ELISA troubleshooting tips

​​Discover practical solutions for your ELISA experiments with this useful troubleshooting guide. 

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Poor standard curve

Improper standard solutionConfirm dilutions are made correctly.
Standard improperly reconstitutedBriefly spin vial before opening; inspect for undissolved material after reconstituting.
Standard degradedStore and handle standard as recommended.
Curve doesn't fit scaleTry plotting using different scales e.g. log-log, 5 parameter logistic curve fit.
Pipetting errorUse calibrated pipettes and proper pipetting technique.

No signal

Incubation time too shortIncubate samples overnight at 4°C or follow the manufacturer guidelines.
Target present below detection limits of assayDecrease dilution factor or concentrate samples.
Incompatible sample typeDetection 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 plateTo 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 compatibilityEnsure assay buffer is compatible with target of interest (e.g. enzymatic activity retained, protein interactions retained).
Not enough detection reagentIncrease concentration or amount of detection reagent, following manufacturer guidelines.
Sample prepared incorrectlyEnsure proper sample preparation/dilution. Samples may be incompatible with microtiter plate assay format.
Insufficient antibodyTry different concentrations/dilutions of antibody.
Incubation temperature too lowEnsure 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 wavelengthVerify the wavelength and read plate again.
Plate washings too vigorousCheck and ensure correct pressure in automatic wash system. Pipette wash buffer gently if washes are done manually.
Wells dried outDo 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 reactionPrepare substrate solution immediately before use. Ensure the stock solution has not expired and is not contaminated. Allow longer incubation.

Large coefficient of variation (CV)

Bubbles in wellsEnsure no bubbles are present prior to reading plate.
Wells not washed equally/thoroughlyCheck that all ports of the plate washer are unobstructed. Wash wells as recommended.
Incomplete reagent mixingEnsure all reagents are mixed thoroughly.
Inconsistent pipettingUse calibrated pipettes and proper technique to ensure accurate pipetting.
Edge effectsEnsure the plate and all reagents are at room temperature.
Inconsistent sample preparation or storageEnsure consistent sample preparation and optimal sample storage conditions (e.g. minimize freeze/thaw cycles).

High background

Wells are insufficiently washedWash wells as per protocol recommendations.
Contaminated wash bufferPrepare fresh water buffer.
Too much detection reagentEnsure 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 buffersIncreasing salt concentrations may reduce non-specific and/or weak off-target interactions.
Waiting too long to read plate after adding stop solutionRead plate immediately after adding stop solution.
Non-specific binding of antibodyUse 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 concentrationTry different dilutions for optimal results.
Substrate incubation carried out in lightSubstrate incubations should be carried out in the dark or as recommended by manufacturer.
Precipitate formed in wells upon substrate additionIncrease dilution factor of sample or decrease concentration of substrate.
Dirty plateClean the plate bottom.

Low sensitivity


Improper storage of ELISA kitStore all reagents as recommended. Please note that all reagents may not have identical storage requirements.
Not enough targetConcentrate sample or reduce sample dilution.
Inactive detection reagentEnsure reporter enzyme/fluor has the expected activity. 
Plate reader settings incorrectEnsure plate reader is set to read the correct absorbance wavelength or excitation/emission wavelengths for fluorescent detection.
Assay format not sensitive enoughSwitch 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 plateCovalently link target to microtiter plate.
Not enough substrateAdd 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 ingredientsCheck 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 kitsAvoid mixing components from different kits.

Matrix effect

ELISA quantification of plasma and serum occasionally encounters problems which are caused by the matrix effect. The matrix effect can arise from a number of matrix components including, but not limited to: interaction between endogenous biological components such as phospholipids, carbohydrates and endogenous metabolites (bilirubin) or an interaction between the analyte of interest and the matrix, such as covalent binding to plasma proteins. This results in erroneous sample readings.

Simply diluting the samples by 2 or 5 folds reduces the matrix effect, when diluting the samples remember to use the same diluent as used for standard curve.

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