Introduction
Immunoassays are indispensable tools in clinical laboratories and research settings, enabling the detection and quantification of analytes ranging from hormones and proteins to drugs and pathogens. The biotin-(strept)avidin system, known for its high affinity and specificity, is commonly utilized in immunoassays due to its robust binding properties. However, recent studies have highlighted the emergence of interference, posing significant challenges to the reliability of immunoassay results. Understanding and addressing these challenges are crucial to maintaining the accuracy and clinical utility of immunoassays.
Fig. 1 Biotin interference and ASA interference in immunoassays (Balzer A.H., Whitehurst C.B. 2023).
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Biotin Interference
Biotin, also known as vitamin B7 or vitamin H, is a water-soluble B vitamin essential for various metabolic processes in the human body. In recent years, biotin supplementation has gained popularity due to its purported benefits for hair, skin, and nail health, as well as its potential role in managing conditions such as type II diabetes, obesity, and inflammatory bowel disease (IBD). However, the increasing use of biotin supplements has led to a rise in biotin interference in immunoassays.
The mechanism of biotin interference in immunoassays involves its high-affinity binding to streptavidin, a protein derived from Streptomyces bacteria. In assays employing the biotin-(strept)avidin system, biotinylated detection reagents bind to streptavidin-coated solid surfaces or conjugate to reporter molecules. However, exogenous biotin from supplements or endogenous biotin from biological samples can compete with biotinylated detection reagents for binding sites on streptavidin, leading to interference and erroneous assay results. Biotin interference can manifest as false-negative or false-positive results, depending on the assay format and detection method used.
The clinical implications of biotin interference are significant, potentially leading to misdiagnosis, inappropriate treatment decisions, and compromised patient care. For example, false-negative results in assays for cardiac biomarkers or thyroid function tests can delay the diagnosis of cardiovascular disease or thyroid dysfunction, respectively. Conversely, false-positive results in assays for tumor markers or infectious disease markers can lead to unnecessary medical interventions, including biopsies, surgeries, or antimicrobial therapies. Therefore, strategies to mitigate biotin interference and improve assay specificity are critical for ensuring accurate diagnostic testing and patient management.
Mitigation Strategies of Biotin Interference
Several strategies have been proposed to mitigate biotin interference in immunoassays and improve assay reliability. These include:
Reducing biotin concentrations in patient samples through sample dilution or pretreatment with avidin or streptavidin to sequester excess biotin.
Modifying assay protocols to minimize the sensitivity of detection reagents to biotin interference, such as using higher concentrations of detection reagents or optimizing incubation times.
Employing alternative detection systems that do not rely on the biotin-(strept)avidin interaction, such as fluorescence-based detection or chemiluminescence assays.
Educating healthcare providers and laboratory personnel about the potential for biotin interference in immunoassays and the importance of verifying assay results in patients taking biotin supplements.
Anti-(Strept)avidin Antibody Interference
In addition to biotin interference, the presence of anti-(strept)avidin antibodies (ASAs) in human serum has emerged as a significant challenge in immunoassays. ASAs can bind to streptavidin, interfering with the biotin-(strept)avidin interaction and leading to inaccurate assay results. Unlike biotin interference, which can be attributed to exogenous biotin supplementation, ASA interference is endogenous and may be present in individuals regardless of biotin intake.
The mechanisms underlying ASA interference are not fully understood but are believed to involve the formation of immune complexes between ASAs and streptavidin. These immune complexes can disrupt the binding of biotinylated detection reagents to streptavidin, resulting in false-negative or false-positive assay results. Furthermore, ASAs may induce conformational changes in streptavidin, altering its binding affinity for biotin and further complicating assay interpretation.
Studies have documented the prevalence of ASAs in the general population, with varying degrees of reactivity observed among individuals. Exposure to soil bacteria, such as Streptomyces avidinii, has been proposed as a possible trigger for ASA production, highlighting the complex interplay between environmental factors and immune responses. However, further research is needed to elucidate the factors influencing ASA production and their clinical significance in immunoassay interference.
Mitigation Strategies of Anti-(Strept)avidin Antibody Interference
Mitigating ASA interference in immunoassays presents unique challenges compared to biotin interference due to the endogenous nature of ASAs in patient samples. Strategies for mitigating ASA interference include:
Developing assays with reduced sensitivity to ASA interference by optimizing assay conditions and reagent concentrations.
Exploring alternative detection systems that minimize reliance on the biotin-(strept)avidin interaction, such as protein A/G-based detection or direct detection methods.
Engineering avidin analogues with reduced immunogenicity or modifying streptavidin sequences to minimize antibody recognition.
Educating healthcare providers and laboratory personnel about the potential for ASA interference in immunoassays and the importance of considering alternative assay formats or validation methods when ASA interference is suspected.
Clinical Implications and Challenges
The presence of biotin interference and ASAs in immunoassays has significant clinical implications, affecting the accuracy and reliability of diagnostic testing. Misdiagnosis or inappropriate treatment decisions resulting from assay interference can have serious consequences for patient outcomes and healthcare resource utilization. Furthermore, distinguishing between biotin interference and ASA interference remains challenging, complicating assay interpretation and result verification.
Addressing these challenges requires collaborative efforts from researchers, clinicians, assay manufacturers, and regulatory agencies to develop standardized protocols, validated assays, and quality control measures. Additionally, education and awareness initiatives are needed to ensure that healthcare professionals are aware of the potential for assay interference and equipped to interpret test results accurately.
Conclusion
Biotin and ASA interference pose significant challenges to the reliability and accuracy of immunoassays in clinical diagnostics and biomedical research. As the prevalence of biotin supplementation and the awareness of ASA interference continue to rise, it is essential to implement robust mitigation strategies and educate healthcare providers and laboratory personnel about these emerging challenges. By advancing our understanding of interference mechanisms and developing innovative assay formats and detection systems, we can enhance the clinical utility of immunoassays and ensure accurate and reliable diagnostic testing for patients worldwide.
Reference
- Balzer A.H., Whitehurst C.B. An analysis of the biotin–(strept) avidin system in immunoassays: Interference and mitigation strategies. Current Issues in Molecular Biology. 2023, 45(11): 8733-54.
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