Enzyme linked immunosorbent assay (ELISA) is an immunoassay method that uses an enzyme-linked immunosorbent assay (ELISA) to detect and quantify biomolecules such as antibodies, proteins, hormones, or peptides, as well as characterize protein-protein and protein nucleic acid interactions. In ELISA, the target used for the experiment (usually an antigen) is immobilized on a solid surface and then detected using enzyme conjugated antibodies of horseradish peroxidase (HRP) or alkaline phosphatase (AP). Quantification is achieved by incubating with substrates catalyzed by enzymes, resulting in measurable byproducts.
The ELISA assay is usually performed in a 96 well plate. These plates are designed to typically bind and immobilize antigens directly onto the surface of microtiter plates or indirectly through pre coated antibodies. After fixation, introduce the primary antibody into the sample to form an immune complex with the antigen. The primary antibody can be covalently labeled onto an enzyme (such as HRP), or if the primary antibody is biotinylated, it can be indirectly detected using enzyme labeled secondary antibodies or streptavidin conjugates. By incubating with a suitable substrate to evaluate the activity of conjugated enzymes, detection can be achieved, and the substrate produces measurable byproducts. Reagent kits vary in sensitivity and compatibility with imaging equipment.
Direct and indirect detection
The method for detecting primary antibody antigen complexes in ELISA can be direct or indirect (Figure 1). In direct detection methods, a single primary antibody conjugated with reporter enzymes (such as HRP or AP) can directly detect the target antigen. Through indirect detection, the target can be sequentially detected using a dual antibody system. Firstly, incubate the sample with unlabeled primary antibodies targeting the target antigen. Then, use a first antibody specific enzyme-linked immunosorbent assay (ELISA) to detect its presence and thus detect the target antigen. If using biotinylated primary antibodies to detect antigens, use enzyme labeled streptavidin conjugates as your secondary detection reagent.
The choice of detection method depends on the expression level of the target antigen. Direct detection is an appropriate method for detecting highly expressed antigens. When sensitivity is crucial, such as detecting low abundance targets or poorly expressed antigens, use indirect detection methods to enhance the signal.
Figure 1. Schematic representation of direct and indirect antigen detection using target specific antibodies.
1.direct method
In direct ELISA, antigens are passively adsorbed onto the surface of the plate and detected using HRP labeled primary antibodies. Introduce a colorless substrate into the sample, which reacts with the enzyme conjugate and produces measurable byproducts. Depending on the substrate selection, the byproduct can be colorimetric, chemiluminescent, or fluorescent. The size of the signal generated is directly proportional to the number of antigens in the sample. Among all ELISA methods, direct ELISA analysis is the simplest, fastest to perform, but has the lowest sensitivity.
Figure 2. Direct method diagram
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2.indirect method
Indirect ELISA is essentially a modified version of direct ELISA. In indirect ELISA, the primary antibody used to detect antigens is uncoupled. Instead, enzyme-linked secondary antibodies that are reactive to the host species of the primary antibody are used to detect the primary antibody antigen complex (indirectly detecting the antigen). Introduce a colorless substrate into the sample, which reacts with the enzyme conjugate and produces measurable byproducts. Depending on the substrate selection, the byproduct can be colorimetric, chemiluminescent, or fluorescent. The size of the signal generated is directly proportional to the number of antigens in the sample. Compared with direct ELISA, using auxiliary detection reagents has significant advantages in signal amplification.
Figure 3. Indirect method diagram.
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3.Sandwich method
Sandwich ELISA analysis is the most commonly used form of ELISA. It requires the use of matching antibody pairs, so that each antibody has specificity for different non overlapping epitopes on the antigen. Firstly, a type of antibody, called a capture antibody, is coated on the surface of a porous microtiter plate to promote the fixation of the target antigen. Then, the second antibody (referred to as the detection antibody) binds to the capture antibody antigen complex. Finally, add enzyme labeled secondary antibody conjugates with specificity for detecting antibodies (rather than capturing antibodies).
Figure 4. Schematic diagram of sandwich method
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