Enzyme-linked Immunosorbent Assay (ELISA) combines the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily assayed enzyme that possesses a high turnover number. ELISA can provide a useful measurement of antigen or antibody concentration.
The direct ELISA uses the method of directly labeling the antibody itself. Microwell plates are coated with a sample containing the target antigen, and the binding of labeled antibody is quantitated by a colorimetric, chemiluminescent, or fluorescent end-point. Since the secondary antibody step is omitted, the direct ELISA is relatively quick, and avoids potential problems of cross-reactivity of the secondary antibody with components in the antigen sample. However, the direct ELISA requires the labeling of every antibody to be used, which can be a time-consuming and expensive proposition. In addition, certain antibodies may be unsuitable for direct labeling. Direct methods also lack the additional signal amplification that can be achieved with the use of a secondary antibody.
The sandwich ELISA measures the amount of antigen between two layers of antibodies. The antigens to be measured must contain at least two antigenic sites, capable of binding to the antibody, since at least two antibodies act in the sandwich. For this reason, sandwich assays are restricted to the quantitation of multivalent antigens such as proteins or polysaccharides. Sandwich ELISAs for quantitation of antigens are especially valuable when the concentration of antigens is low and/or they are contained in high concentrations of contaminating protein. GenWay has successfully applied polyclonal IgY antibodies for development of sandwich ELISA. The technology can expedite development of ELISA with certain throughput and low cost. The ELISA kits are good enough to reach detection senility at sub-nanogram per ml level and are useful for screening protein targets and quantifying their expression in different conditions. For higher detection sensitivity needed, monoclonal antibodies can be further introduced into the ELISA kit to pair with polyclonal IgY as either capture or detection antibodies.
Figure 1. Polyclonal IgY-based Sandwich ELISA.
This technology allows rapidly and cost-effectively developing ELISA kits for target screening and semi-quantification. Polyclonal IgY ELISA can reach detection sensitivity at the level of sub-nanogram per ml.
This type of ELISA is frequently used for the detection of small analyte antigens containing a single epitope. Typically the plate is coated with antibody specific for the single epitope on the analyte. Next, free analyte and analyte ligated to a detection enzyme are incubated on the coated plate. The quantity of the enzyme-ligand conjugate bound to the plate is detected after incubation with an appropriate substrate and the resulting signal measured with a microplate reader. In this type of ELISA, there is an inverse relationship between the signal obtained and the concentration of the analyte in the sample, due to the competition between the free analyte and the ligand-enzyme conjugate for the antibody coating the microplate, i.e. the more analyte the lower the signal.
GenWay offers solutions to protein expression, antibody and assay development. For more detailed information, please see the specific description pages in Protein Expression, and Custom Antibodies