SOLUTION FOR DISSOCIATING VITAMIN D FROM VITAMIN DBINDING
PROTEIN, ASSOCIATED DETECTION METHOD AND USE
The present invention relates to the technical field
of detecting vitamin D. More particularly, the invention
5 provides the use of an association of a fluoalkyl
aurfactant and an alcohol for releasing vitamin D and/or
one of its metabolites from the Vitamin D binding
protein, a solution containing such an association, an in
vitro method of detecting/quantifying vitamin D and/or at
10 least one metabolite of vitamin D using such an
association, and a kit for detecting/quantifying by
immunoassay making use of such an association.
Vitamin D is an important substance having numerous
i-mplications in the biological processes of the human and
15 animal body. Biologically active vitamin D is known to
regulate, amongst other things, the fixing of calcium
from the intestine, and bone mineralization, and it has
an influence on many other metabolic pathways, such as
for example the insulin system. A deficiency or an
20 excess of vitamin D can have various consequences. In
particular, it is known that vitamin D deficiency leads
to severe illnesses such as osteoporosis and rickets.
Furthermore, excess vitamin D, in particular due to
an overdose, is toxic. In particular, a high level of
25 vitamin D can lead to hypercalcemia caused by an increase
in the absorption of calcium by the intestine. Other
toxic effects of vitamin D are manifested by an increase
in blood pressure, gastrointestinal troubles such as
' anorexia, nausea, often followed by excessive production
30 of urine, polydipsia, fatigue, nervousness, itching, or
indeed kidney failure.
As mentioned in application WO 2012/091569, it has
also been discovered in recent years that vitamin D
modulates the immune system and reduces inflammation. It
35 has also been suggested that vitamin D can prevent
cancers of the colon, the ovaries, and the breast.
Consequently, it is important to be able to measure
or quantify vitamin D, i.e. to determine its
concentration, so as to reveal any potential deficiencies
or excesses.
5 Vitamin D is present in the organism in two fo'rms,
namely vitamin D2 (ergocaliferol) and vitamin D,
(cholecalciferol) of formulae that are set out below (in
which the positions in the vitamin D are numbered in
compliance with steroid nomenclature).
10 Vitamin D, (ergocalciferol) :
CH,
Vitamin D, (cholecalciferol) :
Vitamin D, is the exogenous form of vitamin D,.coming
from food. Vitamin D, is the endogenous form of vitamin D
as produced by the organism under the action of
ultraviolet rays from sunlight on the skin. The vitamin
5 D, that is produced by the skin binds with the vitamin D
binding protein, which transports it to the liver. Both
forms may also come from nutritional supplements.
Various metabolites of vitamin D are produced. In
particular, two-step metabolization takes place, the
10 first step consisting in producing 25-hydroxy vitamin D
(D, or D,), followed by producing 1,25-dihydroxy vitamin D
(D, or D,) .
Measuring the quantity of vitamin D itself is of
limited interest, given that its concentration fluctuates
15 strongly as a function of diet. The same applies to
1,25-dihdyroxy vitamin D metabolites which are present in
fairly low concentration and which also fluctuate.
The circulating forms of vitamin D are essentially
metabolites of the 25-hydroxy vitamin D type. Thus, the
20 preferred mode for obtaining information about the
overall concentration of vitamin D in a patient is
assaying 25-hydroxy vitamin D.
The bonding of 25-hydroxy vitamin D, or more
genera1l.y of vitamin D and its metabolites, to the
25 vitamin D binding protein (DBP) complicates assaying its
components. In order to obtain an adequate assay, it is
necessary to release the hapten to be assayed from the
DBP by causing them to dissociate. Thus, various
solutions have been proposed for obtain'ing the release of
30 vitamin D and its metabolites after dissociating DBP and
before detecting them.
Various techniques listed in patent application
WO 2007/039194 and in patent application WO 2012/091569
have been developed. Only some of them are listed below.
3 5 An old technique, used in particular in application
WO 99/67211 consists in preparing a sample of plasma or
serum in order to determine vitamin D by ethanol
precipitation. The precipitates are then eliminated in
order to recover the ethanolic supernatent containing the
soluble metabolites of vitamin D. Precipitation by means
of alcohol or another organic solvent such as
acetonitrile has commonly been used in the past.
Nevertheless, that technique cannot be automated and
requires numerous manual operations (adding solvents to
the serum, mixing, centrifuging, recovering the organic
phase, drying on a column or otherwise, re-suspension in
a li-quid solvent), thus making it obsolete these days
because of the high degree of variation that is observed
between operators.
Patent application WO 2007/039194 proposes using a
solution containing 5% to 30% by volume of one or more
amphi-philic reagents selected from dimethylsulfoxide
(DMSO) and liquid organic amides, and optionally 0.7% to
8% by volume of a short chain alcohol C - C . The
amphiphilic compounds used in that document are
substances that are toxic, or indeed dangerous if it is
DMSO .
Patent applications WO 2011/122948 and
WO 2012/091569 in the name of Future Diagnostics describe
immunoassay and an agent that enable vitamin D to be
released from its binding protein and that uses a
fluoroalkyl surfactant. The Applicant has made use of
that solution, but has nevertheless found that the
dissociation that is obtained is still not sufficient.
The object of the invention is to propose a more
effective dissociation technique that is easy to perform
and that leads to effective release of vitamin D and its
metabolites, after dissociating the vitamin D binding
protein (DBP), subsequently enabling them to be detected
and quantified adequately.
In the context of the present invention, the object
is to provide a new solution for dissociating vitamin D
or one of its metabolites from vitamin D binding protein
(DBP), that is more effective than the techniques
proposed in the prior art by Future Diagnostics in its
patent applications WO 2011/122948 and WO 2012/091569.
In addition, the solution described in the present .
invention does not present the toxicity problems of.the
5 .solutions as proposed in particular in patent application
WO 2007/039194.
