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When using an ELISA kit, you want specific, accurate, sensitive, and reproducible results. To be sure your kit can deliver this, it’s important to have access to the validation data before you make a purchase. However, the kits on the market range from having no validation to a full biological validation. Different levels of validation will influence the type of data available, so it’s important to know what you need at the start. See the table below for the validation break down.
Table 1. The types of product data available with different levels of ELISA kit validation.
|Product data||No validation||Limited validation||Full validation|
|Precision (intra/inter-assay CV)||✓||✓||✓|
|Antibody pair cross-reactivity||✓|
|Alternative species reactivity||✓|
|International standard (NIBSC/WHO)||✓|
What are these data important for my experiment?
|Product data||Why is it useful?|
|Dynamic range||Allows you to see that the kit is capable of detecting and quantifying your expected protein concentrations.|
|Sensitivity||Shows the lowest level of protein that the antibody pair is able to detect. It should always be less than the lowest point of the standard curve.|
|Precision||Shows you how consistent the assay is between runs. A good kit could have a CV < 10%.|
|Standard curve||Visual confirmation that the curve is not a low or flat curve. A poor standard curve means the antibody didn’t bind properly or doesn’t capture the protein standard. The R2 value of the trend line should be >0.99.|
|Spike-and-recovery||Tells you that a target protein is recoverable after being spiked into a matrix. Acceptable results are 80–120%.|
|Linearity||Poor linearity of dilution indicates that the natural sample matrix, the sample diluent and/or standard diluent affect protein detectability differently.|
|Biological samples||Evidence that the antibody pair detects the endogenous protein and not just the recombinant protein.|
|Species cross-reactivity||Gives you an indication of extent to which an antibody pair binds to non-target proteins. This should ideally be <5%.|
|International standard||Calibration to an international control enables cross-comparison between data sets. This helps you switch from an ELISA supplier to another one for the same ELISA kit.|
Example: Human IL-8 SimpleStep ELISA® kit
Human IL-8 ELISA kits are available from various ELISA suppliers but not all of them have been fully validated. When buying a kit, you need to carefully evaluate all of the available data.
Table 2. Comparison of different human IL-8 ELISA kits available on the market
|Product data||Competitor A ELISA kit||Competitor B ELISA kit||Competitor C ELISA kit||Abcam SimpleStep ELISA® kit (ab214030)|
|Dynamic range (pg/mL)||15.6–1,000||1–600||31.2–2,000||3.9–250|
|Assay time||4 h 30 min||5 h||3 h 30 min||90 min|