WO2012104643A1 - Molécule de liaison et procédé de mesure - Google Patents

Molécule de liaison et procédé de mesure Download PDF

Info

Publication number
WO2012104643A1
WO2012104643A1 PCT/GB2012/050222 GB2012050222W WO2012104643A1 WO 2012104643 A1 WO2012104643 A1 WO 2012104643A1 GB 2012050222 W GB2012050222 W GB 2012050222W WO 2012104643 A1 WO2012104643 A1 WO 2012104643A1
Authority
WO
WIPO (PCT)
Prior art keywords
dbp
apo
molecule
binding molecule
vitamin
Prior art date
Application number
PCT/GB2012/050222
Other languages
English (en)
Inventor
David Pritchard
Lesley PRESTON
Margaret Lawlor
Original Assignee
Axis-Shield Diagnostics Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Axis-Shield Diagnostics Limited filed Critical Axis-Shield Diagnostics Limited
Publication of WO2012104643A1 publication Critical patent/WO2012104643A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C401/00—Irradiation products of cholesterol or its derivatives; Vitamin D derivatives, 9,10-seco cyclopenta[a]phenanthrene or analogues obtained by chemical preparation without irradiation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/14—The ring being saturated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2602/00—Systems containing two condensed rings
    • C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
    • C07C2602/14—All rings being cycloaliphatic
    • C07C2602/24—All rings being cycloaliphatic the ring system containing nine carbon atoms, e.g. perhydroindane

