Problem 1：Poor standard curve
Cause	Solution
Improper    standard solution	Confirm    dilutions are made correctly.
Standard    improperly reconstituted	Briefly    spin vial before opening; inspect for undissolved material after reconstituting.
Standard    degraded	Store    and handle standard as recommended.
Curve    doesn't fit scale	Try    plotting using different scales e.g. log-log, 5 parameter logistic curve fit.
Pipetting    error	Use    calibrated pipettes and proper pipetting technique.

 

Problem 2：No signal
Cause	Solution
Incubation    time too short	Incubate    samples overnight at 4℃ or follow the    manufacturer guidelines.
Target    present below detection limits of assay	Decrease    dilution factor or concentrate samples.
Incompatible    sample type	Detection    may be reduced or absent in untested sample types. Include a sample that the    assay is known to detect a positive control.
Recognition of epitope impeded by adsorption to plate	To enhance detection of a peptide by direct or indirect    ELISA, conjugate peptide to a large carrier protein before coating onto the    microtiter plate.
Assay    buffer compatibility	Ensure    assay buffer is compatible with target of interest (e.g. enzymatic activity    retained, protein interactions retained).
Not    enough detection reagent	Increase    concentration or amount of detection reagent, following manufacturer    guidelines.
Sample    prepared incorrectly	Ensure    proper sample preparation/dilution. Samples may be incompatible with    microtiter plate assay format.
Insufficient    antibody	Try    different concentrations/dilutions of antibody.
Incubation    temperature too low	Ensure    the incubations are carried out at the correct temperature. All reagents    including plate should be at room temperature or as recommended by the    manufacturer before proceeding.
Incorrect    wavelength	Verify    the wavelength and read plate again.
Plate    washings too vigorous	Check    and ensure correct pressure in automatic wash system. Pipette wash buffer    gently if washes are done manually.
Wells    dried out	Do    not allow wells to become dry once the assay has started. Cover the plate    using sealing film or tape for all incubations.
Slow    color development of enzymatic reaction	Prepare    substrate solution immediately before use. Ensure the stock solution has not    expired and is not contaminated. Allow longer incubation.

 

Problem 3:Large coefficient of variation (CV)
Cause	Solution
Bubbles    in wells	Ensure    no bubbles are present prior to reading plate.
Wells    not washed equally/thoroughly	Check    that all ports of the plate washer are unobstructed. Wash wells as    recommended.
Incomplete    reagent mixing	Ensure    all reagents are mixed thoroughly.
Inconsistent    pipetting	Use    calibrated pipettes and proper technique to ensure accurate pipetting.
Edge    effects	Ensure    the plate and all reagents are at room temperature.
Inconsistent    sample preparation or storage	Ensure    consistent sample preparation and optimal sample storage conditions (e.g.    minimize freeze/thaw cycles).

 

Problem 4:High background
Cause	Solution
Wells    are insufficiently washed	Wash    wells as per protocol recommendations.
Contaminated    wash buffer	Prepare    fresh water buffer.
Too    much detection reagent	Ensure    the reagent has been diluted properly or decrease the recommended    concentration of detection reagent.
Blocking    buffer ineffective (e.g. detection reagent binds blocker; wells not    completely blocked)	Try    different blocking reagent and/or add blocking reagent to wash buffer.
Salt    concentration of incubation/wash buffers	Increasing    salt concentrations may reduce non-specific and/or weak off-target    interactions.
Waiting    too long to read plate after adding stop solution	Read    plate immediately after adding stop solution.
Non-specific    binding of antibody	Use    suitable blocking buffers e.g. BSA or 5-10% normal serum - species same as    primary antibody if using a directly conjugated detection antibody or same as    secondary if using conjugated secondary. Ensure wells are pre-processed to    prevent non-specific attachment.
High    antibody concentration	Try    different dilutions for optimal results.
Substrate    incubation carried out in light	Substrate    incubations should be carried out in the dark or as recommended by    manufacturer.
Precipitate    formed in wells upon substrate addition	Increase    dilution factor of sample or decrease concentration of substrate.
Dirty    plate	Clean    the plate bottom.

 

Problem 5:Low sensitivity
Cause	Solution
Improper    storage of ELISA kit	Store    all reagents as recommended. Please note that all reagents may not have identical    storage requirements.
Not    enough target	Concentrate    sample or reduce sample dilution.
Inactive    detection reagent	Ensure    reporter enzyme/fluor has the expected activity.
Plate    reader settings incorrect	Ensure    plate reader is set to read the correct absorbance wavelength or    excitation/emission wavelengths for fluorescent detection.
Assay    format not sensitive enough	Switch    to a more sensitive detection system (e.g. colorimeteric to    chemiluminescence/ fluorescence). Switch to a more sensitive assay type (e.g.    direct ELISA to sandwich ELISA). Lengthen incubation times or increase    temperature.
Target    poorly adsorbs to microtiter plate	Covalently    link target to microtiter plate.
Not    enough substrate	Add    more substrate.
Incompatible    sample type (e.g. serum vs. cell extract)	Detection    may be reduced or absent in untested sample types. Include a sample that the    assay is known to detect as a positive control.
Interfering    buffers or sample ingredients	Check    reagents for any interfering chemicals. For example, sodium azide in    antibodies inhibit HRP enzyme and EDTA used as anticoagulent for plasma    collection inhibits enzymatic reactions.
Mixing    or substituting reagents from different kits	Avoid    mixing components from different kits.

 

 

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