ELISAs work on the concept of an antigen-binding to its particular antibody, allowing antibodies or antigens to be detected in samples even if they are present in trace quantities. ELISAs can detect enzymes, proteins, peptides, and antigens, allowing them to be utilized in various applications, including testing in healthcare, quality control in commerce, and science.
Different kinds of ELISAs have been created, each with somewhat other fundamental procedures. However, all of them are linked with the idea that an antibody will attach to a particular epitope. But how to choose which type of ELISA is required for your study? Read on to know the types and usage of different kinds of ELISA tests.
What are the various kinds of ELISA?
Direct, indirect, sandwich and competitive are the four significant kinds of ELISA. Each test has its own set of benefits and appropriateness. Here are some essential qualities of usage of each type of ELISA test:
- Direct ELISA:
These are the most basic types of ELISA tests used by ELISA kit manufacturers. They are termed as direct since the detection method only requires the utilization of one antibody. When the reagent is introduced, the primary detection antibody is immediately labeled with a conjugated enzyme, resulting in a color change. There is no requirement for a secondary antibody. A blocking agent, such as BSA, must be employed when screening for the antigen of concern within a sample to shut off any other possible binding sites.
Although just one antibody is employed, direct ELISAs are known to minimize cross interaction among other antibodies. However, since each of the specific antibodies used in these assays must be enzyme-labeled, the cost of the test and the time it takes to make it may rise. Each new test would require the labeling of a new detection antibody. In contrast to indirect ELISAs, direct ELISAs have a slower response since the signal generated by the test is less magnified. Still, they have a considerably quicker detection speed because only one step is needed for detection.
- Indirect ELISA:
During the detection step of indirect ELISAs, two antibodies are required. This is a two-step procedure that lengthens the time it takes to complete the test. The antigen of concern is initially coated on the plate, introducing the primary antibody into the wells. If the antigen of focus is present in the wells, the target protein will attach to it specifically. After that, the supplementary antibody will be administered, which will have been previously tagged with an enzyme for identification.
The supplementary antibody will attach to the primary antibody, and the primary antibody will then bind to the antigen. The level of antigen may then be calculated based on the observed color shift. The more the color changes, the more antibodies are attached to the antigen since the wash cycle will eliminate minor color changes. This method can increase sensitivity by allowing more than one tagged antibody to bind to the primary antibody.
More than one supplementary detection antibody may be employed with a single primary detection antibody, increasing flexibility. Because only one kind of antibody has to be labeled, it may reduce the cost of performing this test.
- Competitive ELISA:
Tiny compounds such as lipids, hormones, and small peptides are often determined using this method. Researchers might identify larger molecules, but they would need higher concentrations.
Competitive ELISAs are based on the idea that a specimen antigen and an enzyme-coupled form of the identical antigen will fight for a limited number of particular antibody binding locations. Another approach is that the antibody contends for targeted cells on the pre-coated plate with bound antigen. The labeled antibody will interact with the antibody in the test for binding sites.
When measuring antibody concentration in a mixture, the weaker the signal generated by the test, the greater the amount of particular antibody in the sample. The available tagged antibody will contest with the antigen pre-coated to the wells for binding; if more of the antigen in the specimen can attach, it will eliminate more of the tagged antibody after the wash cycles. The same concept applies to marked antigen.
- Sandwich ELISA:
Sandwich ELISAs are the most well-known tests. As the name implies, the antigen of relevance is wedged between two antibodies. These assays may be either direct or indirect.
Because the antigen of interest is positioned between two antibodies, it must be 20 aa long. This guarantees that the antibodies employed may attach to various antigen epitopes and that it will not disrupt the binding of each antibody. The capture monoclonal antibody is the one that absorbs the target antigen into the assay’s wells. Following the addition of the sample, the identification antibody may be added to the wells, attaching to the specific antigen if it is present.
It is critical to utilize matching pairs of antibodies when doing a sandwich ELISA. Antibodies that are tested together to verify that they bind to distinct epitopes of an antigen are matched pairs. This eliminates the possibility of the antibodies recognizing or attaching to the exact location. The detection antibody in a sandwich ELISA may be protein-coupled, or an additional enzyme-conjugated antibody can be added. HRP (horseradish peroxidase) and AP (alkaline phosphatase) are two enzymes often employed in the detection procedure. The inclusion of the substrate is the last stage in the process.
In conclusion
The Enzyme-Linked Immunosorbent Assay (ELISA) is used for medical diagnostic reasons. ELISA can identify HIV, HBV, flu, anemia, Lyme sickness, food allergies, and other diseases. There are now a massive variety of ELISA kits available from producers all over the globe. Therefore, these classifications given above will help you choose your preferred test.
