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7.Дevelop data analysis procedures to use immunoassay data to derive compound potency such as IC 50 or EC 50. 6.Дevelop a sample collection procedure from screening experiments. 5.Дevelop and validate a method for incubation of compounds with a relevant target in the screening mode. Determine compound collections to be tested. 4.Д®stablish stability of the capture antibody or antigen bound to a plate. 3.Д®stablish liquid handling and automation procedures for screening and immunoassay methods. 2.ДЄcquire antibody, antigen/calibrator, label and buffer reagents in quantities needed for HTS.

The critical steps in setting up a screen are as follows: 1.Дevelop a validated immunoassay as described above. Several examples of this approach for using immunoassay procedures have been described in the literature ( 1- 5). Hence the screening procedure will involve incubating compounds with the specified target, usually expressed in cells, and collecting the cell medium or lysates to quantify the activity of the compounds.

These antigens or haptens are characteristic of the disease process and mediated by the target, such as cytokines or growth factors. Immunoassays are used in screening to quantify the production or inhibition of antigens/haptens related to a disease target. If the precision profile limits are not within the desired working range, further optimization of the immunoassay is required prior to validation.Д«asic Steps in Using Immunoassays for High Throughput Screening (HTS) If the precision profile is within the desired working range, then assaying spiked recovery samples over several days completes the validation of the immunoassay. A spiked recovery is then performed by spiking the analyte into the matrix and determining the percent recovery of the analyte in the matrix. 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. Then the immunoassay needs to be developed, which establishes proof of concept.

First, one needs to establish the assay critical success factors. To obtain the most accurate determination of the unknown concentration, an immunoassay must be developed based not only on the usual assay development criteria (standard deviation or optimal signal window) but also on how well the immunoassay can predict the value of an unknown sample. Immunoassays are used when an unknown concentration of an analyte within a sample needs to be quantified. Following these guidelines will increase the likelihood of success in developing a robust immunoassay that will measure consistent values for unknown samples. NET Framework 3.5, then you must uninstall them prior to running this installation by using Add or Remove Programs.The intent of this document is to provide general guidelines to aid in the development, optimization and validation of an immunoassay.

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