ELISA is one of the most widely used immunoassay techniques due to its flexibility, sensitivity, and scalability. During ELISA development, several key factors must be optimized to ensure results are accurate. Here, we explain the relevance of some of those factors and provide tips for getting ELISA development right.
Although ELISA can adopt different formats, all of these involve the same main steps—analyte capture on the surface of microplate wells, blocking to prevent non-specific interactions, and analyte detection/quantification. In a direct ELISA, the analyte is bound to the microplate wells and detected with a target-specific reporter antibody, whereas an indirect ELISA uses a labeled secondary antibody for detection. During a sandwich ELISA, a plate-bound antibody is used for analyte capture, with detection being either direct or indirect. Any of these ELISA formats can be converted to a competitive assay, whereby the target analyte competes with a reference for binding before being detected.
Image: How three types of ELISA work. (a) Indirect ELISA: involves an immobilized antigen interacting with a sample containing the primary antibody and a labeled secondary antibody that recognizes the primary antibody. (b) Sandwich ELISA: involves an immobilized capture antibody with a biotin-labeled detection antibody “sandwiching” the bound target protein. (c) Competitive ELISA (EIA): samples and standards are spiked with biotinylated peptide where the target protein and the labeled peptide interact competitively with the capture antibody.
A broad range of samples is analyzed by ELISA, including tissue culture supernatants, cell or tissue lysates, and bodily fluids such as serum, plasma, or urine. Many of these contain a complex mixture of biomolecules in addition to the analyte of interest, which may cause unwanted background signal or target degradation. It is recommended that conditioned media samples be serum-free; where this is not possible, an uncultured media blank should always be used as a control to assess baseline signals. Buffers for preparing cell or tissue lysates should contain protease and phosphatase inhibitors, and should avoid strongly denaturing detergents, sodium azide, or high concentrations (>10mM) of reducing agents. For plasma and serum samples, whole blood should be centrifuged upon collection before being aliquoted and stored at -80°C until use.
Care is required when selecting buffers for microplate coating, blocking, diluting antibody reagents, and washing the microplate wells. The coating buffer functions to promote adsorption to the microplate surface and stabilize the bound biomolecule and must be protein-free; 0.2M carbonate/bicarbonate at pH >9 is commonly used, although PBS or TBS are viable alternatives. Blocking buffers prevent non-specific binding, and include bovine serum albumin (BSA) and various sera; BSA should be IgG- and protease-free while sera should be derived from the same host species as the labeled antibody. Antibodies are typically diluted in blocking buffer, albeit with a lower concentration of the blocking agent. Tris-buffered saline (TBS) and phosphate-buffered saline (PBS) containing 0.05% (v/v) Tween®-20 are widely used for washing. Other important reagents to consider are the antibodies—that should be validated for ELISA and demonstrate target specificity/selectivity —and any substrates required for detection.
Sandwich ELISA is the most popular ELISA format because it enhances specificity by using two different antibodies to recognize the target analyte. However, to prevent competition for target binding, it is important that each antibody recognizes a distinct epitope. Some antibody manufacturers offer matched pairs of antibodies for ELISA; where these are unavailable, antibody datasheets may provide epitope mapping. Optimizing antibody concentrations is critical and can be achieved using a checkerboard configuration; by titrating one analyte-specific antibody down the microplate and the other across (while keeping all other variables constant), it is possible to determine conditions that produce the maximal signal: noise ratio.
A standard curve is essential for quantifying the amount of target analyte present and should be included on every assay plate, ideally with each concentration being run in duplicate. The curve range can be established by spiking a high concentration of the standard into one of the microplate wells and titrating it down to define the upper and lower limits of detection; a standard curve can then be produced on each assay plate by selecting an appropriate starting concentration and dilution factor. When determining the curve range and actively testing samples, the buffer used for diluting the standard should closely match that of the sample to avoid introducing artefacts.
The more time that is invested in optimizing an ELISA, the less likely things are to go wrong. But, when they do, several common issues are easily addressed. High background can often be overcome by using a more concentrated blocking solution, extending the incubation time for blocking, or increasing the number/duration of wash steps. Weak signals may be boosted with a different coating buffer or a higher concentration of analyte-specific antibody. And tackling edge effects or plate drift may involve little more than ensuring plates are tightly sealed between reagent additions to prevent evaporation. More troubleshooting tips for running routine ELISAs can be found here.
RayBiotech offers a diverse selection of high-quality ELISA kits for detecting analytes that include soluble proteins (cytokines, growth factors, signaling molecules), transcription factors, and post-translational modifications. To learn more, visit raybiotech.com