ELISA Principle Basis and Extension
Enzyme-linked Immunosorbent Assays (ELISAs) combine the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily-assayed enzyme. ELISAs can provide a useful measurement of antigen or antibody concentration. There are two main variations on this method: The ELISA can be used to detect the presence of antigens that are recognized by an antibody or it can be used to test for antibodies that recognize an antigen. A general ELISA is a five-step procedure: 1) coat the microtiter plate wells with antigen; 2) block all unbound sites to prevent false positive results; 3) add primary antibody (e.g. rabbit monoclonal antibody) to the wells; 4) add secondary antibody conjugated to an enzyme (e.g. anti-mouse IgG); 5) reaction of a substrate with the enzyme to produce a colored product, thus indicating a positive reaction. There are many different types of ELISAs. One of the most common types of ELISA is “sandwich ELISA“.
A sandwich ELISA schematic principle
1. To coat the ELISA platewith diluted capture antibody and incubate overnight at 4°C.
2. Wash the plate wells with ddH2O, wash with PBS-Triton twice.
3. Block non-specific binding using 1% BSA/PBS and incubate for 30-60 minutes at RT.
4. Wash plate. Add standards and 100ul of diluted samples to appropriate wells.
5. Incubate for 1 hour at RT. Wash.
6. Add 100ul appropriate dilution of the secondary antibody conjugated with Alkaline Phosphatase (AP) or Horseradish Peroxidase (HRP) and incubate for 1 hour. Wash.
7. Add 100ul of substrate to well and incubate at RT for 1 hour. (Add stopping solution)
8. Read plates on an ELISA microplate reader.