In this context, the invention provides the use of
at least one fluoroalkyl surfactant, and in particular a
perfluoroaklyl surfactant, and of at least one alcohol'
10 having 1 to 4 carbon atoms for dissociating vitamin D
and/or a vitamin D metabolite from vitamin D binding
protein. Such joi.nt use makes it possible to increase
significantly the dissociation rate of vitamin D or one
of its metabolites from vitamin D binding protein, in
15 comparison with a similar use differing solely by the
absence of alcohol. In the use of the invention, the
fluoroalkyl surfactant, and in particular the
perfluoroalkyl surfactant, and the alcohol having 1 to 4
carbon atoms are incorporated in a liquid sample
20 including the vitamin D and/or a vitamin D metabolite
(referred to as an analyte or vitamin D analyte),
associated with vitamin D binding protein. The analyte
for dissociation is thus in contact both with the
fluoroalkyl surfactant and with the. alcohol, which, when
25 used together, enable dissociation to be achieved that is
greater than when using the same fluoroalkyl surfactant
on its own. The joint use of fluoroalkyl surfactant, and
in particular perfluoroalkyl surfactant, and alcohol
having 1 to 4 carbon atoms, leads to effective
30 dissociation between vitamin D binding protein and
vitamin D and/or one of its metabolites.
In particular, the fluoroalkyl surfactant and the
alcohol are used in quantities such that the ratio
multiplied by 100 of the weight of surfactant, when it is
35 solid, or of the volume'of surfactant, when it is liquid,
over the volume of alcohol lies in the range 10% to 60%,
preferably in the range 15% to 40%, and more
in the range 15% to 30%. The solid or liquid nature is
assessed at ambient temperature (in particular at 22"~)
and at atmospheric pressure (in particular at
1013 hectopascals (hPa) ) .
5 Advantageously, the fluoroalkyl surfactant is
selected from perfluorocarboxylic acids,
perfluorosulfonic acids and their salts, and in
particular from perfluorohexanoic acid,
perfluoroheptanoic acid, and perfluorooctanoic acid, and
10 their salts. Perfluorohexanoic acid is also known as
perfluorocaproic acid or indeed as undecafluorohexaonic
acid; its chemical abstract service (CAS) registration
number is 307-24-4. Perfluorooctanoic acid is also known
as perfluorocaprylic acid or indeed as
15 pentadecafluorooctanoic acid; its CAS number is 335-67-1.
In general, the salts of these perfluoroalkyl acids are
solid, whereas the corresponding free acids are liquid.
Perfluorohexanoic acid, possibly in salt form, is
the preferred fluoroalkyl surfactant, since it presents
20 better degradability compared with longer chain
fluoroalkyl surfactants.
In preferred manner, the alcohol used has 1 to 3
carbon atoms and is selected from methanol, ethanol, npropanol,
and isopropanol. Methanol is the preferred
25 alcohol in the invention since it makes it possible to
obtain performance that is better in terms of the
reproducibility of the results of the assaying performed
after dissociation, and provides a good compromise in
terms of improving dissociation and the reproducibility
30 of the results obtained.
In particularly advantageous manner, the
dissociation is performed with perfluorohexanoic acid and
methanol.
In the context of the invention, the dissociation
35 may be performed in the presence of an additional
surfactant selected, in particular from block copolymers
based on ethylene oxide and propylene oxide,
polysorbates, and polyethylene glycol ethers.
By way of example, the invention is performed to
dissociate 25-hydroxy vitamin D, and in part~cular 25-
5 hydroxy vitamin D2 and/or 25-hydroxy vitamin Dj from
vitamin D binding protein.
The alcohol and the fluoroalkyl surfactant may be
incorporated separately in the sample in which it is
desired to obtain dissociation, or they may be
10 incorporated simultaneously. Under such circumstances, a
single solution is used referred to as a "dissociation"
solution, in order to minimize manipulations.
The invention also provides such solutions
comprising at least one fluoroalkyl surfactant, and in
15 particular a perfluoroalkyl surfactant, and at least one
alcohol having 1 to 4 carbon atoms.
Such solutions may be said to be aqueous and they
include a large quantity of water, which generally
represents more than 80% by volume relative to the total
20 volume of the solution.
Advantageously, such a solution comprises
a percentage of fluoroalkyl surfactant expressed by
volume when the surfactant is liquid, or by weight when
it is solid, relative to the total volume of the solution
25 lying in the range 0.1% to 3%, preferably in the range 1%
to 2%. In equally preferred manner, such a solution
comprises a percentage by volume of alcohol relative to
the total volume of the solution lying in the range 0.5%
to lo%, and preferably in the range 2% to 7%.
3 0 The same preferences concerning the choice of the
alcohol and of the fluoroalkyl surfactant and their
relative quantities, stated with reference to the use,
apply to the dissociation solutions of the invention.
In an advantageous variant, the dissociation
35 solutions of the invention also contain another
surfactant selected from block copolymers based on oxide
ethylene oxide and propylene oxide, polysorbates, and
polyethylene glycol ethers. Without seeking to be tied
to any particular interpretation of the results, such an
additional surfactant can improve the solubility of
vitamin D or of its metabolite that is to be assayed.
The use of such an additional surfactant, such as
PluronicB F-127, which is a polyol corresponding to a
block copolymer based on oxide ethylene oxide and
propylene oxide serves in particular to improve the
reproducibility of the results. It is found that the
final assay is more reliable. By way of non-limiting
example, it is possible to use PluronicB F-127 at a
concentration in the dissociation solution that
preferably lies in the range 0.1% to 3% (by volume
relative to the final volume of the dissociation
solution).
In general, the dissociation solutions of the
. .
invention are buffered, in particular to a pH lying in
the range 6 to 8.
The buffers conventionally used in the field of
diagnosis may be incorporated in the solution, so as to
obtain a pH in the desired range and so as to stabilize
it. By way of example, a phosphate-buffered saline (PBS)
buffer or a tris buffer (tris-hydroxyrnethyl aminomethane)
may be used.
A base may be incorporated in order to adjust the
pH. Such a base may be any base that is conventionally
used for this purpose, such as KOH, NaOH, LiOH, or
Na,HPO, .
Such a dissociation solution may be prepared by
mixing a buffer of concentration that preferably lies in
the range 1 millimole (mM) to 500 mM, a fluoroalkyl
surfactant of concentration that preferably lies in the
range 0.1% to 3% (by weight or by volume, ,depending on
the solid or liquid nature of the surfactant, relative to
the final volume of the dissociation solution) and an
alcohol of concentration that preferably lies in the
range 0.5% to 10% (by volume relative to the final volume
of the dissociation solution) in demineralized water.
