Sandwich ELISA

Sandwich enzyme assay

Sandwich ELISA is variant of Enzyme assay that detects presence of antigen and quantify it between two layers of antibodies. It is named so as antigen is sandwiched between two antibodies. It is sensitive and robust method which measures antigen concentration in an unknown sample.


Sandwich enzyme Assay


This assay uses two different antibodies that are reactive with different epitopes on antigen with a concentration that needs to be determined.

Either Monoclonal or polyclonal antibodies can be used as capture and detection antibodies in sandwich ELISA system. Monoclonal antibodies recognize a single epitope that allows quantification of small differences in antigen while polyclonal antibody is often used as capture antibody to pull down as much of antigen as possible.

An antigen to be measured must contain at least two antigenic epitope capable of binding to antibody, since at least two antibodies act in the sandwich..

  • A fixed quantity of one antibody is attached to a series of replicate solid supports, such as plastic microtiter multi-well plate.


  • Test solutions containing antigen at an unknown concentration are added to the wells and allowed to bind. Unbound antigen is removed by washing, and a second antibody which is linked to an enzyme is allowed to bind.


  • This second antibody-enzyme complex constitutes the indicator system of test. Antigen serves as bridge, so more antigen in test solution, more enzyme-linked antibody will bind.


  • Test solution is used in parallel with a series of standard solutions with known concentrations of antigen that serve as control and reference.


The results obtained from the standard solutions are used to construct a binding curve of the second antibody as a function of antigen concentration. The concentration of antigens can be inferred from absorbance readings of standard solutions.

Sandwich ELISA can be direct as well as indirect. In direct method antigen is detected by two antibodies i.e. capture antibody and detection antibody while indirect method involves use of secondary antibody to detect detection antibody bound to antigen immobilized by capture antibody.


Sandwich ELISA



  1.  High specificity since two antibodies are used, antigen is specifically captured & detected.
  2.  Suitable for complex samples as antigen doesn’t require purification prior to measurement.
  3.  Good flexibility and sensitivity, since both direct and indirect detection methods can be used.



  1. Antibody optimization can be difficult as cross-reactivity may occur between capture and detection antibodies.
  2. Need of standardized ELISA kit or tested antibody pair.


Download Link: Sandwich ELISA.pdf

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