luong2019

Biotechnology Advances 37 (2019) 634–641
Contents lists available at ScienceDirect
Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv
Research review paper
Biotin interference in immunoassays based on biotin-strept(avidin)
chemistry: An emerging threat
T
⁎
John H.T. Luonga,b, , Keith B. Malec, Jeremy D. Glennona,b
a
University College Cork, Irish Separation Science Cluster ISSC Ireland, Innovative Chromatography Group, Western Rd, Cork T12 YN60, Ireland
University College Cork, School of Chemistry, College Road, Cork T12 YN60, Ireland
c
CCA Lab, Hampstead, Quebec H3X 1X5, Canada
b
A R T I C LE I N FO
A B S T R A C T
Keywords:
Biotin interference
Avidin/streptavidin
Immunoassays
Sandwiched assay
Competitive assay
Falsely negative
Falsely positive
Biotin removal
Biotinylated antibodies/antigens are currently used in many immunoassay formats in clinical settings for diversified analytes and biomarkers to offer high detection selectivity and sensitivity. Biotin cannot be synthesized
by mammals and must be taken as an essential supplement. Normal intake of biotin from various foods and milk
causes no effect on the streptavidin/biotin-based immunoassays. However, overconsumption of biotin (daily
doses 100–300 mg) poses a significant problem for immunoassays using the biotin-strept(avidin) pair. Biotin
interferences are noted in immunoassays of thyroid markers, drugs, hormones, cancer markers, the biomarker
for cardiac function (β–human chorionic gonadotropin), etc. The biotin level required for serious interference in
test results varies significantly from test to test and cannot easily be predicted. Immunoassay manufacturers with
technologies based on strept(avidin)-biotin binding must investigate the interference from biotin (up to at least
1200 ng/mL or 4.9 μM of biotin) in various formats. There is no concrete solution to circumvent the biotin
interference encountered in blood samples, short of biotin removal. Considering the short half-life of biotin in
the human body, patients must stop taking biotin supplements for > 48 h before the test. However, this scenario
is not considered for patients in emergency situations or those with biotinidase deficiency, mitochondrial metabolic disorders or multiple sclerosis. Apparently, a rapid analytical procedure for biotin is urgently needed to
quantify for its interference in immunoassays using strep(avidin)-biotin chemistry. To date, there is no quick and
reliable procedure for the detection of biotin at below nanomolar levels in blood and biological samples.
Traditional lab-based techniques including HPLC/MS-MS cannot process an enormous number of public
samples. Biosensors with high detection sensitivity, miniaturization, low cost, and multiplexing have the potential to address this issue.
1. Introduction
Immunoassays are based on the ability of an antibody to recognize
and bind its specific macromolecule in a complex mixture of other
molecules. Different assay formats and detection schemes have been
developed for a plethora of target analytes of clinical, environmental,
and biosecurity importance (Vashist and Luong, 2018). Like many
analytical techniques, immunoassays still lack specificity and accuracy,
far from perfection. Besides the antibody binding property, the assay
specificity is subject to the sample matrix, reagent components, and the
assay format. Interfering substances in the specimen alter the measurable analyte level and biotin has become an emerging interferent.
Biotin consists of a tetrahydrothiophene ring containing a valeric acid
side chain, which is joined to an imidazolidone ring to form eight
possible stereoisomers (Scheme 1). Only D(+)-biotin occurs in nature
but cannot be synthesized by mammals. As an essential supplement,
biotin is part of the B complex group of vitamins that metabolizes
amino acids, carbohydrates, and fats. This vitamin biotin might be involved in the regulating transcription or protein expression of different
proteins (Pacheco-Alvarez et al., 2002). It is also a critical nutrient for
women during pregnancy considering its important role for embryonic
growth. Biotin is available from various foods and milk and some intestinal bacteria also synthesize biotin (Combs Jr, 2016). Biotin deficiency results in hair loss (Trüeb, 2008), dry scaly skin, and other
symptoms related to fatigue (Osada et al., 2004), insomnia, and depression. Thus, there is increasing marketing of biotin as a remedy for
common hair and skin problems, weight loss, glucose metabolism, and
boosting energy. High dose biotin therapy has been considered for
⁎
Corresponding author at: University College Cork, Irish Separation Science Cluster ISSC Ireland, Innovative Chromatography Group, Western Rd, Cork T12 YN60,
Ireland.
E-mail address: j.luong@ucc.ie (J.H.T. Luong).
https://doi.org/10.1016/j.biotechadv.2019.03.007
Received 3 January 2019; Received in revised form 21 February 2019; Accepted 8 March 2019
Available online 11 March 2019
0734-9750/ © 2019 Elsevier Inc. All rights reserved.
Biotechnology Advances 37 (2019) 634–641
J.H.T. Luong, et al.
This review article will address the interference and magnitude of
biotin in immunoassays for various biomolecules and biomarkers that
play a crucial role in diagnostics. The removal of biotin from the assay
sample together with biotin withdrawal might be a useful approach to
suppress the interference of biotin and this aspect will also be discussed.
In addition, the sensitive and selective detection of biotin in blood
samples including using biosensors is discussed.
2. Solid phase immunoassays
Scheme 1. Chemical structure of D(+)-biotin, C10H16N2O3S, molar mass of
244.31 g/mol. The human body needs biotin from different food sources to
facilitate the conversion of certain nutrients such as fats, carbohydrates, and
amino acids into energy. As biotin plays an important role in the health of hair,
skin, and nails, it has been formulated in several beauty products. Some bacteria in the intestine of humans also synthesize small amounts of biotin.
