Checkerboard assay

Checkerboard assay Cell migration occurs through a porous filter... 

Checkerboard analysis. The filter assay does not allow one to directly observe migration of cells, and track them to establish... 

The potential interaction between two antimicrobials can be demonstrated using a variety of laboratory procedures, e.g. checkerboard MIC assays where the microorganism is exposed to varying dilutions of each drug alone and in combination, disc diffusion tests (see Fig. 11.4) and kinetic kill curve assays. With the former, results can be plotted in the form of a figure called an isobologram (see Fig. 11.7). 

A prototype method can be obtained from a pharmaceutical company s QC or discovery group, an academic institution, or some other source, although often the scientist may need to design and develop such a method. Checkerboard experiments [8,10] define the optimal concentration of coating material, detector antibody concentration, and approximate range of the calibration curve. A calibration curve generated in assay buffer serves as a reference to assess the suitability of blank matrix for spiking calibrators and QC samples. 

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. 

Sandwich assays require an Ag to contain at least two distinct antigenic sites and as a result are only applicable to large molecular weight compounds. Optimization of the sandwich assay parameters is also commonly done by a checkerboard approach. This is not a saturation assay, free Ab binding sites must remain after the adsorption of Ag. 

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