Sandwich ELISA (Protocol)

Sandwich ELISA process

Sandwich ELISA is used for detection of antigen. In this test, known antibody is coated and immobilized onto wells of microtiter plates. Test sample containing suspected antigen is added to wells and is allowed to react with antibodies in wells after which washing is done.


After washing the wells, a second enzyme-conjugated antibody specific for a different epitope of antigen is added and allowed to incubate. After removing any free secondary antibody by rewashing, specific substrate is added, and ensuing chromogenic reaction is measured.


The chromogenic reaction is then compared with a standard curve to determine exact amount of antigen present in test sample. In a positive test, an enzyme acts on substrate to produce a color, and its intensity can be measured by spectrophotometer or ELISA reader. Change of color can also be observed by the naked eye.


Sandwich ELISA protocol

Steps/ Process:

  1. Coating of surface (multi-well plate or a mobile surface such as a magnetic bead) with capture antibody.
  • Washing off unbound antibody after incubation to increase assay sensitivity


  1. Blocking of unbound protein binding sides on surface to reduce background and nonspecific binding.



  1. Adding sample containing target antigen and incubate to allow binding of target antigens to immobilized capture antibodies.
  • Washing of well to remove unbound antigen.



  1. Adding detection antibody-enzyme conjugate to bind antigen- capture antibody complex.
  • Incubate and washing


  1. Detection by addition of substrate and measuring any chromogenic, chemiluminescent, or fluorescent readout from enzyme-substrate interaction.


If magnetic beads are used as solid support system they may be collected using magnetic separation prior to detection in order to improve signal intensity.


Sandwich ELISA Protocol 


The common procedure for Sandwich ELISA is outlined as follows:

  1. Preparation of surface to which a known quantity of capture antibody is bound.
  2. Blocking of any nonspecific binding sites on surface.
  3. Addition of antigen-containing sample to the wells.
  4. Washing of well, so that unbound antigen is removed.
  5. Addition of specific antibody which binds to antigen (sandwich: Ag is stuck between two antibodies)
  6. Addition of enzyme-linked secondary antibodies as detection antibodies that also bind specifically to antibody’s Fc region (non-specific).
  7. Washing of the plate so that unbound antibody-enzyme conjugates are removed.
  8. Addition of substrate that is converted by enzyme into a color or fluorescent or electrochemical signal.
  9. Measuring absorbance or fluorescence or electrochemical signal (e.g. current) of plate wells to determine presence and quantity of antigen.


Download Link: Sandwich ELISA protocol.pdf

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