Definitions

  • the present invention relates to a method for measuring vitamin D in a sample and a binding molecule, in particular an apo-DBP binding molecule.
  • vitamin D principally operates to maintain the circulating levels of calcium and phosphate in the blood, substances which in vivo affect properties such as bone mineralisation, muscle contraction and nerve conduction as well as other general cellular functions. As such, variation in vitamin D concentration can impact on muscle function and the immune, nervous, heart and circulatory systems, as well as being linked to diverse medical conditions such as bone disease, type II diabetes and cancer.
  • Vitamin D is found in various forms, the principal two of which are considered to be D2 and D3, though the term is recognised by those skilled in the art to refer broadly to the family of related fat soluble molecules as well as metabolites and other analogues of these substances. Accordingly, hereinafter vitamin D will be used to refer to all the members of the group of molecules collectively known in the art as vitamin D.
  • Vitamin D can be acquired in the diet or produced photochemically in the skin through the action of sunlight. Vitamin D 2 is not known to be synthesised by animals and is acquired in the diet from fungal and plant sources. Vitamin D 3 can similarly be acquired by animals via a carnivorous diet or, as said above, can be synthesised de novo in the skin. Regardless of its source, however, vitamin D is principally stored in vivo in the 25 hydroxyvitamin D
  • 25 (OH) D form, which is produced by hydroxylation of vitamin D in the liver.
  • 25 (OH) D is the major form of vitamin D found in blood and is the precursor of 1, 25 dihydroxyvitamin D (hereinafter 1,25 (OH) 2 D), a physiologically active form of vitamin D produced mainly in the kidney.
  • 1,25 (OH) 2 D binds to the vitamin D receptor (VDR) which, upon said binding, can subsequently mediate both rapid and long-term genomic responses.
  • Vitamin D function by binding to the vitamin D receptor (VDR) in target cells, for example in the intestine, bone or kidney.
  • Vitamin D is poorly soluble in water and so, to enable its transport in the blood to its target cells, it is bound in complex with a soluble 52 kDa carrier protein known as vitamin D binding protein or group specific component (Gc) .
  • group specific component Gc
  • DBP group of proteins including all variants known in the art as vitamin D binding protein or Gc will be referred to as DBP.
  • DBP concentration is ordinarily 20- times that of vitamin D in the blood and so, since DBP has very high binding affinity for vitamin D, most blood vitamin D is found bound to DBP.
  • the vitamin D-DBP complex is hereinafter referred to as holo-DBP.
  • Apo-DBP refers to DBP wherein the vitamin D binding site is available for binding to vitamin D.
  • the two main forms of vitamin D which can be measured in bodily fluids are 25 (OH) D and 1,25 (OH) 2 D.
  • 25 (OH) D is normally found at a higher concentration in the blood than 1,25 (OH) 2 D, and, since it also has a relatively long half-life, 25 (OH) D is commonly measured to assess and monitor vitamin D status in individuals.
  • the plasma/serum levels of 1,25 (OH) 2 D tend to be maintained at a constant level, so 25 (OH) D levels are a better indicator of an individual's overall circulating vitamin D status. Determining levels of 1,25 (OH) 2 D, however, serves as an indication of whether there is adequate production in the kidney (s) of the active form of the compound .
  • vitamin D is initially dissociated from its DBP before measuring the level of vitamin D which is poorly soluble in aqueous media. This separation may be achieved by denaturing and removing DBP or using a displacement reagent to release vitamin D and then measuring the resultant free vitamin D. The requirement for dissociation of the DBP obviously necessitates incorporation of such a step in any vitamin D assay and so adds to the time and complexity of conducting the assay.
  • vitamin D Due to the hydrophobic nature of vitamin D, a current assay technique, for example, is to use solvents to release the vitamin D and denature the DBP. Such an approach necessitates subsequent separation of the denatured DBP and solvent extraction before vitamin D content can be assessed.
  • the hydrophobicity of the vitamin D molecule introduces significant problems for the development of assays, for example the molecule has a propensity to bind to plastics surfaces thereby affecting the complete recovery of free vitamin D. This is an issue for most vitamin D assays.
  • the requirement for organic solvents to maintain the vitamin D molecule in solution can be detrimental to and non-compatible with the use of protein molecules such as antibodies in the assay system. Some solvents are also non-compatible with the specialised high throughput instrumentation present in most modern laboratories .
  • the present invention seeks to overcome problems associated with the prior art.
  • a method for measuring vitamin D in a sample comprising facilitating differentiation between holo-DBP and apo-DBP in the sample by binding apo- DBP to an apo-DBP binding molecule to form, or enable formation of, a molecule-DBP complex, such that a recognition molecule binds preferentially to holo-DBP over the molecule-DBP complex.
  • an apo-DBP binding molecule capable of binding apo-DBP to form, or enable formation of, a molecule-DBP complex such that a recognition molecule which binds holo-DBP does so preferentially over the molecule-DBP complex.
  • the recognition molecule may recognise both holo- and apo- DBP (e.g. it may be an anti-DBP antibody that binds both holo- and apo-), but that after the molecule-DBP complex is formed, and thus there is less or no apo-DBP present, the relevant consideration is that the recognition molecule, which may be measured qualitatively or quantitatively, recognises holo-DBP preferentially over the molecule-DBP complex .
  • the apo-DBP binding molecule is an analogue and/or derivative of vitamin D.
  • the term 'vitamin D' may be understood to encompass the different forms of vitamin D including their
  • Analogues and derivatives of vitamin D include modified versions and structural analogues of vitamin D.
  • the recognition molecule does not bind the molecule-DBP complex.
  • the recognition molecule is any of an antibody, antibody fragment, protein, molecular imprinted polymer, a complementarity determining region, oligopeptide, oligonucleotide, aptamer or small organic chemical.