Biotin forms an irreversible complex with avidin and streptavidin
referred to as strept(avidin) and this pair has been extensively used in
diversified techniques including commercial immunoassay platforms
for clinically important analytes and biomarkers. This unique interaction is also widely used for the preparation of biotinylated antibodies,
biotinylated antigens, and strept(avidin)-coated magnetic beads for
diversified applications (https://www.thermofisher.com/ca/en/home/
life-science/antibodies/biotin-binding-protein-conjugates.html).
The
carboxyl group (-COOH) of biotin can be easily bioconjugated with an
amino group (-NH2), notably by the popular carbodiimide procedure as
shown in Scheme 2 (Hermanson, 2013). Among various carbodiimides,
EDC is the most popular choice for conjugating biomolecules containing
carboxylates and amines. Water-soluble EDC permits its direct addition
to an aqueous solution of biomolecules without prior organic solvent
dissolution. The covalent attachment of biotin to proteins, polypeptides, and low molecular weight antigens including thyroid and steroid
hormones has minimal effects on the biological and antigenic activities
of such biomolecules. This distinct feature enables the use of biotin
conjugates as ligands in various immunoassay formats.
The solid-phase approach requires only one step and offers high
detection sensitivity, specificity, and robustness (Fig. 1). In this simple
format, the biotinylated capture antibody is mixed with the specimen
and the labeled antibody (luminescent or fluorescent compound, enzyme, isotope), also known as the detection antibody. The “sandwich”
format is used to measure large molecules, such as thyroid stimulating
hormone, insulin, thyroglobulin, C-peptide, etc. The target is “sandwiched” between the two different antibodies. Consequently, the signal
response increases with increasing analyte concentration. Obviously,
this “one-step” detection scheme is susceptible to free biotin in the
sample since it will compete with biotinylated molecules for streptavidin binding, resulting in falsely low results (Fig. 1A). Based on a very
high binding capacity of strept(avidin) coated microparticles, the assay
format could provide some binding capacity for free biotin in the
sample. The biotin level of healthy subjects is sufficiently low and exhibits negligible interference. However, serious interference becomes
pronounced when the biotin level exceeds the threshold concentration
for each specific assay.
For small analytes, a competitive assay is a better format that involves a capture antibody and a labeled analyte. In this case, both the
analyte and the labeled analyte will compete for the binding site of the
capture antibody. If the capture antibody is conjugated with biotin, it
will bind strept(avidin) coated microparticles, like the sandwich
format. For samples with free or low biotin, a high target concentration
in the sample will lower the signal-labeled analyte bound to the capture
antibody, i.e., the calibration curve is a decreasing curve. Free biotin in
samples with high concentrations will compete favorably with the
biotinylated capture antibody for the binding site of strept(avidin),
resulting in very negligible response signals, known as falsely positive
(Fig. 1B).
A small population with multiple sclerosis and biotin-related diseases are subject to high biotin dose therapy. The monitoring of biotin
in these samples can be accommodated by other analytical procedures
including high-performance liquid chromatography equipped with
mass spectrometry.
various medical problems such as mitochondrial energy metabolism
(Depeint et al., 2006), progressive multiple sclerosis (Sedel et al.,
2015), and muscle cramps in hemodialysis (Oguma et al., 2012)
Despite scientifically unproven benefits, biotin at high doses
(> 1 mg) has been used with the expectation to grow hair and nail as
mentioned previously. Biotin intake of 1.2 mg shows an average plasma
concentration of 15 nM (three hours after an acute dose) and 22 nM
after two weeks of prolonged intake. By extrapolation, uptakes of
5–10 mg biotin should result in peak biotin concentrations of 62 nM to
0.124 μM. Even at these levels, most of the immunoassay platforms are
still resistant to biotin interference except for troponin T (a biomarker
of heart attack) and anti-thyroid antibodies (thyroid disease). The peak
serum biotin levels after single doses of 100 mg are 2.024 ± 0.658 μM
(Samarasinghe et al., 2017). Thus, overconsumption of biotin (500 mg/
day) results in elevated biotin in blood, micromolar levels, which causes
significant interference in immunoassays using the strept(avidin)-biotin
interaction. The misdiagnosis and mismanagement of patients were
reported for the serious problem of biotin interference in thyroidfunction tests (Trambas et al., 2016). Consequently, biotin becomes an
emerging issue in clinical and hospital settings as the immunoassay
platform based on biotin-strept(avidin) accounts for 50% of the total
immunoassays (221 over 374 methods used by the eight most popular
immunoassay platforms in the United States) (Holmes et al., 2017).
Unless this problem has been addressed and overcome, immunoassays
using the strep(avidin)-biotin chemistry for analyses of target biomarkers and analytes in samples with high biotin are expected to produce aberrant test results. As an example, a recent study illustrates the
interference of biotin with a concentration range from 31.35 to
1000 ng/mL or 0.13–4.09 μM (Li et al., 2018) in various immunoassays
based on sandwich and competitive formats. As shown in Tables 1-2,
the biotin level with serious interference in test results varies significantly from test to test and cannot easily be predicted.
Table 1
The concentration of biotin at which interference might be problematic in
various assays (Li et al., 2018).
Target analytes
Serum insulin
C-peptide
CA (cancer antigen)
LH (Luteinizing hormone)
Vitamin B12
Vitamin D
Prolactin, Folate, CA19-9,
CA15-3, CEA
(carcinoembryonic
antigen), and AFP (alphafetoprotein)
Increased (%)
14.48
16.84
Nil
Decreased (%)
Biotin
interference
level ng/ml
14.07
14.26
11.13
16.84
750
1000
750
750
750
250
1000
Nil
635
Biotechnology Advances 37 (2019) 634–641
J.H.T. Luong, et al.
