Conjugated Primary Antibodies

Conjugated primary antibodies are used for direct detection in a broad range of immunoassay techniques, including flow cytometry, enzyme-linked immunosorbent assay (ELISA), and western blotting (WB), as well as immunohistochemistry (IHC) and immunocytochemistry (ICC). A main advantage of direct detection is that it shortens experimental workflows by eliminating the need to perform a secondary antibody incubation step and any associated washes. Direct detection also simplifies panel design for multiplexed experiments by allowing for several antibodies from the same host species to be used in combination, provided the antibodies in question have high specificity for their antigenic targets. In general, conjugated primary antibodies feature similar labels to secondary antibodies. These include enzyme labels such as alkaline phosphatase (AP) and horseradish peroxidase (HRP), which are used for either chromogenic or chemiluminescent detection depending on the type of immunoassay being performed and the choice of substrate. For example, an AP-conjugated primary antibody might be paired with the chromogenic substrate para-nitrophenyl phosphate (PNPP) to produce a yellow reaction product in an ELISA, while HRP-conjugated antibodies are often used with luminol-based substrates for chemiluminescent western blot detection. Primary antibodies labeled with fluorophores such FITC, PE, or Cyanine dyes, or tandem dye products like APC-Cyanine 7 or PE-Cyanine 5, are also widely available. These types of reagents are especially useful for multiplexed flow cytometry experiments, where they allow for building larger panels and also serve to mitigate the risk of secondary antibody cross-reactivities that could cause unwanted background signal. In addition, fluorophore-conjugated primary antibodies are useful for studying protein colocalization via ICC and IHC, and for simultaneously detecting both a protein of interest and its post-translationally modified isoform via western blotting. Other types of conjugated primary antibodies include those labeled with biotin, which binds with high affinity to avidin-, streptavidin-, or neutravidin-based reagents. For example, biotinylated primary antibodies can be used with streptavidin-coated agarose beads for immunoprecipitation (IP) reactions, or with avidin-fluorophore conjugates for fluorescent detection. Primary antibodies can also be conjugated directly to agarose or magnetic beads and used to enrich a protein analyte from solution, either by centrifugation or by capturing the beads with a magnetic field.