ELISA high background or no signal: causes and fixes

Your standards and blanks already say which half of the assay failed. Look at them before repeating the run.

• Too little washing. Residual conjugate is the commonest cause. Fill wells completely, soak briefly if the manual allows, and tap the plate dry on clean paper after the last wash.
• A blocked washer pin. One clogged channel gives one streak of high wells. Test the washer with water and check every channel dispenses.
• Too much detection antibody or conjugate. Recheck the dilution. Do not reuse a working solution from an earlier run.
• Substrate problems. TMB should be almost colourless before use. If it is already blue, it has been contaminated or exposed to light.
• Incubating too long or too warm. Keep to the stated time and temperature, and read soon after adding stop solution.
• Carry-over. Use fresh plate sealers at each step and a fresh tip for each reagent. A trace of HRP on a tip is enough to colour a substrate reservoir.

• A step was missed or done out of order. Work through the manual with a tick sheet on the repeat run.
• Sodium azide in a buffer. Azide inhibits HRP, so azide-preserved wash or sample buffers can abolish the signal.
• Reagents not at room temperature. Cold reagents slow binding and colour development. Bring them up for about 30 minutes first.
• Degraded standard or conjugate. Reconstitute as directed, aliquot, and avoid repeated freeze-thaw. Check expiry and storage.
• Wrong wavelength. TMB with an acid stop reads at 450 nm. A reference at 570 or 630 nm is optional and corrects for plate imperfections.
• Wrong species or sample type. A kit only measures what it was validated for. See how the formats differ.

• Duplicates. Pre-wet tips, use the same technique for every well and remove bubbles before reading. Mix each sample before pipetting.
• Standard curve. Mix every step of the dilution series thoroughly and use a fresh tip at each step, since an error at the top carries through all the points.
• Curve fit. Immunoassay curves are sigmoidal. A four-parameter logistic fit usually describes them better than a straight line.
• Edge effects. Bring the plate and reagents to temperature first, seal it well and avoid stacking plates in the incubator.

A sandwich ELISA can also show a high-dose hook: at extreme concentrations the target saturates both antibodies and the reading falls. If a sample reads lower than expected, rerun it at a higher dilution and see whether the calculated concentration goes up.

If controls pass and your samples still fail, the kit may not suit the sample type or species. Send us the target, species and sample and we can check validated options in the ELISA kit range.