Immunoassay Interference in Clinical Diagnostics

Immunoassays form the backbone of modern clinical diagnostics, offering rapid, sensitive and high-throughput quantification of a vast array of biomarkers. Despite their widespread utility—from hormone panels and cardiac markers to therapeutic drug monitoring—these assays are inherently prone to analytical interference. Such interference may stem from endogenous antibodies, small-molecule supplements or pharmaceutical agents, each capable of disrupting the specific antibody–antigen recognition that underpins assay function. Consequences range from spurious elevations or depressions of analyte concentrations to entirely false reports, potentially leading to misdiagnosis, inappropriate treatment and avoidable healthcare costs. Understanding the mechanistic origins of interference and developing robust detection and mitigation strategies are therefore critical to maintaining assay accuracy. Advances in assay design, auxiliary blocking reagents and orthogonal validation methods are reshaping laboratory practice, underscoring the global imperative for vigilance in immunoassay interpretation.

Research from Nature Portfolio

Recent studies have characterised the nature and diversity of anti-animal antibodies that compromise immunoassay integrity. In one foundational investigation, canine anti-mouse antibodies were shown to target multiple regions of murine immunoglobulin G, cross-react with antibodies from different species and exist in all major canine immunoglobulin classes. Individual variability in antibody specificity and prevalence among breeds was demonstrated, underlining that interference is not simply a by-product of prior immunisation. Importantly, the heterogeneity of these antibodies means that no single blocking approach is universally effective. The work provides a detailed blueprint for combining strategies—such as reagent modification, sample pre-treatment and alternative detection platforms—to neutralise diverse interfering antibodies in both veterinary and human immunoassays.

Immunoassay Interference in Clinical Diagnostics publication trend

The graph below shows the total number of articles in immunoassay interference in clinical diagnostics across all publications each year (not limited to Nature Index journals).

Technical terms

Immunoassay: Analytical method using antibody–antigen binding to detect or quantify substances in a biological sample.

Endogenous antibodies: Patient-derived antibodies that can bind assay components and distort measurement results.

Biotin–streptavidin interaction: A high-affinity binding pair used in many assays that can be disrupted by excess free biotin, leading to false readings.

Sandwich immunoassay: Format in which the target analyte is captured between two antibodies—one immobilised for capture and one labelled for detection.

Competitive immunoassay: Format in which sample analyte competes with a labelled analogue for a limited number of antibody binding sites.

Cross-reactivity: Unintended binding of structurally similar substances to assay antibodies, causing inaccurate quantification.

LC-MS/MS: Liquid chromatography–tandem mass spectrometry, an orthogonal analytical technique that separates and identifies compounds to confirm or refute immunoassay results.

References

1. Characterization of canine anti-mouse antibodies highlights that multiple strategies are needed to combat immunoassay interference. Scientific Reports (2019).
2. Endogenous anti-streptavidin antibodies causing erroneous laboratory results more common than anticipated. Scandinavian Journal of Clinical and Laboratory Investigation (2021).
3. Oestradiol measurement during fulvestrant treatment for breast cancer. British Journal of Cancer (2019).
4. Clinical risk assessment of biotin interference with a high-sensitivity cardiac troponin T assay. Clinical Chemistry and Laboratory Medicine (2020).