WO2003023391A2 - Procede et agents permettant la mesure directe de composes de la vitamine d dans le serum ou le plasma - Google Patents

Procede et agents permettant la mesure directe de composes de la vitamine d dans le serum ou le plasma Download PDF

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Publication number
WO2003023391A2
WO2003023391A2 PCT/EP2002/009740 EP0209740W WO03023391A2 WO 2003023391 A2 WO2003023391 A2 WO 2003023391A2 EP 0209740 W EP0209740 W EP 0209740W WO 03023391 A2 WO03023391 A2 WO 03023391A2
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vitamin
sample
weight
analysis
serum
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PCT/EP2002/009740
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German (de)
English (en)
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WO2003023391A3 (fr
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Franz Paul Armbruster
Sabine Friedl
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Immundiagnostik Ag
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Publication of WO2003023391A2 publication Critical patent/WO2003023391A2/fr
Publication of WO2003023391A3 publication Critical patent/WO2003023391A3/fr

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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Definitions

  • the invention relates to a method for the quantitative determination of vitamin D compounds in serum or plasma.
  • Vitamin D 3 cholecalciferol
  • vitamin D 2 ergocalciferol
  • Vitamin D 2 and D 3 differ only slightly in the side chains.
  • the body stores vitamin D as 25-hydroxyvitamin-D, which is therefore the vitamin D metabolite with the highest concentration in the circulation.
  • D-hormone which regulates calcium absorption from the intestine, bone mineralization, osteoplast differentiation, bone matrix synthesis and also neuromuscular functions.
  • Vitamin D deficiency is an important risk factor for senile osteoporosis. Its early diagnosis and vitamin D supplementation enable effective fracture prevention. A severe vitamin D deficiency of less than 5 micrograms of 25-hydroxyvitamin-D per milliliter of serum (12.5 ⁇ mol / L) leads to rickets in children or to osteomalacia in adults. An excess of vitamin D, for example due to drug overdose, causes hypercalcaemia syndrome.
  • WO 99/67211 (Armbruster et al) teaches the preparation of the serum and plasma sample for the vitamin D determination by ethanol precipitation of the serum or plasma proteins.
  • the protein precipitate is centrifuged off and the ethanolic supernatant with the soluble vitamin D metabolites is used in the binding assay.
  • This object is achieved by a method for the determination of vitamin D compounds in plasma or serum by protein binding analysis, which is characterized in that the plasma or serum before the protein binding analysis contains at least 0.05% by weight of a soluble hydroxylated aromatic carboxylic acid, based on the weight is added to the sample liquid and that the protein binding analysis is based on antibodies against the vitamin D compound to be determined.
  • the pH of the sample is preferably adjusted to 3.0 to 9.0.
  • the salt of a salicylic acid is added to the sample from 0.5 to 15% by weight, preferably from 1 to 12% by weight, particularly preferably from 2 to 8% by weight, based in each case on the weight of the sample.
  • the binding analysis is carried out in the presence of 1 to 7% by weight of sodium or potassium salicylate at a pH between 3.0 and 7.0.
  • 0.1 to 5 preferably 1 to 3% by weight of natural and / or chemically modified cyclodextrin can also be added to the sample, in particular if the serum or plasma sample is lipemic.
  • the sample can also be purified by binding the vitamin D compound to an antibody, preferably a monoclonal antibody against the D hormone.
  • the method according to the invention is particularly suitable for measuring a vitamin D compound selected from 25-hydroxy-vitamin D 2 , 25-hydroxy-vitamin D 3 , 1 ⁇ , 25-dihydroxy-vitamin D 2 , 1 ⁇ , 25 -Dihydroxy-Vitamin-D 3 .
  • the competitive bond analysis is carried out as part of an ELISA, RIA, FIA or LIA.
  • the binding analysis can be carried out using an antibody-coated solid phase, the antibody binding the vitamin D compound to be determined.
  • the vitamin D tracer is preferably coupled to a directly determinable coloring, fluorescent or chemiluminescent marker.
  • Another aspect of the invention relates to a release and analysis buffer for the direct measurement of vitamin D, 25-hydroxy-vitamin D 2 , 25-hydroxy-vitamin D 3l 1 ⁇ , 25-dihydroxy-vitamin D 2 , 1 ⁇ , 25-dihydroxy-vitamin D 3 or another derivative of vitamin D in plasma or serum by protein binding analysis, the buffer having a pH of 3.0 to 9.0 and containing at least 0.05% by weight of a soluble hydroxylated aromatic carboxylic acid ,
  • the buffer preferably contains a salt of salicylic acid in an amount of 1 to 15% by weight, preferably 2 to 8% by weight.
  • the release and analysis buffer according to the invention particularly preferably has a pH between 3.0 and 7.0 and sodium or potassium salicylate in an amount of 1 to 15% by weight, preferably 2 to 8% by weight. It can also contain 0.1 to 5% by weight, preferably 1 to 3% by weight, of natural and / or chemically modified cyclodextrin.