The pH of the dissociation solution is adjusted depending
on the selected buffer, by adding acid or by adding base
so as to obtain a value lying in the range 4 to 8, and
5 preferably around 7. When the dissociation solution
contains an additional surfactant, it may be introduced
at any stage.
Advantageously, such dissociation solutions do not
contain any of the following compounds: dimethyl
10 sulfoxide, dimethylformamide, N,N-dimethylacetamide,
tetramethylurea, N-methylpyrrolidone,
1,3-dimethyl-3,4,5,6-tetrahydro-2 (1H) -pyrimidone,
triamide hexamethyl phosphoric acid.
The invention also provides a detection and
15 quantification method for detecting and quantifying, in
vitro, vitamin D and/or at least one vitamin D metabolite
in a biological sample, the method comprising the
following steps:
a) a step of treating the sample by incorporating at
20 least one fluoroalkyl surfactant and at least one alcohol
having 1 to 4 carbon atoms, so as to dissociate including
at least one fluoroalkyl surfactant and at least one
alcohol having 1 to 4 carbon atoms
from vitamin D binding protein; and
25 b) a step of detecting and quantifying vitamin D
and/or at least one of its metabolites.
The dissociation step a) should be performed before
detection and quantification step b).
Advantageously, in such a detection and
30 quantification method, the treatment of step a) is
performed by mixing the sample with a dissociation
solution of the invention. Usually, 1 to 20 volumes of
dissociation solution, preferably 5 to 10 volumes, more
preferably 6 to 8 volumes, and in particular about 7
35 volumes of solution are used for 1 volume of sample. The
selected volume is a function of the presumed
concentration of vitamin D and/or vitamin D metabolite
that is to be detected (referred to as analyte to be
detected).
Advantageously, the detection and quantification
method of the invention is performed on a sample of
blood, of serum, or of plasma.
The detection and quantification method is suitable
in particular for detecting and quantifying 25-hydroxy
vitamin D, and/or 25-hydroxy vitamin D3.
In step b), detection and quantification are
preferably performed by performing an immunoassay, or
indeed by mass spectrometry.
The invention also provides a kit for detecting and
quantifying vitamin D and/or at least one vitamin D
metabolite by immunoassay and comprising a dissociation
solution of the invention. Such a kit may also include a
binding partner for vitamin D or one of its metabolites
and/or a solid phase on which a hapten analogous to
vitamin D and/or the vitamin D metabolite(s) for
detection is bonded, which hapten is recognized by the
marked antibody.
In the context of the invention, vitamin D
metabolites cover all compounds that contain the skeleton
of vitamin D, or the skeleton of vitamin D3, and in
particular:
25-hydroxy vitamin D, which specifies the
metabolites of vitamin D that are hydroxylated in
position 25, i.e. 25-hydroxy vitamin D2 and 25-hydroxy
vitamin D3; and
the 1,25 and 24,25-dihydroxy vitamin D forms,
which specify the metabolites of vitamin D that are
dihydroxylated, respectively in positions 1 and 25 or in
positions 24 and 25.
In the methods of uses of the invention,
dissociation may be performed by incorporating, in the
sample for analysis, both the fluoroalkyl surfactant and
also the Cl-C, alcohol and preferably C1-C3 alcohol that
have been selected, either together or separately. After
such incorporation, no separation is needed, and
detection can be performed directly on the resulting
sample. Preferably, in order to limit manipulations, a
previously prepared solution containing the fluoroalkyl
5 surfactant and the C1-C, alcohol and preferably C,-C,
alcohol that have been selected is incorporated in the
sample, and in particular a solution in accordance with
the invention.
In order to enhance dissociation, the sample
10 containing the fluoroalkyl surfactant and in particular
the perfluoroalkyl surfactant and C,-C, alcohol and
preferably Cl-C3 alcohol that have been selected is
subjected to mixing. Such mixing may be performed with
any appropriate device, and in particular by means of a
15 reaction cone acting as a pipette, as in the Applicant's
VidasB technology (VidasB Manuel Instrument, 2005,
Chapitre 2 Description fonctionnelle, 2-1 to 2-16,
bioMBrieux France [VidasB Instrument manual, 2005,
Chapter 2, Functional description, 2-1 to 2-16,
20 bioMBrieux France] ) .
The vitamin D or one of its metabolites may be
detected using any technique known to the person skilled
in the art, and in particular by performing a test using
a binding partner of the analyte to be detected, and in
25 particular an immunological test (also known as an
immunoassay test), or indeed by mass spectrometry.
Naturally, the prefix "immuno" in the term
"immunoassay", for example, should not be considered in
the present application as indicating strictly that the
30 binding partner is necessarily a partner of immunological
origin, such as an antibody or an antibody fragment. As
is well known to the person skilled in the art, this term
is used more widely to designate tests and methods in
which the binding partner is not solely a partner of
35 immunological origin and/or nature, but may consist, for
example, of a receiver for the analyte that is to be
detected and/or quantified, the condition being that the
binding partner in question must be capable of binding to
the looked-for analyte, and preferably in specific
manner. Thus, it is known to use the term "enzyme-linked
immunoabsorbant assay (Elisa)" for assays that make use
5 of binding partners that are not strictly speaking
immunological, and that are referred to more broadly as
"ligand binding" assays, even though the term "immuno" is
included in the acronym ~lisa. For the purposes of
clarity and uniformity, the term "immuno" is used in the
10 present application to cover any biological analysis
using at least one binding partner suitable for binding
with the looked-for analyte and for detecting and/or
quantifying it, preferably in specific manner, even when
said binding partner is not strictly speaking of
15 immunological nature or origin.
The immunological test is preferably competition
assaying, which is a form of assaying well known to the
person skilled in the art and which is used when the
analyte is a hapten. It consists in assaying the in-
20 sample analyte, specifically vitamin D and/or at least
one of its metabolites, by setting up competition between
the analyte of the sample and an analog of the analyte.
The immunological reaction is then revealed by the
presence of a tracer.
25 The analyte analog may be used in the competition
reaction without prior coupling or after coupling to a
marker in order to form a conjugate or tracer.
Immunological assay by competition also requires the
use of a binding partner of the analyte relative to which
30 the analyte analog and the analyte enter into
competition. When the analyte analog is not coupled to a
marker (it is not the tracer but the capture partner),
the binding partner is marked in order to constitute the
reaction tracer. When the analyte analog is coupled to a
35 marker (it is then the tracer), the binding partner then
becomes the capture partner.
