Considerations for ELISA kit selection

Ready-to-use ELISA kits are often preferred to developing an ELISA in-house since they can offer faster time to results, more consistent performance, and uninterrupted long-term supply. When selecting an ELISA kit for your analyte of interest, it is important to consider factors such as the assay format, the chosen sample material, and the type of readout produced.

Four main types of ELISA are available to researchers. In a direct ELISA, the target analyte becomes bound to the microplate surface before being detected with an enzyme-labeled primary antibody. An indirect ELISA is similar, but uses both an unlabeled primary antibody and a labeled secondary antibody for detection, which provides signal amplification. During a sandwich ELISA, the microplate wells are coated with an analyte-specific antibody, then a second analyte-specific antibody (which recognizes a different epitope) is used to detect the bound target. All of these assay formats can be converted to a competitive ELISA, in which the analyte of interest competes with a labeled reference for microplate binding; the more analyte that is present in the sample, the lower the signal.

A typical sandwich ELISA begins with plate coating, whereby the capture antibody is diluted in a specialized coating buffer - usually 0.2M carbonate/bicarbonate at pH >9 - and added to the microplate wells. The coating buffer serves to stabilize the antibody, as well as maximize its adsorption to the microplate, and must be protein-free.

Following incubation, the wells are washed several times - usually with Tris-buffered saline (TBS) or phosphate-buffered saline (PBS) containing 0.05% v/v Tween-20 - to remove any unbound antibody, then a blocking solution is applied to prevent non-specific binding events. Common blocking solutions include bovine serum albumin (BSA) and normal serum.

Next, the samples are introduced into the wells, alongside any controls or reference standards. After incubation and further washing, the detection antibody is added. This is typically an antibody labeled with horseradish peroxidase (HRP), alkaline phosphatase (AP), or biotin, of which the latter necessitates a separate incubation step with a streptavidin-enzyme conjugate for detection purposes.

Finally, an appropriate enzyme substrate is added to produce a measurable readout. Common substrates include 3,3′,5,5′-Tetramethylbenzidine (TMB) for HRP-based detection, and p-Nitrophenyl Phosphate (pNPP) for AP-based detection, both of which are converted to soluble colored products that can be measured using a standard benchtop spectrophotometer.

Alternatively, a chemiluminescent substrate can be used to provide higher sensitivity, although this requires access to a dedicated reader. In recent years, ELISA has been configured to use antibodies conjugated to fluorophores, oligonucleotides, and other detection moieties, although these approaches cannot be strictly classified as enzyme-linked.

With so many off-the-shelf ELISA kits to choose from, it can be difficult to know where to start when it comes to product selection.
Factors to consider include checking that the kit has been validated for your chosen sample type, as well as confirming that the expected analyte concentration of your sample material falls within the kit’s dynamic range. It is also important to look for information about known cross-reactivities since this will help to avoid misleading results.
Commercial products should come with a detailed protocol, covering the preparation of samples, controls, reference standards, and buffers, and should always be supplied with a batch-specific certificate of analysis verifying assay performance.
For additional confidence in ELISA results, you may wish to select a kit that has been cited in the literature or that regularly features among the vendor’s best-sellers. For further guidance, speak with technical support teams for assistance with identifying a product that best meets your needs.

Preformulated instant buffer

TMB Substrates

Smith, Brendon & Strakova, Jana & King, Jennifer & Erdman Jr, John & Jr, William. (2010). Validated Sandwich ELISA for the Quantification of von Willebrand Factor in Rabbit Plasma. Biomarker Insights. 5. 10.4137/BMI.S6051.