Advantages of Using a Secondary Antibody
With its single labeling step, the direct method offers a shorter assay time with a simpler workflow. Since it minimizes species cross-reactivity and non-specific binding, the direct method is best used for specific targeting if multiple antibodies are raised in the same species. However, this method demands an abundant supply of expensive conjugated antibodies—with few color selections and limited range of reporter molecules available—and is much less practical than the indirect method.
Although the indirect method requires additional steps, time, and added complexity, it still offers several advantages over the direct method. More than one secondary antibody can specifically bind to different parts of the same primary antibody—increasing the versatility, antigen signal detection, and amplification. The indirect method also contributes to the detection, sorting, and purification of target antigens—providing higher degrees of specificity and sensitivity. Commercially available conjugated secondary antibodies are relatively inexpensive and available in a wider spectrum of colors compared to conjugated primary antibodies—with increased access to several different probes.
By using primary antibodies as a ‘bridge’ to bind with the target antigen, secondary antibodies reduce the possibility of the reporter molecule compromising the binding capability to the antigen epitope. If the target antigen is expressed at a low concentration, using secondary antibodies will allow for multiplexing or multi-labeling across applications (e.g. immunofluorescence, western blot) to validate target antigen detection.
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