  • the recognition molecule is an antibody or antibody fragment, e.g. an anti-DBP antibody or Fab (fragment of antigen binding) .
  • An appropriate anti-DBP antibody or Fab could, for example, be one generated using either apo-DBP or holo-DBP as the antigen, such as one of those mentioned in the Examples below.
  • the molecule-DBP complex when the molecule-DBP complex is compared to holo- DBP, the molecule-DBP complex may have an altered conformation of DBP, and/or reduced access to a 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. This could facilitate development or identification of recognition molecules which are specific for or bind preferentially to holo-DBP or molecule-DBP complex .
  • a region can be an amino acid, a group of amino acids, an epitope or a group of epitopes. It is not necessary that the amino acids or the epitopes are in proximity to each other.
  • the apo-DBP binding molecule is an analogue and/or derivative of 25 (OH) D or 1,25 (OH) 2D, more preferably 25(OH)D.
  • the apo-DBP binding molecule is an analogue and/or derivative of vitamin D 2 or vitamin D 3 . More preferably, the apo-DBP binding molecule is an analogue and/or derivative of vitamin D 3 . [0025] More preferably, the apo-DBP binding molecule is an analogue and/or derivative of 25(OH) D3.
  • the apo-DBP binding molecule is an analogue and/or derivative of vitamin D, including the various forms and metabolites of vitamin D, any or all of the A, C and D rings is/are modified.
  • the A, C and D rings are clearly identified in Figure 1 (the nomenclature being consistent between the vitamin D forms/metabolites).
  • the apo-DBP binding molecule is preferably modified to have an exogenous side chain.
  • the A-ring has been modified.
  • the A-ring is closest to the opening of the binding pocket and, therefore, modifying it may have a significant effect on the ability of molecules to bind DBP around the binding pocket, e.g. by impeding access to the binding pocket or creating a conformational change.
  • the A-ring is modified to have an exogenous side chain, which may conveniently be coupled to the carbon atom at the 3-position (the side chain being in place of the OH group on the 3-position carbon) .
  • a side chain may be attached to another carbon or group of the A ring or an appropriate atom or group on another (C or D) ring.
  • the side chain may protrude from the surface of DBP upon formation of the molecule-DBP complex. In this way, it may be operable to inhibit binding of a holo-DBP recognition molecule.
  • the side chain may comprise a number of carbon atoms in a chain of any suitable length, commonly referred to as a spacer arm.
  • the side chain may comprise from 1 to 500 carbon atoms.
  • the side chain may comprise a hydrophilic polyethylene glycol (PEG) group.
  • PEG polyethylene glycol
  • the side chain terminates in an OH (hydroxyl), SH (thiol) or N3 ⁇ 4 (amine) group.
  • additional components may more easily be coupled to the side chain.
  • the side chain may incorporate or be coupled to, either directly or indirectly, a further component, e.g. a protein, protein fragment, peptide, polypeptide, antigen, complementarity determining region, antibody, antibody fragment, polynucleotide, polymer, steroid, vitamin, carbohydrate, hydrocarbon, lipid or other appropriate substance.
  • a further component e.g. a protein, protein fragment, peptide, polypeptide, antigen, complementarity determining region, antibody, antibody fragment, polynucleotide, polymer, steroid, vitamin, carbohydrate, hydrocarbon, lipid or other appropriate substance.
  • further components include biotin, streptavidin, myelin basic protein, glutathione S-transferase and PEG. In this way, the size of, for example, a protein such as streptavidin may be utilised to inhibit binding site access. Such a coupling may happen before or after binding to apo-DBP.
  • the further component may then optionally be bound by a binding partner to further enhance (or enable) the inhibition of binding of a relevant holo-DBP recognition molecule to a molecule-DBP complex.
  • binding partnerships include biotin and streptavidin, an antigen and antibody, and glutathione and glutathione-S- transferase. This may happen before or after binding to apo-DBP.
  • Preferred side chains have the formula:
  • X OH, SH or NH 2 and n is zero or an integer, and are bound to the apo-DBP binding molecule by the oxygen atom shown as singly bonded.
  • This oxygen atom may be thought of as the oxygen of the OH group attached to the 3- position carbon of the A-ring, the hydrogen atom of the OH group having been replaced by the remainder of the chain, as shown in Figure 3.
  • n is an integer from 1 to 6, more preferably n is 1 or 6.
  • the side chain may comprise a polyethylene glycol (PEG) group.
  • PEG polyethylene glycol
  • the side chain may have the formula H- (OCH 2 OCH 2 ) m -NH-C0 2 - where m is an integer from 1 to 6, preferably 2.
  • the apo-DBP binding molecule comprises a structure selected from structures AH-1 to AH-6 and TG-1 to TG-6, shown in Table 1 below:
  • the side chain may be coupled to a further component as described above by virtue of an appropriate reaction with the ⁇ ⁇ ' group. Such a coupling may happen before or after binding to apo-DBP.
  • the side chain may be coupled to biotin by virtue of an appropriate reaction with the ⁇ ⁇ ' group, for example when the X group is OH.
  • the side chain may then also be coupled to streptavidin via biotin.
  • Streptavidin may bind biotin (and biotin may be coupled to the side chain) before or after the apo-DBP has been bound.
  • biotin may subsequently be coupled indirectly to the side chain via the streptavidin.
  • Other binding partnerships known or developed may similarly be exploited, for example glutathione and glutathione-S-transferase or an antigen and antibody.
  • the apo-DBP binding molecule does not displace vitamin D, or does not significantly displace vitamin D, from holo-DBP.
  • the apo-DBP binding molecule may be more straightforwardly used to facilitate measurement of vitamin D levels in a sample. It will be appreciated by a person skilled in the art, however, that some displacement of vitamin D may occur without adversely affecting the function/utility of the invention.
  • the molecule will displace less than 20% of vitamin D from holo-DBP, more preferably less than 10%, most preferably less than 5%.
  • the apo-DBP binding molecule may further comprise or be coupled to a component such as a tag (e.g. 6-His, FLAG) .
