Quick to Learn Different ELISA Tests
The complete expression of ELISA is an Enzyme-linked Immunosorbent Assay, which uses materials with specific absorption for antigens or antibodies as immunosorbents, and is mainly used for the immunological detection of antibodies and antigens in samples. There are several different ELISA tests: direct method, indirect method, sandwich method, and more. Next, these main three ELISA methods will be introduced to you.
Different ELISA Tests
- Direct ELISA is the simplest of the ELISA detection methods. During operation, the antigen is usually directly immobilized on the surface of the multi-well plate, and an enzyme-labeled antibody specific to the antigen is added to allow the antigen to bind to the plate. Substrates are added next, which catalyze a reaction with their respective substrates, producing a visible colorimetric signal output proportional to the amount of analyte. It can be measured with special instruments.
- Of all ELISA test methods, direct ELISA is the simplest and fastest to perform due to the fewer reagents and steps required. And the assay is also less error-prone since it does not require secondary antibodies that may cross-react, which greatly reduces potential errors.
However, because no secondary antibody is used, there is no signal amplification, so its detection sensitivity is less flexible. It uses only one antibody and is less specific. Also due to antigen fixation, more background noise may be observed.
- Direct methods are best suited for the analysis of immune responses to antigens. Direct methods are also commonly used when assessing antibody affinity and specificity.
- Indirect ELISA detection is similar to direct ELISA at the first step: the antigen is immobilized in the ELISA plate, and then the primary antibody specific to the antigen is added to bind to the specific target antigen. Then, the enzyme-labeled secondary antibody against the host species of the primary antibody is used to bind the conjugated secondary antibody to the primary antibody, and then the substrate is added to generate a signal proportional to the amount of antigen in the well for detection. It involves an additional amplification step than the direct method.
- The indirect ELISA method has more than one labeled secondary antibody that can be combined with the primary antibody, which has higher sensitivity and greater flexibility. And it uses less labeled antibodies and is more economical. It uses a secondary antibody for amplification, and the received signal is more obvious than direct ELISA, which facilitates the detection of results.
- Indirect ELISA is the best assay used to determine the total endogenous antibody concentration in a sample.
- Sandwich ELISA is relatively common and commonly used. Two specific antibodies are required to bind to the protein of interest and they are used to sandwich the antigen known as a matched antibody pair. An antibody coated on the surface of a multiwell plate to facilitate the immobilization of the antigen is called a capture antibody. Another type of antibody is a conjugated antibody that aids in the detection of the antigen. The detection antibody is enzyme-conjugated known as a direct sandwich ELISA. If the detection antibody is not labeled, a secondary antibody, enzyme-linked detection antibody, is called an indirect sandwich ELISA.
- First, the capture antibody is coated on the wells of the ELISA plate to form a capture antibody coating, then the analyte or sample is added, and then the conjugated detection antibody is added directly or indirectly, and finally the substrate is added to detect the presence of the product and the sample. A signal proportional to the amount of analyte.
- The main advantage of sandwich ELISA is that the specificity and sensitivity are the best among several methods. Sensitivity is 2-5 times that of direct method or indirect method. Because of the use of two antibodies to participate in the detection of antigens it has high specificity. It is also very flexible and can be filled directly or indirectly.
- Sandwich ELISA is particularly suitable for the determination of analyte concentrations in biological samples and the analysis of complex samples, since antigens do not require purification prior to measurement.
The above is about the introduction of different ELISA tests. Today, ELISA technology is widely used in various fields such as medicine and scientific research, and it is very efficient and reliable. Especially in the medical field, it is closely related to us. ELISA technology can be used to detect allergens, virus antigens, infectious disease pathogens, etc. If you need related consumables such as ELISA detachable plates, Hawach is willing to provide you with the best products.