  • Another aspect of the invention relates to a reagent set for direct measurement of vitamin D, 25-hydroxy-vitamin D 2 , 25-hydroxy-vitamin D 3 , 1 ⁇ , 25-dihydroxy-vitamin D 2 , 1 ⁇ , 25-dihydroxy -Vitamin-D 3 or another derivative of vitamin D in plasma or serum by protein binding analysis, comprising a release and analysis buffer according to the invention, at least one antibody against the vitamin D compound to be measured and a vitamin D tracer for a competitive protein binding analysis.
  • the tracer can be coupled to a directly determinable coloring, fluorescent or chemiluminescent marker
  • 2A, B are graphical representations of the comparative analysis with measured values from conventional external vitamin D determinations (purified by ethanol precipitation or column chromatography) and from direct determination according to the method according to the invention; 3A-B graphical representation of the recovery of different amounts of added vitamin D in different serum samples and buffers. 4A-B graphical representation of the linearity of the direct determination method according to the invention with different (increasing) vitamin D concentration
  • the method according to the invention differs from the prior art in that at least 0.05% by weight of a soluble hydroxylated aromatic carboxylic acid, based on the weight of the sample, is added to the plasma or serum sample before the protein binding analysis and the protein binding analysis on antibodies against the The determining vitamin D connection takes place and not on the vitamin D binding protein (DBP) or other vitamin D binding proteins.
  • DBP vitamin D binding protein
  • the pH of the sample is preferably set between 3.0 and 9.0 for protein binding analysis on the antibody.
  • a slightly acidic pH in the range from 3.0 to 7.0 has proven to be particularly advantageous.
  • Protein binding analysis on the antibody is very particularly preferably carried out at a pH of about 6.0.
  • a salicylate salt such as sodium or potassium salicylate is added to the sample before the protein binding analysis, preferably 1 to 12% by weight, particularly preferably 2 to 8% by weight of salicylate salt.
  • the binding analysis is particularly preferably carried out in the presence of 1 to 7% by weight of sodium or potassium salicylate at a pH between 3.0 and 7.0. All weight percentages relate to the weight of the sample.
  • the samples can optionally contain 0.1 to 5% by weight, preferably 1 to 3% by weight of natural and / or chemically modified cyclodextrin can be added.
  • foodstuffs such as milk, cheese, etc. can in principle also be examined directly for their vitamin D content.
  • the method according to the invention can be carried out together with conventional sample preparation methods. In individual cases, it may be appropriate to first partially remove the interfering substances and serum proteins from the plasma or serum sample by organic extraction or denaturing precipitation and then to carry out a protein binding analysis according to the invention.
  • the disruptive influence of the vitamin D-binding proteins remaining in the sample after processing is neutralized by the addition of a hydroxylated aromatic carboxylic acid, for example by salicylic acid, a salt or other derivative of salicylic acid, possibly also by acetylsalicylic acid.
  • a previous sample purification is particularly useful if the content of D-hormone in human serum is determined in addition to the form of vitamin D storage.
  • the D hormone can, for example, be easily isolated from the human serum or plasma by binding to an antibody which is specific only for the dihydroxy form. Monoclonal antibodies that specifically recognize the D hormone but not 25-hydroxyvitamin D are known and easy to produce. It is then advisable to carry out the binding between the D hormone and the monoclonal antibody in the presence of a salicylate salt in order to eliminate the simultaneous competing binding to vitamin D binding proteins naturally occurring in human serum, such as DBP (vitamin D binding protein).
  • DBP vitamin D binding protein
  • the method according to the invention is suitable for the quantitative determination of all types of vitamin D compounds. Diagnostically particularly interesting and important are: 25-Hydroxyvitamin-D 2 , 25-Hydroxyvitamin-D 3 , 1 ⁇ , 25-Dihydroxyvitamin-D 2 and 1 ⁇ , 25-Dihydroxyvitamin-D 3 .
  • the competitive protein binding assay according to the invention can be carried out as an enzyme-linked immunoabsorbent assay (ELISA - enzyme linked immunosorbent assay) or radioimmunoassay (RIA).
  • ELISA - enzyme linked immunosorbent assay enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • Non-radioactive detection systems based on fluorescence and chemiluminescence markers are particularly preferred. If the vitamin D tracer is coupled with the enzyme for the detection system, for example with alkaline phosphatase or a fluorescence or chemiluminescence marker, then the fixed phase or the wall of the microtiter plate with antibodies against the vitamin D to be determined -Connection coated.
  • An advantage of the method according to the invention is that the quantitative determination of the vitamin D compounds can be carried out directly in a liquid sample such as serum or plasma.
  • the sample does not have to be prepared by problematic protein precipitation, organic extraction and / or by column chromatography. Instead, the vitamin D compounds to be determined are displaced by the addition of salicylic acid, a salicylic acid salt or a derivative of salicylic acid in vitro by the vitamin D-binding proteins naturally present in the plasma or serum, so they are released in the sample and can then are determined directly using the principle of competing binding to anti-vitamin D antibodies.