  • a tag e.g. 6-His, FLAG
  • the present invention provides a method for measuring vitamin D in a sample, the method comprising facilitating differentiation between holo-DBP and apo-DBP in the sample by binding apo- DBP to an apo-DBP binding molecule to form, or enable formation of, a molecule-DBP complex, such that a recognition molecule binds preferentially to holo-DBP over the molecule-DBP complex.
  • the apo-DBP binding molecule is preferably an apo- DBP binding molecule according to the second aspect of the invention .
  • the method comprises the steps of: incubating the sample with the apo-DBP binding molecule, reacting the sample with a recognition molecule which preferentially binds holo-DBP over any resulting molecule- DBP complex and measuring holo-DBP in the sample.
  • the apo-DBP binding molecule may be incubated with the sample prior or simultaneously to the sample being reacted with the recognition molecule.
  • the appropriate method may be influenced by binding kinetics of the different molecules used.
  • the recognition molecule may also bind (cross-react with) apo-DBP (e.g. it may be an anti-DBP antibody that binds both holo- and apo-) but it is desirable for there to be no or minimal apo-DBP remaining in the sample after incubation with the apo-DBP binding molecule. In this way, even if the recognition molecule can interact significantly with apo-DBP, where the recognition molecule shows binding it should principally be by virtue of holo-DBP present in the sample.
  • apo-DBP e.g. it may be an anti-DBP antibody that binds both holo- and apo-
  • another component may be added (and reacted with the apo-DBP binding molecule) such that it binds the apo-DBP binding molecule to form (or become part of) the molecule-DBP complex.
  • the apo-DBP binding molecule comprises biotin
  • streptavidin may be added after incubation of the sample with the molecule to bind biotin and form part of the molecule-DBP complex.
  • any inhibitory effect streptavidin may have (e.g. due to its relative bulk) on the initial binding of the apo-DBP binding molecule to apo-DBP may be mitigated while still allowing for it to function to enhance differences between holo-DBP and the resulting molecule-DBP complex, such that there is enabled differential binding by a recognition molecule .
  • the apo-DBP binding molecule comprises a side chain ending in, for example, an OH, SH or NH 2 group
  • a further component e.g. a protein, protein fragment, peptide, polypeptide, antigen, complementarity determining region, antibody, antibody fragment, polynucleotide, polymer, steroid, vitamin, carbohydrate, hydrocarbon, lipid or other appropriate substance
  • further components include biotin, streptavidin, myelin basic protein, glutathione S- transferase and PEG.
  • the further component may then optionally be bound by a binding partner to further enhance (or enable) the inhibition of binding of a relevant holo-DBP recognition molecule to a molecule-DBP complex. Examples of binding partnerships are discussed above.
  • a recognition molecule which preferentially binds holo-DBP over molecule-DBP could be immobilised to a solid support, such as a microtitre plate, and an ELISA-type assay could be performed wherein the sample is incubated with the apo-DBP binding molecule prior to (or concurrent with) a step wherein the sample is incubated with the recognition molecule.
  • the method comprises the steps of: coupling to a solid support a recognition molecule which preferentially binds holo-DBP over a molecule-DBP complex, incubating a sample with the apo-DBP binding molecule such that it binds apo-DBP to form or enable formation of the molecule-DBP complex, reacting the sample during or after incubation with the apo-DBP binding molecule with the solid support such that holo-DBP binds the recognition molecule, separating unbound sample material from the solid support, and measuring the holo- DBP.
  • the method may further comprise the step of adding streptavidin to form (or become part of) the molecule-DBP complex.
  • the solid support could, for example, be beads, a membrane, a column, a microtitre plate or a sensor for use with a surface plasmon resonance technology platform for measuring protein interaction (e.g. SensiQ) .
  • a surface plasmon resonance technology platform for measuring protein interaction e.g. SensiQ
  • the recognition molecule used in the method is any of an antibody, antibody fragment, protein, molecular imprinted polymer, a complementarity determining region, oligopeptide, oligonucleotide, aptamer or small organic chemical.
  • the recognition molecule is an antibody or antibody fragment, e.g. an anti-DBP antibody or Fab (fragment of antigen binding) .
  • An appropriate anti- DBP antibody or Fab could, for example, be one generated using either apo-DBP or holo-DBP as the antigen, such as one of those mentioned in the Examples below.
  • a recognition molecule capable of binding holo-DBP for use in the methods according to the present invention .
  • the recognition molecule is any of an antibody, antibody fragment, protein, molecular imprinted polymer, a complementarity determining region, oligopeptide, oligonucleotide, aptamer or small organic chemical.
  • the recognition molecule is an antibody or antibody fragment.
  • the apo-DBP binding molecule may also be used to remove apo-DBP from (or sequester it in) a sample. In this way, any recognition molecule which recognises DBP may more easily be subsequently used to measure holo-DBP, irrespective of its relative degree of interaction with the molecule-DBP complex.
  • a method for measuring vitamin D in a sample comprising: reacting a sample with an apo-DBP binding molecule according to the second aspect of the invention, separating the sample from any resulting molecule-DBP complex and measuring the amount of vitamin D in the sample.
  • this method comprises: attaching the apo-DBP binding molecule to a solid support, reacting the sample with the apo-DBP binding molecule on the solid support, separating the remainder of the sample from any resulting molecule-DBP complex, and measuring the amount of vitamin D in the sample.
  • the apo-DBP binding molecule binds the apo-DBP to the solid support and the holo-DBP remains in the sample to be measured.
  • the solid support could, for example, be beads, a membrane, a column or microtitre plate .
  • the recognition molecule is any of an antibody, antibody fragment, protein, molecular imprinted polymer, a complementarity determining region, oligopeptide, oligonucleotide, aptamer or small organic chemical.
