Secondary Antibodies

Secondary antibodies are useful tools that can help increase the effectiveness of a variety of experimental methods including Western blot, immunohistochemistry (IHC), flow cytometry, and ELISA. They are generated by immunizing host animals with antibodies from a different species. The antibodies that are generated recognize and bind to the immunoglobulin (Ig) domain of the immunizing primary antibody and assist in purifying, sorting, and detecting target antigens. Secondary antibodies are typically conjugated to a label. The downstream application will determine the conjugation state and choice of label of the secondary antibody. 

Indirect detection of target analytes using the combination of primary and secondary antibodies offers multiple advantages over directly detecting the proteins using only conjugated primary antibodies.

•  Amplifies the Signal

Multiple secondary antibodies can bind to a single primary antibody. Each bound secondary antibody is conjugated to a reporter molecule. This results in an amplification of the signal and an increase in the sensitivity of the assay. For some assays, the signal can be increased further using a biotinylated secondary antibody and the streptavidin-conjugated reporter molecule.

• Allows for Multiplexing Experiments

Since secondary antibodies are species specific, experiments can be designed to detect multiple analytes using primary antibodies derived from different species. The different analytes are detected using secondary antibodies conjugated to fluorochromes that emit light at different wavelengths.

• Preserves the Paratope

Conjugating a primary antibody to a reporter molecule has the potential of compromising its antigen-binding site, also called its paratope. This would reduce the primary antibody’s ability to bind to its target antigen. Using a conjugated secondary antibody allows an experimenter to use an unconjugated primary antibody in their assay.