ELISA kits: choosing between sandwich, competitive and direct elisa kits, picking among human elisa kits for your matrix, and the validation evidence a supplier should publish

Reading the data sheet properly

1. Format follows the analyte. Sandwich assays need two non-competing epitopes and give the best specificity for proteins; competitive formats handle small molecules and short peptides where two antibodies cannot bind at once. The format is determined by the analyte rather than chosen for convenience.
2. Check the sample type by name. A kit validated in serum is not validated in plasma with a particular anticoagulant, in culture supernatant or in tissue lysate. Matrix effects are real and large, so confirm your exact sample type appears in the validation and, if it does not, plan a spike and recovery experiment.
3. The standard decides what the number means. Kits calibrated against recombinant standards and kits calibrated against an international reference material give different absolute values. Where results must be compared across studies or over years, prefer a kit traceable to a recognised reference material and record which was used.
4. Sensitivity, range and precision. Look for the working range rather than the lowest detectable figure, and for within-run and between-run precision at concentrations spanning that range. A sensitivity figure on its own says nothing about whether the assay is reliable where your samples sit.
5. Lot variation and bridging. Ask whether the supplier provides lot-specific standard curves and whether lots are bridged. For a study running across lots, order enough of one lot or plan a bridging experiment, because an unbridged lot change mid-study is a confound.

Research use only, and what it excludes

Most research kits are labelled for research use only, which means they are not for use in diagnostic procedures and have not been reviewed for that purpose. Using one to inform a clinical decision is outside what the labelling permits, whatever the assay's technical performance.

If results will support a regulated submission, confirm what quality system the kit was manufactured under and what documentation the supplier will provide. This varies enormously between suppliers at similar prices.

Validating a kit in your own hands

Whatever the supplier validated, run spike and recovery and a dilution linearity check in your own matrix before committing a study. These two experiments catch most matrix problems and cost one plate.

Include a quality control sample of known concentration on every plate and track it. Plate to plate drift is the most common source of unexplained variance in ELISA data and is invisible without a control.

Common questions

What is the difference between sandwich and competitive ELISA?

A sandwich assay captures the analyte between two antibodies binding different epitopes, which needs a molecule large enough to present both. A competitive assay measures displacement of a labelled analogue and suits small molecules and short peptides.

Why do two ELISA kits give different values?

Different antibody pairs recognise different epitopes and forms of the analyte, and kits are calibrated against different standards. Absolute values are therefore kit specific, which is why the kit and lot should be recorded with the data.

What does research use only mean?

The kit is labelled for laboratory research and has not been reviewed for diagnostic use, so it may not be used to inform clinical decisions regardless of how well it performs technically.

Should I validate a kit myself?

Yes, at least spike and recovery plus dilution linearity in your own matrix, and a quality control sample on every plate. Supplier validation was performed in their matrix, not yours.

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