Reoptimizing Reagent Concentrations

The checkerboard approach is the common way a microtiter plate assay is developed and optimized. In terms of commercial kits, this approach could be used to change a kit component or, in the case of development kits (e.g., R + D systems Duo Sets ), to optimize the assay range for the specific requirements of the program. 

On a checkerboard, multiple concentrations of each reagent are added to a plate. Multiple replicates of a limited standard curve are added to the microtiter plate to see how the reagent concentration differences affect the assay range. If a very sensitive method is required, then the biggest difference between zero (blank) and the lowest standard would be required. The checkerboard should allow the assay developer to assess the ideal reagent concentration to obtain the required assay range. Checkerboards can also be run under various incubation conditions to compare the effect of these on assay response. 

A checkerboard example that can be run for an ELISA is shown in Fig. 7.1. This is a sandwich ELISA using two antibodies against the analyte. Detection is achieved using an anti-species conjugate. Actual concentrations used should be at the discretion of the method developer. 

A Blank Blank Blank Blank Blank Blank Blank Blank Bunk Blank Blank Blank 

C Medium Mtdlum Mtdlum Medium Medium Medium Medium Medium Medium Medium Medium Medium 

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