A sandwich ELISA is a useful assay to detect an analyte in samples. Generally, two antibodies such as polyclonal – monoclonal, polyclonal – polyclonal or monoclonal – monoclonal paired for capture antibody on ELISA plates – detection antibody conjugated with peroxidase or biotin. To obtain high sensitivity and assay specificity, the monoclonal – monoclonal combination is ideal to develop ELISA. Since a surface area of well in ELISA plates is limited, 5-10 ug/ml antibody solution at 100 ul per well converted effective area of the plate. This means there is no chance to improve the binding, which is the number of antibodies in wells. On the other hand, we can increase detection antibody concentration as much as we can. However, higher concentration will develop high OD values in blank wells which only receive a buffer and detection antibody. The noise reaction reduces the assay sensitivity and accuracy.
To obtain a better concentration of standards and detection antibody, a checkerboard ELISA is highly recommended. For the assay, the standard titration can be made in rows from A to G and no standard in H (blank), then detection antibody titration can be made in column from 1 to 12. We can obtain expected standard range and detection antibodies to compare OD values of an analyte and blank. This is a start point, then we can change a buffer system (pH, salt concentration, and blocking agents), incubation time from 30 minutes to overnight, assay temperature etc. (1)