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ELISA Troubleshooting Tips

ContentsProductsRelated Reading
Weak Signal, No Signal, Low Sensitivity
High Signal, Saturated Signal, High Background
Poor Replicate Data, Inconsistent Assay to Assay Results, Large Coefficient of Variation (CV)
Poor Standard Curve
Matrix Effect
Custom Services
References
FlexLISA
Lightning-Link® Antibody, Protein & Peptide Labeling Kits
Nanoparticles Colloidal GOLD and Innovacoat GOLD
Conjugation Kits
ELISA
ELISA Principles, Formats & Streamlining
FlexLISA® Kits
Lightning-Link® Antibody, Protein & Peptide Labeling Kits
Check&Go! Conjugation Validation Kits

ELISA is a well know term that is an abbreviation of Enzyme Linked Immunosorbent Assay. This microplate based technique relies on the use of an antibody that has been linked to an enzyme. In the presence of an appropriate substrate, enzymatic activity produces a color change as the ELISA readout, which can be measured and provides information about the presence and quantity of the target antigen in the sample material.

Although ELISAs were originally developed to exploit enzymatic activity, as assay technologies have evolved, alternative readouts such as fluorescence and chemiluminescence have been developed. As a result, the term ELISA is now broadly used to describe any microplate based immunoassay in which a molecule has been adsorbed on to a solid surface.

Although the ELISA technique is unique, there are several steps during the experimental process where issues may occur. The most commonly encountered problems are detailed here, along with potential solutions. Please note that the following, although comprehensive, is not exhaustive:

Problem: Weak Signal, No Signal, Low Sensitivity

Possible CausesSolution
Coating buffer contains blocking protein• Ensure that no blocking protein is in the coating buffer (e.g. BSA)
Incorrect preparation of sample• Ensure correct sample preparation / buffer / dilution
• Sample may be incompatible with microplate assay format
Poor binding of capture antibody to microplate


• Ensure that a high binding microplate is used
• Try a different coating buffer
• Increase the incubation time for the coating step
Incomplete adsorption of target analyte to microplate• Perform covalent linkage of target to microplate
Target antigen is below the limits of detection of the ELISA / insufficient amount of target• Decrease dilution factor of samples
• Use more concentrated samples
Sample type is incompatible (e.g. cell extract vs. serum)• Detection in untested sample types may be reduced / absent
• Include a positive control i.e. a sample that the assay is known to detect
Incompatible pairing of capture antibody and detection antibody• Ensure that the capture and detection antibodies do not recognize the same epitope (unless the target is a multimer) (FlexLISA® Kits; Lightning-Link® Antibody, Protein & Peptide Labeling Kits)
• Try different antibody pairings
Incompatible / interference from buffers or sample preparations • Incompatible / interference from buffers or sample preparations
• Ensure compatibility of assay buffer with target antigen (e.g. retainment of enzymatic activity / protein interactions if appropriate)
• Confirm that the reagents contain no interfering additives, e.g. sodium azide inhibits HRP, EDTA inhibits a number of enzymes when used as a protease inhibitor or blood / plasma anticoagulant
Epitope recognition is impeded by adsorption to plate• Conjugate the target molecule to a large carrier protein prior to adsorption onto the microplate to enhance detection of the target molecule by direct or indirect ELISA (FlexLISA® Kits; Lightning-Link® Antibody, Protein & Peptide Labeling Kits)
Incorrect amount of capture antibody used• Try different concentrations / dilutions of capture antibody
Incorrect amount of detection antibody used• Use the detection antibody at the optimized solution
Insufficient amount of substrate / detection reagent used• Increase the concentration / amount of substrate / detection reagent according to manufacturer’s guidelines (FlexLISA® Kits; Lightning-Link® Antibody, Protein & Peptide Labeling Kits)
Improper storage of ELISA kit / reagents• Ensure that all assay components are stored appropriately, taking note that different components of the kit may require different storage conditions
• Avoid repeated freeze / thaw of test samples
Microplate wells allowed to dry out• Do not let the wells become dry once the assay has started.
• Use plate seals, or wrap the plate, during incubations
Sub-optimal incubation temperature• Ensure incubation is performed at the correct temperature
• Ensure that the plate and all reagents are at room temperature / the recommended temperature before starting
• Use an incubator if room temperature fluctuates
Sub-optimal incubation time• Follow the manufacturer’s instructions
Incorrect plate reader settings / wavelength used• Confirm that the appropriate absorbance or emission / excitation wavelengths have been selected
Inappropriate detection method used• Change to a more sensitive readout, e.g. from colorimetric to fluorescent / chemiluminescent (FlexLISA® Kits; Lightning-Link® Antibody, Protein & Peptide Labeling Kits)
Interfering buffer components / enzyme inhibitors present• Confirm that the assay reagents contain no interfering additives, e.g. sodium azide inhibits HRP, phosphate inhibits AP, EDTA inhibits a number of enzymes
Vigorous plate washings• Ensure correct pressure set for the automatic plate washer used
• If plate wash is performed manually, ensure gentle pipetting of wash buffer
Poor / slow development of color for the enzymatic reaction• Prepare substrate solution immediately before use
• Ensure stock solution is not contaminated or has expired
• Incubate for longer
Substrate / detection reagent not working• Check that the substrate is correct for the enzyme
• Ensure that the reporter enzyme / fluorophore is working and has the expected activity (FlexLISA® Kits; Lightning-Link® Antibody, Protein & Peptide Labeling Kits)
Insensitive assay format• Change to a more sensitive detection readout, e.g. from colorimetric to fluorescent / chemiluminescent
• Change to a more sensitive assay type. e.g. direct ELISA to sandwich ELISA
• Increase incubation times / increase temperature
Use of components from different kits• Do not use / mix components from different kits

