The enzyme-linked immunosorbent assay or ELISA is a technique based on a plate that is engineered to detect proteins, hormones, antibodies, and peptides. In an ELISA test, there’s a need to immobilize the antigen to solid surfaces. It would then involve complexing an antibody linked to a specific enzyme.
The detection process is achieved by assessing the conjugated enzyme activities where the incubation of a substrate is involved. A measurable product is then produced from the incubation, measured by the ELISA kit and the experts handling it. The most important factors of this detection strategy are the interaction of antigen and antibody in humans.
How ELISA is Performed
Typically, this procedure is performed through the 384-well or 96-well plates made from polystyrene. They passively bind the proteins and antibodies in the plates, which will immobilize reagents put into them. Overall, this is a technique that many people can perform, and the process is easy and simple.
When the reactants are starting to immobilize in the ELISA plates, the microplate in the surfaces allows for the separation of the non-bound from bound material in the process of the assay. The ones handling the plates can wash away the non-bound clothes, which can be a powerful tool in measuring specific analytes, even if the preparation is considered crude.
4 Types to Know About
Several modifications are usually done on the primary test. These are the sandwich, competitive, direct and indirect techniques. One of the critical differences that they have is the antigen’s immobilization process, which can be done with the adsorption directed to the plate or the assay. The indirect way may involve the capture of the antibody attached to the plate.
The antigen is detected indirectly through the enzyme-labeled secondary antibody or directly through the primary antibodies. Overall, detection is often accompanied by labels like horseradish peroxidase (HRP) or alkaline phosphatase (AP). You can learn more about the alkaline phosphatase test on this page here.
Some substrates or a mass selection of them are available in some laboratories when performing ELISA for either the AP or HRP conjugate. The substrate choice can depend on the sensitivity of the assay and the instruments used for signal detection. These instruments can be fluorometer, spectrophotometer, or luminometer.
About the Direct ELISA
This is for the extensive and more direct detection where antibodies detect an antigen. The entire process has been involved with a conjugated enzyme. This is an excellent method and option if you don’t have commercially available kits that will be compatible with your target protein. Some of its advantages include quicker results because of fewer steps, and only a single antibody is used, and there’s cross-reactivity that’s eliminated.
Usually, an indirect detection may involve a coated antigen that’s placed into well plates. There are two layers or stages included in the multi-well. The first step is to have an unlabeled primary antibody applied to the plate. These are the antibodies where they are specific to an antigen. The second layer is an enzyme-labeled antibody bound to the first.
The advantages of the direct step are it can have various secondary antibodies that can be available commercially. There are versatile options where the second antibody is often used for the detection process. The sensitivity can be significantly increased because of several epitopes present, allowing for amplification of signals.
Sandwich Type of ELISA
The sandwich ELISA may typically require pairs of matching antibodies. Each of them is specific for non-overlapping epitopes or parts of any chosen antigen molecule. Read more info about antigen here: https://www.britannica.com/science/antigen. The capture or first antibody is the one coated with the wells.
This will require matching pairs that have higher specificities in analytes and antigens. They can be detected and captured specifically for a purpose. The crude samples are suited for complex tests where the advantages include an antigen that does not need purification before the measurement.
This is a kit that’s also called an inhibition format. This is a plate or surface-based assay where the plates are coated with captured antibodies. They can be highly reactive to specific molecules as well. Samples contain native molecules that are often conjugated with proteins. These recombinants or competing molecules are added on top of the wells.
Since there’s a constant amount of enzyme-conjugated molecules on each well, the levels of the natives will determine the overall ratio of the substance. After the period of incubation, any unbound antibodies are washed off. The substrates like HRP and TMB are added into the wells, and they can transform into blue precipitates.
The amount is usually proportionate to the enzymes in the well. Precipitates may turn yellow when you add an acid stop solution. The concentration of this outcome is read by utilizing 450 nm of light absorbance.