Due to their rapid and straightforward procedure, ELISA immunoassays are one of the most widely used in research and diagnostics for the identification and/or quantification of analytes of a protein nature such as peptides, proteins, antibodies, and hormones.
Depending on how antigen-antibody interactions occur, ELISAs can be classified into four main categories.
We will tell you the details about the different types of ELISA, the differences between them, and the advantages and disadvantages of each one.
Types of ELISA
As we have already commented in the introduction, based on how antigen-antibody interactions occur, ELISAs are classified into four types: direct ELISA, indirect ELISA, sandwich ELISA, and competitive ELISA.
Let’s look at each of these types of ELISA in more detail:
1. Direct ELISA
The direct ELISA is the simplest and fastest ELISA of all. A primary antibody labeled with an enzyme will bind directly to the antigen of interest, allowing its detection and/or quantification.
The simplified procedure would be the following:
- The antigen is immobilized on a plate
- A primary antibody labeled with an enzyme is added that will bind to the antigen of interest
- The substrate is added that when reacting with the enzyme will provide a visible signal that will allow the detection and/or quantification of the antigen of interest.
2. Indirect ELISA
It is an assay similar to the direct ELISA but in two steps, which allows amplifying the signal obtained. In this case, two antibodies are used, one primary and the other secondary, and it is the latter that will be conjugated to an enzyme.
The simplified procedure would be the following:
- The antigen is immobilized on a plate
- An unlabeled primary antibody is added that binds to the antigen of interest
- A secondary antibody labeled with an enzyme is added that will bind to the primary antibody
- The substrate is added that when reacting with the enzyme will provide a visible signal that will allow the detection and/or quantification of the antigen of interest.
3. ELISA sandwich-type
In the sandwich ELISA, the antigen is immobilized between two antibodies, one for capture and the other for detection, also known as pairs of antibodies, which will bind to two different epitopes of the same antigen.
The simplified procedure would be the following:
- The capture antibody is immobilized on the plate
- The sample containing the antigen of interest that will bind to the capture antibody is added.
- The detection antibody is added that will bind to the antigen linked in turn to the capture antibody.
- In case the detection antibody is conjugated to an enzyme. We will proceed directly with the 5th step. If not (which is most common in sandwich ELISAs), it will be necessary to add an enzyme-labeled secondary antibody that will bind to the detection antibody.
- The substrate is added that when reacting with the enzyme will provide a visible signal that will allow the detection and/or quantification of the antigen of interest.
4. Competitive ELISA
The competitive ELISA is a more sophisticated variant of the ELISA technique. It is also known as inhibition ELISA due to the use of a reference antigen that will compete with the sample antigen for binding to the primary antibody.
It is generally used to detect and/or quantify antigens present in meager amounts.
The simplified procedure would be the following:
- The reference antigen is immobilized on the plate.
- On the other hand, an excess of unlabeled primary antibody is incubated with the sample containing the antigen of interest, giving rise to the formation of antigen-antibody complexes.
- The antigen-antibody mixture is added to the plate, where the reference antigen will compete with the antigen in the sample to bind to the antibody.
- The plate is washed, eliminating the soluble antigen-antibody complexes.
- An enzyme-labeled secondary antibody that will bind to the primary antibody anchored to the reference antigen is added to the plate.
- The substrate is added that when reacting with the enzyme will provide a visible signal that will be inversely proportional to the amount of antigen of interest present in the sample.
ELISA kits by Boster Bio
Boster Bio is a well-recognized name of a biotechnology corporation, manufacturing an array of products used in clinical trials and scientific research. Boster Bio primarily manufactures ELISA kits, antibodies, recombinant proteins, and reagents.
The company’s long commitment to the work of scientists is worth every mention. The products by Boster Bio have successfully been incorporated in medical researches owing to more than 20,000 plus citations.
The manufacturing process of ELISA kits follows a carefully thought and devised plan and implements proprietary coating and blocking technology in order to achieve “picogram” level of sensitivity. This condition of sensitivity is crucial in the success of research and development trials and practical experiments.