ELISA Sandwich Method
ELISA can be used to measure antibodies and antigens, and different detection methods can be used according to different situations. Common ELISA methods include direct method, indirect method, competition method, and double-antibody sandwich method, among which the most widely used is the double-antibody sandwich method. This article will mainly introduce this ELISA detection method.
Sandwich Method Fundamental of ELISA
- The principle of the sandwich method is that the antibody-antigen specificity will bind. It is divided into double-antibody sandwich method and double-antigen sandwich method, which are used to detect antigen and antibody respectively.
- Basic method: After the coated antibody is adsorbed on the surface of the ELISA plate, the sample to be tested is added, then the detection antibody and the enzyme-labeled secondary antibody are added respectively, and finally the color development is observed by adding the substrate. The depth of the color presented in the ELISA plate is positively correlated with the concentration of the analyte.
- The origin of the name of the double antibody sandwich method is that the antigen to be detected is coated between the capture antibody and the detection antibody on the well.
- Applicable conditions: It is a macromolecular substance containing multiple antigenic epitopes.
- Necessary reagents required for the double-antibody sandwich method: solid-phase antigen or antibody, enzyme-labeled antigen or antibody, and enzyme-acting substrate.
- Result: A colored hydrolyzate will be produced.
The Operation of Sandwich Method
- Firstly, coat the specific antibody in the wells of the ELISA plate, and the specially treated material of the ELISA plate can adsorb the antibody on its surface. Unadsorbed excess antibody is then washed with buffer.
- Then add the sample to be tested, make it contact with the antibody on the well plate and react for a period of time under a suitable temperature environment. This is to allow the antigen in the specimen to combine with the antibody on the ELISA plate to form a solid-phase antigen complex. Unbound material is then removed by washing.
- Add another antibody specific for the antigen. Stay for a suitable time to allow it to bind to the antigen to be tested in the ELISA plate, and then wash the unbound antibody.
- The next step is to add an antibody with the enzyme to bind the complexes in the previous ELISA plate. Wash away unbound excess enzyme-labeled antibody.
- Now add the substrate. Enzymes in sandwich complexes catalyze substrates to produce colored products. The results of this antigen are read according to the color results.
- The same operation method are used for the determination of antibodies in specimens by the double-antigen sandwich method.
ELISA method is one of the most mature immunoassay methods at present. It is widely used and has made contributions in medical experiments, clinical diagnosis and biopharmaceuticals. Therefore, it is necessary to select a high-quality ELISA plate for the experiment to be carried out in the application. If you have consumable-related needs, Hawach, who has 96 well elisa strip plates with constant praise, hopes to be your partner.