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QUICK ANSWER On a Human I-FABP/FABP2 ColorStep ELISA, the top of the standard curve looked perfect while the three lowest standards showed duplicate %CVs of 37–450% — because their optical density (OD) sat right at the blank. Near the bottom of the working range, a few milli-OD of well-to-well noise becomes a huge relative error, and dual-wavelength (OD450–620) subtraction pushed near-zero wells negative. The fix: keep the curve inside the validated 0.156–10 ng/mL range, retain single-wavelength OD450, and bring low samples up into the reliable mid-curve (run neat or concentrate). QC confirmed no kit defect. On this page The pro …

Quick answer: A researcher subtracted the blank from a competitive ELISA (a Serotonin kit) and every result turned negative. In a competitive assay the signal is inversely proportional to concentration, so the blank (zero standard) gives the highest optical density — subtracting it drives all readings below zero. Do not subtract the blank in a competitive ELISA. Blank subtraction belongs to sandwich ELISAs (like the researcher’s Zonulin kit), where signal rises with concentration and the blank is the lowest value. On this page The problem The data Why it happens Our analysis Root cause What we recommended Ke …

Quick answer On the Dog Beta-endorphin (BEP) ELISA Kit, a researcher ran the same canine serum aliquots on two plates from the same lot and saw roughly double the concentration on one plate versus the other. The standard curves on every run were valid and the lot’s QC precision was strong (inter-assay CV ≈ 6.8%), so the kit chemistry was working. The discrepancy traced to sample-level handling and an undiluted-serum matrix effect on a single run — not a kit fault. Diluting serum into the mid-range of the curve and tightening pipetting resolved it. On this page Click any section below to jump straight to it ↓ The pr …

Quick answer On the Periaxin (PRX) ELISA, a researcher found every serum sample reading above the top standard — optical density (OD) around 2.8–3.0 at 450 nm, well beyond the highest calibrator. The kit was not faulty. The samples simply contained far more analyte than a 1:6 dilution could bring into the assay’s 0.156–10 ng/mL range. The fix is to confirm the standard curve, then dilute samples enough (here, closer to 1:100–1:1000) to land them inside the linear region. On this page Click any section below to jump straight to it ↓ The problem › Snapshot of the data › Why it ha …

Quick answer On the Fatty Acid Oxidation (FAO) Assay Kit (BR00001), a researcher found that a subset of skeletal-muscle samples returned negative values once the control well was subtracted from the reaction well, while other samples on the same plate read clearly positive. The negatives were not a kit failure: they mark samples whose FAO activity sits at or below the assay's detection floor, where ordinary well-to-well optical noise tips the background-subtracted value just below zero. The fix is interpretation, not a repeat run — report these as ND (not detectable), confirm blood was washed from the tissue, and let the valid positive samples validate the plate. …

Quick answer A researcher preparing a Human IL-18 ELISA wasn't sure whether the protocol's “300-fold” HRP-Streptavidin step meant adding 40 µL of concentrate to 12 mL of diluent, or to a total of 12 mL. A fold dilution always describes the final total volume, not the diluent alone. For a 300-fold dilution you combine 1 part concentrate (40 µL) with 299 parts diluent (11,960 µL) to reach 12 mL total — so 40 µL HRP-Streptavidin + 11,960 µL Assay Diluent = 12 mL. The worked example's rounded “12 mL diluent” wording was the source of the confus …

Quick answer A competitive ELISA is running correctly when the blank (zero standard) gives the highest OD, the standard curve drops smoothly with rising concentration, and your samples land in the middle of that curve. If your interpolated values already sit comfortably inside the kit's range — for the Bovine PGE2 (PGE2) ELISA that is 15.6–1000 pg/mL — you generally do not need to dilute. Fit the standards with a 4-parameter logistic (4PL) regression and keep duplicate CVs at or below 20%. On this page The problem The data Why it happens Our analysis Root cause What we recommended Key takeaway FAQ The problem A researcher reached out before starting a new …

QUICK ANSWER If your ELISA returns sample concentrations that are orders of magnitude too high, the assay itself is usually fine — the error is almost always in data analysis. The most common culprit is entering the wrong standard concentrations when building the standard curve. In a recent porcine MAP (PigMAP) sandwich ELISA case, a researcher assigned a 5000–78 ng/mL range to standards that should have been 10–0.156 ng/mL. The optical density (OD) readings were valid; only the concentrations tied to them were wrong. Re-assigning the correct values to the same ODs brought results back into a sensible range. On this page The problem Snapshot of t …

Quick answer On the GDF-6 (Growth Differentiation Factor 6) ELISA Kit, a researcher saw an elevated blank (OD ≈ 0.41) with the two lowest standards reading at or below it, and no signal for a recombinant GDF-6 sample. The cause was a background/handling issue — most likely an over-concentrated HRP conjugate, incomplete washing, or stale wash buffer — not a fault in the antigen or kit. Fresh reagents, thorough washing, and a rebuilt low-end curve resolve it. On this page Click any section below to jump straight to it ↓ The problem › Snapshot of the data › Why it happens › Our a …