  • the recognition molecule is an antibody or antibody fragment, e.g. an anti-DBP antibody or Fab (fragment of antigen binding) .
  • An appropriate anti-DBP antibody or Fab could, for example, be one generated using either apo-DBP or holo-DBP as the antigen, such as one of those mentioned in the Examples below.
  • Figure 1 shows the structure of vitamin D 3 , identifying the A, C and D rings.
  • Figure 2 illustrates how an apo-DBP binding molecule may inhibit binding of recognition molecules which recognise holo-DBP to a molecule-DBP complex.
  • Figure 3 shows an example of an apo-DBP binding molecule according to the present invention wherein X is a hydroxyl (OH), thiol (SH) or amine (NH 2 ) and n indicates zero or an integer.
  • A-ring modified vitamin D is through a convergent route.
  • a CD-ring/side chain fragment and an A-ring fragment are synthesised separately. Then, both fragments are coupled using a Wittig reaction to prepare the vitamin D analogue.
  • the mixture is then removed from the Dewar flask, 20 ml of the solvent Me 2 S is added, and the mixture is incubated overnight with continuous stirring while nitrogen is bubbled through it until no peroxides are detected in the mixture.
  • the solution is concentrated to 50 ml by evaporation, and the remaining yellow concentrate is washed with hexane (3 x 10ml), distilled H 2 0 (2 x 25ml) and again with hexane (2 x 10ml) .
  • the washes are collected into a separatory funnel.
  • the product is extracted with hexane, dried over a 2 S0 4 and evaporated to yield a ketone.
  • a solution of ergocalciferol in ethanol is cooled to -20°C and potassium permanganate (KMnC ⁇ ) is added dropwise to the solution, which is stirred continuously, ensuring that the temperature remains ⁇ 10°C.
  • the mixture is stirred for another hour and then heated to 45°C with occasional stirring by hand.
  • the solid is allowed to settle at room temperature (RT) then the yellow upper layer is decanted and the remaining brown suspension filtered to separate the solid from the ethanol.
  • the yellow solution is evaporated to dryness and the solid residue is recrystallised in hexane to obtain a white triol compound.
  • a solution containing the triol compound, imidazole and 4-Dimethylaminopyridine ( DMAP ) ) in CH 2 C1 2 (40 ml) is added dropwise to tert-butyldimethylsilylchloride , the protecting group, in CH 2 CI 2 (40 ml) and stirred at room temperature (RT) for 48 hours.
  • the solution is then washed with H 2 0 (3 x 80ml) and saturated sodium chloride solution
  • Tetrabutylammonium fluoride (1.5 ml of 1.0 M in THF, 1.5 mmol) is added dropwise to a solution of phosphine oxide in THF (4 ml) at 0°C and the reaction is then stirred overnight at RT .
  • the crude residue is poured into 3 ⁇ 40/ EtOAc and the aqueous layer extracted with EtOAc. The combined organic fractions were subjected to flash chromatography (80-100% hexane/EtOAc gradient elution) to yield the phosphine alcohol product.
  • the vitamin D analogues/derivates may optionally be biotinylated.
  • the heterofunctional crosslinker N- [p- maleimidophenyl ] isocyanate (PMPI), a sulfhydryl- and hydroxyl-reactive crosslinker, is used to couple a thiolated biotin molecule to a hydroxy vitamin D analogue (e.g. AH-1, AH-3, AH-5, TG-1, TG-3, TG-5) .
  • a thiolated biotin molecule e.g. AH-1, AH-3, AH-5, TG-1, TG-3, TG-5) .
  • the coupling is performed in DMSO with a 1.1 molar ratio of PMPI to the hydroxy vitamin D molecule.
  • the thiolated biotin, in DMSO is added at 1.1 molar ratio to the PMPI-vitamin D molecule reaction and mixed for an additional 30 min.
  • a biotinylated ⁇ -Hydroxysuccimide (NHS) ester containing a hydrophilic polyethylene glycol spacer arm is coupled to an amine vitamin D analogue (e.g. AH-2, AH-4, AH- 6, TG-2, TG-4, TG-6) .
  • an amine vitamin D analogue e.g. AH-2, AH-4, AH- 6, TG-2, TG-4, TG-6 .
  • the coupling is performed in DMSO with a 1.5 molar excess of the biotin.
  • the reaction is incubated for 1 hour.
  • molecule-DBP To prepare molecule-DBP, excess vitamin D analogue (5-10 molar excess) is incubated with mixed type vitamin D binding protein (Merck Chemicals Ltd, catalogue number 345802) overnight at 2-8°C. Molecule-DBP is separated from unreacted apo-DBP using a chromatofocusing technique on a mono P column from GE Healthcare. The mono P column is equilibrated with the start buffer, 0.025M Methylpiperazine pH 5.7. The elution buffer (7 mis of 10% polybuffer 74 pH 4.0) is applied then the molecule-DBP mixture, which is preadjusted to the same pH as the start buffer, is applied. The molecule-DBP fractions are collected while running the elution buffer.
  • Holo-DBP may be prepared in a similar way.
  • Example 4 Screening of antibodies or Fabs generated using either apo-DBP or holo-DBP as the antigen for their ability to bind holo-DBP and molecule-DBP complex.
  • vitamin D used to make holo-DBP was 25 (OH) D 3 and the DBP used was mixed type DBP, but it will be apparent that other forms of vitamin D and their metabolites may be used, and specific forms of DBP could be used.
  • the antibodies/Fabs used in this Example (which bind DBP) were obtained from Abeam and AbD Serotec.
  • Either holo-DBP or molecule-DBP (50-100 ⁇ / ⁇ at pH 4.5) is covalently coupled onto a COOH sensor for use on the SensiQ, which is a surface plasmon resonance technology based instrument for measuring protein interaction.
  • Streptavidin (O.lmg/ml) is optionally passed over the holo- DBP or molecule-DBP coated sensor. An increase in signal is observed for the molecule-DBP coated sensor when the streptavidin binds the biotin on the molecule-DBP.
  • Antibodies for DBP which are commercially available, or Fabs generated from a phage display screen with holo-DBP (obtained from AbD Serotec) , are tested at a concentration of 0.5 to 5 ⁇ in a hepes buffered saline, pH 7.4 solution, for binding to both the holo-DBP and molecule-DBP complex.
  • Tables 2 and 3 show the specificity of the antibodies tested for holo-DBP and molecule-DBP complexes.
  • Table 2 shows data produced using sensor coated in biotinylated AH-1 (the short hydroxy 25 (OH) D3 analogue) over which streptavidin had been passed.
  • Table 3 shows data produced using sensor coated in biotinylated AH-4 (the long amine 25 (OH) D3 analogue) both with and without streptavidin having been passed over.
  • Table 2 Data produced using sensor coated with biotinylated short hydroxy 25 (OH) D3 analogue (AH-1) which had streptavidin (O.lmg/ml) passed over it. Hug of each antibody was used. Holo-DBP and molecule-DBP were coated at a concentration of 50 ⁇ g/ml
