The competitive ELISA format is based on the binding competition in the initial insulin RIA and IgG EIA methods. The competition between the target analyte from the sample and the reference analyte and the binding of the analyte-specific antibody is the key to this format.
Either antibody or target analyte can be labeled with enzyme and used in the binding reaction. When using labeled antibodies (Figure 3Ai), the reference analyte is first passively adsorbed into the wells of the microplate, and then samples containing the target analyte, such as cell lysate, serum, etc., are added and incubated with a fixed concentration of labeled antibody . The target analyte in the sample competes with the immobilized analyte to bind a limited number of labeled antibodies, and the signal generated by the subsequent enzymatic reaction is inversely proportional to the concentration of the target analyte in the sample. Alternatively, a solid-phase adsorbed antibody and labeled analyte can be used. The target analyte in the sample is incubated with a fixed amount of labeled analyte and competes with each other to fix the binding of the antibody. As in the previous example, the signal generated is inversely proportional to the concentration of the target analyte in the sample.
Direct ELISA is the simplest ELISA format. The target analyte in the sample is adsorbed into the wells of the microplate, and the target analyte is detected directly through enzyme coupling reagents or labeled detection antibodies.
Analyte represents the target protein to be determined. In a competitive ELISA, the target analyte or antibody can be labeled, while in the direct or indirect ELISA format, only the detection antibody is labeled.
Indirect ELISA is similar to direct ELISA, the main difference between the two is that the primary antibody is not labeled. The detection of the target analyte is achieved by adding an enzyme coupling reagent or labeling the detection antibody again to bind it to the main antibody.
Direct ELISA is faster because it has one step less than indirect ELISA. It is usually used for routine tests that need to be completed quickly, and commercial home pregnancy tests are an example of this format. Although indirect ELISA has one more step than direct ELISA, signal amplification is usually better than direct ELISA. Therefore, indirect ELISA is usually more sensitive than direct ELISA and can measure lower abundance proteins.
As shown in Figure 3C, the additional steps of indirect ELISA compared to direct ELISA are the direct adsorption of the target analyte from the sample to the microplate, but it is also difficult to achieve such an effect. Because the target analyte may be washed away during the plate washing step, it may increase the variability of the test. In addition, other proteins that bind non-specifically to the microplate may cause false positive results. In order to overcome the above-mentioned challenges, other more reliable formats have been derived. Among them are usually called sandwich, bridge or competitive ELISA (in the sandwich or bridge ELISA format). Among these formats, one can be compared with The biological reagent that specifically binds to the target analyte is first adsorbed into the microplate.
Schematic diagram of sandwich ELISA (A) and bridge (B) ELISA
Sandwich ELISA requires two different reagents or antibodies, one for capturing and the other for detecting the target analyte. These reagents bind to different epitopes (binding sites) of the target analyte to form a sandwich format. The resulting interaction has a particularly high specificity (because the capture and detection steps require specific The detection signal can only be generated by the recognition of the epitope).
Bridge-linked ELISA is often used to determine the concentration of antibodies or other target analytes with two identical antigen binding sites (divalent). It can label and capture the same reagents to detect a bivalent target analyte. Therefore, the target The analyte can be viewed as a bridge between capture and detection reagents (Figure 4B). Either sandwich or bridge ELISA format can be designed as a competitive ELISA.
The above format is the basic design of ELISA. All formats can be adjusted using competition or inhibition conditions to determine antigen or antibody. All methods require pre-reaction/incubation with reagents to achieve the best state. These optimal conditions can be challenged by adding antigens or antibodies. As the free antigen (antibody) in the solution increases, the amount of antibody (antigen) that can bind to the immobilized substrate decreases. After the washing step, a chromophore substrate is added to generate a signal (color change or light emission). The signal change caused by the antibody/antigen challenge reveals information about the competing antigen/antibody. Competitive ELISA is very useful for determining the concentration of antigen in complex mixtures, especially in unknown samples that may contain antigens and similar samples that contain known amounts of purified antigens.