The measured signal as emitted by the tracer is then
inversely proportional to the quantity of analyte in the
sample.
The term "marker" is used to mean any molecule
5 containing a group that reacts with the capture partner
or the analyte analog, depending on the format, directly
without chemical modification, or after chemical
modification to include such a group, which molecule is
capable of generating a detectable signal either directly
10 or indirectly. Such a reactive group may in particular
be a primary amine. A non-limitlng list of such direct
detection markers is as follows:
enzymes that produce a signal that is detectable,
e.g. by colorimetry, fluorescence, luminescence, such as
15 horseradish peroxydase, alkaline phosphatase,
P-galactosidase, and glucose-6-phosphate dehydrogenase;
chromophores such as fluorescent, luminescent, and
dye compounds;
radioactive molecules such as 32P, 35S, or lZ5I;
20 fluorescent molecules such as Alexa or
phycocyanines; and
electrochemiluminescent salts such as organometallic
derivatives based on acridinium or ruthenium.
Indirect detection systems may also be used, such as
25 for example ligands capable of reacting with an antiligand.
The ligand then corresponds to the marker for
acting with the analyte analog or the binding partner to
constitute the tracer.
Ligand/anti-ligand pairs are well known to the
30 person skilled in the art as applies for example to the
following pairs: biotin/streptavidin, hapten/antibody,
antigen/antibody, peptite/antibody, sugar/lectin,
polynucleotide/polynucleotide complement.
The anti-ligand can then be detected directly by the
35 above-described direct detection markers or may itself be
detectable by using some other ligand/anti-ligand pair,
and so on.
Under certain conditions, these indirect detection
systems can lead to the signal being amplified. This
signal amplification technique is well known to the
person skilled in the art, and reference may be made to
5 prior patent applications FR 2 781 802 or WO 95/08000 in
the name of the Applicant.
Depending on the type of marker used, the person
skilled in the art adds reagents enabling the marking to
be viewed or a signal to be emitted that is detectable by
10 any appropriate type of measuring apparatus, such as for
example: a spectrophotometer; a spectrofluorometer; or
indeed a high definition camera.
The term "binding partner" for vitamin D or one of
its metabolites, or indeed for a plurality of these
15 compounds, is used to mean any molecule capable of
binding.with vitamin D or one of its metabolites, or
indeed with several of these. compounds (referred to in
general manner as "vitamin D analytes"). As an example
of a vitamin D analyte binding partner, mention maybe
20 made of antibodies, antibody fractions, nanofitins,
vitamin D analyte receivers, or any other protein that is
known to interact with vitamin D analyte.
By way of example, the binding partner antibodies
may be either polyclonal antibodies or monoclonal
25 antibodies.
Polyclonal antibodies may be obtained by immunizing
an animal with the target vitamin D analyte as the
immunogen, followed by recovering the looked-for
antibodies in purified form by taking serum from said
30 animal, and separating said antibodies from the other
constituents of the serum, in particular by affinity
chromatography on a column having fixed thereon an
antigen that is specifically recognized by the
antibodies, in particular the immunogen.
3 5 Monoclonal antibodies may be obtained by the
hybridoma technique that is well known to the person
skilled in the art. Monoclonal antibodies may alsobe
recombinant antibodies obtained by genetic engineering,
using techniques well known to the person skilled in the
art.
As examples of antibody fragments, mention may.be
made of Fab, Fab', F(ab1)2 fragments and of single chain
variable fragments (scFv) and double-stranded variable
fragments (dsFv). These functional fragments may be
obtained in particular by genetic engineering.
Nanofitins (trade name) are small proteins that,
like antibodies, are capable of binding to a biological
target, thus making it possible to detect it, capture it,
or merely to target it within an organism.
The binding partners used may be specific or nonspecific
to the vitamin D analyte. They are said to be
"specific" when they are capableof binding in exclusive
or almost exclusive manner with the vitamin D analyte.
They are said to be "non-specific" when the selectivity
of the binding with the vitamin D analyte is weak so they
are also capable of binding with other ligands, such as
other proteins or antibodies. In a preferred
implementation, specific binding partners are preferred.
Anti-vitamin D analyte antibodies are known, and
they are described in particular in Hollis, Clin. Chem.
31/11, 1815-1819 (1985) and Holis, Clin. Chem. 39/3,
529-533 (1993) and in patent EP 1 931 711. They may also
be obtained from various suppliers such as Bioventix
( U K ) .
Binding partners or vitamin D analogs, when they are
used in capture, may optionally be bound to a medium,
such as microtitration plates, latexes, reaction cones,
beads having a diameter of the order of one hundred
micrometers to a nanometer, using any technique well
known to the person skilled in the art. The vitamin D
analogues are preferably immobilized on a solid phase.
It is possible in particular to make use of a medium
that has been functionalized with avidin and/or
streptavidin and to bind on the solid phase a
biotinylated analyte analog. Functionalization
techniques are well known to the person skilled in the
art who can make reference thereto.
In conventional manner, in order to determine the
5 quantity of vitamin D and/or of at least one of its
metabolites, the signal, which is inversely proportional
to the quantity of analyte in the %ample, may be compared
with a calibration curve previously obtained using
techniques that are well known to the person skilled in
10 the art. Thus, by way of example, the calibration curve
may be obtained by performing an immunological assay
using the same binding partner together with increasing
known quantities of vitamin D. A curve is thus obtained
with concentration of vitamin D plotted along the
15 abscissa axis and the corresponding signal obtained after
immunological assay plotted up the ordinate axis.
The detection/quantification method of the invention
may be applied directly to the format of commercial tests
available for detecting/quantifying vitamin D. Such
20 formats for assaying vitamin D and/or one of its
metabolites are sold in particular by Abbott (Architect
25-OH vitamin D, ref. 3L52), DiaSorin (Liaison@ 25-OH
vitamin D total assay, ref. 310600), IDS (IDS-iSYS
25-hydroxy vitamin D assay, ref. IS-2700), Siemens (Advia
25 Centaur@ vitamin D total, ref. 10491994), Roche (Elecys
vitamin D total).