  • Derivatives or salts thereof not only effectively displace the 25-hydroxy or 1 ⁇ , 25-dihydroxy-vitamin D from the binding sites on the vitamin D binding protein (DBP), but generally from all binding sites in the plasma or Serum-occurring proteins that can bind vitamin D
  • the vitamin D-binding proteins include not only the highly specific, highly affine binding DBP (Gc-globulin), which is also known as Gc-globulin or group-specific component, but also abundant proteins such as albumin, ⁇ -fetoprotein and many others more. These proteins can also bind 25-hydroxy- and 1 ⁇ , 25-dihydroxy-vitamin-D with more or less high specificity and affinity.
  • the purification of the vitamin D compounds is comparatively incomplete as in the case of ethanol or denaturing protein precipitation, residual amounts of vitamin D binding proteins such as serum albumin are present in the binding assay despite the purification.
  • the proteins remaining in the sample solution can then not only bind the vitamin D or the vitamin D tracer in the competitive binding assay, but also go to the hydrophobic wall of the sample vessel or the solid phase, so that the quantitative vitamin D determination of Randomness depends on how complete the protein precipitation and the cholesterol level of the sample is.
  • the method according to the invention offers the advantage that the vitamin D compounds do not have to be isolated and purified, but rather the competitive protein binding assay can be done directly in plasma or serum.
  • Z is the oxygen or sulfur atom of an ether group or a carbon atom
  • X is a substituted or unsubstituted hydrocarbon group of 0.8 to 4.2 ⁇ m in length, preferably a C 8 - to C 2 group, which may optionally have the heteroatoms S, O, N or P, very preferably a hexamido, octamido or Decamido-amidopropyl linker group;
  • Y is hydrogen or a hydroxy group;
  • A is a functional group selected from groups bound by a high affinity protein, for example antibodies or streptavidin, reactive groups for coupling the functional vitamin D derivative to a solid support such as beads, labeled solid particles or magnetic particles; functional proteins such as green fluorescent protein (GFP), enzymes such as alkaline phosphatase or peptides and signaling groups such as fluorescent or chemiluminescent groups;
  • GFP green fluorescent protein
  • enzymes such as alkaline phosphatase or peptides
  • signaling groups such as fluorescent or chemiluminescent groups
  • R is the side group of a vitamin D metabolite, preferably the side group of
  • Vitamin D 2 or D 3 preferably the 25-hydroxylated side group of vitamin D 2 or D 3 .
  • the dissociation constant K should here be greater than 10 8 , dissociation constants greater than 10 16 are preferred.
  • A is selected from biotin, digoxigenin, tyrosine, substituted tyrosine, substituted amino acids, characteristic amino acid and peptide sequences, FITC, proteins and protein groups such as protein A or protein G or another vitamin D derivative.
  • the vitamin D tracer is particularly preferably covalently coupled to a color or signal-giving group such as a ruthenium complex, fluorescein, etc.
  • the spacer group X is preferably selected from substituted and unsubstituted C bodies with a length of 0.8 to 4.2 nm, a spacer group of 0.12 nm in length is particularly preferred.
  • An aminocarboxylic acid in particular an aminoundecanoic acid, peptide and keto groups or a substituted or unsubstituted aminopolyether radical with a length of 0.8 to 4.2 nm, preferably about 0.9 to 1.5 nm, is very particularly preferred. This distance between group A and the binding or recognition site for the vitamin D residue allows the binding proteins to bind to their respective binding site and not interfere with each other.
  • vitamin D tracer derivatives which contain a further vitamin D group as functional group A.
  • the advantage of these dimeric vitamin D derivatives as tracers is that they do not introduce non-systemic groups into the assay and allow increased sensitivity and reliability of the competitive binding analysis. Symmetrical vitamin D dimers are particularly favorable.
  • Vitamin D tracers in which A is a solid phase or a fluorescent or chemiluminescent group are also suitable.
  • examples include: fluorescein, ruthenium complex compounds, etc.
  • vitamin D In the direct determination of vitamin D in serum, all vitamin D that is bound to DBP (Gc-globulin) or other vitamin D-binding proteins such as albumin is first displaced from their binding sites and used for binding with the vitamin D Antibodies made available. According to the invention, this is done by adding a hydroxylated aromatic carboxylic acid, for example a salicylic acid compound, preferably sodium or potassium salicylate.
  • the salicylic acid compound has the property of displacing the vitamin D from the DBP / Gc globulin and other vitamin D-binding proteins such as albumin.
  • the salicylate does not block the binding of anti-vitamin D antibodies to 25-hydroxyvita- min-D or 1 ⁇ , 25-dihydroxyvitamin-D, so that a direct quantitative determination in serum or plasma is possible in a competitive binding analysis. Because of the omitted sample preparation steps, the method according to the invention can be automated easily, quickly and easily. In addition, the measured values are not disturbed by the individual properties of the sample and randomnesses in the preparation of the sample.
