Biological Sample Not in Detectable Range (ELISA (R&D Guide))

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Standard curve appears normal, but sample wells show no signal or signal below the lowest standard. This occurs when the analyte concentration in samples is below the assay detection limit. Common causes: 1) Sample contains analyte at concentration below assay detection limit 2) Sample matrix masking or interfering with detection 3) Excessive sample dilution reducing analyte below detectable levels 4) Standard has degraded (if signal present in samples but not standards) Solutions: 1) Perform serial dilution starting with more concentrated sample (less dilution) 2) Spike sample with known analyte concentration to check for matrix interference 3) Dilute samples at least 1:2 in appropriate diluent; perform dilution series to assess recovery 4) Prepare fresh standard according to instructions; centrifuge lyophilized standard before reconstitution