HK1124870A - Antibodies against 25-hydroxyvitamin d - Google Patents

Description

Antibodies against 25-hydroxyvitamin D

Background

The present invention relates to a method for producing an antibody against 25-hydroxyvitamin D, an antibody produced according to the method of the present invention and a method for detecting 2-hydroxyvitamin D using the same.

The term "vitamin" implies that an adequate supply of vitamin D is vital. Vitamin D deficiency can lead to serious diseases such as rickets or osteoporosis. Although vitamin D was still considered as a single substance in the beginning of the last century, the vitamin D system has evolved into a complex and diverse vitamin D metabolite system in the last 30 years. More than 40 vitamin D metabolites are now known (Zercrkh, J.E., Ann. Clin. biochem.41(2004) 272-281).

Vitamin D is produced by ultraviolet radiation when the human body is irradiated on the skin only by sunlight₃Or a calciferol. Vitamin D production in skin₃Combined with so-called vitamin D binding proteins and transported to the liver where they are converted to 25-hydroxyvitamin D by 25-hydroxylation₃. In addition to the two organs of skin and liver mentioned above, a large number of other tissues are now also proven to be involved in vitamin D metabolism (reviewed by Schmidt-Gayk, H., et al, (eds.), "Calcium regulating hormones, vitamin D metabolites and cyclic AMP (Calcium that regulates hormones, vitamin D metabolites and cyclic adenylate)", Springer Verlag, Heidelberg (1990), pp.24-27). 25-hydroxyvitamin D, specifically 25-hydroxyvitamin D, in total₂And 25-hydroxyvitamin D₃Is the main form of vitamin D storage in the human body. When required, these precursors can be converted in the kidney into the biologically active 1 α, 25-dihydroxyvitamin D, the so-called hormone D. Active vitamin D in the body not only regulates calcium intake, among others from intestinal and bone mineralization, it also affects a number of other metabolic pathwaysSuch as the insulin system.

Measuring vitamin levels by itself does not help to determine the vitamin D status of a patient, since vitamin D (vitamin D) does not help₂And vitamin D₃) The concentration of (a) may fluctuate greatly according to the absorption of food. In addition, the biological half-life of circulating (24 hours) vitamin D is relatively short, which is another reason why it is not a suitable parameter for determining the vitamin D status of a patient. The same applies to the physiologically active form of vitamin D (1, 25-dihydroxyvitamin D). The concentrations of these bioactive forms are also relatively small and fluctuate widely compared to 25-hydroxyvitamin D. In summary, quantification of 25-hydroxyvitamin D is a suitable means for the comprehensive analysis of a patient's total vitamin D status.

Due to the high clinical value of 25-hydroxyvitamin D, a number of more or less reliable methods for the determination of 25-hydroxyvitamin D have been proposed in the literature.

The determination of the concentration of 25-hydroxyvitamin D in blood samples by means of High Performance Liquid Chromatography (HPLC) is described, for example, in Haddad, J.G.et al, J.Clin.Endocrinol.Metab.33(1971)992-995 and Eisman, J.A.et al, anal.biochem.80(1977) 298-305.

One of the other ways to measure 25-hydroxyvitamin D utilizes vitamin D binding proteins, e.g. present in milk. A vitamin D assay based on the use of vitamin D binding proteins to bind hydroxy vitamin D and dihydroxy vitamin D and determining their concentration by a competitive assay procedure is described in holck, m.f. and Ray, R. (US 5,981,779) and DeLuca et al. (EP 0583945). However, the prerequisite for this method is that the vitamin D metabolites to be determined must be in a form isolated by organic extraction from the original blood or serum sample and must be purified, for example, by means of a chromatographic column.

Armbruster, f.p. et al (WO 99/67211) teach the need to prepare samples of serum or plasma by ethanol precipitation for vitamin D determination. In this method, the protein precipitate is removed by centrifugation, leaving an ethanol supernatant comprising soluble vitamin D metabolites. These can be determined by competitive binding assays.

