What Causes High Background in ELISA Tests?

High background in an ELISA test is a common and often difficult issue for assay developers to overcome. High background in ELISA’s can be caused by several factors including, non-specific binding, contamination of samples, cross-reactivity, and more. In the following overview, the potential causes of high background in ELISA tests will be reviewed along with how assay developers can overcome high background when building immunoassays. 

Causes of High Background in ELISA Tests and How to Solve Them:

In developing an immunoassay, assay developers may experience high background which can negatively impact the accuracy and reliability of the application. High background in ELISA’s can be caused by several factors. Before understanding how best to overcome high background in an ELISA, it is important to understand the fundamentals of an ELISA Test. 

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 protein targeted. The most common ELISA application 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 Solutions & Protocols

Surmodics™ IVD provides assay developers a “one-stop-shop” for immunoassay test development, including the components needed for development of ELISA’s. This can be seen in Surmodics™ IVD’s full line of dried protein stabilizers and blockers, sample or assay diluents, in-solution protein stabilizers, ELISA substrates, stop solutions and accessory reagents, and supply of DIARECT™, now part of BBI™ Solutions’ antigens and antibodies.

Causes of High Background and Recommended Solutions: 

Now that the fundamentals of an ELISA have been reviewed, the following section will cover one of the most common obstacles in immunoassay development, high background and how assay developers can overcome high background when building an immunoassay. 

1. 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.

  • How non-specific binding most commonly occurs: 
    • 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.
    • Non-specific binding can result in a false positive and in turn, can negatively impact the proper diagnosis and treatment of disease. For immunodiagnostic assays, a false positive or false negative result can lead to a misdiagnosis of a disease or condition and improper treatment decisions. Clinicians and patients depend on the in vitro diagnostics (IVD) industry to develop high quality assays that deliver accurate results consistently.
    • With the increased use of antibody therapy, and animal-derived products being used to treat patients, the prevalence of heterophilic antibodies, human anti-mouse antibodies (HAMA), human anti-animal antibodies (HAAA), and rheumatoid factors, is becoming an increasing problem for assay developers. With immunoassays pursuing lower detection limits, multiplexing analytes, and higher sample through-put, the negative effects of heterophilic antibodies, HAMA and other matrix effects needs to be addressed. Surmodics™ IVD’s sample/assay diluents allow for the dramatic reduction of false positives in both in-house and commercially available kits, without having to sacrifice assay sensitivity.
  • How to Overcome Non-Specific Binding: 
    • Surmodics™ IVD's Protein Stabilizers, Blockers & Diluents help to cut back non-specific binding while maintaining and even increasing signal-to-noise ratios. Review the following list of Surmodics™ IVD’s immunoassay reagents designed to tackle non-specific binding in immunoassays.
    • Dried Protein Stabilizers & Blockers: Surmodics™ IVD’s dried protein stabilizers and blockers, StabilCoat™, StabilGuard™ and StabilBlock™ offer assay developers unmatched stabilization of dried proteins and blocking efficacy in a one-step process. These immunoassay reagents are designed to preserve the conformation and activity of dried proteins coated on a wide range of surfaces, keeping antibodies and antigens at peak performance for long durations. At the same time, the blocking mechanisms in these reagents reduce non-specific binding of interfering proteins to maximize assay sensitivity. Our dried protein stabilizers and blockers are the industry gold standard for stability and blocking efficacy. The result is immediate improvement of assay performance in a one-step process for streamlined manufacturing.

      Assay developers need numerous blocking mechanisms while selecting the antibody titration during assay optimization. Surmodics™ IVD’s dried protein stabilizers and blockers provide alternative blocking mechanisms to achieve the best signal-to-noise ratios for each assay. With the introduction of StabilBlock™, Surmodics™ IVD now offers the best available reagent for reducing non-specific binding to boost immunoassay signal-to-noise ratios.



