SOME VALIDATION CONSIDERATIONS FOR IMMUNOASSAYS

Guidelines have been proposed for the validation of bioanalytical meth ods applied to the support of clinical and preclinical pharmacokinetic studies. These guidelines deal primarily with accuracy, precision, se nsitivity, and establishment of the calibration curve. Immunoassays di ffer from chromatographic assays because they employ one or more antib odies as the key reagent in the assay. Introduction of this biological reagent brings a relative shift in the importance of some assay perfo rmance parameters, such as specificity. Specificity may be defined for small molecules by measuring the crossreactivities of known metabolit es, co-administered drugs, or any relevant endogenous compounds in the assay. However, it is normally impossible to study all metabolites in this way since most drugs have minor unidentified metabolites that ma y also be present in the milieu under analysis. Thus, comparison with a separate, and, usually chromatographic, method is the preferred rout e for confirmation of specificity. The advent of liquid chromatography -mass spectrometry (LC/MS)-based methods of high sensitivity now allow s this approach to be realized in the calibration range of many small molecule immunoassays. Immunoassay calibration curves are inherently n onlinear, so that special considerations arise in determining lower an d upper limits of quantitation. The current surge of interest in devel oping protein molecules as therapeutic agents also brings new and inte resting considerations for validation of immunoassays for these analyt es, since this is still the method of choice for quantitation of these agents in plasma or serum. For example, specificity relative to the p roducts of metabolism or catabolism cannot be defined as easily as for small molecules. Assays to detect antibodies to these therapeutic pro teins have also been developed, prompting the question of how extensiv ely these antibody detection assays can and should be validated.