WO2003023391A2 - Method and agents for direct measurement of vitamin d compounds in serum or plasma - Google Patents

Abstract

The invention relates to a method and a sample buffer for a direct measuring assay in order to determine vitamin D compounds such as 25-hydroxyl-vitamin-D2, 25-hydroxy-vitamin-D3. 1 alpha ,25-dihydroxy-vitamin-D2, 1 alpha ,25-dihydroxy-vitamin-D3 .in plasma or serum. The assay is based on a protein binding analysis on antibodies in place of the vitamin-D binding protein- in relation to the vitamin D compound to be determined, and the sample and analysis buffer contains at least 0.05 wt. % soluble hydroxylated aromatic carboxylic acid or a salt thereof for a slightly acid pH, preferably 1-7 wt. % sodium or potassium salicylate for a pH ranging between 3.0 und 7.0, and optionally cyclodextrin.

Description

 METHOD AND MEANS FOR DIRECTLY MEASURING VITAMIN-D COMPOUNDS IN SERUM OR PLASMA

FIELD OF THE INVENTION

The invention relates to a method for the quantitative determination of vitamin D compounds in serum or plasma.

BACKGROUND OF THE INVENTION Humans can only use D vitamins or calciferols with the help of

Form sunlight in the skin. Both the cholecalciferol (vitamin D ₃ ) in human skin and the occasional added ergocalciferol (vitamin D ₂ ) in food are bound in the circulation by the vitamin D binding protein (DBP), transported to the liver and to 25-hydroxyvitamin -D ₂ or -D ₃ metabolized. 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. If necessary, it is hydroxylated in the kidney to form biologically active 1α, 25-dihydroxyvitamin-D, the so-called D-hormone, which regulates calcium absorption from the intestine, bone mineralization, osteoplast differentiation, bone matrix synthesis and also neuromuscular functions.

Even a slight deficiency of less than 15 micrograms of 25-hydroxyvitamin-D per milliliter of serum (37.5 μmol / L) leads to an increase in the parathyroid hormone level and an increased bone resorption due to the lower calcium intake. 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 μmol / 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.

In Germany, up to a third of the normal population over 50 years of age suffers from vitamin D deficiency in winter. The reasons are the lack of sunlight and the reduced vitamin D synthesis in the skin in older people. People with dark skin tones represent a special risk group. In geriatric patients, approximately 3% of the treatment cases show a vitamin D deficiency. Younger people can also suffer from vitamin D deficiency: in gastrointestinal diseases, liver function Disorders, maladsorption or increased drug metabolism, e.g. due to antiepileptics. The content of vitamin D in human serum and plasma is therefore an important diagnostic parameter.

The current laboratory methods for determining vitamin D in human serum and plasma are complex and tedious (see Tanner et al. (1988), J. Assoc. Of Analyt. Chem., 17, 607-710).

US 5,981,779 (Holick et al) and WO 89/01631 (DeLuca et al) describe vitamin D assays for hydroxy and dihydroxyvitamin D by analyzing their binding to DBP in comparison to a competing vitamin D compound. Before this, the vitamin D metabolites must first be isolated from the blood and serum sample by organic extraction and purified by chromatography.

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.

However, all of the aforementioned methods are error-prone and cumbersome because of the necessary sample preparation. It is an object of the invention to provide a method for the direct quantitative measurement of vitamin D compounds in serum or plasma.

SUMMARY OF THE INVENTION

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.

In one embodiment according to the invention, 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. In a preferred embodiment, 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. If necessary, 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 , 1α, 25-dihydroxy-vitamin D ₂ , 1α, 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 1α, 25-dihydroxy-vitamin D ₂ , 1α , 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 , 1α, 25-dihydroxy-vitamin D ₂ , 1α, 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

Further advantages, features and embodiments of the invention result from the examples below and the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS It shows:

1 A-D ELISA calibration curves with various 0, 0.05, 0.5 and 5 percent by weight sodium salicylate in the analysis buffer;

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

DETAILED DESCRIPTION OF THE INVENTION

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.

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.

In the method according to the invention, 0.5 to 15% by weight of 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.

So that the binding analysis is not disturbed by free fatty acids, cholesterol and other lipidic molecules, 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.

Instead of the plasma or serum sample, 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).

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 , 1α, 25-Dihydroxyvitamin-D ₂ and 1α, 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). Non-radioactive detection systems based on fluorescence and chemiluminescence markers (FIA or LIA) 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. Surprisingly, the addition of salicylic acid or one

Derivatives or salts thereof not only effectively displace the 25-hydroxy or 1α, 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, α-fetoprotein and many others more. These proteins can also bind 25-hydroxy- and 1α, 25-dihydroxy-vitamin-D with more or less high specificity and affinity. If 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. But also the organic extraction and / or the chromatographic purification of the vitamin D compounds do not improve the quantitative determination, because these steps also influence and change the amount of biologically active vitamin D compound that can be detected, depending on the properties of the sample - among other things, which and how much vitamin D binding proteins are present in the sample, whether the sample is lipemic, etc.

