Since COVID-19, changes in the immune system, specifically increased levels of autoantibodies such as rheumatoid factor (RF), have begun to impact diagnostic testing by increasing the potential for interference in certain immunoassays.
While the pandemic’s most visible clinical impact was respiratory disease, COVID-19 also left behind a complex immunological footprint. Even after full recovery, many individuals show signs of immune dysregulation, including elevated levels of autoantibodies – self-reactive antibodies commonly associated with autoimmune conditions.1 These changes are now being recognized as a potential source of interference in immunoassays, prompting assay developers to revisit performance considerations in the post-COVID landscape.
Among these, rheumatoid factor (RF) has been particularly notable. It is a common biomarker found in up to 80% of rheumatoid arthritis (RA) patients2 and is a known source of immunoassay interference. A 2021 study by Wang et al.3 demonstrated that RF levels increased after SARS-CoV-2 infection, even in individuals with no prior autoimmune disease. This has important implications for assay design and interpretation, particularly in maintaining reliability and clinical accuracy.
The Evidence: COVID-19 and Rising RF Levels
Respiratory viral infections have been linked to an increased risk of autoimmune inflammatory arthritis.4 While RF has generally been considered functionally similar across diseases, new evidence suggests this may not be the case. A 2024 study published in the Journal of Autoimmunity5 identified polyreactive forms of RF in COVID-19 patients, uniquely capable of binding to a wide range of protein targets, including viral antigens.
There are five immunoglobulin isotypes of RF: IgM, IgG, IgA, IgD, and IgE, each with distinct immunological roles. Among these, IgM-RF is the most commonly measured and clinically relevant type, particularly in the context of RA diagnosis and assay interference. IgM-RF, which is typically the first to appear during immune activation, is also the most strongly associated with false-positive results in immunoassays due to its ability to bind animal-derived antibodies.6 Its high avidity and pentameric structure enable it to bind multiple IgG molecules simultaneously.
Rheumatoid Factor and Fc-Mediated Assay Interference
Overall, RF is found in approximately 5–10% of the general population and in about 70% of rheumatoid arthritis patients.7 There is significant homology between the Fc domains of RF antibodies and those of several animal species, which may explain why RFs can bind to animal-derived antibodies.8,9
RF’s cross reactive properties, along with its tendency to bind nonspecifically to IgG, make it a well-recognized source of interference in immunoassays, particularly those that depend on high antibody-antigen specificity. In sandwich-based formats, RF can form false immune complexes by bridging detection and capture antibodies, mimicking a true signal even in the absence of the target antigen (refer to image 1). This mechanism can lead to false positives, misleading clinical interpretations, and reduced confidence in test results.10
IgM Rheumatoid Factor (RF) Interference
In the context of SARS-CoV-2 antibody detection, this type of interference is well documented: RF can bind to the Fc portion of surface-bound antibodies, generating nonspecific signals that may result in misdiagnosis or inaccurate assessment of immune status.11
Strategies for Mitigating Antibody-Mediated Interference in Immunoassays
As the diagnostic field adapts to the post-COVID era, it faces a range of emerging challenges—including the increasing relevance of assay interference due to post-infectious immune changes. To ensure that immunoassays remain accurate, reliable, and clinically actionable, several mitigation strategies should be considered:
- Use recombinant antibodies or Fab fragments to eliminate Fc-based binding
RF binds specifically to the Fc region of IgG. By replacing conventional antibodies with recombinant formats, such as truncated Fab fragments or Fc-free recombinant antibodies, developers can remove the primary binding site for RF and significantly reduce the risk of interference. These engineered antibodies are increasingly used in next-generation assays due to their improved consistency, reduced cross-reactivity, and better lot-to-lot reproducibility. - Incorporate interference-blocking reagents such as mouse IgG or active blockers such as human anti-mouse antibody (HAMA) and RF-blocking agents
These reagents work by competitively binding interfering antibodies – such as RF or heterophilic antibodies – before they can interact with the capture or detection antibodies in the assay. The use of animal-free blocker formulations further improves assay robustness by eliminating cross-reactivity with animal-derived proteins, reducing the risk of heterophilic antibody interference and enhancing reproducibility across diverse patient samples. - Validate assays using post-COVID sample panels
Validation protocols should now include testing with post-COVID clinical samples, especially those from individuals with long COVID or post-acute sequelae. These samples more accurately reflect the current immunological landscape and help developers identify and resolve potential sources of signal distortion that might not have been evident in pre-pandemic populations.
