Elisa Troubleshooting -Technical Issues

Addressing Technical Issues

Overcoming non-specific binding in an assay:

• Non-specific binding is going to be an issue in many assay formats, especially those utilizing complex biological samples. Surmodics Protein Stabilizers, Blockers & Diluents help to cut back non-specific binding while maintaining and even increasing signal-to-noise ratios. 

• Poster: Novel Formulation for Eliminating Non-Specific Binding to Microspheres Luminex Assays and Other IVD Applications
• Learn more about the Pillars of Non-Specific Binding

Overcoming matrix interference in an assay:

• Matrix interferences can include HAMA (Human Anti-Mouse Antibodies), RF (Rheumatoid Factor) interference, non-specific binding and more. Surmodics Diluents help to significantly reduce matrix interferences and in turn, reduce the risk for false positives.

• White Paper: Reducing Immunoassay Interferences through Innovative Blocking Technology
• Learn more about What is a Diluent

Addressing lot-to-lot inconsistency in an assay:

• Lot-to-Lot inconsistencies can be a very common issue for assay developers that can directly lead to false positives or false negatives. Surmodics’ dedication to quality is demonstrated by the ISO 9001:2015 and ISO 13485:2016 certifications and helps ensure lot-to-lot consistency of our high performance reagents.

Overcoming false positives in an assay:

• 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. Surmodics Protein Stabilizers, Blockers and Diluents help to scale back cross reactivity and in turn, reduce the risk for false positives. Surmodics NEW MatrixGuard Diluent is our latest tool for reducing false positives in immunoassays.

Increasing the sensitivity and specificity of an assay:

• Sensitivity and specificity are critical measurements when determining the accuracy and reliability of an assay. Surmodics Protein Stabilizers, Blockers & Diluents help to increase the sensitivity and specificity of the assay and in turn increase the diagnostic accuracy of the assay.

Increasing the stability of an assay:

• Stability represents the shelf life of an assay. Surmodics Conjugate Stabilizers and TMB Substrates help to increase the overall shelf life of the diagnostic kit.
• Learn More About How To Determine Protein Stability

Choosing the most effective substrate for an assay:

• Detection limit, dynamic range, and reproducibility are cornerstones in the development of a successful immunoassay application. Surmodics’ portfolio of BioFX™ colorimetric and chemiluminescent substrates offers the stability, low background and sensitivity needed to meet the demands of assay manufacturers.

• Poster: Detection, Detection, Detection: Considerations for Enzyme/Substrate Selection in Immunoassay Development
• White Paper: Substrate Selection in Immunoassay Development

Immobilizing biological molecules to diagnostic surfaces:

• Modifying the functionality of a diagnostic surface can provide efficient attachment of the biological assay component and reduce non-specific background for improved sensitivity. Surmodics TRIDIA™ surface coatings optimize DNA, RNA, protein, and cell attachment for molecular diagnostic/microarray and point of care device applications, reducing non-specific background and improving sensitivity.

• Poster: An Assay Developer's Tool to Enhance Diagnostic Surfaces and Improve Assay Performance

Choosing the most effective antigen/antibody for an assay:

• During the optimization process, antibody/antigen systems are carefully chosen to provide the specificity and sensitivity of the desired analyte measurement.  Surmodics’ supply of DIARECT™ antibodies and recombinant and native antigens have a proven track record for quality and sensitivity in ELISA, Western Blot and lateral flow applications.

Learn More about ELISA Reagents

ELISA Troubleshooting Guide

Further Optimization and ELISA Troubleshooting Tips:

Some of the most common troubleshooting areas for ELISA kits include:

Weak Signal

• There are many reasons as to why to you may be experiencing low signal in your ELISA, including poor protein to surface binding, poor stability of the dried surface protein and insufficient reagent titers. When looking at the signal-to-noise ratios, it is important to understand the ways in which you can increase the sensitivity of your assay. Surmodics IVD offers several reagents that are designed to increase the signal-to-noise ratios of immunoassays, including ELISAs. 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.
• 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.
• Ensure you are using an ELISA plate, not a tissue culture plate.
• Visit the link below to contact us to request free your samples.
• Learn more about how immunoassay tests work.

