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ELISA Main Experimental Methods

Popularity:381 Published Date:2022-03-25 10:44

What is ELISA? 

Enzyme-linked immunosorbent assay (ELISA) is an immunoassay technology established by combining the specific immune response of antigens and antibodies with the catalytic reaction of enzymes.

ELISA can be used to measure antigens or antibodies. The main types are: double-antibody sandwich method, indirect method, competitive antibody detection, capture method, and avidin-biotin method.

 Double-Antibody Sandwich Method

The double-antibody sandwich method is often used to detect antigens, and the principle is as follows:

1.The antibody is bound to the surface of the solid phase carrier to form a solid phase antibody;

2.The antibody is bound to the surface of the solid phase carrier to form a solid phase antibody;

3.The enzyme-labeled antibody binds to the test antigen immobilized on the solid support to form a complex;

4.The amount of enzyme is positively correlated with the antigen to be tested in the sample, the substrate is added, and the enzyme catalyzes the substrate to generate a colored product;

Qualitative or quantitative analysis of antigens is performed according to the degree of color reaction.

 

Indirect Method

The indirect method is often used to detect antibodies, and the principle is as follows:

1.The antigen is bound to the solid phase carrier to form a solid phase antigen;

2.The antibody to be tested is specifically bound to the solid-phase antigen and fixed on the solid-phase carrier;

3.The enzyme-labeled antibody combines with the antibody to be tested immobilized on the solid support to form a complex;

4.The substrate is added, the enzyme and the substrate are used for color reaction, and the qualitative or quantitative analysis of the antigen is carried out according to the degree of the color reaction.

Competitive Antibody Detection

When the interfering substances in the antigen material are difficult to remove, or it is difficult to obtain enough purified antigen, the specific antibody can be detected by the competitive method.

Small molecule antigens or haptens lack more than two sites that can be used as sandwich method, so double antibody sandwich method cannot be used, and competition method can also be used.

The competitive method for detecting antibodies, the principle is as follows:

Coat the antigen in a microplate to form a solid-phase antigen, add the antibody to be tested and the enzyme-labeled antibody for competitive binding, incubate at a suitable temperature and a certain period of time, wash, and then add a substrate for color reaction. The intensity of the color in the medium is negatively correlated with the concentration of the analyte.

Biotin-Avidin Elisa Method

The biotin-avidin method also detects the antigen or antibody through the color reaction of the enzyme and the substrate. The difference from the previous method is that the enzyme is not labeled on the antigen or antibody but on biotin. One molecule of avidin can have a specific affinity with four biotin small molecules. Since avidin can bind to multiple biotins, one antigen-antibody complex can be labeled with multiple enzymes, which can play a role in multi-level signal amplification. This method can be used for detection when the antigen-antibody content in the test sample is low.

Instruments Required for ELISA Experiments

In the ELISA experiment, the enzyme labeled on the antibody or antigen reacts with the substrate to produce colored substances. Different color reactions have different detection wavelengths. For ELISA experiments, the detection wavelength is within the range of 300-700.

 

 

 

ALLSHENG microplate reader can meet all ELISA experimental applications. We have a whole series to give you one-stop solution.

 

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