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”.

Types of ELISA

Frequently there are 3 types of ELISA on the basis of binding structure between the Antibody and Antigen.

  1. Indirect ELISA
  2. Sandwich ELISA
  3. Competitive ELISA

1. Indirect ELISA

Antibody can be detected or quantitatively determined by indirect ELISA. In this technique, antigen is coated on the microtiter well. Serum or some other sample containing primary antibody is added to the microtiter well and allowed to react with the coated antigen. Any free primary antibody is washed away and the bound antibody to the antigen is detected by adding an enzyme conjugated secondary antibody that binds to the primary antibody. Unbound secondary antibody is then washed away and a specific substrate for the enzyme is added. Enzyme hydrolyzes the substrate to form colored products. The amount of colored end product is measured by spectrophotometric plate readers that can measure the absorbance of all the wells of 96-well plate.

Procedure of Indirect ELISA

  1. Coat the micro titer plate wells with antigen.
  2. Block all unbound sites to prevent false positive results.
  3. Add sample containing antibody (e.g. rabbit monoclonal antibody) to the wells and incubate the plate at 37°c.
  4. Wash the plate, so that unbound antibody is removed.
  5. Add secondary antibody conjugated to an enzyme (e.g. anti- mouse IgG).
  6. Wash the plate, so that unbound enzyme-linked antibodies are removed.
  7. Add substrate which is converted by the enzyme to produce a colored product.
  8. Reaction of a substrate with the enzyme to produce a colored product.

2. Sandwich ELISA

Antigen can be detected by sandwich ELISA. In this technique, antibody is coated on the microtiter well. A sample containing antigen is added to the well and allowed to react with the antibody attached to the well, forming antigen-antibody complex. After the well is washed, a second enzyme-linked antibody specific for a different epitope on the antigen is added and allowed to react with the bound antigen. Then after unbound secondary antibody is removed by washing. Finally substrate is added to the plate which is hydrolyzed by enzyme to form colored products.

Procedure of sandwich ELISA

  1. Prepare a surface to which a known quantity of antibody is bound.
  2. Add the antigen-containing sample to the plate and incubate the plate at 37°c.
  3. Wash the plate, so that unbound antigen is removed.
  4. Add the enzyme-linked antibodies which are also specific to the antigen and then incubate at 37°c.
  5. Wash the plate, so that unbound enzyme-linked antibodies are removed.
  6. Add substrate which is converted by the enzyme to produce a colored product.
  7. Reaction of a substrate with the enzyme to produce a colored product.

3. Competitive ELISA

This test is used to measure the concentration of an antigen in a sample.

In this test, antibody is first incubated in solution with a sample containing antigen. The antigen-antibody mixture is then added to the microtitre well which is coated with antigen. The more the antigen present in the sample, the less free antibody will be available to bind to the antigen-coated well. After the well is washed, enzyme conjugated secondary antibody specific for isotype of the primary antibody is added to determine the amount of primary antibody bound to the well. The higher the concentration of antigen in the sample, the lower the absorbance.

Procedure

  1. Antibody is incubated with sample containing antigen.
  2. Antigen-antibody complex are added to the microtitre well which are pre-coated with the antigen.
  3. Wash the plate to remove unbound antibody.
  4. Enzyme linked secondary antibody which is specific to the primary antibody is added.
  5. Wash the plate, so that unbound enzyme-linked antibodies are removed.
  6. Add substrate which is converted by the enzyme into a fluorescent signal.

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