Making the right choice for your ELISA kit is made simple by comparing all the parameters in our easy-to-follow guide.

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Choose the right ELISA kit for your sample

​Careful evaluation of assay performance is an important first step in choosing a new ELISA kit. Important parameters include sensitivity, dynamic range, and precision which are reported for most ELISA kits. Other parameters are more predictive of ELISA performance for typical sample types. Percent (%) recovery and linearity of dilution measure the target protein in real samples such as plasma, serum or cell culture media.

This short guide explains how to interpret the different parameters to help you find the right ELISA kit for your sample.

Summary of parameters to look for when choosing an ELISA kit

* it is important to know what levels of the target protein are expected in a sample to check that the sensitivity and dynamic range of a given ELISA are appropriate. Samples with a high concentration of the target protein can be diluted so that the raw signal falls within the dynamic range of the assay.

Sensitivity

Sensitivity is the lowest level of protein that the antibody pair used in the ELISA kit can accurately detect.

Dynamic range

The dynamic range is defined as the upper and lower concentrations of the target protein that the assay can accurately quantify.

Reported values for sensitivity and dynamic range can be misleading because they are often determined by using standard protein in simple buffers. This may not represent the kinetic of detection of an endogenous protein in a biological sample.

CV

The CV (%) or coefficient of variability shows how consistent the assay is.

CV is generally calculated to evaluate the inter-assay or plate-to-plate consistency and the intra-assay precision or consistency between duplicates run in the same experiment.

• Inter-assay % CVs of less than 15 are generally acceptable.
• Intra-assay % CVs should be less than 10.

Specificity

It is important that the antibodies used in the ELISA kit do not cross-react with non-target proteins. These can be proteins of high homology across species. For instance, Human Factor IX SimpleStep ELISA® kit (ab188393) does not react with mouse, rat, rabbit, goat, guinea pig, hamster, cow, dog or pig sample.

Percent recovery

Percent (%) recovery is determined by spiking a known amount of purified target protein into a biological sample type (also called sample matrix). For secreted proteins such as cytokines, typical sample matrices are plasma and serum; for intracellular proteins such as kinases, samples are cell culture lysates.

The spiked sample is measured in the ELISA and the concentration is calculated from the standard curve. This calculated concentration is compared to the known concentration of the protein and is expressed as a percentage. For example, 100% recovery means that the observed concentration was the same as the actual concentration of spiked protein in the sample. It suggests that the proteins and other molecules in the sample type did not interfere with the quantification of the protein.
If % recovery measures less than 80% for a specific sample type then a different ELISA kit should be selected for quantification.

Linearity of dilution

The linearity of dilution is a good companion to % recovery because it measures native rather than spiked protein in biological samples. The linearity of dilution is determined by measuring multiple dilutions of known positive samples by ELISA. The concentration of the target protein is determined by multiplying the dilution factor by the calculated concentration. For best results, the concentration of the samples should be similar for all dilutions. If a difference of more than 20% from the undiluted sample is observed then a different ELISA kit should be chosen for accurate quantification.

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Standard curve

The standard curve is used to determine the concentration of the sample used. A poor standard curve, such as one that is low or flat, may be indicative of an antibody that did not bind properly or did not capture the protein standard.

In general, a good standard curve should have an R2 value above 0.99, a blank well optical density lower than 0.25, and a maximum absorbance value higher than 0.8.

Biological samples

Testing an ELISA with a biological sample ensures sensitivity to the endogenous protein, not just the recombinant protein.

Species cross-reactivity

Species cross-reactivity measures the extent to which an antibody pair binds to non-target proteins. This measure should ideally be below 5%.

International standard

Calibration to an international control enables cross-comparison between data sets. This helps you switch from one ELISA supplier to another one for the same ELISA kit.