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A. Hernández-Martín et al. / Bioorg. Med. Chem. 21 (2013) 7779–7789
Antibodies recognising holo-DBP or an epitote close to 25-OH-D3 binding pocket
25
25
R1
R1
8
H
H
and/or
+
+
7
25-OH-D3
Antibodies
R2
HO
1
R2
1
Apo-DBP
Holo-DBP
HO
2, R1= R2= H Vitamin D3
4, R1= OH; R2= H 25-OH-D3
6, R1= R2= OH 1α,25-(OH)2-D3
1, R1= R2= H Vitamin D2
3, R1= OH; R2= H 25-OH-D2
5, R1= R2= OH 1α,25-(OH)2-D2
Modified vitamin D analogues prevent antibodies binding to molecule-DBP complex
Chart 1. Vitamin D related structures.
and/or
+
+
sunlight exposure, dietary intake, and supplements whenever
required.
Antibodies
Vitamin D
analogue
Apo-DBP
Molecule-DBP complex
In recent years several methods to determine vitamin D levels for
diagnostic purposes were described and performance characteris-
tics and limitations have been reviewed.7,12 There are two types of
methods: competitive immunoassay and those based on chroma-
tography separation followed by non-immunological direct detec-
tion. Currently, immunoassays are performed on automated
platforms, which use a chemiluminescent label. These methods are
limited by specificity in relation to the nonequimolar recognition
of the D2 and D3 forms of 25-OH-D. Meanwhile liquid chromatogra-
phy/tandem mass spectrometry methods have the advantage of
being able to measure both25-OH-D2 (3) and 25-OH-D3 (4) indepen-
dently. However, they require expensive equipment and restrict
sample throughput in the large clinical laboratory. Current assays
to determine 25-OH-D require initial dissociation from its DBP
which may be achieved by denaturing and removing DBP or using
a displacement reagent to release free 25-OH-D. Solvents are used
to release the 25-OH-D and denature the DBP but subsequent
separation of the denatured DBP and solvent extraction of the
sample is required prior to assay. The hydrophobic nature of
25-OH-D introduces significant problems, for example the molecule
has a propensity to bind to plastics surfaces thereby affecting
the complete recovery of free 25-OH-D. The requirement for organic
solvents is non-compatible with the use of protein molecules, such
as antibodies, in the assay system, or with the specialised high
throughput instrumentation present in most modern laboratories.
High variability is a common issue with vitamin D assays13 because
of the multi-stage nature of the existing assays, including 25-OH-D
displacement, liquid additions and removals, and multiple wash
steps, precision suffers accordingly. Consequently, there is a need
for a simplified vitamin D assay that is likely to provide higher
precision.
Figure 1. The modified vitamin D analogues when complexed to apo-DBP could
prevent antibodies binding to holo-DBP and/or antibodies recognising epitotes
close to the vitamin D binding pocket. The analogues could also be used to remove
apo-DBP from the samples prior to measurement of holo-DBP using either an
antibody capable of recognising either holo- or apo-DBP.
7, R=
HO
H2N
HO
8, R=
OH
9, R=
6
H
H2N
HO
10, R=
11, R=
12, R=
6
O
O
R
2
N
O
H
O
H2N
2
Chart 2. Target vitamin D analogues with C-3 A-ring modifications.
to holo-DBP or molecule-DBP complex, the latter may have an
altered conformation of DBP, and/or introduces steric hindrance
in the region around the vitamin D binding pocket of DBP, and/or
an altered conformation of a region around the vitamin D binding
pocket of DBP.
In our on going research related with vitamin14 and previtamin
D3,15 we have developed several vitamin D3 derivatives
(7–12, Chart 2) substituted at 3-position with a carbamate function
as apo-DBP binding molecule. In holo-DBP the A-ring is closest to
the opening of the binding pocket16 and, therefore, modifying it
may have a significant effect on the ability of molecules to bind
DBP around the binding pocket, for example by impeding access
to the binding pocket or creating a conformational change.
An advantage of these derivatives is that they contain hydrophilic
groups such as alcohol or amino at the end of the exogenous alkyl
chain of the carbamate and a spacer group of different length.
Among the spacer arms, polyethylene glycol (PEG) has been
incorporated to increase solubility and flexibility. Thus, the side
chain could protrude from the surface of DBP upon formation
of the molecule-DBP complex. In this way, it may inhibit binding
of a holo-DBP recognition molecule. To further facilitate differentia-
tion between holo-DBP and molecule-DBP complex the side
chain will be coupled to biotin or streptavidin thus potentially
further increasing the steric hindrance of the molecule when bound
to DBP.
2. Results and discussion
With this background in mind, here we report a novel method
to determine the relative amounts of the vitamin D–DBP complex
(hereinafter referred to as holo-DBP, Fig. 1) and free DBP (referred
to as apo-DBP) by binding apo-DBP to a binding molecule (ana-
logue) to form a molecule-DBP complex, such that a recognition
molecule (e.g., an antibody) binds preferentially to holo-DBP over
the molecule-DBP complex. Since most of the 25-OH-D3 is bound
to DBP, using a recognition molecule, which preferentially binds
holo-DBP over molecule-DBP complex, allows assessment of the
vitamin D level in a sample. The relative holo-DBP/vitamin D level
may be determined by reference to control measurements. The
recognition molecule can be an antibody or antibody fragment,
for example an anti-DBP antibody or fragment of antigen binding
(Fab). In order to facilitate development or identification of
recognition molecules that are specific for or bind preferentially