  • the method according to the invention thus enables a non-radioactive, quantitative determination of 25-hydroxy- and 1 ⁇ , 25-dihydroxyvitamin-D in serum or plasma without complex sample preparation steps.
  • the direct method is therefore particularly suitable for routine examinations in the context of osteoporosis prophylaxis, in the case of suspected D-hypovitaminosis or D-hypervitaminosis, for general diagnosis and in research.
  • Another aspect of the invention relates to a set of reagents for the determination of vitamin D metabolites such as 25-hydroxy and 1 ⁇ , 25-dihydroxyvitamin-D, which contains, inter alia, a functional vitamin D derivative and salicylic acid or a salicylic acid compound in a binding or dilution buffer.
  • the reagent set optionally includes anti-vitamin D antibodies, that is to say antibodies to 25-hydroxyvitamin D 2 3 or 1 ⁇ , 25-dihydroxyvitamin D2 / 3 or other metabolites of vitamin D, coated microtiter plates and / or magnetic or other microparticles and reagents.
  • the reagent set also contains cyclodextrin (CD) for sequestering and masking disruptive hydrophobic molecules such as fatty acids, cholesterol, etc. in the plasma or serum sample.
  • CD cyclodextrin
  • the alpha, beta or gamma cyclodextrin is preferably used in amounts of 0.1 to 10% by weight, preferably about 0.2 to 7.5% by weight, particularly preferably in an amount of 1 to 5% by weight. based on the final concentration in the sample.
  • the aqueous cyclodextrin stock solution is preferably neutral to slightly alkaline, since cyclodextrins are usually split by acid.
  • the cyclodextrin (CD) can also be chemically modified, for example with methyl, ethyl, propyl, hydroxyethyl, 2-hydroxypropyl, glycosyl, maltosyl, carboxymethyl. Natural cyclodextrins and 2-hydroxypropyl-beta-cyclodextrin are particularly preferred. Examples
  • Example 1 Determination of 25-hydroxyvitamin-D in serum or plasma (i) Binding of vitamin D tracer to a solid phase. 25-Hydroxyvitamin-D-3ß-3- [6-N- (biotinyl) hexamidojamidopropyl ether was converted into Streptavidin bound to a solid phase. For this purpose, 100 ng streptavidin was first added to the wells of a microtiter plate, dissolved in 200 ⁇ l 60 mmol sodium hydrogen carbonate, pH 9.6, and the plate was incubated at 4 ° C. overnight. The streptavidin solution was removed and the wells washed five times with 200 ⁇ l wash buffer (PBS, pH 7.4 with 0.05% Tween-20).
  • PBS pH 7.4 with 0.05% Tween-20
  • the serum and plasma samples were only stored briefly at room temperature. The sample was stored at 4 to 8 ° C if the determination was made within 24 hours after collection. Otherwise, the samples were frozen at -20 ° C until determined. Multiple freezing and thawing of the sample was avoided. Hemolysis did not interfere with the result. Whole blood, however, could not be used as sample material. Lipemic samples were centrifuged at approx. 30,000 x G for 10 minutes until the fat phase floated upwards and the aqueous phase could be removed with the pipette through the fat layer. In the case of strongly lipemic material, use was made of a delipidation kit for lipemic sample material.
  • cyclodextrin was added to the sample, either as natural cyclodextrin or as chemically modified cyclodextrin. However, not every cyclodextrin proved to be suitable.
  • concentration of natural alpha-cyclodextrin in the sample was preferably set to about 2.5% by weight.
  • the sample, serum or plasma was diluted 1:20 in sample dilution buffer, e.g. B. 20 ul serum to 400 ul phosphate buffer, pH 6.0, 0.1% gelatin with 0.05% by weight, 0.5% by weight and 5% by weight sodium salicylate. The samples were mixed well and left for 5 to 10 minutes at room temperature (21 to 25EC).
  • concentration of salicyl compound in the binding buffer was 0.05% by weight, preferably 0.5% by weight, particularly preferably 5% by weight sodium salicylate in phosphate buffer, pH 6.0.
  • the micro titer plate was 24 hours at 4 ! ° C shaken in the dark.
  • the 25-hydroxyvitamin-D present in the sample then competed with the vitamin D tracer for the binding sites of the antibody.
  • the solutions were then removed from the wells and the wells were washed five times with 200 ⁇ l of washing buffer.
  • the presence of the salicyl compound in the assay meant that the vitamin D-binding proteins present in the sample (such as, for example, the serum albumin) could not bind to the vitamin D tracer, which leads to false high vitamin D contents in the prior art , Bonds of proteins that bind vitamin D directly to the hydrophobic wall of the microtiter plate also had no influence on the measurement values, because the 25-hydroxyvitamin-D to be determined in the sample could not, due to the presence of the salicyl compound, also not bind to the vitamin adhering to the wall -D bind binding proteins.
  • the 2-hydroxyvitamin D values in the sample are false low. According to the invention, both binding processes are largely blocked by the added salicyl compound.
  • FIGS. 1A-D the calibration curves shown in FIGS. 1A-D were created.
  • the ordinate shows the optical density as the mean of the two measurements at 450 nm; the abscissa shows the concentration of 25-hydroxyvitamin-D in nmoles.
  • the results show that the best conditions are achieved with pH 6.0 and 0.1% gelatin and 5% sodium salicylate.
  • Example 3 Recovery in different serum samples.
  • sample preparation or the competing binding analysis was carried out as stated in the example at pH 6.0, 0.1% gelatin, 5% sodium salicylate in serum.

