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Substrate Selection in Immunoassay Development

Assay development requires that many components such as antibody/antigen systems, blocking and tabilization, and enzyme detection substrates be evaluated to produce the most robust assay possible. While the choice of antibody or antigen is often the primary consideration, the choice of substrate also has a substantial aeffect on performance and deserves equal attention for optimal selection. This article examines several key areas that can influence your choice of colorimetric substrate including substrate kinetics, stop reagent, dynamic range, and assay timing. The terms “sensitivity” and “detection limit” are defined here and used to illustrate how these key parameters affect assay performance. Our goal is to provide the assay developer with the tools necessary to make an informed selection of tetramethylbenzidine (TMB) substrate by understanding its impact on assay performance.

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