  • Vitamin D 25-OH-D3
  • Vitamin D Analogues using tritiated [ 3 H]Vitamin D ( [ 3 H] 25-OH-D3 ) to monitor displacement
  • vitamin-D analogues do not displace vitamin D from vitamin D binding protein.
  • Experiments to study the effect of analogues upon the displacement of vitamin D from vitamin D binding protein were performed using tritiated- 25-OH-D3.
  • apo-DBP To 1 mg of apo-DBP was added 750 ⁇ of PBS, 50 ⁇ 1 of 25-OH-D3 (lmg/ml) and 200 ⁇ , of hydroxyvitamin D3, 25- [26, 27- 3 H]-. This mixture was incubated overnight at 2-8°C, and a ⁇ aliquot was taken to obtain a radioactivity reading.
  • Table 7 shows averaged data for duplicated samples of Table 6 and indicates the % of [ 3 H] vitamin D displacement .
  • IRMA enzyme linked immunosorbent assays
  • MEIA microparticle enzyme immunoassays
  • a sample may be first incubated with excess apo-DBP binding molecule which is allowed to bind to sample apo-DBP.
  • the holo-DBP and thus the vitamin D levels in the sample, typically plasma or serum, are then measured by capturing the holo-DBP, but not the molecule-DBP complex, on a solid surface such as, but not limited to, the surface of a microtitre (e.g. 96-well) plate or magnetic or latex microparticle pre-coated with a holo-DBP recognition molecule (conveniently an antibody or antibody fragment) .
  • a microtitre e.g. 96-well
  • magnetic or latex microparticle pre-coated with a holo-DBP recognition molecule (conveniently an antibody or antibody fragment) .
  • Holo-DBP is then measured using a holo-DBP recognition molecule (which may also recognise apo-DBP but which binds holo-DBP preferentially over molecule-DBP complex) , which may be the same or different to that used to capture the holo-DBP, conjugated to a label or signal molecule as described below.
  • a holo-DBP recognition molecule which may also recognise apo-DBP but which binds holo-DBP preferentially over molecule-DBP complex
  • a label or signal molecule as described below.
  • the holo-DBP recognition molecule on the surface of the microtitre plate captures the holo-DBP in the sample but not the molecule-DBP.
  • the microtitre well/microparticle may then be washed to remove unbound sample material and an appropriate, optionally labelled, holo-DBP recognition molecule (for example an anti-DBP antibody, anti-vitamin D antibody or anti-holo-DBP antibody) , which may be the same or different to that used for capturing the holo-DBP, is incubated with the captured holo-DBP. If necessary, substrate is then added and the presence of the label is measured.
  • a secondary recognition molecule e.g.
  • the amount of signal generated is related to the amount of holo-DBP in the sample.
  • the amount of holo-DBP in the sample is related to the concentration of vitamin D in the sample.
  • the sample may first be incubated with excess apo-DBP binding molecule which is allowed to bind to sample apo-DBP to form molecule-DBP .
  • Molecule-DBP is then removed from the sample and the remaining holo-DBP can be measured using a recognition molecule which binds holo-DBP (though it may also bind apo-DBP) .
  • the molecule- DBP can be removed from the sample using a recognition molecule, capable of binding molecule-DBP, pre-coated onto a solid surface such as, but not limited to, a magnetic or latex microparticle.
  • the apo-DBP binding molecule may also itself be pre-coated onto a solid surface such as a magnetic or latex microparticle and used to remove the apo- DBP from the sample.
  • a microtitre plate or microparticles may be coated with the apo-DBP binding molecule either directly or indirectly, e.g. via a recognition molecule to molecule-DBP.
  • Apo-DBP binding molecule captures the apo-DBP in the sample (typically plasma or serum) but not the holo-DBP.
  • the sample, containing holo-DBP may then be removed/separated from the captured apo-DBP and incubated with an appropriate recognition molecule etc, and an appropriate procedure akin to that described above may be performed.
  • a label or signal molecule may be conjugated to the recognition molecule.
  • labels/signal molecules which can be conjugated to the recognition molecule are, but are not limited to, enzymes such as horseradish peroxidase, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase , ribonuclease , urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholine esterase, chemiluminescent compounds such as acridinium ester, suitable fluorescent labelling compounds include fluorescein isothiocyanate , rhodamine, phycoerythrin, phy
  • the recognition molecule may otherwise be radio- labelled or electrochemically labelled.
  • Anti-vitamin D binding protein antibody (17355, AbD Serotec) is diluted to l g/ml in carbonate buffer (16mM sodium carbonate anhydrous, 35mM sodium hydrogen carbonate pH 9.5) . A microtitre plate is coated with the antibody solution at ⁇ /well. The plate is incubated overnight at 2-8°C.
  • Patient samples are prepared by adding hydroxy vitamin D analogue (50 g/ml) and vortexed before being incubated for 1 hour at 18-25°C.
  • a further component for example, streptavidin (e.g. if the analogue/derivative is biotinylated) may, if relevant, also be added at this or a subsequent stage.
  • ⁇ /well TMB substrate is added and developed for 15 minutes before the reaction is stopped with the addition of ⁇ /well 0.25M Sulphuric acid.
  • the recognition molecule which preferentially binds holo-DBP over molecule-DBP complex comprising DBP and apo-DBP binding molecule may also be used in direct detection of holo-DBP and thus, for example, 25(OH)D.
  • techniques such as Surface Plasmon Resonance, Surface Acoustic Wave and Quartz Crystal Microbalance methodologies (Suzuki M, Ozawa F, Sugimoto W, Aso S. Anal Bioanal Chem 372:301-304 2002; Pearson J E, Kane J W, Petraki-Kallioti I, Gill A, Vadgama P.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne un procédé et une molécule permettant de mesurer la vitamine D dans un échantillon. Ladite molécule est capable de se lier à l'apo-DBP pour former un complexe molécule-DBP, ou en permettre la formation, de manière à ce qu'une molécule de reconnaissance se lie préférentiellement à l'holo-DBP plutôt qu'au complexe molécule-DBP.
PCT/GB2012/050222 2011-02-02 2012-02-02 Molécule de liaison et procédé de mesure WO2012104643A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB1101801.7 2011-02-02
GBGB1101801.7A GB201101801D0 (en) 2011-02-02 2011-02-02 Binding molecule and method for measuring