In conventional manner, performing an immunoassay
thus requires the reagents necessary for immunological
detection as described aboye, which reagents are to be
30 incorporated in the sample. Advantageously, the
dissociation and thus the incorporation in the sample for
study, both of the fluoroalkyl surfactant and of the C,-C,
alcohol, preferably a C,-C, alcohol, that have been
selected, are performed before incorporating such
35 reagents.
It is also possible to use mass spectrometry for
performing the detection/quantification step once the
dissociation has been obtained. This technique is an
analysis technique that makes it possible to determine
the molar masses of the compounds being analyzed, and
also enables their molecular structure to be identified,
5 and even enables them to be quantified. When applied to
a complex mixture such as a biological fluid, it needs to
be coupled to a separation technique that enables the
complexity of the fluid to be reduced. Usually that
comprises gas chromatography (GC) or liquid
10 chromatography (LC). Tandem mass spectrometry (MS/MS)
combines two analyzers and can be used for
detection/quantification purposes. The ionic compounds
selected in the first analyzer are analyzed more finely
in the second. Such double analysis serves to increase
15 significantly the specificity of the method. For this
technique, reference may be made in particular to Van den
Broek et al., J. Chromatogr. B 929 161-179 (2013) .
The biological sample with which the method of the
invention can be performed is any animal and preferably
20 human biological sample that might contain the analyte
(vitamin D or one of its metabolites), in which an
irnrnunoassay or a mass spectrometry analysis can be
performed. Such samples are well known to the person
skilled in the art. The sample used in the assay method
25 may optionally be modified prior to being used. As
examples of such samples that are not previously
modified, mention may be made of biological fluids such
as total blood, and as examples of samples that have been
previously modified, also known as sample derivatives,
30 mention may be made of serum, plasma, cells recovered
from a biopsy, or from a surgery, and then cultured in
vitro. The concentration of vitamin D or of one of its
metabolites may then be assayed in the culture
supernatent, or indeed in the cellular lysate.
35 The examples below serve to illustrate the
invention, but they have no limiting character.
For each experimentally tested condition, the tables
given in the examples give the relative fluorescence
value (RFV) signal as determined by the VidasB machine
(bioM6rieux). Often, a plurality of independent
measurements were taken for each of the conditions.. The
"mean RFV" corresponds to the arithmetic mean of such
independent measurements.
In order to verify the effectiveness of the
dissociation solutions, the results obtained with two
biological samples having different concentrations of
25-OH vitamin D were compared by calculating the % ratio
between the two signals obtained (written RFVN,,2/RFV,,,,,
sample No. 1 being the sample rsiith the lower
concentration of 25-OH vitamin D). For repeated
independent measurements, the ratio was calculated from
mean RFVs. The smaller this ratio, the better the
dissociation.
The coefficient of variation (CV) is defined as the
ratio between the standard deviation and the mean. It is
often expressed as a percentage (CV%). The CV% is a
measure of relative dispersion and it reflects the
reproducibility of the results. A reduction in the value
obtained is indicative of an improvement in
reproducibility.
Some of the experiments described form part of
experiment optimization plans that were constructed by
using Tagushi tables. When the optimum is a nominal
value, as for a reproducibility study (fixed signal
value), variance around the value may be considered as
being the result of noise factors, and thus as being
detrimental to reproducibility. On the basis of repeated
experimental data, the signal-to-noise ratio (S/N) may be
calculated and it is defined using the formula
lOxlog,, (mean2/standard deviation2) . The S/N ratio,
sometimes known as the Taguchi constant, is indicative of
the reproducibility of the results. An increase in the
value obtained is indicative of an improvement in
reproducibility.
The ratio B/BO% is the signal obtained for the
tested range point divided by the signal obtained for the
5 range point having 0 nanograms per millimeter (ng/mL) of
analyte, multiplied by 100.
In the various tables that follow, with the
exception of Table 5, the perfluoroalkyl acids used were
liquid, their percentages being given by volume relative
10 to the total volume of the dissociation solution. For
the alcohol used, in all cases, the percentages are given
by volume relative to the total volume of the
dissociation solution.
15 EXAMPLE 1 - Advantage of dissociation using a methanol
and perfluorohexanoic acid mixture compared with
perfluorohexanoic acid alone
Preparation of dissociation solutions
Comparative dissociation solution: the components
20 for preparing the PBS buffer (5 mM of sodium hydrogen
phosphate (Na,HPO,), 1.5 mM of potassium dihydrogen
phosphate (KH,PO,) , 131 mM of NaC1) and 0.75%
perfluorohexanoic acid were dissolved in demineralized
water by stirring for about 30 minutes (min). The pH was
25 adjusted to 7.2 using 6N NaOH.
Solution of the invention: the components for
preparing the PBS buffer (5 mM of sodium hydrogen
phosphate (Na,HPO,) , 1.5 mM of potassium dihydrogen
phosphate (KH,PO,) , 131 mM of NaC1) , 0.75%
30 perfluorohexanoic acid, and 5% of methanol were dissolved
in demineralized water by stirring for about 30 min. The
pH was adjusted to 7.2 using 6N NaOH.
Method of quantifying total 25-OH vitamin D
3 5 Immunological assays were performed using a VidasO
immunoanalysis machine (from bioM6rieux). The single-use
cone serves both as the solid phase for the reaction and
as a pipetting system. The cartridge was made up of ten
wells covered in a sealed and labeled aluminum sheet.
The first well had a cut-out portion to facilitate
inserting the sample. The last riel1 rias an optical
5 cuvette in which the fluorescence of the substrate was
measured. The various reagents needed for analysis were
contained in the intermediate wells. A11 of the steps of
the test were performed automatically by the instrument.