In contrast, 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.

The invention also includes the use of labeled vitamin D derivatives and also the following non-radioactive vitamin D derivatives of the formula I:

where is:

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 μm 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;

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 .

For the in vitro determination directly in serum or plasma it is important that either there is a high affinity between the functional group A and its binding protein, for example an antibody. The dissociation constant K should here be greater than 10 ⁸ , dissociation constants greater than 10 ¹⁶ are preferred. In preferred embodiments, 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.

25-Hydroxyvitamin-D-3ß-3- [6-N- (biotinyl) hexamido] amidopropyl ether and the corresponding 1α, 25-dihydroxyvitamin-D-biotin compound are preferred. Also suitable are 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.

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. Surprisingly, on the other hand, the salicylate does not block the binding of anti-vitamin D antibodies to 25-hydroxyvita- min-D or 1α, 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 1α, 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 1α, 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 1α, 25-dihydroxyvitamin D2 / ₃ or other metabolites of vitamin D, coated microtiter plates and / or magnetic or other microparticles and reagents. In a preferred embodiment, 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. 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 μl 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 μl wash buffer (PBS, pH 7.4 with 0.05% Tween-20). Then 250 ocl block buffer (phosphate buffer pH 7.4 with 0.5% casein, 1% thimerosal) was added to each well, incubated for one hour at room temperature, the assay buffer was removed and each well was washed five times with 200 μl wash buffer. 10 ng of 25-biotinhydroxyvitamin-D in 200 μl of washing buffer were then added to the wells, incubated for one hour at room temperature in the dark and with shaking, the biotin-vitamin-D solution was removed from the wells and each well 5 times with 200 μl wash buffer washed. This was followed in the liquid phase by competitive binding of a monoclonal mouse antibody (ID2) against vitamin D in the presence of 25-hydroxyvitamin D from the standard or sample. (ii) Sample preparation and handling

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. To increase the reproducibility of the binding assay, cyclodextrin was added to the sample, either as natural cyclodextrin or as chemically modified cyclodextrin. However, not every cyclodextrin proved to be suitable. The 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). During this time, the sodium salicylate was able to displace the 25-hydroxyvitamin-D from its binding to the DBP (Gc-globulin) and other vitamin D-binding proteins, so that the 25-hydroxyvitamin-D and the vitamin D tracer around the binding sites of the antibody could compete. (iii) Competitive binding analysis 100 .mu.l monoclonal antibody (2D8) from mouse (1: 20000) was in each case

Wash buffer together with 100 μl standard, control or sample (20 μl serum in 400 sample dilution buffer) were added to the wells. The 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 μl 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. In the prior art, on the other hand, when vitamin D-binding proteins come into contact with the wall of the microtiter plate, 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. (iv) Determination of the competitive bond

In each case 200 μl conjugate (goat anti-mouse MAB antibody peroxidase labeled) was diluted 1: 2500 in washing buffer, added to the wells and incubated for half an hour at room temperature in the dark and with shaking. The solutions were removed and the wells were washed five times with 200 μl wash buffer. For the color reaction, 200 μl of tetramethylbenzidine (TMB) substrate solution (from NOVUM Diagnostika GmbH, Dietzenbach, DE) in the wells. After 30 minutes the color development was stopped by adding 50 ul 2 MH ₂ SO per well. The optical density was measured at 450 nm.

Solutions of 25-hydroxyvitamin D ₃ assay buffer with the following concentrations, 0, 6.4, 16, 40, 100 and 250 nmol / l (see calibration curve) were used as standard.

Sera from patients from a normal population served as controls or samples. Solutions in which the respective concentration of 25-hydroxyvitamin-D was known from other determinations served as a further control. From the mean values and the known concentrations of 25-hydroxyvitamin

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 2: Comparative bond analysis

Unless otherwise stated, all reagents and buffers and materials were the same as in the previous example except for sample preparation. On the one hand, certain measurements were used on our sera in external laboratories. On the other hand, additional vitamin D determinations were made. For the competitive binding analysis, 50 μl serum in a 1.5 ml Eppendorf reaction vessel was mixed with 200 μl absolute ethanol (precooled to -20 ° C.) by vortexing and precipitated at -20 ° C. for 20 minutes. The samples were centrifuged in a table centrifuge at maximum speed, the supernatants were removed and used in the ELISA. The tables below show the correlation between the ELISA values and the direct determinations in tabular and graphic form.

TABLE I

Correlation of routine samples (external laboratory): ELISA vs direct correlation (n = 47)

Tabelle II

Table II

Correlation 25-vitamin D: direct vs CPBA normal collective (n = 40)

Die vergleichende Analyse bzw. Vitamin-D-Bestimmung aus Serum mit verschiedenen Verfahren zeigt eine gute Übereinstimmung für eine Vielzahl von Seren. Allerdings waren auch erhebliche Diskrepanzen festzustellen, welche die Zuverlässigkeit der Vitmain-D-Bestimmungen in Frage stellten. Die Nachanalyse und Nachschau einzelner Werte legte aber dabei, dass die erfindungsgemäße Direkt- Bestimmung die zuverlässigere Methode ist. Mit anderen Worten, die Diskrepanzen waren zumeist eine Folge der bereits oben angesprochenen systematischen und serumprobenbedingten Fehlerquellen. Diese zeigte insbesondere die nachstehende Analyse der Wiederfindung in verschiedenen Proben.