In addition, EP 0753743 teaches the use of periodate to separate proteins from blood or serum samples. In this case, the samples were treated with periodate and the protein-free supernatants of the samples were assayed for vitamin D compounds. In certain commercial assays, the use of acetonitrile as a medium for extraction from serum or plasma samples is recommended (e.g., radioimmunoassay by DioSorin or vitamin D assay by "Immundiagnostik").

In recent years a large number of different release agents have been planned to be released, which are in principle suitable for releasing vitamin D compounds from binding proteins in a sample. However, such release or isolation should be performed under relatively mild conditions so that the sample treated with the released reagent can be used directly in the binding assay. (see, e.g., WO 02/57797 and US 2004/0132104). Despite recent enormous efforts, all existing vitamin D detection methods suffer from drawbacks such as labor intensive sample preparation, poor standardization, lack of consistency between test procedures or low yield of spiked (spiked) vitamin D. (see, in particular, Zerwekh, J.E., supra).

In particular, there is no mention in the prior art of the production of a reliable method for the determination of 25-hydroxyvitamin D. It was therefore an object of the present invention to find a reliable method for producing antibodies suitable for use in 25-hydroxyvitamin D assays. The methods described below, both methods for producing antibodies, and methods and kits for determining vitamin D using these antibodies are also included.

Summary of The Invention

The present invention relates to a method for producing antibodies against 25-hydroxyvitamin D comprising the steps of:

a) will contain 25-hydroxy vitamin D₃Or 25-hydroxy vitamin D₂Conjugates as haptensImmunizing and inoculating into an experimental animal body,

b) separating the serum or plasma of the experimental animal, and

c) by immunoadsorption to a mixture of vitamin D compounds each containing 25-hydroxy vitamin D₂Or 25-hydroxy vitamin D₃Thereby purifying the antibodies in the serum or plasma.

In addition, the invention relates to anti-25-hydroxy vitamin D₃Can be mixed with 25-hydroxy vitamin D of the order of 10% to 1000%₂And (4) carrying out cross reaction.

The application also describes how to detect 25-hydroxyvitamin D in an automated assay using the antibodies produced by the present invention.

In addition, a test kit is disclosed which can be used to detect 25-hydroxyvitamin D, including reagent compositions required in the test procedure and an anti-25-hydroxyvitamin D antibody included in the present invention.

Detailed Description

The present invention relates to a method for producing antibodies against 25-hydroxyvitamin D comprising the steps of:

a) will contain 25-hydroxy vitamin D₃Or 25-hydroxy vitamin D₂As a conjugate of a hapten, is immunized into an experimental animal,

b) separating the serum or plasma of the experimental animal, and

c) by immunoadsorption to a mixture of vitamin D compounds each containing 25-hydroxy vitamin D₂Or 25-hydroxy vitamin D₃Thereby purifying the antibodies in the serum or plasma.

The term "vitamin D" is understood to include vitamin D according to the following structural formulae I and II, unless otherwise stated₂And vitamin D₃Two forms.

Formula I

Formula II

The respective positions of vitamin D in structural formulae I and II are indicated according to steroid nomenclature. 25-hydroxyvitamin D represents a vitamin D metabolite having the C-25 position of the structural formulae I and II hydroxylated, i.e., 25-hydroxyvitamin D₂And 25-hydroxyvitamin D₃. As mentioned above, 25-hydroxyvitamin D₂And 25 hydroxy vitamin D₃Is a diagnostically significant form of vitamin D.

The active form of vitamin D, 1, 25-dihydroxyvitamin D (the so-called hormone D), is mentioned to be formed by hydroxylation of the C-1 and C-25 positions in the formulae I and II.

Another known vitamin D metabolite is 24-dihydroxyvitamin D₂25-dihydroxyvitamin D₂24-dihydroxyvitamin D₃And 25-dihydroxy vitamin D₃。

All known vitamin D metabolites are not able to generate an immune response by themselves. It is not an easy matter to chemically activate the vitamin D metabolic components and to bind them to carrier molecules or reporter groups. Thus, for successful immunisation it is a prerequisite that a conjugate is prepared, e.g. comprising 25-hydroxyvitamin D as hapten. The term hapten is herein understood by those skilled in the art as a substance that is not immunogenic in itself, but which, upon binding to a larger carrier molecule, exhibits antigenicity and can therefore be used to raise antibodies directed against it. Suitable carrier materials for the production of hapten conjugates are known to those skilled in the art. Is usually used asThe carrier material is bovine serum albumin, beta-galactosidase or so-called keyhole limpetHemocyanin (KLH).

