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Review
. 2023 Oct 31;45(11):8733-8754.
doi: 10.3390/cimb45110549.

An Analysis of the Biotin-(Strept)avidin System in Immunoassays: Interference and Mitigation Strategies

Amy H A Balzer  1 , Christopher B Whitehurst  1
Affiliations
Review

An Analysis of the Biotin-(Strept)avidin System in Immunoassays: Interference and Mitigation Strategies

Amy H A Balzer et al. Curr Issues Mol Biol. .

Abstract

An immunoassay is an analytical test method in which analyte quantitation is based on signal responses generated as a consequence of an antibody-antigen interaction. They are the method of choice for the measurement of a large panel of diagnostic markers. Not only are they fully automated, allowing for a short turnaround time and high throughput, but offer high sensitivity and specificity with low limits of detection for a wide range of analytes. Many immunoassay manufacturers exploit the extremely high affinity of biotin for streptavidin in their assay design architectures as a means to immobilize and detect analytes of interest. The biotin-(strept)avidin system is, however, vulnerable to interference with high levels of supplemental biotin that may cause elevated or suppressed test results. Since this system is heavily applied in clinical diagnostics, biotin interference has become a serious concern, prompting the FDA to issue a safety report alerting healthcare workers and the public about the potential harm of ingesting high levels of supplemental biotin contributing toward erroneous diagnostic test results. This review includes a general background and historical prospective of immunoassays with a focus on the biotin-streptavidin system, interferences within the system, and what mitigations are applied to minimize false diagnostic results.

Keywords: avidin; biotin; immunoassays; interference; streptavidin.

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Conflict of interest statement

Amy Balzer is an employee of Siemens Healthcare Diagnostics.

Figures

Figure 1
Figure 1
Procedural differences of homogeneous assays and heterogeneous assays for the quantitation of thyroid stimulating hormone (TSH) in a sample. Created using BioRender.com accessed on 29 October 2023. (a) Homogeneous immunoassay procedure. (1) Anti-TSH antibodies conjugated with a donor fluorophore (D) are added to a well (2) followed by the addition of sample containing TSH analyte. (3) TSH analyte from the sample binds to the anti-TSH antibodies. (4) Immunocomplex-specific antibodies conjugated with an acceptor (A) fluorophore are added and (5) bind to the TSH–anti-TSH immunocomplex, bringing the donor and acceptor fluorophores in close proximity, inducing FRET. Fluorescence is directly proportional to TSH present in sample. (b) Heterogeneous immunoassay procedure. (1) Biotinylated anti-TSH antibodies bound to immobilized streptavidin on magnetic beads are added to a well (2) followed by the addition of sample containing TSH analyte. (3) TSH analyte from the sample binds to the anti-TSH antibodies. (4) Anti-TSH antibodies labeled with acridinium ester (AE) are then added and (5) bind to TSH analyte complex. (6) A magnet induces solid phase separation by immobilizing the magnetic beads with the bound immunocomplex while (7) unbound molecules are washed and removed from the well. (8) An addition of reagents induces a chemical reaction activating the AE label and (9) AE activity is measured. AE activity is directly proportional to the TSH present in sample.
Figure 2
Figure 2
Design possibilities for quantifying thyroid stimulating hormone (TSH) in serum using heterogeneous methods. Created using BioRender.com accessed 29 October 2023. (a) Represents a direct design where the target antigen TSH is immobilized on the solid support along with other untargeted antigens from the sample matrix. An anti-TSH antibody labeled with a fluorescent probe binds specifically to the target TSH antigen only. Fluorescence is directly proportional to the amount of TSH present in sample. (b) Represents an indirect design where a primary antibody (mouse anti-TSH antibody) binds to TSH bound to a capture antibody (human anti-TSH antibody) immobilized on a solid support. A secondary antibody (anti-mouse antibody) labeled with a fluorescent probe binds to the mouse anti-TSH antibody. Fluorescence is directly proportional to the amount of TSH in the sample. (c) Represents a sandwich design where TSH is bound (“sandwiched”) between an anti-TSH capture antibody and an anti-TSH detection antibody. Capture antibody is in excess. Fluorescence is directly proportional to the amount of TSH present in sample. (d) Represents a competitive design where TSH in the sample competes with a TSH analogue labeled with a fluorescent probe for binding to an immobilized anti-TSH antibody. Detection analogue is in excess. Fluorescence is inversely proportional to the amount of TSH in the sample.
Figure 3
Figure 3
Methods of BRAB (a) and LAB (b). Created using BioRender.com accessed 29 October 2023.
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