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Making the right choice for your ELISA kit is made simple by comparing all the parameters in our easy-to-follow guide.
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
|Sensitivity||Depends on the target protein*|
|Dynamic range||Depends on the target protein*|
|CV (%) for intra-assay precision||≤ 10%|
|CV (%) for inter-assay precision||≤ 15%|
|Specificity||Check reactivity with highly homologous proteins|
|% Recovery||≥ 80%|
|Linearity of dilution||≤ 15% difference from undiluted samples|
* 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 is the lowest level of protein that the antibody pair used in the ELISA kit can accurately detect.
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.
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.
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 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.