Table 2
The potential effect of biotin on immunoassays https://www.healthcare.uiowa.edu/path_handbook/Appendix/Chem/BiotinImmunoassayTables.pdf
Thyroid markers
Thyroid Stimulating Hormone, Reflexive, Thyroid Stimulating Hormone
Free T4, Free T3, Total T4, Total T3, Thyroid Peroxidase Antibody, Thyroglobulin Antibodies
Hormones
Follicle stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, prolactin, growth hormone, insulin, C-peptide
Cortisol, estradiol, testosterone, progesterone, dehydroepiandrosterone sulfate
Tumor markers
Alpha fetoprotein, cancer antigen, carcinoembryonic antigen, carbohydrate antigen, prostate specific antigen, total prostate specific antigen, screening,
prostate specific antigen, free, HCG – tumor marker or pregnancy
Cardiac markers
Troponin T, NT-proBNP
Nutritional markers
Ferritin
Vitamin D, 25-hydroxy, vitamin b12, vitamin b12, reflexive, folate
Infectious disease serologies
HIV antigen/antibody combo, hepatitis c virus antibody, hepatitis a antibody, total, hepatitis a antibody, IgM, hepatitis b surface antigen, hepatitis b surface
antibody, hepatitis b antigen, hepatitis b core antibody, IgM.
Hepatitis B core antibody, total (LAB622)
Pregnancy-related markers
Pregnancy screen, qualitative, HCG – pregnancy, HCG – tumor marker or pregnancy
Therapeutic Drugs
Digoxin
Other proteins
Immunoglobulin E, myoglobin, sex hormone binding globulin
R
O
Falsely decreases
Falsely increases
Falsely decreases
Falsely decreases
Falsely decreases
Falsely increases
Falsely decreases
Falsely increases
Falsely decreases
Falsely increases
Falsely decreases
O
R
N=C=N
NH2
R1
Falsely decreases
Falsely increases
N
R1
OH
H
Biomolecule
CH3
Cl
NH
N=C=N
H
H3C
CH3
EDC = 1-Ethyl-3-3(dimethylaminopropyl) Carbodiimide
Hydrochloride, MW 191.7
Scheme 2. Covalent attachment of the carboxyl group to the amino group by the popular carbodiimide coupling procedure.
3. Avidin and its analogs
isolated from Streptomyces avidinii is smaller (53 kDa) compared to
avidin and lacks the glycoprotein portion found on avidin. Captavidin
has a nitrated tyrosine in its biotin-binding site (Takakura et al., 2009),
resulting in its significantly lower affinity to biotin, compared with
other avidin analogs (Table 3). The captavidin-biotin pair can be dissociated at pH 10 instead of the necessity of 8 M guanidine hydrochloride, pH 1.5 for the avidin-biotin complex. A recombinant avidin,
known as tamavidin, is derived from E. coli-expressed mushroom
(Pleurotus cornucopiae) with much lower affinity to biotin, compared to
Tetrameric avidin (MW = 67–68 kDa), commonly found in egg
whites, is synthesized in the oviducts of various animals, e.g., birds,
reptiles, and amphibians whereas dimeric avidin is also found in some
bacteria, e.g., Rhizavidin from Rhizobium etli (Helppolainen et al.,
2007). Neutravidin or deglycosylated avidin retains its affinity to biotin
with minimal nonspecific binding properties, particularly, the nonbinding of sugar binding proteins. Like neutravidin, streptavidin
Fig. 1. (A) (Upper) Microparticles are coated with strept
(avidin). The capture antibody for a target analyte is bioconjugated with biotin. The specific binding between
biotin-strept(avidin) leads to the formation of a tertiary
complex. The capture antibody then binds the target compound. (Lower) Free biotin in the sample will compete with
the biotinylated capture antibody for binding to strept
(avidin) coated on microparticles, leading to erroneous results. This format is known as “sandwich” assays for large
molecules. (B). The target analyte is labeled with a detectable signal and this format is applicable for detecting small
molecules. Without biotin in the sample, a high target
concentration in the sample will lower the signal-labeled
analyte bound to the capture antibody. For samples with high biotin levels, free biotin will compete favorably with the biotinylated capture antibody for the binding
site of strept(avidin), resulting in very negligible response signals, known as falsely positive.
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J.H.T. Luong, et al.
Table 3
Some key properties of avidin and its analogs.
Properties
Avidin
Neutravidin
Streptavidin
Captavidin
Tamavidin
MW (kDa)
Affinity for biotin (Kd,M−1)
Binding sites for biotin
Isoelectric point (pI)
67–68
10−15
4
10
60
10−15
4
6.3
53
10−15–10−14
4
6.8–7.5
67–68
10−9
4
60
2.8–4.4 × 10−7 (Ref. 4)
4
crosslinker” since EDC does not become part of the final amide-bond
crosslink between the two molecules (Hermanson, 2013). The attachment of biotin to various molecules is termed as “biotinylation”, which
plays an important role in other analytical techniques to probe protein
localization-interaction, protein, DNA transcription, replication, etc.
The BCAb forms a stable complex with streptavidin and offers accessible binding sites for both the analyte and the conjugated analyte.