KLH has proven particularly suitable as a vector in the methods provided by the present invention. Therefore, conjugates of 25-hydroxyvitamin D with KLH are preferred for immunization.

As shown in formulae I and II, each position of the structure is in principle suitable for activation and coupling to a support material. For example, 25-hydroxyvitamin D has been demonstrated₂Or 25-hydroxy vitamin D₃The 3-position coupling of (a) facilitates the production of antibodies that bind 25-hydroxyvitamin D in a suitable manner. Thus in a preferred embodiment, the conjugates for immunization according to the invention each comprise 25-hydroxyvitamin D coupled via the 3-position of the backbone₃Or 25-hydroxy vitamin D₂(compare formulas I and II).

In a series of experiments performed as part of the work of the present invention, an attempt was made to use 25-hydroxyvitamin D₃Immunoabsorption of immunogen into 25-hydroxy vitamin D₃The antibodies produced on the substrate are purified and used in the corresponding tests. Although these experiments were not successful, it was surprisingly found that 25-hydroxyvitamin D was adsorbed by immunoadsorption₂Suitable antibodies can be obtained from the same serum on the substrate. This method has proven to be reliable and reproducible. The method of the invention therefore comprises a step in which an antibody directed against 25-hydroxyvitamin Dx (where x is 2 or 3) is purified by immunoadsorption onto a matrix comprising conjugates of the respective complementary forms of 25-hydroxyvitamin D. In this sense, 25-hydroxyvitamin D₃With 25-hydroxyvitamin D₂Complementation, in turn 25-hydroxyvitamin D₂Also with 25-hydroxyvitamin D₃And (4) complementation. This indicates that when 25-hydroxyvitamin D is used₃When immunized, the mixture is mixed with 25-hydroxy vitamin D₂The immunoadsorption of (4); when 25-hydroxy vitamin D is used₂When immunized, the mixture is mixed with 25-hydroxy vitamin D₃The immunoadsorption of (1).

Furthermore, it has proved advantageous to use the same position on the vitamin D backbone for chemical coupling, whether in the 25-hydroxyvitamin D conjugate used for immunization or in the matrix used for immunoadsorption. 25-hydroxyvitamin D in immune₃The conjugate preferably binds to 25-hydroxyvitamin D₃At position 3 of (A), likewise, 25-hydroxyvitamin D₂Also preferably bound at the 3-position of the matrix.

The reverse procedure is also possible, i.e.using 25-hydroxyvitamin D₂Conjugate immunization using coupling of 25-hydroxyvitamin D₃The substrate is immunoadsorpted. In another preferred embodiment of the present invention, 25-hydroxyvitamin D₂The conjugates are used as immunogen conjugates for immunoabsorbing antibodies generated by such immunogens to 25-hydroxyvitamin D₃On a substrate.

EAH-Sepharose (Sepharose) proved to be particularly suitable as a matrix material for immunoadsorption. In a preferred embodiment, anti-25-hydroxyvitamin D is produced in serum or plasma by immunization₃Or 25-hydroxy vitamin D₂By using an antibody comprising 25-hydroxyvitamin D₂Or 25-hydroxy vitamin D₃The substrate is purified by immunoadsorption. EAH-Sepharose is a preferred column packing.

