Unconjugated Secondary Antibodies

Secondary antibodies are best known for their utility as indirect detection reagents, meaning they allow the detection of a protein analyte to which an analyte-specific primary antibody is already bound. To achieve this, secondary antibodies must have specificity for the host species and isotype of the primary antibody, and be labeled with a detectable tag such as an enzyme or fluorescent dye. For example, if a polyclonal primary antibody raised in goat was used to recognize and bind an analyte of interest in a human tissue section, an anti-goat IgG (heavy and light chains) secondary antibody featuring an appropriate label would be required for detection purposes. A main advantage of using secondary antibodies for indirect detection is that the assay sensitivity is improved compared to direct detection with labeled primary antibodies. This results from multiple secondary antibodies binding to each primary antibody and can be especially beneficial when studying low abundance analytes. Labeled secondary antibodies are used for a broad range of immunoassay techniques, including western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and immunocytochemistry (ICC), as well as immunohistochemistry (IHC), flow cytometry, and lateral flow assay (LFA). Despite being synonymous with indirect detection, secondary antibodies can also be used label-free for scientific research, albeit in slightly different ways. One approach involves using unconjugated secondary antibodies as coating reagents for ELISA, where they enable the capture of antibodies from solution. Another method uses unconjugated secondary antibodies as linker reagents to provide signal amplification. For example, a sample might be incubated with an analyte-specific primary antibody, followed by an unconjugated secondary antibody, after which a labeled tertiary antibody (raised against the secondary antibody and featuring a relevant label) could be added for detection. Unconjugated secondary antibodies can also be custom conjugated if the desired product is not available commercially. Researchers performing complex flow cytometry assays or multiplex assays may choose to go down this route to increase flexibility for panel design.