The activity of the conjugated analyte is measured and related to the
analyte concentration. The response signal is high for low analyte
concentration and vice versa as the response signal is inversely proportional to the analyte concentration. In the presence of elevated
biotin, these free molecules bind streptavidin strongly and pull it out of
the beads. The biotinylated Ab still binds the analyte and the conjugated analyte, but all materials will be washed out during the
washing step. The response signal becomes very small or negligible,
reflecting a high level of the analyte in the calibration curve, falsely
positive. This format is used to target small molecules such as testosterone, estradiol, cortisol, steroids, T3 (triiodothyronine) and T4
(thyroxine)-hormones produced by the thyroid gland, hydroxyvitamin
D, etc.
In sandwich assays, the Ab for a target analyte is biotinylated, designated as the capture or primary Ab. The Ab is also conjugated with
an enzyme, designated as detection or secondary antibody. With biotinfree samples, the biotinylated capture Ab binds streptavidin and the
antigen at two different binding sites. The labeled Ab then forms a
tertiary complex with the antigen and the capture Ab. The activity of
the labeled enzyme is quantified and related to the amount of the antigen. Based on a double epitopic recognition, sandwich assays are also
known as “two-site immunoassays”. In the presence of free biotin in a
sample, this molecule competes with the biotinylated capture antibody
for binding streptavidin. As a result, the density of the tertiary complex
is greatly reduced, resulting in an underestimation of the antigen in the
sample or false negative (Fig. 1A). In clinical settings, the sandwich
assay is applied to measure fairly large molecules to very large molecules with molecular weights ranging from 3 kDa to 650 kDa: stimulating hormone (TSH, thyrotropin: MW = 28,000), pituitary glycoprotein hormones (PGH), human chorionic gonadotropin (HCG:
MW = 36,700), parathyroid hormone (PTH, MW = 9500), insulin-like
growth factor-1 (IGF1, MW = 7649), insulin (MW = 5808), thyroglobulin (MW = 650,000), and C-peptide (MW = 3020). There are
three human pituitary glycoprotein hormones: luteinizing hormone
(LH, MW = 30,00 for animals), follicle-stimulating hormone (FSH,
MW = 35,500), and thyrotropin (TS, MW ~ 28,000). A guidance for
tests of serious diseases such as HIV and hepatitis C virus needs to be
developed to provide proper results. This concern is also extended to
the assays of cardiac biomarkers and the assessment of tumor recurrence. Slight skewing of the results might lead to erroneous treatment
and devastating emotions.
its analogs (Takakura et al., 2010; Takakura et al., 2013). Of importance is the stability of the biotin-strept(avidin) complex under extreme pH and temperature and its resistant to various organic solvents
and denaturing agents. Detailed dimensional (3D) structures of both
avidin and streptavidin in complex with D-biotin can be found in the
literature (Livnah et al., 1993a, 1993b). Both tetrameric avidin and
streptavidin have four identical subunits, with each subunit containing
a single biotin-binding site. However, avidin is glycosylated with one
disulfide bridge and two methionine residues whereas streptavidin is a
non-glycoprotein without sulfur-containing residues. The amino acid
compositions of these two proteins are also very different. Avidin has a
single tyrosine (Tyr) residue whereas streptavidin has six Tyr per subunit. The avidin (Tyr-33) is in the primary sequence (30Thr-Gly-ThrTyr-Ile-Thr-Ala-Val) and located in a position similar to one of the Tyr
residues (Tyr-43) of streptavidin (40Thr-Gly-Thr-Tyr-Glu-Ser-Ala-Val).
Upon the binding of biotin, strept(avidin) changes its conformation,
stability, and flexibility (Livnah et al., 1993a, 1993b; Weber et al.,
1992; Gonzalez et al., 1999). Biotin interacts strongly with avidin in a
loop located between β-strands 3–4, its most flexible part. Of importance is the role of the single tyrosine residue (Tyr-33) in the avidin
subunit, which is attributed to the biotin-binding site. Modification of
avidin by p-nitrobenzenesulfonyl fluoride, a tyrosine-specific reagent
(Liao et al., 1982) results in the complete loss of its biotin-binding
(0.5 mol of tyrosine residue/mol of avidin subunit) (Tsou, 1962). The
tyrosine residues may also stabilize the native protein structure. For
streptavidin, the modification of 3 mol of tyrosine residue/mol of subunit is required to nullify its biotin-binding activity (Livnah et al.,
1993a, 1993b). Apparently, Tyr-43 (major fraction) and Tyr-54 (minor
fraction) are also involved in the binding of biotin.
Streptavidin is preferred over avidin in immunoassays due to its
lower non-specific binding properties (Chivers et al., 2010) and comparable affinity to biotinylated molecules. Captavidin and tamavidin
with much lower affinity to biotin offer the possibility to dissociate the
complex when formed. They might be more receptive in biosensing
schemes, bioseparation or other applications that require the dissociation of the avidin-biotin pair for repeated uses. Commercial avidin,
streptavidin, and neutravidin with high purity bind 12 μg (0.05 μmol)
of biotin (MW = 244) per mg protein or 1.4 × 10−2 μmol
(MW = 50 kDa–70 kDa), corresponding to a biotin to avidin binding
ratio of 3.6, approaching the maximum value of 4 (Table 3).
4. Immunoassays using the biotin/streptavidin interaction
Streptavidin is preferred over avidin to minimize nonspecific protein binding despite its higher cost and there are several formats based
on the streptavidin-biotin chemistry in immunoassays. In competitive
assays, streptavidin-coated beads, a conjugated analyte (normally with
an enzyme, e.g., horseradish peroxidase), and a biotinylated capture
antibody (BCAb) are prepared. Compared to avidin, biotin is a significantly smaller molecule (MW = 244) with a carboxylic group. The
presence of this group has been exploited to conjugate with a plethora
of proteins, enzymes, and other molecules for diversified applications.