Problem: High Signal, Saturated Signal, High Background

Possible CausesSolution
Inadequate washing / wells not washed equally / thoroughly• Wash as per the protocol / manufacturer’s recommendations
• Increase the number and / or duration of washes
• Add detergent to the wash buffer
• Check that the plate washer nozzles are not obstructed
Inefficient blocking• Try a different blocking reagent
• Increase the concentration of blocking reagent
• Add blocking reagent to the wash buffer
Contaminated wash buffer• Prepare fresh wash buffer
Addition of the substrate results in a precipitate in the wells• Reduce the concentration of the substrate
• Increase sample dilution factor
Prolonged incubation times• Use the incubation times which were optimized during assay development or as stated in the manufacturer’s recommended protocol
• Consider overnight incubation at 4°C
Incorrect amount of detection antibody used (i.e. too high)• Use the detection antibody at the optimized or recommended dilution / concentration
• Titrate the antibody / perform antibody titration assay to determine optimal concentration
Plate seals not used• Use plate seals, or wrap the plate during all incubations to prevent evaporation and subsequent concentration of reagents
Samples diluted incorrectly • Ensure that the samples have been diluted appropriately
Incubation / wash buffer salt concentration• Nonspecific and / or off target interactions can be reduced by increasing the salt concentration
Incorrect plate reader settings / wavelength used• Confirm that the appropriate absorbance or emission / excitation wavelengths have been selected
Concentration of sample is too high• Use a higher sample dilution factor
• Perform a titration assay to determine the optimal sample dilution
Excessive quantities of substrate• Reduce the concentration / amount of substrate
• Use as per manufacturer’s recommendations (NB: A substrate provided in the ELISA kit may require further dilution)
Substrate changed color prior to use• Do not prepare substrate solution until immediately before use
Too long before reading assay plate after substrate addition / too long before adding stop solution• Read the plate within 30 minutes after adding the substrate to allow sufficient color to develop in the plate
• If color develops quickly, stop the reaction by adding stop solution to the plate and immediately read the plate
• Refer to manufacturer’s recommendations
Incubation step with substrate performed in daylight• Substrate should be incubated in the dark or as per manufacturer’s recommendations
Nonspecific binding of antibody• Ensure plate wells are preblocked to prevent nonspecific binding
• Blocking buffers should contain either BSA or 5–10% normal serum (NB: If using a directly conjugated detection antibody then the serum species should be the same as the primary antibody. If using a conjugated secondary antibody, then the serum species should be the same as the secondary antibody)
• Use a blocking buffer with a different formulation

Problem: Poor Replicate Data, Inconsistent Assay to Assay Results, Large Coefficient of Variation (CV)