They were constituted by a succession of suck/blow cycles
10 of the reaction medium.
a) Sensitizing and passivating the cone
The cones were sensitized with 300 microliters (pL)
of a carrier anti-protein antibody solution diluted to
15 10 micrograms per milliliter (pg/mL) in a 50 mM MES
buffer of pH 6.1. After 6 hours (h) of incubation at
+18/25"C, washing was performed with a 9 grams per liper
(g/L) solution of NaC1. Thereafter, there were added
300 pL of a solution of vitamin D coupled to the carrier
20 protein and diluted to 150 nanograms per milliliter
(ng/mL) in a 200 mM tris buffer of pH 6.2 containing
human albumin. Sensitization/passivation continued at
+18/25"C overnight. The cones were emptied, dried, and
then stored at t-4'C while sheltered from moisture until
25 they were used.
b) Pretreatment of the sample
The sample for assay (100 pL) was introduced into
the first well of the cartridge. The sample and the
30 pretreatment reagent (comparative dissociation solution
or solution of the invention) were put together for
separating the vitamin D contained in the sample from its
binding protein. The VidasB machine mixed 48 IIL of
sample with 340 pL of dissociation solution; the mixture
35 was incubated at 37°C for 5 min.
c) Immunoassay reaction procedure
The pretreated sample (about 0.9 volumes) was
transferred into the well containing 1 volume of an antivitamin
D antibody marked with alkaline phosphatase
5 (conjugated, bioM6rieux). The alkaline phosphatase
antibody conjugate was diluted beforehand to about
10 pg/mL in 100 mM tris buffer of pH 7.1, 300 mM NaC1,
containing human albumin. The sample/conjugate mixture
was incubated in the well for about 5-7 min. Thereafter
10 the sample/conjugate mixture was incubated in the cone
for an additional 5-7 min approximately, during which
competition took place between the antigen present in the
sample and the vitamin D antigen fixed on the cone for
sites of the antibody specific to conjugated anti-vitamin
15 D. Thereafter, three successive washes with 200 mM tris
buffer of pH 8.4, 300 mM NaC1, Tween@ 20 0.2%, were
performed in order to eliminate the non-fixed compound.
During the final development step, the
4-methylombelliferyl phosphate substrate was sucked out
20 and then delivered into the cone; the enzyme of the
conjugate catalyzes the reaction of hydrolyzing the
substrate into 4-methylombelliferyl, and the fluorescence
it emits was measured at 450 nanometers (nm). The value
of the fluorescence signal is inversely proportional to
25 the concentration of the antigen present in the sample.
Table 1 below summarizes the fluorescence signals
(RFV) determi-ned by the VidasB machine as a function of
the dissociation solution used, usin'g three different
solutions of human serum. For each experimental setup,
30 four independent measurements were made.
T a b l e 1
RFV signal t---
I Mean RFV
0.75% perfluorohexanoic
acid WITHOUT methanol
Comparative dissociation
solution
0.75% perfluorohexanoic acid
5% methanol
Solution of the invention
I I
sample No. 1 at 11 nglmL
3356
3504
CV%
RFV signal
Mean RFV
CV%
SIB ratio
% ratio RFVNo21RFVNo.l
RFV signal
Mean RFV
CV%
It can be seen that adding alcohol improves
5 dissociation (the signal ratio in % decreases), while
4%
sample No. 2 at 20 nglmL
improving reproducibility (increase in the signal-tonoise
ratio and decrease in CV%).
3%
SIB ratio
2863
3290
3496
3072
31 80
9%
21.32
91%
sample No. 3 at 36 nglmL
32.43
63%
SIB ratio
EXAMPLE 2 - Comparing different alcohols
10 The dissociation solutions used in this example were
prepared using the procedure explained for Example 1, in
3430
3189
3316
3149
3271
4%
28.17
85%
2642
2440
2761
2815
2655
6%
24.1
a PBS buffer with pH of 7.2. The natures and the
28.58
2420
2349
2490
2433
2423
2%
30.82
% ratio RFVNo.3/RFVNo,l 76%
concentrations of the fluoroalkyl surfactant and of the
alcohol were varied and are set out in Tables 2 and 3.'
Otherwise, the procedure was as in Example 1.
5 Table 2: Use of perfluorohexanoic acid
Comparative dissociation solution: PBS + 1%
perfluorohexanoic acid without alcohol
sample No. 1
at I I nglmL
Dissociation solution of the invention: PBS + 1%
perfluorohexanoic acid + 5% methanol
sample No. 2
at 35 nglmL
I I
Mean RFV 2648
sample No. 1
at 11 nglmL
Dissociation solution of the invention: PBS + 1%
perfluorohexanoic acid + 5% ethanol
3224
sample No. 2
at 35 nglmL
I I
% ratio RFVN~.ZIRFVNO.~
Dissociation solution of the invention: PBS + 1%
perfluorohexanoic acid + 5% isopropanol
82%
Mean RFV 2323
Mean RFV
% ratio RFVN,Z/RFVN,<
3118
% ratio RFVN~.Z/RFVN~.~
sample No. 1
at I I nglmL
2707
.
Mean RFV
% ratio RFVN,~/RFVN~,
sample No. 2
at 35 ng/mL
1925
71%
-
sample No. 1
at 11 nglmL
2633
75%
sample No. 2
at 35 nglmL
2003
76%
It can be seen that adding alcohol, regardless of
the alcohol, leads to an improvement in dissociation (the
signal ratio in % decreases).
5 Table 3: The use of perfluoroctanoic acid
Comparative dissociation solution: PBS + 0.75%
perfluoroctanoic acid without alcohol
Dissociation solution: PBS + 0.75% ~erfluorooctanoic acid
Mean RFV
% ratio RFVN,~IRFVN,,
sample No. 1
at I I ng1mL
2684
L + 5% methanol
sample No. 2
at 35 nglmL
1197
45%
sample No. 1
at I I nglmL
I I
It can be seen that adding alcohol, regardless of
10 the alcohol, leads to an improvement in the dissociation
(signal ratio in % decreases).
sample No. 2
at 35 nglmL
Mean RFV
Dissociation solution: PBS + 0.75% perfLuorooctanoic acid
+ 5% ethanol
EXAMPLE 3 - Influence of perfluoroalkyl acid
concentration
15 The dissociation solutions used in this heat
exchanger were prepared using the procedure explained for
Example 1, in a PBS buffer with pH of 7.2.
-Mean RFV
% ratio RFVN~~IRFVN~~
Otherwise the procedure was as in Example 1.
% ratio RFVN,Z/RFVN,., I I 31%
2574 787
sample No. 1
at I I nglmL
2612
-
sample No. 2
at 35 nglmL
759
29%
.
Table 4
1.25%
perfluorohexanoic acid
without methanol
Mean RFV
% ratio
RFv~o.21
RFv~o.1
1.25%
perfluorohexanoic acid
+ 5% methanol
1.6%
perfluorohexanoic acid
without methanol
It can be seen that the dissociation increases in
the presence of alcohol under all circumstances. It can
5 also be seen that by increasing the concentration of
perfluoroalkyl acid, the dissociation is increased
further .