Publications (1)

Publication Number Publication Date
WO2012104643A1 true WO2012104643A1 (fr) 2012-08-09

Family

ID=43824998

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2012/050222 WO2012104643A1 (fr) 2011-02-02 2012-02-02 Molécule de liaison et procédé de mesure

Country Status (2)

Country Link
GB (1) GB201101801D0 (fr)
WO (1) WO2012104643A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014083427A3 (fr) * 2012-11-30 2014-07-24 Siemens Healthcare Diagnostics Inc. Compositions et procédés de détection de la vitamine d
JP2016531306A (ja) * 2013-09-17 2016-10-06 ビオメリューＢｉｏｍｅｒｉｅｕｘ ビタミンｄ結合タンパク質からビタミンｄを解離するための溶液、その関連検出法及び使用
EP2759550B1 (fr) * 2013-01-28 2018-08-15 DiaSorin S.p.A. Procédé et kit de détection de 1,25-dihydroxyvitamine D et anticorps associés

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5981779A (en) * 1995-12-29 1999-11-09 A And D Assay, Incorporated Labeled vitamin D compounds and the use thereof
WO1999067211A1 (fr) * 1998-06-25 1999-12-29 Immundiagnostik Gesellschaft Für Produktion Und Vertrieb Von Labordiagnostika Mbh DERIVES FONCTIONNELS DE LA VITAMINE D ET PROCEDE POUR LA DETERMINATION DE 25-HYDROXY- ET 1α, 25-DIHYDROXY-VITAMINE D
DE19840435A1 (de) * 1998-06-25 1999-12-30 Immundiagnostik Gmbh Funktionelle Vitamin-D-Derivate und Verfahren zur Bestimmung von 25-Hydroxy- und 1a, 25-Di-Hydroxy-Vitamin-D
WO2002046746A2 (fr) * 2000-12-06 2002-06-13 Immunodiagnostic Systems Ltd Procede de detection de metabolites de la vitamine d
US20050014211A1 (en) * 1998-06-25 2005-01-20 Armbruster Franz Paul Functional vitamin D derivatives and method of determining 25-hydroxy- and 1alpha, 25-dihydroxy vitamin D
WO2010100487A1 (fr) * 2009-03-02 2010-09-10 Axis-Shield Diagnostics Limited Dosage pour la détection de la vitamine d et anticorps correspondants
EP2372365A1 (fr) * 2010-04-01 2011-10-05 Future Diagnostics B.V. Analyse directe pour la vitamine D

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5981779A (en) * 1995-12-29 1999-11-09 A And D Assay, Incorporated Labeled vitamin D compounds and the use thereof
WO1999067211A1 (fr) * 1998-06-25 1999-12-29 Immundiagnostik Gesellschaft Für Produktion Und Vertrieb Von Labordiagnostika Mbh DERIVES FONCTIONNELS DE LA VITAMINE D ET PROCEDE POUR LA DETERMINATION DE 25-HYDROXY- ET 1α, 25-DIHYDROXY-VITAMINE D
DE19840435A1 (de) * 1998-06-25 1999-12-30 Immundiagnostik Gmbh Funktionelle Vitamin-D-Derivate und Verfahren zur Bestimmung von 25-Hydroxy- und 1a, 25-Di-Hydroxy-Vitamin-D
US20050014211A1 (en) * 1998-06-25 2005-01-20 Armbruster Franz Paul Functional vitamin D derivatives and method of determining 25-hydroxy- and 1alpha, 25-dihydroxy vitamin D
WO2002046746A2 (fr) * 2000-12-06 2002-06-13 Immunodiagnostic Systems Ltd Procede de detection de metabolites de la vitamine d
WO2010100487A1 (fr) * 2009-03-02 2010-09-10 Axis-Shield Diagnostics Limited Dosage pour la détection de la vitamine d et anticorps correspondants
EP2372365A1 (fr) * 2010-04-01 2011-10-05 Future Diagnostics B.V. Analyse directe pour la vitamine D

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
CHOU S F; HSU W L; HUANG J M; CHEN C Y, CLIN CHEM, vol. 48, 2002, pages 913 - 918
GOTOR-FERNANDEZ VICENTE ET AL: "Chemoenzymatic synthesis and biological evaluation of C-3 carbamate analogues of 1alpha,25-dihydroxyvitamin D3", BIOORGANIC & MEDICINAL CHEMISTRY, vol. 12, no. 20, 15 October 2004 (2004-10-15), pages 5443 - 5451, XP002677435, ISSN: 0968-0896 *
HADDAD J G ET AL: "Selective, rapid removal of the vitamin D-binding protein and its sterol ligands from human and bovine plasma", ANALYTICAL BIOCHEMISTRY, ACADEMIC PRESS INC, NEW YORK, vol. 146, no. 1, 1 April 1985 (1985-04-01), pages 96 - 102, XP024823286, ISSN: 0003-2697, [retrieved on 19850401], DOI: 10.1016/0003-2697(85)90401-4 *
OVES DANIEL ET AL: "Novel A-ring homodimeric C-3-carbamate analogues of 1 alpha,25-dihydroxyvitamin D-3: Synthesis and preliminary biological evaluation", BIOORGANIC & MEDICINAL CHEMISTRY, vol. 14, no. 22, November 2006 (2006-11-01), pages 7512 - 7519, XP002677436, ISSN: 0968-0896 *
PEARSON J E; KANE J W; PETRAKI-KALLIOTI I; GILL A; VADGAMA P, J IMMUNOL METHODS, vol. 221, 1998, pages 81 - 94
SUZUKI M; OZAWA F; SUGIMOTO W; ASO S, ANAL BIOANAL CHEM, vol. 372, 2002, pages 301 - 304
SWAMY N ET AL: "Affinity purification of human plasma vitamin D-binding protein", PROTEIN EXPRESSION AND PURIFICATION, ACADEMIC PRESS, SAN DIEGO, CA, vol. 6, no. 2, 1 January 1995 (1995-01-01), pages 185 - 188, XP002229873, ISSN: 1046-5928, DOI: 10.1006/PREP.1995.1023 *
SWAMY N ET AL: "Roles of the structure and orientation of ligands and ligand mimics inside the ligand-binding pocket of the vitamin D-binding protein.", BIOCHEMISTRY 17 JUN 1997 LNKD- PUBMED:9200691, vol. 36, no. 24, 17 June 1997 (1997-06-17), pages 7432 - 7436, XP002677437, ISSN: 0006-2960 *
WEISCH W; KLEIN C; VON SCHICKFUS M; HUNKLINGER S, ANAL CHEM, vol. 68, 1996, pages 2000 - 20004

