Product Name or Catalog Number :
 
 

Sandwich ELISA

        A sandwich ELISA. (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form.

        A less-common variant of this technique, called "sandwich" ELISA, is used to detect sample antigen. The steps are as follows:

  1. Prepare a surface to which a known quantity of capture antibody is bound.
  2. Block any non specific binding sites on the surface.
  3. Apply the antigen-containing sample to the plate.
  4. Wash the plate, so that unbound antigen is removed.
  5. Apply primary antibodies that bind specifically to the antigen.
  6. Apply enzyme-linked secondary antibodies which are specific to the primary antibodies.
  7. Wash the plate, so that the unbound antibody-enzyme conjugates are removed.
  8. Apply a chemical which is converted by the enzyme into a color or fluorescent or electrochemical signal.
  9. Measure the absorbance or fluorescence or electrochemical signal (e.g., current) of the plate wells to determine the presence and quantity of antigen.

        The image to the right includes the use of a secondary antibody conjugated to an enzyme, though technically this is not necessary if the primary antibody is conjugated to an enzyme. However, use of a secondary-antibody conjugate avoids the expensive process of creating enzyme-linked antibodies for every antigen one might want to detect. By using an enzyme-linked antibody that binds the Fc region of other antibodies, this same enzyme-linked antibody can be used in a variety of situations. The major advantage of a sandwich ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present. Without the first layer of "capture" antibody, any proteins in the sample (including serum proteins) may competitively adsorb to the plate surface, lowering the quantity of antigen immobilized.