Optimizing concentrations

When setting up an assay for the first time, it is important to optimize the capture and detection antibodies' of concentrations. In the following, we describe a basic procedure of how to optimize a standard set-up based on adsorbed capture antibody and chemically conjugated detection antibody/DNA. The optimal concentrations depend on the type of antibodies used, but are typically in the range 1-20 ^g ml-1 for the capture antibody and 1-100 ng ml-1 of detection antibody. Using antibodies with higher affinity and specificity for the antigen usually results in more sensitive assay. To determine optimal concentrations of capture and detection antibodies, an evaluation set-up with serial dilutions of both antibodies and antigen, as shown in Figure 12.3, can be performed. In a first attempt the real-time immuno-PCR protocol below can be used.

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Figure 12.3

Microtiter plate set-up for capture and detection antibody concentration optimization. 1-3 represent different concentrations of antigen covering the range of interest. BC is the background control. A1-A3 is a ten-fold dilution series of capture antibody, and B1-B4 is a five-fold dilution series of detection antibody/DNA conjugate.

The combination of capture and detection antibodies' concentrations that give largest Ct difference between the lowest antigen concentration and the BC sample is the best choice, of course, provided they are within the linear range of Ct versus the logarithm of antigen concentration. If this is not the case one must choose different concentrations of the antibodies or replace them.

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