The comparative analysis or vitamin D determination from serum using different methods shows good agreement for a large number of sera. However, there were also significant discrepancies that questioned the reliability of the Vitmain D determinations. However, the post-analysis and review of individual values indicated that the direct determination according to the invention is the more reliable method. In other words, the discrepancies were mostly a result of the systematic and serum sample-related error sources mentioned above. This showed in particular the following analysis of the recovery in different samples.

Example 3: Recovery in different serum samples.

The 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.

The bond analysis in recovery; measured in 1 sample - ELISA with ID2

TABLE

Recovery measured in 2 plasmas (ID2)

Example 4 Investigation of linearity

The linearity of the method according to the invention in various plasmas and sera was then investigated in an analogous manner. The determinations were carried out as indicated in Example 1 at pH 6.0, 0.1% gelatin, 5% sodium salicylate. Table IVa Linearity in Plasma 8 (ID2)

Linearity in Plasma 4 (ID2)

Excellent linear regression is obtained.

Claims

PATENT CLAIMS 1. A method for determining vitamin D compounds in plasma or serum by protein binding analysis, characterized in that the sample of the plasma or serum at least before the protein binding analysis 0.05% by weight of a soluble hydroxylated aromatic carboxylic acid, based on the weight of the sample liquid, is added and that the protein binding analysis is based on antibodies against the vitamin D compound to be determined. 2. The method according to claim 1, characterized in that the pH of the sample is adjusted to 3.0 to 9.0. 3. The method according to claim 1 or 2, characterized in that the sample 0.5 to 15 wt.%, Preferably from 1 to 12 wt.%, Particularly preferably from 2 to 8 wt.%, Each based on the weight of the sample , the salt of a salicylic acid is added. 4. The method according to claim 2 or 3, characterized in that the binding analysis is carried out in the presence of 1 to 7 wt.% Sodium or potassium salicylate at a pH between 3.0 and 7.0. 5. The method according to any one of claims 1 to 4, characterized in that 0.1 to 5, preferably 1 to 3 wt.% Natural and / or chemically modified cyclodextrin is added to the sample. 6. The method according to any one of claims 1 to 5, characterized in that the sample is also purified by binding a type of a vitamin D compound to an antibody, preferably a monoclonal antibody against the D hormone. 7. The method according to any one of the preceding claims 1 to 6, characterized in that the vitamin D compound to be determined is selected from 25-hydroxy-vitamin D ₂ , 25-hydroxy-vitamin D ₃ , 1α, 25-dihydroxy - Vitamin D ₂ , 1 α, 25-dihydroxy vitamin D ₃ . 8. The method according to any one of the preceding claims 1 to 7, characterized in that the competitive binding analysis is an ELISA, RIA, FIA or LIA. 9. The method according to any one of the preceding claims 1 to 7, characterized in that the binding analysis using an antibody-coated Solid phase takes place, with the antibody binding the vitamin D compound to be determined. 10. The method according to claim 9, characterized in that the vitamin D tracer is coupled to a directly determinable coloring, fluorescent or chemiluminescent marker. 11. Displacement and analysis buffer for the direct measurement of vitamin D, 25-hydroxy-vitamin D ₂ , 25-hydroxy-vitamin D ₃ , 1 α, 25-dihydroxy-vitamin D ₂ , 1α, 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. 12. Displacement and analysis buffer according to claim 11, wherein the buffer contains a salt of salicylic acid in an amount of 1 to 15% by weight, preferably 2 to 8% by weight. 13. displacement and analysis buffer according to claim 11, wherein the buffer contains at a pH between 3.0 and 7.0 sodium or potassium salicylate in an amount of 1 to 15 wt.%, Preferably 2 to 8 wt.%. 14. Displacement and analysis buffer according to one of claims 11 to 13, wherein the buffer contains 0.1 to 5% by weight, preferably 1 to 3% by weight, of natural and / or chemically modified cyclodextrin. 15. Reagent set for direct measurement of vitamin D, 25-hydroxy-vitamin D ₂ , 25-hydroxy-vitamin D ₃ , 1α, 25-dihydroxy-vitamin D ₂ , 1α, 25-dihydroxy-vitamin D ₃ or another derivative of vitamin D in plasma or serum by protein binding analysis, comprising a displacement and analysis buffer according to any one of claims 11 to 14; at least one Antibodies against the vitamin D compound to be measured; and a tracer for competitive protein binding analysis. 16. The reagent set according to claim 15, wherein the tracer is coupled to a directly determinable staining, fluorescence or chemiluminescence marker.