Of interest is the use of EDC (1-ethyl-3-(−3-dimethylaminopropyl)
carbodiimide hydrochloride) and other carbodiimides to effect direct
conjugation of the biotin carboxyl to the amino group of a second
biomolecule (protein, enzyme, etc.). This is called a “zero length
5. Issues of overconsumption and high biotin dose therapy
Biotin (vitamin H), literally stems from German “Haut und Haar”,
i.e., “skin and hair”. To date, over-consumption of biotin is inspired by
lofty claims that biotin helps grow healthy and strong hair, skin and
nails. Supporting scientific evidence for this claim is very limited,
however, biotin improves the keratin infrastructure, a basic protein of
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Biotechnology Advances 37 (2019) 634–641
J.H.T. Luong, et al.
Keenan, 2013). The risk of side effects can be reduced by taking biotinsupplements with foods. Its negative side effects are also rare since it is
easily excreted in urine and feces. In contrast, biotin might improve
cognitive function, reduce inflammation, lower blood sugar in diabetes,
decrease “bad” LDL cholesterol and increase “good” HDL cholesterol
(https://www.healthline.com/health/biotin-hair-growth#otherbenefits). Nevertheless, significant scientific proofs are needed to support or refute the side-effects of this water-soluble vitamin as beauty
supplements in high dosage.
Table 4
Biotin interference and its threshold concentration reported for seven commercial systems.
Commercial automated
system
Total test
Total
vulnerable
Biotin concentration
(nM)
Roche Elecsys®
Ortho Vitros®
Siemens Dimension®
Siemens Centaur®
Beckman Coulter Access®/
DXI®
Abbott Architect i2000®a
Diasorin Liaison XL®b
81
43
26
67
48
81
29
21
18
14
29–491
10–82
205–8200
41–4090
41–1000
46
42
2
0
120
Not available
6. Interference of biotin and the removal of biotin
Biotin ranging from 0.12 to 0.36 nM (Zempleni et al., 2009) in blood
of healthy subjects falls into the tolerant level of the assay format and
has a negligible interfering effect on the avidin/streptavidin-biotin
technology (Kwok et al., 2012). Elevated biotin in blood had been
limited to a small population until biotin megadoses became commonplace for non-medical purposes. Enhanced biotin levels in plasma
have been observed for a daily consumption of 300 μg of biotin for
1 week and then 900 μg for another week (Bitsch et al., 1989). Over
1 mg/day of biotin consumption often causes falsely-low or falsely-high
test results, depending on the assay format. The magnitude of interference and the threshold of biotin concentration, however, are also
variable and dependent upon the type of assay and the target molecules. For instance, the assay of anti-TPO (thyroid peroxidase) is vulnerable to plasma biotin above 40.9 nM, compared to 491 nM for carcinoembryonic antigen (CEA) (https://www.exeterlaboratory.com/
biotin-alert-potential-assay-interference). Table 4 illustrates the results
of biotin interference in all immunoassays performed by seven commercial immunoassay systems. In general, the test is vulnerable to
biotin presence when the procedure is based on having a streptavidin/
biotin reaction, an anti-biotin/biotin reaction, or a pre-bound avidin/
streptavidin or biotin/anti-biotin reagent as part of the analysis. In
general, the interference is considered when the spiked biotin in a
sample causes ≥ ± 10% variation of the expected result. The two
systems (Abbott and Diasorin) are not based on strept(avidin)-biotin
technology or involve any chemistry related to biotin.
At first glance, biotin from samples can be removed by avidin/
streptavidin immobilized on insoluble matrices such as magnetic particles, polymers (e.g. agarose beads), silica, etc. Such matrices, commercially available from different suppliers, are added to the microplate together with the sample. This step is time-consuming, about 1 h
for incubation and the removal of the biotin-avidin complex. Strept
(avidin) bound polymers could be packed in a small syringe to facilitate
the loading of a biotin-containing sample. Of course, such modifications
will require extensive revalidation which is time-consuming and expensive. The “full-proof” biotin-removal capacity of streptavidinagarose beads is still a subject of future endeavors. Nevertheless, clinical and hospital settings must be aware of elevated biotin levels in the
assay samples to prevent any misdiagnosis and/or inappropriate
therapy. Other solutions include the dilution of the sample with a validated assay diluent or using a different platform known to be unaffected by biotin interference.
Biotin withdrawal is the easiest option to reduce the financial burdens associated with high-dose biotin on the health care system. With a
half-life of ~ 2 h in low dose consumption, most of biotin should be
cleared from the body within 4–5 h. However, a period of two to five
days might be required for patients with high biotin dose. It should be
noted that this period can be over 15 days, e.g., thyroid function tests
(TFTs) (Koehler et al., 2018) or months in other cases. This strategy is
not an option for patients with MS or other biotin related diseases. Of
course, clinical or hospital settings are equipped with radio immunoassay (RIA) and gas chromatography–mass spectrometry/liquid
chromatography-mass spectrometry to handle such analyses.
