The fundamental requirements for an ELISA under development are discussed. ELISA development is presented as a series of steps, starting with understanding the analyte. Options for assay format are reviewed next. Antibody (or antigen in tests for antibody) selection is reviewed in detail, including monoclonal vs. polyclonal antibody properties and capture vs. detection antibody requirements. Microtiter plate coating and blocking, and separation and washing, are explained. In the signal generation and detection section, the simplest option of using a secondary antibody is described. The selection of enzyme and signal type is reviewed. Assay optimization is explained, including adjustment of incubation time, selection of appropriate buffers, blocking, formulation, titration, and sample volume to optimize the assay kinetics and minimize interferences. Hardware, software and curve-fitting are briefly mentioned. Assay management considerations (standardization, calibration, quality control and validation) are reviewed. Finally, ELISA tips and common troubleshooting situations are listed.

Jianwen He (M.D. & Ph.D.) is currently the R&D Sr. Manager for BioMerieux Shanghai Biotech with responsibility for managing rapid testing and other immunoassay development. Prior to joining BioMerieux, Jianwen worked as a staff scientist and R&D manager for assay development in the Immunoassay Diagnostics Center of Beckman Coulter, Inc for 10 years, where he helped design and develop many CLIA assays including Access Inhibin A. Jianwen also worked as North Asia commercial manager for DiaSorin S.p.A. for a couple of years, where he managed the RIA, ELISA, and CLIA immunoassay business for a matured market. Jianwen has gone through immunoassay from discovery, research and development to commercialization. Jianwen has a Ph.D. in molecular virology.

Enzyme-linked immunosorbent assay, ELISA, in-house, microtiter plate, development, optimization, validation, immunometric, competitive, indirect, immunocapture, sensitivity, specificity, cross-reactivity, stability, safety, analyte, immunogenicity, antigenicity, epitope, conformation, hapten, solid phase, antibody, antigen, affinity, monoclonal antibody, polyclonal antibody, capture, coating, blocking, separation, washing, signal generation, signal detection, secondary antibody, enzyme, horseradish peroxidase, alkaline phosphatase, colorimetry, chemiluminometry, fluorescence, kinetics, buffers, blocking, formulation, volume, hardware, instrumentation, software, curve-fitting, standardization, calibration, quality control, validation, troubleshooting.