High Signal

• If you are experiencing a high signal, this might occur for a few reasons. Some of the most common reasons you 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.
• Furthermore, if you are experiencing too much signal, make sure that you 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 Surmodics NovaStop Stop Solution (NSTP) that has the ability to maintain signal over time once stopped.
• Ensure wells are washed with an ELISA plate washer.
• Additionally, if the blocking buffer is ineffective, try different blocking buffer reagents. Learn more about Surmodics’ Blocking Reagents.
• You may also find that a high signal is a result of a soiled plate. In this case, simply clean the bottom of the plate.
• Learn more about TMB substrates and Stop Solutions

Out of Range

• If you are receiving a result that is out of range, this may also be due to insufficient washing. 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 your reagents and samples. This can lead to the loss of data because you might not be able to see your results after the dilution.
• 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.
• Make sure the detection reagent is not reduced by including a sample that the assay can detect within the positive control.
• If the substrate incubation temperature is too low or the incubation time too short, ensure the incubations are carried out at correct temperatures.
• For no signal, switch to a more sensitive assay type like a sandwich ELISA.
• At the end of every washing step, invert the plate on tissue to allow it to completely drain. Tap to remove any residual fluid.

Learn more about Surmodics’ sample diluents and wash buffers.

High Variation

• Determining the optimal captured protein coating concentration should first be addressed. In addition, focusing on captured protein stability and sufficient blocking can reduce variation within an ELISA. Additional considerations to reduce plate to plate variation include: consistent assay incubation temperature, incubation times, and shaker speed.
• High variation in an ELISA assay can be a very common issue. If this issue arises, it might be due to issues you had while you were preparing your reagents and You might have had mixing errors or pipetting errors while you use a fresh stock solution and the reagent may not be homogeneous. You might also not have agitated the plate enough. If you have data that has a high degree of variation, this can skew your results and make it hard to draw sufficient conclusions based on your work. Always double check calculations.
• In the event of improper calculations of standard curve dilutions, check calculations and make new standard curve dilutions.
• 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.

High Background

• You may also see a high amount of background on your 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 your results.
• For non-specific antibody binding, ensure a block step and suitable blocking buffer are being used.
• Waiting too long to read the plate after adding a stop solution may cause a high background. Read immediately after adding a stop solution.

Poor Standard Curve

• If you have a poor standard curve, you will not be able to publish your results with any degree of validity. If you don’t mix your reagents well or if the standard has degraded, this could lead to a lack of a standard curve. There may have also been pipetting errors that led to this issue on the plate.
• Ensure you are using an ELISA plate, not a tissue culture plate.

Poor replicate data in ELISA Kits

• If there is poor replication data in the ELISA kits, ensure that appropriate incubation time is being used. Make sure that all samples are incubating with an incubation temperature at the same room temperature according to the order of standard protocols. All reagents should be at room temperature from the beginning of the assay. Room temperature can typically be achieved within 1 hour on the bench. Ensure that the buffer component concentrations are the same among all experiments to avoid poor replicate data information.
• Ensure you are using an ELISA plate, not a tissue culture plate.

Lot-to-Lot Consistency:

• The lot-to-lot consistency of immunoassay reagents when building an ELISA is very important. Inconsistency can lead to false positives and false negatives. Surmodics IVD is ISO 13485:2016 and 9001:2015 certified allowing us to ensure a level of quality and lot-to-lot consistency that is unmatched. Visit the link below to learn more about our quality standards.
• Learn more about our quality standards.

Shelf Life:

• Increasing the shelf life of an ELISA or any immunoassay is an important factor to consider when building your immunoassay. Surmodics IVD’s dried protein stabilizers and blockers (immunoassay stabilizers) and in-solution protein stabilizers (conjugate stabilizers) offer assay developers outstanding long-term stability up to 2 years and help to increase the overall shelf life of the assay. Visit the link below to learn more about our protein stabilizers.
• Learn more about our protein stabilizers.

Edge effects in ELISA:

• Edge effects in an ELISA are common among microtiter plates as variation in incubation temperature could impact binding kinetics. To avoid this, 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 and reagent reservoirs during every step. Finally, examine pipetting technique to ensure that the same amount of reagent is being placed in every well.

Tips on washing and pipetting technique:

• Do not touch the reagents on the plate while using a multichannel pipette wash, as this can lead to high background on the ELISA. Furthermore, make sure that all pipettes have been calibrated appropriately, as this can lead to a poor standard curve. Finally, ensure that all pipette tips are tightly secured, as this can lead to poor consistency in the plate washing process.