Lastly, the replacement of biotin-strept(avidin) by a new pair with
the same binding constant opens a new approach in immunoassays.
a
NB The Abbott Architect i2000® is not based on strept(avidin)-biotin
chemistry.
b
As an example, the method for hCG is a sandwich chemiluminescence
immunoassay. A specific mouse monoclonal antibody is coated on the magnetic
particles (solid phase), another monoclonal antibody is linked to an isoluminol
derivative (isoluminol-antibody conjugate (https://www.accessdata.fda.gov/
cdrh_docs/pdf13/K131037.pdf).
hair, skin, and nails. As a result, women and “bald” men have become
obsessive toward biotin consumption as high as 1250–2500 μg twice
daily to beautify their skin, nail, and hair. The recommended dose of
biotin for adults by the Institute of Medicine (US) is only 30 μg per day
(Ross et al., 2016).
Another subject of debate is the efficacy of high-dose biotin therapy
for patients with relapsing and progressive multiple sclerosis (MS).
Myelin is impaired in MS patients, a “lipid-rich” compound that protects the nerve cells. Dimethyl fumarate (DMF), used for the treatment
of psoriasis, is approved to treat patients with relapsing-remitting
multiple sclerosis (MS) (Bomprezzi, 2015). DMF exposure offers the
potential cytoprotection of neurons, oligodendrocytes, and glial cells.
Another FDA-approved immunosuppressive drug for progressive MS in
2017 is Ocrelizumab (trade name Ocrevus). This humanized anti-CD20
monoclonal antibody targets the CD20 marker on B lymphocytes
(McGinley et al., 2017). Clinical trials are still in progress to establish its
efficacy, safety cancer risks and any adverse effects on pregnant women
and children they might bear. This drug is exorbitant, priced at $65,000
(annual cost, for two infusions per year (Ron Winslow, March 28,
2017). “After 40-year odyssey, first drug for aggressive MS wins FDA
approval”), thus, the search is on for more effective treatments and
affordable drugs.
Biotin is very inexpensive and has not been known for any reported
serious side effects or cytotoxicity. Biotin promotes the production of
myelin and provides energy for neurons. Many people with multiple
sclerosis (MS) use vitamins, particularly B vitamins to manage their
symptoms to convert food into energy for supporting the nervous
system. Biotin (B7 or vitamin H), is one of the B complex vitamins and
is essential for human health. Biotin activates several key enzymes and
helps the body to produce more myelin for cell-cell communication,
reducing the level of disability. MS patients often take 300 mg per day.
Biotin therapy is extended to people with biotinidase deficiency and
this disease often commences within months of birth. People with this
disorder lack biotinidase, an enzyme that recycles biotin several times
before its discharge as waste. Other rare genetic diseases that cause
biotin deficiency include holocarboxylase synthetase deficiency, biotin
transport deficiency, and the more common phenylketonuria. The daily
dose of such patients is 10–40 mg (Henry et al., 1996; Wijeratne et al.,
2012).
The plausible side effect of biotin deserves a brief comment here
considering the very high levels of biotin in some beauty products. In
most cases, no serious adverse effects have been reported but minor side
effects include nausea, cramping, and diarrhea. Biotin consumption in
high doses might affect the reproductive female performance by interfering with the synthesis of estrogen and progesterone (Wallig and
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et al., 1991), also resulting in an absorption peak around 500 nm. Excess albumin in the sample might interfere with the assay. In blood
plasma, 81% is free, 12% is covalently bound, and 7% is reversibly
bound (Mock and Malik, 1992). Thus, the measurement of biotin in
blood plasma without sample treatment will represent 80% of the total
biotin. Of notice are two major biotin metabolites: biotin sulfoxide
(BSO) and bisnorbiotin (BNB). In the cerebrospinal fluid of children,
biotin accounts for 42 ± 16%, BSO for 41 ± 12%, and BNB for
8 ± 14% of the total biotin (Bogusiewicz et al., 2008).
High-performance liquid chromatography (HPLC) on a bonded
phase C18 column can be used to resolve biotin and its analogs. Biotin
exhibits no absorption or fluorescence properties, so such compounds
must be derivatized to ω,4-dibromoacetophenone esters for UV detection or 4-bromomethylmethoxy-coumarin derivatives for fluorometric
detection (excitation/emission wavelengths at 360 nm and 410 nm)
(https://www.thermofisher.com/ca/en/home/life-science/cell
analysis/fluorophores/coumarin.html.). HPLC (C18 column) with postcolumn derivatization, using o-phthalaldehyde and 3-mercaptopropionic acid (3-MPA) analyzes for biotin in pharmaceutical preparations
with a detection limit of 10 ng per injection (Nojiri et al., 1998). Direct
determination of biotin in multivitamin pharmaceutical preparations is
feasible by HPLC using electrochemical detection (the type of electrode
is not specified) (Kamata et al., 1986). The ultimate system in clinical
and hospital settings is HPLC-MS/MS. Some pre-treatments are required
and electrospray with positive ionization has been proven as the most
sensitive ionization method. There are two prominent SRM (specialized
pro-resolving mediators) transitions observed for biotin, m/z 245 →
227, and 245 → 114 but only the first one is sufficiently intense for
quantification (Holler et al., 2006). HPLC using reversed-phase and
anion-exchange chromatographic conditions can also resolve biotin and
its analogs. Anion-exchange separations give generally shorter retention times and greater resolution between biotin I- and d-sulfoxide,
compared to reversed-phase separations. Such lab-based methods are
time-consuming and expensive but provide accurate levels of the target
analytes in the plasma of MS patients. This disease is no longer rare
with the global median prevalence of 33/100,000 and could be higher
in specific parts of the world (Thompson et al., 2014). Electroanalysis
with low cost, ease of fabrication/miniaturization, and high detection
sensitivity might be useful for the detection of biotin in blood. However, the progress in this area is very limited. There are only a few
literature reports related to electroanalysis/electrocatalysis of biotin
(Lauw et al., 2013; Marin et al., 1977; Serna et al., 1973). Recently, a
simple Nafion modified boron-doped diamond (BDD) electrode detects
biotin as low as 5–10 nM (Buzid et al., 2018). However, it lacks detection selectivity as other endogenous species in biological samples,
e.g., amino acids, vitamins, dopamine, etc. are also oxidized in this
detection scheme. Thus, the issue of detection selectivity is still very
challenging considering the presence of endogenous electroactive species such as vitamins, drugs, and their metabolites, amino acids, etc. in
blood samples.
