therapeutic drug candidates. Currently available guidance documents dealing with the validation of bioanalytical methods address immunoassays in only a limited way. This review highlights some of the differences between immunoassays and chromatographic assays, and presents some recommen- dations for specific aspects of immunoassay validation. Immunoassay calibration curves 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 immunoassays are not preceded by extraction of the analyte from the matrix of interest. Since the core of the assay is an antigen – antibody reaction, immunoassays may be less precise than chromatographic assays; thus, criteria for accuracy (mean bias) and precision, both in pre-study validation experiments and in the analysis of in-study quality control samples, should be more lenient than for chromatographic assays. Application of the SFSTP (Societe Francaise 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 acceptance/rejection of an immunoassay procedure resulting from validation experiments. These recommendations for immunoassay validation are presented in the hope that their www.elsevier.com/locate/jpba * Corresponding author. Fax: +1-847-982-4857. E-mail address: ira.das@monsanto.com (I. Das) 0731-7085/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII:S0731-7085(99)00244-7