2% perfluorohexanoic
acid without methanol
sample
No.1
at 15
nglmL
4724
1.6%
perfluorohexanoic acid
+ 5% methanol
sample
No.1
at 15
Mean RFV
% ratio
RFVN,.Z/
RFv~o.1
EXAMPLE 4 - Influence of alcohol concentration
10 The dissociation solutions used in this example were
prepared using the procedure explained for Example 1, in
a PBS buffer with pH of 7.2. The natures and
concentrations of the fluoroalkyl surfactant and of the
alcohol were varied and they are set out in Table 5.
15 Since ammonium perfluorooctanoate is solid, the
percentages associated therewith are given by weight of
ammonium perfluorooctanoate relative to the total volume
of the solution.
Otherwise, the procedure was as in Example 1.
sample
No.2
at 35
nglmL
3844
81%
2% perfluorohexanoic
acid + 5% methanol
sample
No.2
at 35
sample
No.1
at 15
ng1mL
4054
sample
No.1
at 15
nglmL
3896
sample
No.1
at 15
nglmL
3352
sample
No.2
at 35
nglmL
2885
71%
sample
No.2
at 35
sample
No.1
at 15
sample
No.2
at 35
nglmL
2252
67%
sample
No.2
at 35
nglmL
2589
66%
nglmL
3245
-
nglmL
1802
56%
nglmL
2564
-
nglmL
1335
52%
2 6
Table 5
1 0.5% ammonium 1 0.5% ammonium 1 0.5% ammonium I
perfluorooclanoate perfluorooctanoate perfluorooclanoate
wilhout methanol I + .. . -. .. .- -.- - .. . .. . . .. .. 5.. % . . . .m . e.l.h ..a . .n . o ..l. . . . . . I + 10% methanol
It can be seen that dissociation increases with
increasing concentration of methanol, sometimes to the
5 detriment of reproducibility, if the concentration is too
great. The methanol concentration should therefore be
adjusted by the person skilled in the art so as to find a
compromise between the quantity of perfluoroalkyl
surfactant and alcohol.
10
EXAMPLE 5 - Adding Pluronic@ F-127
The dissociation solution of the invention used in
this example was prepared using the procedure explained
for Example 1, in a 50 mM Tris buffer with pH of 7.5,
15 with the exception that the fluoroalkyl surfactant was
1.5% perfluorohexaonic acid and the alcohol was 5%
methanol. The PluronicB F-127 was used at a
concentration of 0.25%, or else none was used. The
results are given in Table 6 below.
20 Otherwise the procedure was as in Example 1.
sample
No.1
at 15
Mean RFV
CV%
SIB ratio
% ratio
RFVNO.Z/RFVNO.I
sample
No.2
at 35
sample"
1
at 15
nglmL
2932
4%
27.37
.
sample
No.2
at 35
sample
No.1
at 15
sample
No.2
at 35
nglmL
2341
3%
29.77
80%
nglmL
3944
4%
28.62
ng1mL
2765
1%
38.04
70%
nglmL
4512
2%
35.85
-
nglmL
2697
3%
29.92
60%
Table 6
Without 1 0.25% Pluronic F-127 I
19 nglmL
oint
30 nglmL 1492 2024
oint 3%
102 nglmL 295 413
I 0 nalmL
Adding an additional surfactant such as PluronicO F-
127 serves to improve reproducibility (reduction in CV%).
5 The improvement in reproducibility is greater for samples
having a high concentration of vitamin D.
Pluronic F-127
Mean RFV
CV%
5388
EXAMPLE 6 - Influence of the buffer used
The comparative dissociation solution and the
10 dissociation solution of the invention with the mention
PBS as used in this example were prepared using the
procedure explained for Example 1 in a PBS buffer with pH
of 7.2. The natures and the concentrations of the
fluoroalkyl surfactant and of the alcohol, if any, are
15 given in Table 7 (comparative solution) and in Table 9
(solution of the invention).
The comparative dissociation solution and the
dissociation solution of the invention bearing the
mention Tris contained 50 mM of Tris, the fluoroalkyl
20 surfactant, with or without alcohol. The components were
dissolved in demineralized water by stirring for about
Mean RFV
CV%
5155.5
1597
2%
2914
2%
401
1 %
1328
2%
sample
No.1
sample
No.2
' sample
No. 3
sample
No. 4
1069
3%
2179
4%
188
1%
625
6%
30 min. The pH was adjusted to 7.5 with 6N NaOH. The
natures and the concentrations of the fluoroalkyl
surfactant and of the alcohol, if any, are given in
Table 8 (comparative solution) and in Table 9 (solution
5 of the invention).
Table 7
Comparative dissociation solution: PBS +
1.5% perfluorohexanoic acid
Table 8
Comparative dissociation solution: Tris t
1.5% perfluorohexanoic acid
Mean RFV
% ratio RFVNo.2/RFV~o.l
Table 9
Dissociation solution of the invention: PBS buffer or
Tris buffer + 1.5% perfluorohexanoic acid + 5%
methanol
sample No. 1
at I I nglmL
2510
Mean RFV
% ratio RFVNo.2/RFVNo.l
sample No. 2
at 30 nglmL
1509
60%
10
sample No. 1
at 11 nglmL
2779
Buffer
Mean RFV
It can be seen that the nature of the buffer has no
significant influence on the RFV ratio, and thus on the
sample No. 2
at 30 nglmL
1618
58%
I I I I
15 resulting dissociation.
% ratio RFVNo.2/RFVNo.l
The sole figure plots the B/BO% ratio obtained over
an ng/mL range of analyte for both buffers (PBS buffer
and Tris buffer). The B/BO% ratio is the signal obtained
for the range point under test divided by the signal
sample No. 1
at 11 ng1mL
PBS
sample No. 2
at 30 nglmL
-
TRlS PBS
- 1 40% 1 42%
4181 1 4874
TRlS
1665 1 2030
obtained for the range point having 0 ng/mL of analyte,
multiplied by 100.
It can be seen that the choice of buffer used has no
significant influence on the results obtained, whether in
5 terms of dissociation or in terms of reproducibility.