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Abstract

L'invention concerne un procédé et un tampon échantillon pour une analyse en mesure directe permettant le dosage de composés de la vitamine D, tels que 25-hydroxy-vitamine-D2, 25-hydroxy-vitamine-D3. 1 alpha ,25-dihydroxy-vitamine-D2, 1 alpha ,25-dihydroxy-vitamine-D3 dans le plasma ou le sérum. Le dosage est basé sur une analyse des liaisons protéine à des anticorps à la place de la protéine de liaison à la vitamine D contre le composé de vitamine D à déterminer. L'invention est caractérisée en ce que le tampon d'échantillon et d'analyse renferme, pour un pH légèrement acide préféré, au moins 0,05 % en poids d'un acide carboxylique aromatique hydroxylé soluble ou d'un sel de celui-ci, de préférence 1 à 7 % en poids de salicylate de sodium ou de potassium pour un pH compris entre 3,0 et 7,0 et, éventuellement, de la cyclodextrine.
PCT/EP2002/009740 2001-09-12 2002-08-30 Procede et agents permettant la mesure directe de composes de la vitamine d dans le serum ou le plasma WO2003023391A2 (fr)

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DE10144905.4 2001-09-12
DE2001144905 DE10144905C2 (de) 2001-09-12 2001-09-12 Bestimmung von Vitamin-D direkt in Serum oder Plasma

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Cited By (11)

