What are Some Potential Errors that can Occur in an ELISA?

There are several potential errors that can occur when building, troubleshooting, and optimizing ELISA immunoassays. It is imperative that assay developers overcome these potential errors in order to build accurate and reliable ELISA applications. The following overview will cover the most common potential errors in ELISA’s and how assay developers can overcome these challenges. 

ELISA Troubleshooting: Understanding and Analyzing ELISA Test Results 

What is an ELISA Test: 

Enzyme-linked immunosorbent assays (ELISA’s) are solid phase-based enzyme assays used for identifying and quantifying sample proteins, peptides, antigens, and antibodies that are targets in research of a specific compound. The identification of a specifically targeted protein in an ELISA array is completed by using antibodies to immobilize the protein and detect the presence of the targeted protein. The most common ELISA format is often referred to as a Sandwich assay because the measured analyte is between two antibodies. One of the antibodies serves as the capture antibody, while the other is the detection antibody. Alternative formats of the ELISA include Direct and Competitive assays. 

  • Direct ELISA: antigen coated on the solid phase detected by a conjugated antibody. 
  • Competitive ELISA: a conjugated antigen which competes with the sample antigen to bind to the capture antibody.

Learn more about ELISA’s and Surmodics™ IVD's recommended ELISA Protocols & Solutions

ELISA Troubleshooting Guide – Understanding and Overcoming Potential Errors in ELISA’s

In the following section, the most common errors that can occur in an ELISA will be reviewed as well as Surmodics™ IVD’s best practices and product recommendations for overcoming these technical obstacles. For further support, please contact the team today to connect with one of Surmodics™ IVD’s R&D scientists directly. 

Non-Specific Binding: Non-Specific binding (NSB) refers to an occurrence of an antibody binding to unintended proteins, receptors, or transporters. This binding of assay antibodies is not correlated with the specificity of the antibodies. Another source of NSB is from the sample where proteins bind to the coated antibody or to the solid phase. Moreover, attraction of a primary or secondary antibody to Fc receptors (FcRs) is considered the leading contributor to unwanted binding events. In general, when an antibody binds to unintended proteins with considerably similar epitopes, non-specificity occurs.

Weak Signal: There are many reasons as to why assay developers may be experiencing low signal in ELISA’s, including poor protein to surface binding, poor stability of the dried surface protein and insufficient reagent titers. 

  • Overcoming Weak Signal: When looking at the signal-to-noise ratios, it is important to understand the ways in which assay developers can increase the sensitivity of an immunoassay. Surmodics™ IVD offers several reagents that are designed to increase the signal-to-noise ratios of immunoassays, including ELISA’s. Essentially, these products help to reduce the background noise while maintaining and even increasing the intended signal. Surmodics™ IVD’s protein stabilizers, blockers, diluents and TMB substrates have proven to be useful tools in increasing the signal-to-noise ratios in immunoassays
  • Additional Recommendations: 
    • If a cause is related to a plate reader showing incorrect wavelengths, a solution is to ensure the plate reader is set accurately for the sample type of substrate being used during plate reading setup.
    • Be sure to use an ELISA plate, not a tissue culture plate.

High Signal: high signal may occur for a few reasons. Some of the most common reasons assay developers might have this problem include insufficient plate washing, conjugate oversaturating the wells, standard curve range is too high, adding too much detection reagent, and not stopping the reaction at the appropriate time. Having a high signal can lead to incorrect data in the form of frequent false positives and/or an incorrect standard curve. 

  • Overcoming High Signal: make sure to use an optimal TMB ELISA substrate solution. The substrate solution should be clear and colorless immediately before use with the substrate solution into the various wells. Also, make sure that the reaction is completely stopped. If the color keeps developing, the reaction has not stopped. The need to quickly read the plate can be eliminated with the use of the BioFX™ Liquid Nova-Stop Solution (NSTP) which has the ability to maintain signal over time once stopped.
  • Additional Recommendations:

High Background: Assay developers may also see a high amount of background in an ELISA assay. This can be due to the cross-reactivity of samples or contamination of the samples themselves. This could also be due to inadequate washing of the wash buffer itself, leading to false positives or false negatives, impacting the accuracy of test results.

  • Overcoming High Background: For non-specific antibody binding, ensure a block step and suitable blocking buffer are being used. 
  • Additional Recommendations:
    • Waiting too long to read the plate after adding a stop solution may cause a high background. Read immediately after adding a stop solution.

Out of Range: Getting out of range results can happen based on insufficient washing and incorrect dilution preparation. Processes that are prepared incorrectly result in insufficient washing, which might also occur due to incorrect dilution of the reagents and samples. This can lead to the loss of data as assay developers might not be able to see the results after the dilution.

  • Overcoming Out of Range Results: During substrate incubation time, cover assay plates by using a plate sealer. Use a new sealer each time the plate is opened to prevent wells from contamination.
  • Additional Recommendations
    • Make sure the assay includes a positive control and that the positive control reads within range.
    • Ensure the incubations are carried out at correct temperatures and timings.
    • At the end of every washing step, tap to remove any residual fluid.
    • Learn more about Surmodics™ IVD’s sample diluents and wash buffer.

