The ELISA test creates a special correlation between the amino acids present in an antigen, epitope and an antibody creating site. The antibodies can either be monoclonal or polyclonal in nature. The monoclonal ones are basically derived from the unique antibody known as the hybridomas, whereas the polyclonal is the particular pool made of antibodies out of the animal sera.
Formats Of ELISA Tests:
Most of The ELISA tests are simple in nature, but there are also some complex formats which can provide more specific results with the enhanced analysis. Let us discuss the different formats.
Direct ELISA: In this particular test, a multiwall plate, coated with antigen, is used for incorporating the enzyme and detecting the antibody. The process can be reversed where the plate can be coated with antibody and the antigen can be used for detection. The benefits of this test are:
- As less steps are required, the process is very fast
- Few reagents and steps are involved and so the test is more accurate
Indirect ELISA: Mainly two stages are involved for this specific test. An unlabeled antibody , which is associated with the specific antigen, is applied at first. A secondary antibody, labeled with enzyme is being combined with the first antibody. The polyclonal secondary antibody is generally the anti-species antibody. The advantages of this method are:
- With the single secondary antibody, several primary antibodies can be used with high amount of flexibility
- As small number of labeled antibodies are required, cost savings is also possible
- With the primary antibody, more than a single labeled antibody is generally combine , therefore provides with increased sensitivity
Inhibition or Competition ELISA: For measuring the amount or the concentration of an antigen or an antibody, this particular process is generally used. As the test is determined by the signal output, it is often known as the inhibition ELISA. This test is mostly used in the situations where the analyte is small and only a single antibody is accessible to the antigen.
Sandwich ELISA Test: This particular method is mainly used for combining the different pairs of the antibodies. In this case, the different antibodies are specific to the different molecule of the antigen.
The primary antibody, known as the capture antibody is generally coated with the polystyrene plate. After that, the sample solution or the analyte is being added to the well. Then, the second layer of the antibody is applied for measuring the analyte concentration.
For detection within sandwich ELISA, polyclonals are generally used for capturing. It is to be checked that the variability is present within the polyclonals for allowing the detection as well as the capture. This process will help in the detection of the analyte with the different epitopes.
When the antibody, which is to be detected, is combined with the enzyme, it is known as the direct sandwich ELISA. If the unlabeled antibody is detected, a second antibody will be requiring for the result of the indirect sandwich ELISA. The advantages of this assay are:
- As antigen do not require purification process , it is suitable for the samples which are complex
- Specific antibodies are associated with specific analyte or antigen, therefore high amount of specificity is present
- As both the indirect and direct methods can be used, the process is highly flexible with high sensitivity
Therefore both the monoclonals and the polyclonals can be used in ELISA, but they are generally used for the secondary layer of the detection. The various formats of the tests are therefore very reliable, flexible and highly sensitive in nature.
Author Bio: David Johnson is a scientist who is performing his research on the SESN1 Antibody. In this article, he is discussing with the various formats of the ELISA tests.