Researching Biotin Interference in Bead-Based Assays

High doses of biotin can be part of a treatment regimen for some diseases, for example, inborn errors of metabolism or multiple sclerosis.1,2 Additionally, the general population are increasingly using biotin oral supplements as they are marketed to improve hair, nails, and skin.3

A large number of immunoassays use the high-affinity interaction of streptavidin and biotin as part of the immunocomplex created to analyze the analyte of interest. As such, the presence of biotin has the possibility to affect the assay.

Simoa bead-based assays utilize a biotinylated detection antibody to bind the reporter enzyme conjugate, streptavidin-βgalactosidase (SbG). False-positive signal for Simoa assays related to Biotin may be a result of non-specific biotin binding to the capture bead reagent, and thus causing SbG to bind.

False-negative signals may be a result of biotin in solution binding to unbound SbG, thereby interfering with the enzyme’s ability to bind to a detection antibody that is part of the immunocomplex.

Biotin interference on the bead-based Simoa platform was analyzed using Neurofilament-Light (NF-L), Cytokine 3-Plex B (C3PB: IL-6, IL-17A, TNFα), and Neurology 4-Plex A (N4PA: Tau, NF-Light, GFAP, UCHL1).


The NF-L, N4PA, and C3PB assays were utilized in line with the kit instructions. Biotin (Sigma-Aldrich, Cat# B4639) was added into pooled serum (BioIVT), pooled plasma (BioIVT), or kit sample diluent at concentrations varying from 80 µM to 80 pM.

The impact of residual biotin at the time of SbG addition was tested by employing the N4PA assay. Biotin was directly spiked into the SbG reagent at concentrations ranging from 1 nM to 8 pM to simulate residual biotin left in solution from a sample.


When observing biotin in the samples (plasma, sample diluent, or serum), all three of the assays analyzed (NF-L, N4PA, C3PB) do not display a dose-dependent variation in AEB signal correlating to the unspiked biotin control condition (Figures 1 to 6).

This indicates that up to 80 µM of biotin in samples can be accepted without having a critical impact on AEB signal. Even though it is highly unlikely under the standard operations of the Simoa HD-1, the residual biotin left in solution when SbG addition takes place may have an effect if biotin is present at picomolar (pM) concentrations throughout the SbG incubation.

The N4PA assay at 40 pM of biotin in SbG displays a slight reduction in signal between 9.5% and 17.5% over the four assay plexes. Almost all SbG binding has been inhibited by 200 pM showing decreases by 97.4% to 98.2%.

The effect of biotin presence when the SbG step occurs is dependant on the biotin amount in the sample, the specific assay being run, and the molar ratio between the SbG and biotin. Standard Simoa assays have an SbG range of 50 pM to 300 pM.

As such, low picomolar concentrations of biotin may have a great effect on assay performance if deviated conditions are present.


In normal operating conditions, biotin presence of up to 80 µM (19.52 µg/mL) in a sample does not affect the performance of N4PA, C3PB, or NF-L. The biotin concentrations measured in this study are greater than those found in typical or frequently encountered scenarios. 5,6

Aside from the assay-specific interactions, the data indicates that the Simoa assay process is not prone to signal alteration where biotin is present in the concentrations tested.

It is worth noting that under exceptional conditions, residual biotin during SbG addition may cause an impact. A concentration of 40 pM of biotin demonstrated a slight impact on the N4PA assay. A concentration this extreme is not likely to be present at the time of SbG addition on the Simoa HD-1 unless there is the presence of several compounding factors.

For biotin to have a negative impact, there would need to be an unusually high amount of biotin in the sample, an egregious failure in washing along with a 2-step assay. This kind of failure is much more likely to impact an assay that utilizes a 2-step incubation scheme as there is one less cycle of washes in comparison to a 3-step assay.


The standard dietary recommendations for biotin intake in adults is 30 µg per day.3 Dietary supplements that can be purchased over the counter can be as high as 10mg per day.4 For inherited metabolic disease or in the treatment of multiple sclerosis, high dose treatments can go up to 300 mg per day.

At the highest doses of biotin (300 mg), plasma and serum concentrations at Cmax were close to 1200 ng per mL (4.92 µM).5 This article has demonstrated that concentrations 16 fold higher (80 µM) than the highest possible plasma concentrations do not impact the Simoa assays for C3PB, NF-L, and N4PA.

References and Further Reading

  1. “Biotinidase Deficiency.” Genetic and Rare Diseases Information Center, U.S. Department of Health and Human Services, 2015, ase-deficiency.
  2. Birnbaum, Gary, and Jessica Stulc. “High Dose Biotin as Treatment for Progressive Multiple Sclerosis.” Multiple Sclerosis and Related Disorders, vol. 18, 2017, pp. 141–143., doi:10.1016/j.msard.2017.09.030.
  3. “Office of Dietary Supplements - Biotin.” NIH Office of Dietary Supplements, U.S. Department of Health and Human Services, 7 Sept. 2018,
  4. “Dietary Supplement Label Database (DSLD).” U.S. National Library of Medicine, National Institutes of Health,
  5. Paul, Laure Peyro Saint, et al. Pharmacokinetics and Pharmacodynamics of MD1003 (HighDose Biotin) in the Treatment of Progressive Multiple Sclerosis.” Expert Opinion on Drug Metabolism & Toxicology, vol. 12, no. 3, 2016, pp. 327–344., doi:10.1517/17425255.2016.1136288.
  6. Apple, Fred. “Hemolysis and Biotin Interference on Cardiac Troponin and Natriuretic Peptide Assays.” Committee on Clinical Applications of Cardiac Biomarkers, International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), 26 July 2018

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Last updated: Nov 24, 2020 at 8:38 AM


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