Immunoassay assay parameters

The LCEC method in conjunction with the optimal assay parameters resulted in a very sensitive immunoassay for digoxin in plasma throughout its therapeutic range, with a detection limit of 50 pg/ml. A standard curve is shown in Fig. 7. The relative standard deviation of ip obtained for any given digoxin standard ranged from 4% to 12% on various days and is comparable to results ordinarily obtained by other heterogeneous enzyme immunoassays. Approximately 20 samples can be injected per hour. 

The only published immunoassay method submitted to date to EPA OPPTS as an enforcement method for a range of substrates (water, sediment, crops, processed crop fractions, and animal tissues) is the spinosad method, developed by Young et al. This method uses the spinosad RaPID Assay (Strategic Diagnostics) for determination of total spinosad residues (TSR). This discussion will be limited to crop and animal tissues, because the water and soil analyses are analogous to the triasulfuron method. The extraction, cleanup, and method parameters are summarized in Table 2. 

L7. Lim, C. S., Miller, J. N., and Bridges, J. W., Energy-transfer immunoassay A study of the experimental parameters in an assay for human serum albumin. Anal. Biochem. 108, 176-184 (1980). 

The Immunoassay submitted for evaluation must be "mature," because expensive evaluation studies cannot be undertaken on unoptimized methods. The developer must first optimize and evaluate the immunoassay in-house, preferably by individuals most familiar with the assay system. Parameters addressed should include bias, precision (repeatability and reproducibility at the detection levels of interest), and rate of false positive and false negative results. The Agency should be supplied the raw data documenting assay performance. When possible, this information should be submitted on an IBM compatible floppy disk. A review of the data must be conducted before undertaking the evaluation study to verify interpretations of the data made the developer. 

The optimization and validation of immunoassays for immunogenicity (ADA) testing has been described in detail in several publications [9,14,33,34]. In this section, we will describe the evaluation of relevant performance characteristics (validation parameters) that require the most effort. Some of these are different from the validation of traditional bioanalytical pharmacokinetic (PK) methods for macromolecules [35 37]. Precision, specificity, robustness, and ruggedness are determined similarly between ADA and PK methods. However, recovery/accuracy, sensitivity, stability, linearity, system suitability controls, and selectivity are treated differently between these two types of assays. 

During early immunoassay development, it is important to assess the intended use of the assay. When the intended use is to support pharmacokinetics in either nonclinical PK/TK or clinical bioequivalence (BE) studies, developing a validat-able assay is of utmost importance. A well-thought-out validation plan and fully documented assay methods and results that support the validation are crucial. To withstand the scrutiny of the authorities, and to guarantee the optimal method is applied to your drug development process, the following parameters should be assessed critically, validated to specific criteria, and fully documented. 

Bioassays are frequently used as an alternative or in addition to immunoassay techniques. Bioassays, in contrast to immunoassays, quantify not the pharmacologically active substance, but its biological activity, for example, in cell culture models based on cell differentiation, cell proliferation, or cytotoxicity as well as gene expression assays or whole animal models. Frequent major problems with bioassays comprise a high variability in the measured parameters, lack of precision, and their time- and labor-intensive performance. Furthermore, bioassays oftentimes also lack specificity for the measured compound, as they may also detect the response to bioactive metabolites [16,17]. 

To ensure the reliability of analytical techniques, they need to be validated. Validation provides information on the overall performance of the assay as well as on individual parameters and factors that can be used to estimate the degree of uncertainty associated with an assay (Ellison et al., 2000). An adequate validation procedure assesses, and therefore ensures, that the immunoassay performs within an acceptable range of established criteria. Parameters used to evaluate the performance of the assays may be affected by (1) factors inherent to the analytical technique, such as antibody specificity and antibody cross-reactivity, and (2) external factors such as environmental conditions (temperature) and type of sample (matrix, processed food vs. raw ingredients). A... 

The first step in the validation process involves the establishment of acceptance criteria, which are assay dependent, for the various validation parameters. Assays must meet these criteria in order to receive validation status. The most relevant validation parameters are specificity, accuracy, precision, sensitivity, and robustness. In this section we describe these parameters as they apply to the evaluation of immunoassays. 

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