Sample Values Above Assay Range (Hook Effect Excluded) (ELISA (R&D Guide))

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Sample absorbance readings exceed the top standard, reading off the curve, while the standard curve itself appears normal with proper shape and dynamic range. Common causes: 1) Sample contains analyte (cytokine, protein) at concentration above the upper limit of quantification (ULOQ) 2) Sample not diluted or dilution factor insufficient for analyte concentration Solutions: 1) Dilute samples to bring values within standard curve range (typically 1:5 to 1:100 depending on analyte) 2) Re-run samples with serial dilutions (e.g. 1:2, 1:10, 1:50) to identify optimal dilution 3) Multiply final result by dilution factor to calculate actual concentration