Using the detailed procedure described previously, i.e. using 25-hydroxyvitamin D₃Immunization with the conjugate with 25-hydroxyvitamin D₂Immunoadsorption of the conjugate makes it possible to repeatedly produce a conjugate capable of binding 25-hydroxyvitamin D₂And 25-hydroxyvitamin D₃An antibody reactive with both 25-hydroxyvitamin D forms. The cross-reactivity of the antibodies obtained in this way is of the order of 10% to 1000%. Thus in a preferred embodiment the invention relates to, for example, anti-25-hydroxyvitamin D₃With 25-hydroxyvitamin D₂10% to 1000%And (4) carrying out cross reaction. Preferably, there is a cross-reaction with the complementary 25-hydroxyvitamin D form in the range of 20% to 500%. The extent of cross-reactivity is determined in an immunological assay using the antibodies produced by the present invention. If the same analyte concentration of 25-hydroxyvitamin D is used₂Or 25-hydroxy vitamin D₃When 25-hydroxy vitamin D is used₃Only one tenth of the 25-hydroxyvitamin D was read on the calibration curve drawn₃Against 25-hydroxyvitamin D as hapten₃The produced antibody is to 25-hydroxy vitamin D₂The cross-reactivity of (a) is 10%.

The antibodies against 25-hydroxyvitamin D obtained by the method of the invention proved suitable for the automated testing of 25-hydroxyvitamin D. The invention therefore preferably relates to the use of an antibody against 25-hydroxyvitamin D in an immunological assay for the detection of 25-hydroxyvitamin D. The testing of 25-hydroxyvitamin D is preferably fully automated. The antibodies produced according to the invention are particularly preferably used in automated Elecsys, available from Roche diagnosticsIn one test conducted in an analyzer.

One skilled in the art will be able to combine all of the components required for 25-hydroxyvitamin D detection into a single test kit in accordance with the teachings of the present invention. A preferred 25-hydroxyvitamin D test kit is characterized in that the test kit contains anti-25-hydroxyvitamin D and recognizes two 25-hydroxyvitamin D forms of antibody, i.e., 10% to 1000% cross-react with each complementary form of 25-hydroxyvitamin D.

Preferably, the assay is performed as a competitive immunoassay, wherein antibodies against 25-hydroxyvitamin D produced by the present invention are preferably used as detection reagents. In this competitive assay, 25-hydroxyvitamin D "muramyl antigen" is added in an amount to compete with 25-hydroxyvitamin D in the sample for the binding site of the detection antibody. The more 25-hydroxyvitamin D in the sample, the less signal detected.

It has furthermore proved advantageous if the form of 25-hydroxyvitamin D, which is present in the competitive assay in the form of a muramyl antigen, corresponds to the form used for immunoadsorption. If one of the immunization reagents contains 25-hydroxyvitamin D₃The immunogen of (2) is in 25-hydroxy vitamin D₂Immunoadsorption on a substrate, and 25-hydroxyvitamin D₂The derivatives of (a) are preferably used in the test as mural antigens. The modification site of the mural antigen is preferably the same as the ring site of the immunogen and of the 25-hydroxyvitamin D used on the immunoadsorbent matrix.

Another preferred embodiment of the present invention relates to a method for the immunological detection of 25-hydroxyvitamin D, wherein the polyclonal antibody used in the method is obtained by immunization with a 25-hydroxyvitamin D conjugate and immunoabsorption on a complementary 25-hydroxyvitamin D conjugate, wherein in a competitive assay a 25-hydroxyvitamin D derivative complementary to the immunogen is used as the mural antigen.

The following examples and figures further illustrate the invention. The true scope of the invention is defined in the claims appended hereto.

Brief Description of Drawings

FIG. 1: 25-hydroxy vitamin D₃Scheme for synthesis of immunogens.

Vitamin D₃Activated via position 3 of the main chain of formula II and associated keyhole limpet as a carrierHemocyanin (KLH) coupling.

FIG. 2, 25-Hydroxyvitamin D₂Scheme for the synthesis of immunoadsorbent.

Vitamin D₂Activated via position 3 on the backbone of formula I and coupled to the matrix material EAH-Sepharose.

FIG. 3, biotinylated vitamin D₂Synthetic scheme (2).

Illustrating the availability of 25-hydroxyvitamin D as a mural antigen₂The synthesis step (2).

FIG. 4: prior art antibodies were used for immunoassays.