Shelf-Life Limitations: The stability of an ELISA determines the shelf life of the assay. Assay developers should use optimal protein stabilizers to improve and extend the shelf life of an ELISA’s and other immunoassay applications.

High Variation: High variation in an ELISA assay can be a very common issue. If this issue arises, it might be due to issues had while preparing the reagents. Moreover, assay developers might have had mixing errors or pipetting errors while using a fresh stock solution and the reagent may not be homogeneous. Additionally, the plate may not have been agitated enough. If the data has a high degree of variation, this can skew test results and make it hard to draw sufficient conclusions based on the work. Always double check calculations.

  • Overcoming High Variation: In the event of improper calculations of standard curve dilutions, check calculations and make new standard curve dilutions.
  • Additional Recommendations:
    • Surmodics™ IVD’s sample or assay diluents can be great tools for addressing high variations in samples.
    • If the buffers are contaminated, use fresh buffers and use a fresh solution.
    • Ensure that no bubbles in any wells are present before reading the plate.

Matrix Interferences: Matrix interferences can include HAMA (Human Anti-Mouse Antibodies), RF (Rheumatoid Factor) interference, non-specific binding and more.

  • Overcoming Matrix Interferences: Surmodics™ IVD’s sample/assay diluents help to significantly reduce matrix interferences and in turn, reduce the risk for false positives.

False Positives: Cross reactivity is a common and troublesome issue in assay development. Sensitivity and specificity are critical measurements when determining the accuracy and reliability of an assay.

Poor Standard Curve: If a poor standard curve is present, assay developers will not be able to publish the test results with any degree of validity. If assay developers don’t mix reagents well or if the standard has degraded, this could lead to a poor standard curve. Additionally, there may have been pipetting errors that led to this issue on the plate.

  • Overcoming Poor Standard Curve: Be sure to use an ELISA plate, not a tissue culture plate.

Lot-to-Lot Inconsistencies: The lot-to-lot consistency of immunoassay reagents when building an ELISA is very important. Inconsistency can lead to false positives and false negatives.

  • Overcoming Lot-to-Lot Inconsistencies: Surmodics™ IVD is ISO 13485:2016 and 9001:2015 certified, offering assay developers a level of quality and lot-to-lot consistency that is unmatched. Visit the link below to learn more about Surmodics™ IVD’s quality standards.
  • Learn More

Edge Effects in ELISA: Edge effects in an ELISA are common among microtiter plates as variation in incubation temperature could impact binding kinetics. 

  • Overcoming Edge Effects: Use a uniform room temperature surface and do not stack ELISA plates. Transferring environments of a specific incubation temperature from the benchtop to a shaker can reduce edge effects. Ensuring the plate sealer is well sealed around the edges provides additional confidence. During incubations, cover plates with plate sealers. Use new plate sealers every time the plate is opened. Reusing plate sealers may lead to presence of residual HRP, resulting in non-specific color changes of TMB. Use fresh plate sealers, pipette tips and reagent reservoirs during every step. Finally, examine pipetting technique to ensure that the same amount of reagent is being placed in every well.

How Surmodics™ IVD Can Help: 

Surmodics™ IVD acts as a “one stop shop” for building immunoassays. Surmodics™ IVD’s immunoassay reagents are designed to increase the sensitivity, specificity, and stability of immunoassays, including ELISA’s, point-of-care devices and additional diagnostic applications. These immunoassay reagents are critical for the accuracy and reliability of immunoassays.

Surmodics™ IVD’s immunoassay reagents are the gold standard in product performance and offer assay developers a complete set of tools for building consistent and reliable immunoassays

Please review the immunoassay reagents below for further information on how Surmodics™ IVD can help assay developers optimize immunoassay applications. 

  • Dried Protein Stabilizers/Blockers (Immunoassay Stabilizers):
  • Sample/Assay Diluents: 
  • In-Solution Protein Stabilizers (Conjugate Stabilizers): 
    • Surmodics™ IVD’s StabilZyme™ Stabilizers offer assay developers the gold standard for stability of protein conjugates at working strength concentration for ELISA/EIA, ELISpot, RIA and immunoblot/western blot applications.
    • Learn More about In-Solution Protein Stabilizers
  • ELISA Substrates:
  • Stop Solutions:
    • Gold Standard BioFX™ stop solutions for colorimetric substrates are available as dry blends or in liquid formulations including Nova-Stop Solution that provides minimal drift and a safer alternative to assay developers as it is non-corrosive to both skin and eyes.
    • Learn More About Stop Solutions
  • DIARECT™ now part of BBI™ Solutions Antigens and Antibodies:
    • DIARECT™ recombinant and native antigens have a proven track record for quality and sensitivity in ELISA, Western Blot and lateral flow applications. The lot-to-lot consistency of DIARECT™ antigens reduces development time and increases productivity of immunoassay developers.
    • Learn More About Antigens and Antibodies

Visit the link below to request free samples for evaluation and/or connect directly with one of Surmodics™ IVD’s R&D scientists today!

Contact The Surmodics™ IVD Team Today