Understanding sandwich ELISA; its advantages and steps

The sandwich enzyme-linked immunosorbent assay (ELISA) is the most efficient laboratory procedure, which is helpful to detect the presence and measure the concentration of the target antigen in samples. This test is done for counting the antigens between the two layers of antibodies (i.e., capture antibody and detection antibody). In sandwich ELISA, the two antibodies must be paired and tested before use. Also, while performing the sandwich ELISA, the capture antibody and detection antibody mustn’t interfere with each other’s binding capability. One of the major benefits of sandwich ELISA is the ability to specifically measure antigen from impure samples. The extremely robust nature of the sandwich assay makes it a great diagnostic tool for medical purposes and especially for identifying potential food allergens or testing certain drugs.

Knowing the basics of the sandwich ELISA test

As mentioned earlier, this test is useful for identifying and quantifying the antigen concentration in the unknown samples. Since the procedure of this test uses at least two antibodies, the antigens to be measured must also have at least two non-overlapping epitopes (antigenic determinants) capable of binding to the antibodies. In sandwich ELISA, monoclonal and polyclonal antibodies are used as the detection and capture antibodies. The monoclonal antibodies are used as detection antibodies as they help in more accurate detection and quantification, whereas; the polyclonal antibodies are utilized as the capture antibody since they are helpful in binding antigens as much as possible.

What are the advantages of the sandwich ELISA test?

  • The sandwich ELISA test involves the use of two antibodies. And thus, the antigens are captured and detected more specifically with the help of sandwich ELISA.
  • This ELISA test is more suitable for complex samples as antigens do not require purification before the measurement.
  • The sandwich ELISA is 2 to 5 times more sensitive than the direct and indirect ELISA test. It also gives fast and precise results of the antigens in an unknown sample.
  • You can use both direct and indirect detection methods in this, and thus, sandwich ELISA has good sensitivity and flexibility.

Steps to be followed for sandwich ELISA

  • The first step is to prepare the surface (such as a multi-well plate) with a specific quantity of capture antibodies.
  • Then, you must block the unbound protein binding sides on the surface to reduce background and nonspecific binding.
  • After this, add the sample that contains the target antigen to the plate. The target antigen should be allowed to incubate so that it can bind to the immobilized capture antibodies.
  • Wash off the plate so that the unbound antigen gets removed.
  • Now, the detection antibody is added while ensuring that it binds to a different location on the antigen than the capture antibody.  
  • Again, after the incubation, wash the plate so that the joined unbound antibody-enzyme is removed.  
  • In this step, add the substrate which is changed by the enzyme into a color, electrochemical signal, or fluorescent. Now measure this fluorescence or electrochemical signal on the multi-plate well to determine the presence and quantity of antigens. 

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