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WO2008092917A1 (fr) 2007-02-01 2008-08-07 Immundiagnostik Ag Mesure directe de la vitamine d dans le sérum ou le plasma
EP2372365A1 (fr) * 2010-04-01 2011-10-05 Future Diagnostics B.V. Analyse directe pour la vitamine D
WO2012091569A1 (fr) 2010-12-28 2012-07-05 Future Diagnostics B.V. Réactif de libération pour la vitamine d
WO2014158864A1 (fr) * 2013-03-14 2014-10-02 Enzo Life Sciences, Inc. Analyses de vitamine d
US9341552B2 (en) 2005-09-29 2016-05-17 Roche Diagnostics Operations, Inc. Release reagent for vitamin D compounds
WO2017039574A1 (fr) * 2015-08-28 2017-03-09 Enzo Life Sciences, Inc. Dosages de vitamine d
US9746483B2 (en) 2009-10-27 2017-08-29 Diasource Immunoassays S.A. Process for the production of a hybridoma and antibody obtained therefrom, able to recognize more than one vitamin D metabolite
US10073103B1 (en) 2013-03-11 2018-09-11 Theranos Ip Company, Llc Rapid measurement of total vitamin D in blood
WO2019134948A1 (fr) 2018-01-03 2019-07-11 Immundiagnostik Ag Procédé de mesure du statut de la vitamine d endocytique
US10451639B2 (en) 2010-05-20 2019-10-22 Roche Diagnostics Operations, Inc. Release reagent for vitamin D compounds
US11187709B2 (en) 2011-11-18 2021-11-30 Roche Diagnostics Operations, Inc. Release reagent for vitamin D compounds

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WO2002046746A2 (fr) * 2000-12-06 2002-06-13 Immunodiagnostic Systems Ltd Procede de detection de metabolites de la vitamine d

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US5981779A (en) * 1995-12-29 1999-11-09 A And D Assay, Incorporated Labeled vitamin D compounds and the use thereof
WO2002046746A2 (fr) * 2000-12-06 2002-06-13 Immunodiagnostic Systems Ltd Procede de detection de metabolites de la vitamine d

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US9341552B2 (en) 2005-09-29 2016-05-17 Roche Diagnostics Operations, Inc. Release reagent for vitamin D compounds
AU2008209736B2 (en) * 2007-02-01 2011-03-03 Immundiagnostik Ag Direct determination of vitamin D in serum or plasma
US7964363B2 (en) 2007-02-01 2011-06-21 Immundiagnostik Ag Direct determination of vitamin D in serum or plasma
WO2008092917A1 (fr) 2007-02-01 2008-08-07 Immundiagnostik Ag Mesure directe de la vitamine d dans le sérum ou le plasma
US9746483B2 (en) 2009-10-27 2017-08-29 Diasource Immunoassays S.A. Process for the production of a hybridoma and antibody obtained therefrom, able to recognize more than one vitamin D metabolite
CN102985826A (zh) * 2010-04-01 2013-03-20 未来诊断有限公司 游离的维生素d的免疫分析
US11927595B2 (en) 2010-04-01 2024-03-12 Future Diagnostics B.V. Immunoassay for free vitamin D
JP2013524208A (ja) * 2010-04-01 2013-06-17 フューチャー・ダイアグノスティックス・ベーフェー 遊離型ビタミンｄのイムノアッセイ
US10935557B2 (en) 2010-04-01 2021-03-02 Future Diagnostics B.V. Immunoassay for free vitamin D
AU2011233816B2 (en) * 2010-04-01 2015-02-12 Future Diagnostics B.V. Immunoassay for free vitamin D
CN102985826B (zh) * 2010-04-01 2015-12-16 未来诊断有限公司 游离的维生素d的免疫分析
WO2011122948A1 (fr) * 2010-04-01 2011-10-06 Future Diagnostics B.V. Immunodosage de la vitamine d libre
EP2372365A1 (fr) * 2010-04-01 2011-10-05 Future Diagnostics B.V. Analyse directe pour la vitamine D
US9897615B2 (en) 2010-04-01 2018-02-20 Future Diagnostics, B.V. Immunoassay for free vitamin D
US10451639B2 (en) 2010-05-20 2019-10-22 Roche Diagnostics Operations, Inc. Release reagent for vitamin D compounds
US10422805B2 (en) 2010-12-28 2019-09-24 Future Diagnostics B.V. Release reagent for vitamin D
CN103282390A (zh) * 2010-12-28 2013-09-04 未来诊断有限公司 对于维生素d的释放剂
CN108267602A (zh) * 2010-12-28 2018-07-10 未来诊断有限公司 对于维生素d的释放剂
WO2012091569A1 (fr) 2010-12-28 2012-07-05 Future Diagnostics B.V. Réactif de libération pour la vitamine d
CN108267602B (zh) * 2010-12-28 2020-12-08 未来诊断有限公司 对于维生素d的释放剂
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