Section11:Introduction

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The intent of this document is to provide general guidelines to aid in the development, optimization and validation of an immunoassay. Following these guidelines will increase the likelihood of success in developing a robust immunoassay that will measure consistent values for unknown samples.

Immunoassays are used when an unknown concentration of an analyte within a sample needs to be quantified. To obtain the most accurate determination of the unknown concentration, an immunoassay must be developed based not only on the usual assay development criteria (SD window) but also on how well the immunoassay can predict the value of an unknown sample. First, one needs to establish the assay critical success factors. Then the immunoassay needs to be developed which establishes proof of concept. During the optimization phase the quantifiable range of the immunoassay method is determined by calculating a precision profile in the matrix in which the experimental samples will be measured. If the precision profile is within the desired working range, then assaying spiked recovery samples over several days completes the validation of the immunoassay. If the precision profile limits are not within the desired working range further optimization of the immunoassay is required prior to validation.

Basic Steps for Developing and Running an Immunoassay

1. Establish assay critical success factors.
2. Ensure appropriate antibody and antigen reagents are available.
3. Adsorb antigen or primary antibody to a solid surface.
4. Block nonspecific binding sites to reduce background.
5. Incubate the primary antibody with the sample.
6. Wash off unbound reagents.
7. Incubate secondary antibody-conjugate with sample.
8. Wash off unbound reagents.
9. Incubate substrate to generate signal.
10. Calibration curve fitting, data analysis and quantitation by non-linear regression.

Basic Steps in Using Immunoassays for HTS

Immunoassays are used in screening to quantify the production or inhibition of antigens/haptens related to a disease target. These antigens or haptens are characteristic of the disease process, mediated by the target such as cytokines or growth factors. Hence the screening procedure will involve incubating compounds with specified target, usually expressed in cells, and collecting the cell medium or lysates to quantify the activity of the compounds. Several examples of this approach for using immunoassay procedures have been described in the literature (1-4). The critical steps in setting up a screen are as follows:

1. Develop a validated immunoassay as described above.
2. Acquire antibody, antigen/calibrator, label and buffer reagents in quantities needed for HTS.
3. Establish liquid handling and automation procedures for screening and immunoassay methods.
4. Establish stability of the primary capture antibody bound to a plate or antigen plate stability.
5. Determine compound collections to be tested.
6. Develop and validate a method for incubation of compounds with relevant target in the screening mode.
7. Develop sample collection procedure from screening experiments.
8. Develop data analysis procedures to use immunoassay data to derive compound potency such as IC50 or EC50.