Possible CausesSolution
Bubbles in wells• Before reading the plate confirm that there are no bubbles in the wells
Poor binding of capture antibody to plate• Ensure that a high binding microplate is used
• Try a different coating buffer
• Increase the incubation time of the coating step
• Use a precoated microplate
Incomplete mixing of reagents• Ensure that all reagents are mixed thoroughly
Edge effects• Use plate seals, or wrap the plate during all incubations to prevent evaporation
• Do not stack plates during incubation steps
• If an incubator is used for incubations, place plates in the center of the shelves
• Ensure that all reagents are thoroughly mixed
• Pipette all reagents accurately, using calibrated pipettes and good pipetting technique
Inefficient blocking• Try a different blocking reagent
• Increase the concentration of blocking reagent
• Add blocking reagent to the wash buffer
Sample preparation / storage is inconsistent• Ensure optimal sample storage conditions
• Ensure consistent sample preparation
• Minimize freeze / thaw cycles
Contamination between wells• Ensure no carryover when pipetting
• Ensure clean pipette tips / good pipetting technique
• Ensure plate sealers are not contaminating wells
Poor pipetting technique• Pipette all reagents accurately, using calibrated pipettes and good pipetting technique
Wells not washed equally / thoroughly• Check that the plate washer nozzles are not obstructed
• Wash wells as recommended
Use of components from different kits• Do not use / mix components from different kits

Problem: Poor Standard Curve

Possible CausesSolution
Incorrect reconstitution of standard reagent• Check that the correct buffer was used to reconstitute the standard
• Briefly spin vial before opening to loosen standard
• Ensure that the standard was reconstituted in the correct volume
• Confirm that no undissolved standard was left following reconstitution
Inappropriate storage of standard / standard degradation• Ensure that the standard has been stored correctly, according to the manufacturer’s instructions
• If the standard is stored at sub-zero temperature, produce aliquots to avoid repeated freeze / thaw cycles
Poor pipetting technique• Pipette all reagents accurately, using calibrated pipettes and good pipetting technique
Standard curve does not fit scale• Plot curve using different scales, e.g. log:log, 5-parameter logistic curve fit

Matrix Effect

Occasionally, the ELISA quantification of plasma and serum samples can experience problems caused by a phenomenon known as the ‘matrix effect’, which results in reduced affinity of the antibody for the target protein leading to erroneous sample readings. The matrix effect can arise from interactions between your target protein / analyte of interest and other components in the assay. Although not an exhaustive list, the following are examples that are likely to Possible Causes a matrix effect:

  • Interaction between endogenous biological components (e.g. phospholipids, carbohydrates and endogenous metabolites, such as bilirubin)
  • Interaction between the analyte of interest and the matrix (e.g. covalent binding to plasma proteins).

Some tips to reduce potential matrix effects are detailed below:

  • Centrifugation of the sample can separate matrix components from soluble antigens, thereby partially eliminating or at least reducing the concentration of matrix components and any potential subsequent effects on the results
  • By increasing the dilution factor by 2–5 fold the overall matrix component binding will be reduced and this should mitigate the matrix effect. However, one thing to note is that when diluting the samples, you must remember to factor this in to your calculations, and also to use the same diluents as for the standard curve.

Expedeon produce a range of products to assist you with your ELISAs. Please click here to view our extensive range that can assist you further in your ELISA application.

Custom services from Expedeon

Expedeon are experts in bioconjugation, and we offer a range of custom services to assist you in the development and implementation of your ELISA. These include our:

  • Antibody Microoptimization Service
  • Custom Antibody Conjugation Service
  • Bulk Ordering
  • Inventory Control.

For further information on any of our products or services, please contact us.

References

  • Brandon, DL., Adams, LM. Milk matrix effects on antibody binding analyzed by enzyme-linked immunosorbent assay and biolayer interferometry. J Agric Food Chem. 2015 Apr 8;63(13):3593–8. doi: 10.1021/acs.jafc.5b01136. Epub 2015 Mar 30.
  • Kohl TO, Ascoli CA. Indirect Immunometric ELISA. Cold Spring Harb Protoc. 2017 May 1;2017(5):396–401. doi: 10.1101/pdb.prot093708.
  • Methods in Enzymology n.d. Accessed February 2019. Available at:http://www.sciencedirect.com/science/journal/00766879
  • Thway, T., Salimi-Moosavi, H. Evaluating the impact of matrix effects on biomarker assay sensitivity. Bioanalysis. 2014 Apr;6(8):1081–91. doi: 10.4155/bio.14.55.