Weak or no signal |
Enzyme conjugate was not added. |
Add Enzyme conjugate.
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Substrate Reagent was not added. |
Add Substrate.
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Very low incubation temperature or agitation. |
Control your room (lab) temperature to bring it within the recommended range (Refer to the Package Insert). Set your plate shaker to 600rpm.
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High background across the plate. Standard curve is saturated.
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Wrong conjugate dilution used. |
Dilute the conjugate at the recommended dilution.
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The assay was incubated for too long in one or all steps. |
Strictly follow the assay incubation time in all steps according to the procedure.
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The substrate is contaminated (not fresh). |
Check the color of the substrate - it should be colorless.
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High incubation temperature. |
Control your room (lab) temperature to bring it within the recommended range (Refer to the Package Insert).
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Positive signal (high background) in negative controls or standard wells.
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Contaminated Pipette tips. |
Use clean pipette tips and change tips for every sample and/or standard.
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Cross contamination has occurred between wells. |
Carefully wash wells. The wash step is very critical.
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High CV across the plate (high variation in samples / standards).
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Pipetting problem. |
When using: Single Channel Pipette: Check tips for bubbles between replicates. Multichannel Pipette: Calibrate the pipette. Check tips for bubbles before dispensing.
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Non-Uniform Washing. |
All the wells should be uniformly washed. Check your washing system and/or method. |
Non-homogeneous reagents (samples/standards) |
Vortex samples to ensure homogeneity before pipetting.
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Sample Readings are out of the assay dynamic range.
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Sample contains undetectable analysis level, i.e. below assay limit of detection (LOD).
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Sample contains high analyte level: i.e. above assay highest standard point.
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Samples should be diluted and re-assayed.
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