Of notice is the use of captavidin as a regenerable biorecognition
element on boron-doped diamond for biotin sensing (Buzid et al.,
2019). The biotin-avidin binding occurs via hydrogen bonding between
the carbonyl group on the ureido ring of the former and the single
tyrosine (Tyr-33, pKa = 10.5) of the latter at pH below this pKa (Morag
et al., 1996). For captavidin, three of the four tyrosine moieties are
nitrated to nitroso-avidin with a pKa value of ~7.2. Thus, its binding to
biotin is weaker compared to avidin at pH below 10. As shown in
Table 3, the association constant of the captavidin-biotin pair is only
10−9 M compared to 10−15 M (González et al., 1997) of the strept
(avidin)-biotin counterpart. Above pH 10, the hydroxyl groups of the
tyrosine moieties ionize, affecting the hydrogen disruption to release
biotin. This behavior has been exploited for the fabrication of a regenerable biosensor for the detection of biotin in blood plasma with a
detection limit of below 1 nM. In this impedance sensing scheme,
captavidin irreversibly adsorbs on carboxymethyl cellulose to form a
Cucurbiturils represent emerging molecules (Mock, 1995) that can bind
several molecules, e.g., diamantane diammonium ion, with
Ka = 7.2 × 10−17 M−1 in D2O. Cucurbiturils with different sizes can be
synthesized from the acid-catalyzed condensation of glycoluril and
formaldehyde. Albeit the chemistry of cucurbiturils have received
considerable attention (Shetty et al., 2015; Hwang et al., 2007), the
implementation of these materials and their counterparts is a long-term
endeavor. First, it remains to investigate their non-specific protein
binding and plausible interferences from biotin, vitamins, and other
endogenous molecules in clinical and biological samples.
The use of magnetic particles (micro or nanoscale), consisting of an
inorganic core of iron oxide (magnetite (Fe3O4), maghemite (Fe2O3) or
other insoluble ferrites), deserves a brief comment here due to their
commercial availability and facile bioconjugation. Coating magnetic
particles with a polymer, organic molecule or biomolecule imparts
functional groups such as amino and carboxylic acids to facilitate
subsequent conjugations. Such materials conjugated with strept(avidin)
are also attractive for their use in the removal of biotin in the sample.
Nanoparticles of iron oxides (5–15 nm in diameter) are superparamagnetic whereas microparticles are ferromagnetic. Coating magnetic particles with a polymer alters their size and magnetic properties.
For applications in the separation technique or biotin removal, small
magnetic monodomain nanoparticles are preferred because they do not
possess remanence (remanent magnetization or residual magnetism)
when the magnetic field is removed (Leslie-Pelecky and Rieke, 1996).
Nanoparticles of metal oxides, e.g., magnetite Fe3O4 and maghemite γFe2O3 are simply prepared by the alkaline coprecipitation of ferric and
ferrous salts (Massart and Cabuil, 1987). Magnetic polymer beads with
various functional groups can be prepared by coprecipitation of iron
salts directly in a pertinent polymer matrix. In general, the size of
magnetic particles becomes smaller than in the absence of polymer
(Pardoe et al., 2001).
In principle, immunoassays using these “pre-bound” reagents followed by sample addition are expected to be resistant to, or insignificantly affected by biotin interference. However, thorough investigation
and validation are still needed to ascertain that these expectations are
supported by the results of interference studies. Immunoassay manufacturers are expected to conduct this protocol for various target analytes to establish the tolerable biotin concentration for each analyte.
Similarly, the pre-removal of biotin in the sample using strep(avidin)
coated polymers or magnetic particles is still subject to intensive investigation to iron out any plausible interference due to non-specific
interaction/adsorption of the target analyte or other endogenous species with strept(avidin) coated carriers.
7. Measurement of biotin
Rapid screening of biotin is desirable before the sample is subject to
immunoassays to provide a warning to plausibly falsely negative or
falsely positive outcomes. Such information may be used for more accurate risk assessment in predicting the effects of biotin. The HABA (4′hydroxyazobenzene-2-carboxylic acid) method provides a colorimetric
method to estimate the biotin concentration in a solution. HABA has an
absorption peak at 348 nm and binds avidin relatively weakly
(Kd = 5.8 × 10−6 M) (Green, 1970) to form an emerging peak at
500 nm. HABA binds to avidin as a hydrazone tautomer, involving an
intramolecular hydrogen bond and the loss of planarity (Livnah et al.,
1993a, 1993b). Similarly, streptavidin binds HABA as the hydrazone
tautomer with a lower Kd (10−4 M (Green, 1970). Some HABA derivatives bind avidin more strongly but their absorption (extinction)
coefficients are low (Repo et al., 2006). Biotin can easily dissociate
HABA from the HABA-avidin complex due to its significantly higher
affinity for avidin (Kd = 10−15 M). The decrease of the absorption peak
at 500 nm is followed and related to the biotin concentration. This assay
has a linear range from 2 to 16 μM. Of importance is the non-specific
binding of avidin with other proteins, e.g., serum albumin (Tarnoky
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presence of elevated biotin in bloods affects immunoassays using the
biotin-strept(avidin) pair for heart failure, pregnancy, cancer, and irondeficiency anemia. This issue is “somewhat” complicated as the level of
biotin interference is dependent on the tested analyte. As a few examples (Schauss, 2018), the hCG (human chorionic gonadotropin), PTH
(parathyroid hormone), and NTproBNP (N-terminal pro–B-type natriuretic peptide) assays are significantly interfered by > 5 ng/mL
(20 nM) of biotin concentration whereas the Troponin-T assay experiences no interference from biotin up to 100 ng/mL (0.4 μM).