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014083427A3 (fr) * 2012-11-30 2014-07-24 Siemens Healthcare Diagnostics Inc. Compositions et procédés de détection de la vitamine d
CN104937422A (zh) * 2012-11-30 2015-09-23 西门子医疗保健诊断公司 用于检测维生素d的组合物和方法
US9244083B2 (en) 2012-11-30 2016-01-26 Siemens Healthcare Diagnostics Inc. Compositions and methods for detecting vitamin D
KR20160047422A (ko) * 2012-11-30 2016-05-02 지멘스 헬쓰케어 다이아그노스틱스 인크. 비타민 ｄ를 검출하기 위한 조성물 및 방법
EP2926143A4 (fr) * 2012-11-30 2016-07-13 Siemens Healthcare Diagnostics Compositions et procédés de détection de la vitamine d
US10203344B2 (en) 2012-11-30 2019-02-12 Siemens Healthcare Diagnostics Inc. Compositions and methods for detecting vitamin D
KR102124352B1 (ko) * 2012-11-30 2020-06-18 지멘스 헬쓰케어 다이아그노스틱스 인크. 비타민 ｄ를 검출하기 위한 조성물 및 방법
EP2759550B1 (fr) * 2013-01-28 2018-08-15 DiaSorin S.p.A. Procédé et kit de détection de 1,25-dihydroxyvitamine D et anticorps associés
JP2016531306A (ja) * 2013-09-17 2016-10-06 ビオメリューＢｉｏｍｅｒｉｅｕｘ ビタミンｄ結合タンパク質からビタミンｄを解離するための溶液、その関連検出法及び使用

Also Published As

Publication number Publication date
GB201101801D0 (en) 2011-03-16

Similar Documents

Publication Publication Date Title
US11231424B2 (en) Levetiracetam immunoassays
JP4187929B2 (ja) 機能性ビタミンＤ誘導体の製造方法および２５−ヒドロキシおよび１α，２５−ジヒドロキシビタミンＤ代謝物の検出用試薬セット
EP3623811B1 (fr) Procédé pour mesurer la capacité d'absorption de cholestérol de lipoprotéines
Liu et al. Preparation of polyclonal antibodies to a derivative of 1-aminohydantoin (AHD) and development of an indirect competitive ELISA for the detection of nitrofurantoin residue in water
EP1360507A2 (fr) Dosage de la vitamine d
US9664691B2 (en) Molecularly imprinted polymer for detection of pentraxin protein and method for preparing the same
JP7398366B2 (ja) 対称性ジメチルアルギニンの検出
WO1994017408A1 (fr) Dosages et materiels pour la detection d'analytes en presence de substances de reaction croisee
EP0475783B1 (fr) Anticorps contre les ligand-analogues et leur usage dans les dosages ligand-recepteurs
EP2754672A1 (fr) Anticorps apte à se lier à une région spécifique de la périostine, et procédé de mesure de la périostine à l'aide de celui-ci
WO2008059837A1 (fr) Anticorps dirigé contre les aflatoxines, support utilisant ledit anticorps, procédé de détection immunologique des aflatoxines et procédé de concentration et de purification des aflatoxines
WO2012104643A1 (fr) Molécule de liaison et procédé de mesure
CA2834432C (fr) Procede d'inhibition d'une reaction non specifique dans un reactif de dosage pivka-ii
KR20170027791A (ko) 비타민 d에 관한 조성물
KR20170016009A (ko) 비타민 d 검정에서 비타민 d 에피머에 특이적인 결합 파트너
KR102309377B1 (ko) 비타민 d 에피머에 특이적인 결합 파트너
EP3881072A1 (fr) Molécules et procédés d'immunodétection améliorée de petites molécules, telles que l'histamine
JP2000502793A (ja) Ｅｌｉｓａ試験システム
EP1988094B1 (fr) Compose haptene et anticorps
EP3427054A2 (fr) Étalons internes pour dosages immunologiques et leur utilisation
JP5690514B2 (ja) アゾキシストロビン誘導体、アゾキシストロビンに対する抗体またはそのフラグメント、ならびにそれらの抗体またはフラグメントを用いた測定キットおよび測定方法
KR20140072264A (ko) C-반응성 단백질 검출용 합성항체
Hendrickson et al. Competitive photometric enzyme immunoassay for fullerene C 60 and its derivatives using a fullerene conjugated to horseradish peroxidase
JP5880029B2 (ja) ２またはそれ以上の種類の形態で存在する物質の測定方法
JP3995316B2 (ja) ホルモンレセプターに対する自己抗体の測定による癌の検出方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12714346

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12714346

Country of ref document: EP

Kind code of ref document: A1