Section11:Immunoassay Parameters

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It is important to establish a set of critical success factors before one begins the development, optimization and validation of an immunoassay:

1. Analyte (hapten or antigen) to be measured.
2. Sample matrices in which measurements will be made (serum, plasma, cell lysates, culture media etc.)
3. Source of antibody, analyte standards and detection reagents (labeled antibody, enzyme substrates etc). Availability of these reagents is a critical requirement.
4. Detection mode (colorimetric, fluorescence or chemiluminescence) and appropriate plate readers.
5. Type of immunoassay to develop: Sandwich, competitive or antigen-down formats.
6. Expected analyte concentration ranges to be measured: pg/ml, ng/ml or µg/ml in the sample matrix of choice. This would determine the detection limits and the measurable range that should be achieved in a validated assay.
7. Data analysis models and format for reporting results.
8. Validation and optimization criteria using statistical experimental design tools.
9. Recovery, accuracy and precision expected at the limits of quantification and the measurable range.
10. Sample throughput, frequency of use, automation and the number of laboratories that would run the assay.
11. Control samples that would be used for optimization, validation and quality control runs.