Meridian’s Blocking Solutions: Designed for Today’s Diagnostic Challenges
To support assay developers navigating these post-COVID challenges, Meridian Bioscience offers a range of high-performance immunoassay blockers specifically engineered to reduce interference from rheumatoid factor, HAMA, and other heterophilic antibodies. Meridian’s portfolio includes both traditional blockers—such as highly purified mouse IgG and human anti-mouse antibody blockers—as well as animal-free formulations designed to minimize variability and cross-reactivity. Meridian’s leading proprietary blocker, TRU Block™, is available in three different strengths for HAMA interference reduction. It is widely used in commercial diagnostic kits across ELISA, lateral flow, and chemiluminescent platforms, and can be easily integrated into assay development workflows. Whether you are developing new diagnostics or troubleshooting interference in existing formats, Meridian’s blocker solutions provide a reliable, scalable approach to maintaining assay accuracy in the evolving immune landscape.
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参考文献:
1. Mohan, A., et al. (2023). Navigating the post-COVID-19 immunological era: Understanding long COVID-19 and immune response. Life (Basel), 13(11), 2121.
https://doi.org/10.3390/life13112121
2. Shapiro, S. C. (2021). Biomarkers in rheumatoid arthritis. Cureus, 13(5), e15063.
https://doi.org/10.7759/cureus.15063
3. Wang, E. Y., et al. (2021). Diverse functional autoantibodies in patients with COVID-19. Nature, 595(7866), 283–288.
https://doi.org/10.1038/s41586-021-03631-y
4. Joo, Y. B., et al. (2019). Respiratory viral infections and the risk of rheumatoid arthritis. Arthritis Research & Therapy, 21(1), 199. https://doi.org/10.1186/s13075-019-1977-9
5. Shelef, M., et al. (2024). Polyreactive rheumatoid factors in COVID-19 patients. Journal of Autoimmunity, 112, 102110https://doi.org/10.1016/j.jaut.2024.102110
6. Nayeemuddin, S. N., et al. (2024). Heterophilic interference of rheumatoid factor in TSH immunometric assay: A cross-sectional observational study. Indian Journal of Endocrinology and Metabolism, 28(1), 29–34.
https://doi.org/10.4103/ijem.ijem_99_23
7. Todd, D. J., et al. (2011). Erroneous augmentation of multiplex assay measurements in patients with rheumatoid arthritis due to heterophilic binding by serum rheumatoid factor. Arthritis & Rheumatism, 63(4), 894–903.
https://doi.org/10.1002/art.30213
8. Nakamura, M., et al. (1988). Human monoclonal rheumatoid factor-like antibodies from CD5 (Leu-1)+ B cells are polyreactive. Journal of Immunology, 140, 4180–4186.
9. Burastero, S. E., et al. (1988). Monoreactive high affinity and polyreactive low affinity rheumatoid factors are produced by CD5+ B cells from patients with rheumatoid arthritis. Journal of Experimental Medicine, 168(6), 1979–1992.
https://doi.org/10.1084/jem.168.6.1979
10. Gehin, J. E., et al. (2021). Rheumatoid factor and falsely elevated results in commercial immunoassays: Data from an early arthritis cohort. Rheumatology International, 41(9), 1657–1665.
https://doi.org/10.1007/s00296-021-04865-9
11. Kharlamova, N., et al. (2021). False positive results in SARS-CoV-2 serological tests for samples from patients with chronic inflammatory diseases. Frontiers in Immunology, 12, 666114.
https://doi.org/10.3389/fimmu.2021.666114