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Optimizing the ELISA (Enzyme-Linked Immunosorbent Assay) Test

How to Best Design an ELISA Test

The design and development of an ELISA assay depends on the various components, including capture antibody, detection antibody, blocking buffer, diluent, and sample concentration, just to name a few. One must first choose which format of ELISA they want to use and then develop the protocol. Systematically adjusting and testing the various components is critical to obtaining reliable ELISA results.

CST has developed, produced, and thoroughly validated in-house ELISA products to ensure the highest possible product quality and support. CST buffers are optimized to be used with other kit components as well as any CST ELISA antibody pair, and they are sold individually as well as in kits.

Pathscan® and FastScan™ ELISA kits are available for a wide selection of antibody assays measuring important cellular signaling nodes and other cellular target proteins, including phosphorylated forms. FastScan™ ELISA kits incorporate a single-wash assay protocol, which reduces hands-on time, assay complexity, and lets you detect targets in as little as 90 minutes.

While CST provides researchers with an extensive portfolio of validated and optimized ELISA kits, there are a number of considerations to take into account when designing your own ELISA.

How to Choose the Right Antibody

The choice of antibody pairs is critical for ELISA and, therefore, should be assessed for its specificity and sensitivity.

Choosing between monoclonal or polyclonal requires the following points to be considered:

  • Monoclonal antibodies exhibit the highest degree of specificity, because they are specific to a single epitope and, therefore, are less likely to bind, nonspecifically to other antigens. Since they are clonal there should not be supply issues associated with these antibodies.
  • Polyclonal antibodies yield more robust signals because they are able to bind at multiple epitopes. There can be sourcing issues so, for larger projects, it is advisable to check availability. Reactivity for polyclonal antibodies can change over time, so long-term standardization is important.


CST provides a comprehensive catalog of validated antibody pairs to ensure optimal ELISA results.

Determine the Best Antibody Concentration for an ELISA Test

Proper titration of antibody concentration is achieved by serial dilutions of capture and detection antibodies. These are compared to proper controls to determine the dynamic range of detection as well as the presence of any artifacts. The ideal concentration would provide the highest signal-to-noise ratio and largest linear range for the assay.

Increase Signal to Background

To reduce background noise or increase the signal in an ELISA assay, a number of factors should be considered:

  • Desired assay sensitivity
  • Specificity of the antibody
  • Detection modality


Additionally, one may need to optimize:

  • Incubation time and temperature
  • Blocking buffers
  • Washing steps, such as number of washes


How to Choose the Right Buffers for an ELISA Test

The following factors should be considered in order to have the best impact on well-to-well consistency and improve the signal over background noise:

  • Coating buffers - used to preserve the biological activity and stability of any proteins; however, this buffer must not change the epitopes or interfere with binding.
  • Blocking buffers - concentration of nonionic detergent and/or nonspecific proteins of the buffer should be optimized for binding to open sites on the coated plate.
  • Washing buffers - the correct amount of salts and detergents should be at the optimal pH to reduce background noise and stabilize the antibody-antigen complexes.
  • Lysis/binding buffers - the lysis buffer should be optimized so as to lyse the cells without interfering with the antigen/antibody binding.


Importance of Well-to-Well Consistency

In order to obtain reliable results, all wells in the ELISA test should be subject to the same considerations. To further avoid well-to-well variability, pipetting of the sample and washing should be performed accurately and consistently. Use of multichannel or automated pipetting and wash systems may improve results. Additionally, attention should be paid to the well plate conditions, including temperature and humidity.