Checkerboard titration

Biomolecules like antibodies attach to surfaces via a variety of mechanisms. This attachment phenomenon is controlled by the chemical properties of the surface, but can be influenced by factors such as pH and temperature. In the case of antibody coating to a solid support the use of so-called medium-binding plates is to be recommended. Coating conditions can be optimized by performing a checkerboard titration (in the following example the optimal coating antibody concentration is determined) ... 

The optimal concentration of labeled antigen can also be determined via a checkerboard titration protocol ... 

Fig. 6. Checkerboard titration of the antigen concentration used for coating and of antibody concentration. (A) Enzyme activity as a function of antigen concentration at coating for antiserum diluted 1 25 ( ), 1 125 (x), 1 625 (O), and 1 3, 125 (A). (B) Enzyme activity as a function of antiserum dilution in wells coated with a-fetoprotein 1 /ag/ml ( ), 300 ng/ml (X), 100 ng/ml (O), and 30 ng/ml (A).

Figure 4. Typical checkerboard titration for antisera to 8b from rabbit number 658 with coating antigen 12c (System D in Tablell).

In two-layer methods, exemplified by the indirect and polymer-based labels, each of the separate immune reagents must be applied at optimal dilution. In addition, the dilutions of the primary and secondary antibodies (or labels) are interdependent in terms of contrast developed by the procedure as a whole. This fact necessitates comparison of the results obtained using several dilutions of the labeling reagent (secondary antibody) with several different dilutions of the primary antibody comparison is achieved by checkerboard titration (Table... 

PAP methods and other multi-step procedures require more complex checkerboard titrations of each of these separate steps sensitive polymer-based methods have superseded the use of these methods, and we will not discuss them further. Refer to previous editions of... 

TABLE 1.2 Determination of Optimal Titers for Indirect Immunoperoxidase Method—Checkerboard Titration ... 

A key feature in helping this process is through the use of chessboard or checkerboard titrations (CRTs). The use of microtiter plates is an important feature of ELIS As. This chapter describes this type of format. CBTs can be accomplished in any format in which reagents can be diluted, but the microtiter plate, with its associated equipment for ease of pipetting, is ideal. It will become clear that CBT is not the only method for optimizing reagents, and that often concentrations must be adjusted with reference to actual test conditions. 

Figure 2. ELISA checkerboard titration of PCS coating antigen and PCS antibody. Conditions Varying concentrations of PCS antisera diluted in carbonate/bicarbonate buffer (pH 9.5), plates were washed with phosphate buffer pH 7.2, PCS antibody was diluted in PBS Tween 20, seconday antibody was IgG goat anti-rabbit with alkaline phosphatase label.

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