Related: IgG, streptavidin, biotin, fluorophore, western blot, ELISA
Secondary antibody conjugates are utilized for the indirect detection of a target antigen. They do so by detecting and binding to unconjugated primary antibodies targeted to a specific antigen. This indirect detection method is advantageous as it improves detection sensitivity by promoting signal amplification.
For example, multiple secondary antibodies conjugated to fluorophores will bind to a primary antibody-antigen complex. This interaction results in several fluorophores indirectly binding to the target antigen generating an amplified signal. This allows for the detection of a target of interest, even if the target is low in abundance in a complex biological sample.
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HeLa cells were incubated with (Tubulin+) or without (Tubulin-) mouse anti-tubulin followed by iFluor™ 488 goat anti-mouse IgG conjugate (Green, Left) or Alexa Fluor® 488 goat anti-mouse IgG conjugate (Green, Right), respectively. Cell nuclei were stained with Hoechst 33342 (Blue).
Time-resolved fluorescence energy transfer (TR-FRET) is the practical combination of time-resolved fluorometry (TRF) combined with Förster resonance energy transfer (FRET) that offers a powerful tool for drug discovery researchers. TR-FRET combines the low background aspect of TRF with the homogeneous assay format of FRET. The resulting assay provides an increase in flexibility, reliability and sensitivity in addition to higher throughput and fewer false positive/false negative results. FRET involves two fluorophores, a donor (such as trFluor Eu and trFluor Tb) and an acceptor. Excitation of the donor by an energy source (e.g. flash lamp or laser) produces an energy transfer to the acceptor if the two are within a given proximity to each other. The acceptor in turn emits light at its characteristic wavelength. The FRET aspect of the technology is driven by several factors, including spectral overlap and the proximity of the fluorophores involved, wherein energy transfer occurs only when the distance between the donor and the acceptor is small enough. In practice, FRET systems are characterized by the Förster's radius (R0): the distance between the fluorophores at which FRET efficiency is 50%. For many FRET parings, R0 lies between 20 and 90 Å, depending on the acceptor used and the spatial arrangements of the fluorophores within the assay. Through measurement of this energy transfer, interactions between biomolecules can be assessed by coupling each partner with a fluorescent label and detecting the level of energy transfer. Acceptor emission as a measure of energy transfer can be detected without needing to separate bound from unbound assay components (e.g. a filtration or wash step) resulting in reduced assay time and cost.
HL-60 cells were incubated with (Red, +) or without (Green, -) Anti-human HLA-ABC (W6/32 mAb), followed by iFluor™ 405 goat anti-mouse IgG (H&L). The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the Pacific Blue channel (Ex/Em=405/445 nm).
HeLa cells were incubated with (Tubulin+) or without (Tubulin-) mouse anti-tubulin followed by AAT’s Cy3® goat anti-mouse IgG conjugate (Red, Left) or Jackson’s goat anti-mouse IgG conjugated with Cy3® (Red, Right), respectively. Cell nuclei were stained with Hoechst 33342 (Blue, Cat# 17530).
Signal amplification can also be achieved using the labeled streptavidin-biotin (LSAB) method. LSAB exploits biotin’s high affinity to streptavidin as a detection method. Biotinylated secondary antibodies bind to streptavidin conjugates tagged with either an enzyme or fluorophore. Streptavidin conjugates can bind four biotins per molecule in order to amplify the signal. The LSAB method is flexible as it may be integrated into any immunoassay.
Secondary antibody conjugates also provides researchers with an efficient technique for immunolableing. Primary antibodies can be raised to have the same structural constant region but different variable regions to recognize various antigens. A secondary antibody can then be raised to recognize the constant region of these primary antibodies to indirectly detect various antigens. As a result, only a singly secondary antibody requires labeling versus labeling every primary antibody which reduces both time and cost.
AAT Bioquest offers a diverse selection of labels such as fluorophores, phycobiliproteins and enzymes already conjugated to secondary antibodies. Our secondary antibody conjugates are well-suited and optimized for use in various immunoassays such as flow cytometry or fluorescence microscopy.