  • Sample/Assay Diluents: Due to the variability within patient samples, different blocking strategies are needed to dilute samples to achieve maximum assay performance. Both MatrixGuard™ and Surmodics™ Assay Diluent (Protein-Free) provide the gold standard in reducing false positives in immunoassays. 

    MatrixGuard™ (protein-containing) and Surmodics™ Assay Diluent (protein-free) formulations provide two options to use when different methods are needed to block matrix interferences, while maintaining the clinical utility of the assay.

    For IVD kit manufacturers requiring a strong, consistent blocking diluent across a variety of assays, Surmodics™ IVD’s MatrixGuard™ Diluent provides unsurpassed blocking whereby matrix interferences are effectively blocked while the intended assay signal is maintained.

    Unlike other diluents that either are marginally effective at blocking matrix interferences or alternatively block out true assay signal, MatrixGuard™ Diluent achieves the goal of maximum blockade of matrix interferences while simultaneously allowing signal to be maintained.



2. Sample Contamination: Samples can be vulnerable to contamination in immunoassays which can result in higher background levels. 

3. Cross Reactivity: Cross reactivity is a common and troublesome issue in assay development that occurs when compounds with a similar form to the target analyte are present in the immunoassay. 

  • How to Overcome Cross Reactivity: Sensitivity and specificity are critical measurements when trying to overcome cross reactivity in an immunoassay. Selecting the capture antibody with the highest specificity to the analyte is the best way to reduce cross reactivity. Using Surmodics™ IVD’s dried protein stabilizers/blockers and sample/assay diluents can help eliminate the risk for cross reactivity by blocking the weakly-bound cross reactants, and in turn, reduce the cause of high backgrounds.

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

  • Innate Substrate Color: when a substrate has an innate color, this can cause an increase in background. Be sure to use an optimal substrate when building an immunoassay. 
  • Substrate Background: Waiting too long to read the plate after adding a stop solution may cause a high background. Read immediately after adding a stop solution.
  • How to Choose the Right Substrate: Choosing the best substrate for an assay will allow assay developers to create a robust assay with superior performance. Click the link below to review Surmodics™ IVD’s White Paper on substrate selection.

White Paper: Substrate Selection in Immunoassay Development

5. Inadequate Washing: inadequate washing can be caused by incorrect washing techniques and not using the right wash buffers

  • How to Overcome Inadequate Washing: Do not touch the reagents on the plate while using a multichannel pipette wash, as this can lead to high background on the ELISA. Moreover, make sure that all pipettes have been calibrated appropriately, as this can lead to a poor standard curve. Ensure that all pipette tips are tightly secured, as this can lead to poor consistency in the plate washing process. Finally, remove excess wash buffer from the plate by tapping on a paper towel or by aspiration for bead assays. 

6. Turbidity: turbidity can be attributed to many factors including, incompatible components, incompatibilities with the sample, occurrence of precipitation, contamination of samples/reagents, and degradation issues. Turbidity will increase the baseline backgrounds and cause incorrect readings. 

7. Poor water quality in reagents or wash buffer: the water quality of immunoassay components can be susceptible to contamination. Ensuring the quality of the water used in reagents, including the wash buffer is important when reducing the risk for high background in an ELISA. 

  • How to overcome poor water quality: Use distilled or deionized water to dilute wash buffer and reagents. Be sure to use optimal wash practices and wash buffer reagents in the development of an immunoassay. Visit the link below to learn more about Surmodics™ IVD’s recommended wash buffer protocols and products. 

Learn More

How can Surmodics™ IVD help? 

As part of Surmodics™ IVD’s mission to improve the detection and treatment of disease, Surmodics™ IVD offers assay developers the gold standard in immunoassay reagents designed to improve the sensitivity, stability and specificity of diagnostic applications. In doing so, Surmodics™ IVD helps assay developers overcome difficult technical issues such as high background in ELISA’s. 

For further assistance, please contact the team to connect directly with one of Surmodics™ IVD’s R&D scientists or visit the IVD Library to learn more about ELISA troubleshooting and optimization! 

Contact The Surmodics™ IVD Team Today