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Posted on Thu, Nov 15, 2018

Direct and Indirect Elisa protocol

For the indirect and direct elisa, the antigen is applied to the surface of the elisa plate, but for a capture antibody is attached directly to the surface of the plate for sandwich elisa. Aside from this major difference the indirect and elisa protocol is very similar to the sandwich elisa protocol. There are plenty of blocking and washing steps to avoid non-specific binding, and there are incubation times to allow antibodies and antigens to bind properly. The indirect elisa requires two antibodies—a primary antibody to bind to the antigen, and a secondary antibody conjugated to an enzyme or fluorophore. The direct elisa uses a primary antibody that is directly conjugated to an enzyme or fluorophore. Either way, both of these methods—and indeed every elisa protocol, is a labeled assay. The antibody-antigen binding event cannot be quantified without the presence of the enzyme or fluorophore.

Choosing plates for an elisa protocol

The most commonly used plate is a 96-well polystyrene plate. These are used because they provide many wells for performing replicate side-by-side reactions. Also, proteins passively bind to the polystyrene surface.  Additionally, the plates come as translucent plastic or as dark black plastic. The translucent plates are ideal for colorimetric detection systems that produce a color change that can be quantified by a spectrophotometer. The dark plates are useful for chemiluminescent systems where the light production is detected by a luminometer. They are also used for fluorescent systems, which are quantified by a fluorometer. An elisa can be performed with a plain uncoated 96-well plate, but many pre-coated plates are commercially available to improve binding specificity, optimize concentrations, and to decrease the overall working time.

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Some examples of pre-coated plates:

  • protein A/G
  • maleic anhydride coated plates improve binding of amino acids rich in primary amines
  • maleimide coated plates improve binding of amino acids rich in sulfydryl groups
  • Streptavidin or biotinyated plates
  • Nickel or copper chelate coatings to preferentially bind histidine-tagged proteins


Basic steps in a direct or indirect elisa protocol

  1. Optimize concentrations of antigen and antibody:
This is easy to accomplish in a 96 well plate where serial dilutions of antigen and antibody can be applied. The optimum concentrations are identified by the sensitivity of the enzyme-substrate reaction and detection.

2. Bind antigen to plate surface:
  • can use an uncoated or coated plate
  • an overnight incubation at 4°C or a 1 hour incubation at 37°C is standard

3. Wash away unbound antibody and block unbound sites on the plate
  • this prevents non-specific binding of antibodies to the plate
  • milk is a common blocking agent, but proteins such as BSA, ovalbumin, aprotinin, or others are also used. Animal serum is also a useful blocking agent, but it contains far more proteins and antibodies than single-source protein blocks.

4. Wash away block

5. Select and add antibodies and antibody labels. Incubate for at least 1 hour.
  • direct elisa: a conjugated antibody-enzyme is applied
  • indirect elisa: a primary antibody is added, followed by an extra wash and incubation with a labeled secondary antibody


6. Wash away unbound antibody

7. Apply substrate to the wells of the plate

  • Alkaline phosphatase (AP) and horseradish peroxidase(HRP) are two commony used enzymes
  • AP catalyzes the hydrolysis of phosphate groups from the substrate to produce color or light.
  • HRP catalyzes the oxidation of substrates by hydrogen peroxide to produce color or light.


8. Detect the resulting color change, fluroescence, or light production from the interaction of the substrate with the antibody-linked enzymes.

The elisa is the gold standard of immunoassays, but it is time-consuming, label-dependent, expensive, and difficult to make into a multiplex assay (testing for multiple antigens or antibodies at once). Cutting edge research in biosensing and immunoassay development is trending toward label-free assays that do away with enzyme reactions and conjugated fluorophores. These devices will be compact and will enable multiplex analysis of samples. However, until these devices hit the market, the trusty elisa will be the standard diagnostic tool in every clinical laboratory.


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