Tips for ELISA

 

ELISA represents one of the most widely used antibody applications from basic research to diagnostics. This assay is the preferred method to determine the titer of an antibody but can also be successfully used to quantify antigens or analytes in a sample. Moreover, ELISA provides an economical, rapid, and highly sensitive method for screening a large number of samples, often referred to as high throughput screening (HTS). The assay is based upon an antigen-antibody interaction and subsequent enzymatic action on a substrate yielding a soluble colored product. Variations of the basic method exist for specialized applications including detection and quantification of antibodies, proteins, peptides, and even small molecules. Here, you will find some important recommendations and useful tips to achieve outstanding ELISA results.


1 Determine the best ELISA format for your purposes

  • Indirect ELISA is the method of choice for determination of antibody titers and can be used with either anti-serum, hybridoma supernatants, or purified antibody. In this assay, the observed changes in signal intensity are proportional to the amount (dilution) of the tested antibody.

  • Direct ELISA is a faster format in which the coated antigen is directly incubated with a conjugated antibody. It is commonly used for titering conjugated secondary antibodies and is very useful to estimate antigen cross-reactivity.

  • Sandwich ELISA is an ideal format to quantify soluble antigen either pure or in a complex matrix. It is usually a very sensitive antigen assay that requires a pair of specific antibodies (capturing and detecting antibodies). In this assay, one antibody is used to coat the plate and capture the antigen present in the sample solution. Then, the second specific antibody (conjugated to a reporter system) is used to detect the captured antigen. Probably the most sensitive ELISA format.

  • Competitive ELISA is an excellent approach to assess the specificity of an antibody. This assay is a slight variation of the direct and indirect formats, where the antibody is pre-incubated with pure antigen and then added to the antigen pre-coated plate. It can be also used to quantify antigens or cross-reactivity between secondary conjugated antibodies and antigens.

2 Determine the optimal reagent concentrations (before you start!)

It is highly recommended to optimize the amount of a reagent (antigen and/or antibody) in the assay by performing a checkerboard titration. This is accomplished by serial dilution of one reagent across the plate and serial dilution of the other reagent down the plate. This design permits you to analyze different concentrations of the two reagents in each well and to obtain the optimal combination of both reagents.


3 Optimizine the plate coating

It is important that the coating solution is absolutely free of detergents because competition for binding may cause low and/or uneven binding. Excessive concentrations of coating protein may actually lead to less coating. Although the coating solution doesn’t need to be a pure antigen, this shouldn’t be less than three percent of the total protein in the coating solution. Also, the sensitivity of some assays might benefit from including BSA in the coating solution.


4 Choose the best blocking buffer for genuine intensity signals and reduced backgrounds

For this purpose and to account for unspecific binding between conjugated secondary antibody and the antigen, it is also necessary to include controls of antigen coated wells incubated with the conjugate only. Efficient blocking buffers for ELISA include BSA and gelatin.


5 Optimize the amount of conjugated secondary antibodies to use

Different conjugates will provide different ranges of signal depending on how much antibody they will bind. Because the goal is to bind the entire antigen-antibody complex present in each well, you want to make sure you will use the right amount of conjugate in your assay by performing a serial dilution assay in advance (see tip #2). Always use high-quality antibody conjugates. For alkaline phosphatase conjugates use TBS as a replacement for PBS.


6 Adjust incubation times to improve the performance of your assay

In general, antigen-antibody complexes will form within two hours of incubation at room temperature but in some cases, significantly stronger specific signal might be obtained with longer incubation times. Following the addition of the substrate, it is important to read the plate within the recommended time for the specific reporter system being used. Chromogenic and chemiluminescent substrates, particularly those used by peroxidase-conjugated antibodies can saturate the signal intensity and reduce the dynamic range of the assay.


7 Always keep the conditions unmodified throughout all steps after the assay is optimized

Because ELISA is a very sensitive assay, even minor changes in buffer composition, volumes, washing times, antigen/antibody concentrations, temperature, or incubation times can have a significant impact on the performance of the assay. It is highly advisable to run all assays in replicates (duplicates or triplicates) and include controls that help to account for plate to plate variability.