Substrates are critical for the detection and visualization steps of an ELISA. The choice of substrate depends on a number of factors, including the degree of sensitivity required, different instrumentation and filter requirements, safety issues, and choice of enzyme. Most immunoassays use antibodies/proteins conjugated to enzymes in order to generate signal through the catalytic properties of the enzyme. Most commonly used enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (AP). HRP has been shown historically to be more sensitive than AP primarily due to its faster catalytic rate. However, HRP reactions may be self-limiting due to substrate inhibition of enzyme. In contrast, AP exhibits a slower catalytic rate, but is not self-limiting. In addition, reaction rates remain linear over long periods of time; therefore, sensitivity can be improved by allowing the reaction to proceed for a longer time. AP substrates also tend to be less toxic and thus easier to handle.

KPL offers a variety of standard and proprietary chromogenic and chemiluminescent substrates for ELISA applications that can be used with alkaline phosphatase or horseradish peroxidase.  Please see KPL's Overview of ELISA Substrates for peroxidase and phosphatase for assistance in comparing and choosing the best substrate for your application.

See our Technical Guide for ELISA for an understanding of the many factors influencing ELISA performance.