Biotin is an emerging interferent but manufacturers for immunoassay equipment and chemicals are still looking for some concrete
solutions. It will take time and money to find a bold solution and
clinical validation. At best, the subject must abstain from biotin intake
for 2 days or longer before the test, whereas clinical laboratories try
their best by running the same sample on different immunoassay
platforms to validate the results. Sample dilution can alleviate this
problem but not beyond the detection sensitivity for the target analyte.
Patients should declare whether they have consumed biotin-containing
supplements prior to having sample collection, considering a quick and
inexpensive method for analysis of plasma biotin is still not available.
Biotin metabolites may interfere in some assays and this task deserves
further studies. This issue of biotin interference is particularly critical
for patients in emergency situations considering a quick test for biotin is
still not available to confirm whether the patient has been subjected to
high-dose biotin. Apparently, an extensive communication campaign to
educate physicians, laboratorians, and patients must be fostered to
publicize the emerging issue of elevated biotin interference in immunoassays.
Supraphysiologic biotin intake will not be diminished any time
soon, particularly for MS patients and for patients with other inherited
metabolic diseases, who are subject to high dose biotin therapy.
Furthermore, over-consumption of biotin also stems from claims of its
benefit for healthy hair and nail growth. Thus, elevated biotin in blood
remains problematical for immunoassays using the avidin/streptavidinbiotin chemistry.
regenerable biorecognition element for biotin. This layer is then retained and stabilized on a boron-doped diamond electrode by a Nafion
film. The biosensor has two distinct features: captavidin confers detection specificity and regenerability whereas the negatively charged
Nafion and carboxymethylcellulose layers circumvent the diffusion of
endogenous electroactive species.
Indeed, the literature also covers the use of captavidin in surface
plasmon resonance, SPR (Garcia-Aljaro et al., 2009) and carbon nanotube (SWNT) field-effect transistors, FETs (Munzer et al., 2014). With
SPR, biotinylated antibodies can be successfully subject to up to nine
serial capture-release events from the captavidin-functionalized surface, i.e., captividin is not exploited for the binding and detection of
biotin. For the work related to FET, the reversible captavidin binding
with pyrene-biotin functionalized SWNT FETs has been demonstrated.
This biosensing scheme offers to probe the dissociation constant of
captavidin and differentiate between two different biotin-binding molecules, streptavidin and Neutravidin based on the pH-dependent sensor
response. Gold nanoparticle decorated SWNT FETs are functionalized
with biotin to display reversible captavidin binding. This sensing
scheme could be a promising platform for the detection of proteins
based on enhanced Raman spectroscopy (Munzer et al., 2014).
Gold nanoparticles (AuNPs) have been used together with magnetic
beads in a competitive immunoassay format for the detection of biotin
(Lin et al., 2019). The assay is based on anti-biotin antibody-modified
magnetic beads (Ab-MBs) and biotinylated thiol-DNA AuNPs (biotinAuNPs). Without biotin in the sample, biotin-AuNPs bound to Ab-MBs
are retained by an external magnetic field and the solution is transparent. For the assay sample with biotin, free biotin will compete with
biotin-AuNPs and bind the Ab-MBs and the complex is retained by the
magnetic field. Unbound biotin-AuNPs are still floating in the solution,
resulting in a red color or surface plasmon resonance at 530 nm. The
detection limit of this method for biotin is only 2 pmol/100 μL of the
total sample, i.e., 20 nM. Several proteins, amino acids, and biomolecules will bind AuNPs to cause significant interferences (Zhong et al.,
2004). Albeit this method has been tested for some food samples, its
applicability for the analysis of biotin in blood or blood plasma remains
to be confirmed. The colorimetric assay strategy for avidin and biotin
interactions can be probed by a colorimetric assay using AuNPs (Shi
et al., 2018). In brief, biotin-ssDNA specifically bound to avidin with
strong affinity is protected from hydrolysis by exonuclease I (Exo I). The
biotin-ssDNA (negative charge) would attach to the surface of AuNPs
(positive charge) in high salt solution through electrostatic interactions
to prevent the aggregation of AuNPs. The surface plasmon resonance of
AuNPs at 520 nm increases gradually with a red color with increasing
avidin in the assay sample. This method aims for the detection of avidin
with a detection limit of 4 × 10−3 μg/mL. Therefore, considerable efforts are still needed to use this concept for the detection of biotin.
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8. Concluding remarks
Despite no claimed benefit having been proven, the use of vitamin
and mineral supplements for non-medical treatment continues to grow.
Such vitamins and biotin including their metabolites will become pervasive interferents. It is estimated that the US supplement industry is
now costing consumers over $30 billion annually (Kelly, 2017). Laboratorians have long had to contend with potential analytical interferences due to ingested substances. Now, biotin has become a pervasive interferent, increasingly insidious and problematic to clinical
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