CLAIMS
1. The use of at least one fluoroalkyl surfactant and of at least one alcohol having 1 to 4 carbon atoms for dissociating vitamin D and/or a vitamin D metabolite from 5 vitamin D binding protein.
2. The use according to claim 1, characterized in that the fluoroalkyl surfactant and the alcohol are used in quantities such that the ratio multiplied by 100 of the
10 weight of surfactant, when it is solid, or of the volume of surfactant, when it is liquid, over the volume of alcohol lies in the range 10% to 60%, preferably in the range 15% to 40%, and more preferably in the range 15% to
30%.
15
3. The use according to claim 1 or claim 2, characterized
in that the fluoroalkyl surfactant is selected from
perfluorocarboxylic acids, perfluorosulfonic acids and
their salts, and in particular from perfluorohexanoic
20 acid, perfluoroheptanoic acid, and perfluorooctanoic
acid, and their salts.
4. The use according to any one of claims 1 to 3,
characterized in that the alcohol has 1 to 3 carbon atoms
25 and is selected from methanol, ethanol, n-propanol, and
isopropanol.
5. The use according to any one of claims 1 to 4,
characterized in that perfluorohexanoic acid is used with'
30 methanol.
6. The use' according to any one of claims 1 to 5,
characterized in that the dissociation is performed in
the presence of an additional surfactant selected from
35 block copolymers based on ethylene oxide and propylene
oxide, polysorbates, and polyethylene glycol ethers.
7. The use according to any one of claims 1 to 6 for
dissociating 25-hydroxy vitamin D, and in particular 25-
hydroxy vitamin D, and/or 25-hydroxy vitamin D, from
vitamin D binding protein.
5
8. A solution comprising at least one fluoroalkyl
surfactant and at least one alcohol having 1 to 4 carbon
atoms.
10 9. A solution according to claim 8, characterized in that
the fluoroalkyl surfactant is selected from
perfluorocarboxylic acids, perfluorosulfonic acids and
their salts, and in particular from perfluorohexanoic
acid, perfluoroheptanoic acid, and perfluorooctanoic
15 acid, and their salts.
10. A solution according to claim 8 or claim 9,
characterized in that the alcohol has 1 to 3 carbon atoms
and is selected from methanol, ethanol, n-propanol, and
20 isopropanol.
11. A solution according to any one of claims 8 to 10,
characterized in that it contains methanol as the alcohol
having 1 to 4 carbon atoms and at least one fluoroalkyl
25 surfactant selected from perfluorocarboxylic acids,
perfluorosulfonic acids, and their salts, the fluoroalkyl
surfactant preferably being perfluorohexanoic acid.
12. A solution according to any one of claims 8 to 11,
30 characterized in that it comprises a percentage of
fluoroalkyl surfactant by volume when the surfactant is
liquid, or by weight when it is solid, relative to the
total volume of the solution lying in the range 0.1% to
3%, preferably in the range 1% to 2%, and/or a percentage
35 by volume of alcohol relative to the total volume of the
solution lying in the range 0.5% to lo%, and preferably
in the range 2% to 7%.
13. A solution according to any one of claims 8 to 12,
characterized in that the fluoroalkyl surfactant and the
alcohol are present in quantities such that the ratio
5 multiplied by 100 of the %sleight of surfactant, \,?hen it is
solid; or of the volume of surfactant, when it is liquid,
over the volume of alcohol lies in the range 10% to 60%,
preferably in the range 15% to 40%, and more preferably
in the range 15% to 30%.
10
14. A solution according to any one of claims 8 to 13,
characterized in that it further contains an additional
surfactant selected from block copolymers based on
ethylene oxide and propylene oxide, polysorbates, and
15 polyethylene glycol ethers.
15. A sol.ution according to any one of claims 8 to 14,
characterized in that it is a buffer solution.
20 16. A solution according to any one of claims 8 to 15,
characterized in that it is buffered to a pH lying in the
range 6 to 8.
17. A solution according to any one of claims 8 to 16,
25 characterized in that the it does not contain any of the
following compounds: dimethyl sulfoxide,
dimethylformamide, N,N-dimethylacetamide,
tetramethylurea, N-methylpyrrolidone,
1,3-dimethyl-3,4,5,6-tetrahydro-2 (1H) -pyrimidone,
30 triamide hexamethyl phosphoric acid.
18. A detection and quantification method for detecting
and quantifying, in vitro, vitamin D and/or at least one
vitamin D metabolite in a biological sample, the method
35 comprising the following steps:
a) a step of treating the sample by incorporating at
least one fluoroalkyl surfactant and at least one alcohol
havi-ng 1 to 4 carbon atoms, so as to dissociate the
vitamin D and/or its metabolite(s) to be detected from
vitamin D binding protein; and
b) a step of detecting and quantifying vitamin D
5 and/or at least one of its metabolites.
19. A detection and quantification method according to
claim 18, characterized in that the treatment of step a)
is performed by mixing the sample with a dissociation
10 solution according to any one of claims 8 to 17.
20. A detection and quantification method according to
claim 19, characterized in that 1 to 20 volumes of
dissociation solution, preferably 5 to 10 volumes, more
15 preferably 6 to 8 volumes are used for 1 volume of
sample.
21. A detection and quantification method according to
any one of claims 18 to 20, characterized in that the
20 biological sample is a sample of blood, of serum, or of
plasma.
22. A detection and quantification method according to
any one of claims 18 to 21, characterized in that in step
25 b), 25-hydroxy vitamin D, and/or 25-hydroxy vitamin D,
is/are detected.
23. A detection and quantification method according to
any of claims 18 to 22, characterized in that in step b),
30 detection and quantification are performed by an
immunoassay.
24. A detection and quantification method according to
any one of claims 18 to 22, characterized in that in step
35 b), detection and quantification are performed by mass
spectrometry.
25. A detection and quantification kit for detecting
vitamin D and/or at least one vitamin D metabolite by
immunoassay, the kit comprising a solution according to
any one of claims 8 to 17.
5
26. A detection and quantification kit according to claim
25, characterized in that it further comprises a binding
partner for vitamin D or one of its metabolites.
10 27. A detection and quantification kit according to claim
25 or claim 26, characterized in that it further
comprises a solid phase on which there is bonded a hapten
analogous to vitamin D and/or to the vitamin D
metabolite(s) for detection, which hapten is recognized
15 by the marked antibody.