The content of 25-hydroxyvitamin D was determined by immunoassay and HPLC analysis of a total of 32 samples. The values of the immunoassay are indicated on the Y-axis and the values of the HPLC are indicated on the X-axis.

FIG. 5: comparison of High Performance Liquid Chromatography (HPLC) and LC-MS-MS.

The content of 25-hydroxyvitamin D was determined by analysis of a total of 66 samples by liquid chromatography tandem mass spectrometry LC-MS-MS and High Performance Liquid Chromatography (HPLC). The values for LC-MS-MS are indicated on the Y-axis and the values for HPLC on the X-axis.

FIG. 6: immunoassays with the antibodies of the present invention were compared to LC-MS-MS.

The content of 25-hydroxyvitamin D was determined by analysis of a total of 66 samples by HPLC method and immunoassay based on the antibody of the present invention. The values of the immunoassay are indicated on the Y-axis and the values of the HPLC are indicated on the X-axis.

Example 1

25-hydroxy vitamin D₃Synthesis of (E) -3-hemisuccinate-KLH

In this synthesis, 25-hydroxy vitamin D is chemically activated₃And coupled to KLH as the immunogen support. This synthesis is via 25-hydroxy vitamin D₃-3-hemisuccinate and 25-hydroxyvitamin D₃Intermediate steps of-3-hemisuccinate-N-hydroxysuccinimide ester, etc., are illustrated in FIG. one.

1.125-Hydroxyvitamin D₃Preparation of (E) -3-hemisuccinate

10mg (25. mu. mol) of 25-hydroxyvitamin D are taken₃(Sigma-Aldrich, No. H-4014) was dissolved in 1ml of anhydrous pyridine, which was stirred with 125mg (1.25mmol) of succinic anhydride at room temperature in the dark for 4 days. The reaction mixture is taken up in 10ml of ethyl acetate, washed with 2X 10ml of water, 0.1M hydrochloric acid and then again with water. The organic phase was dried over about 1g anhydrous sodium sulfate, filtered and the solvent removed in vacuo. The remaining solid was dried with high vacuum. 10.5mg (yield: 84%) of a colorless solid was obtained.

1.225-Hydroxyvitamin D₃Preparation of (E) -3-hemisuccinate-N-hydroxysuccinimide ester

10.0mg (20. mu. mol) of 25-hydroxyvitamin D are taken₃-3-hemisuccinate was dissolved in 7ml of anhydrous dichloromethane and mixed with 2.76mg (24. mu. mol) of N-hydroxysuccinimide and 3.72mg (24. mu. mol) of N (3-dimethylaminopropyl) -N' -ethyl-carbodiimide (EDC). Stirring was carried out overnight under argon, and the organic phase was washed twice with 10ml of water, dried over about 1g of anhydrous sodium sulfate and filtered. The solvent was removed in vacuo and the remaining reaction product was dried under high vacuum for 3 hours. 11.3mg (yield: 94%) of N-hydroxysuccinimide ester were obtained which did not require further purification and were useful for conjugation.

1.325-Hydroxyvitamin D₃Synthesis of (E) -3-hemisuccinate-KLH

Taking 150mg keyholeHemocyanin (KLH; Sigma-Aldrich No. H8283) was dissolved in 25ml of 0.1M potassium phosphate buffer pH 8.0, and 2ml of a DMSO solution containing 11.3mg of N-hydroxysuccinimide ester was added. The mixture was stirred at room temperature overnight, and then the product was purified by gel column method (AcA 202, column volume 0.5L; 0.1M potassium phosphate buffer pH 7.0). The conjugated protein-containing fractions were detected by uv absorption spectroscopy (λ ═ 256nm) and collected. Adding 10% glycerol and mixingThe milk-white solution is used for immunization.

Example 2

Production and isolation of anti-25-hydroxy vitamin D₃The antibody of (1).

2.1 immunization

Antibodies are produced in sheep. 25-hydroxy vitamin D obtained in example one₃-3-hemisuccinate-KLH conjugates for immunization. The immunization dose was 0.1mg per animal. The first immunization was performed in complete Freund's adjuvant. The subsequent immunization was carried out in incomplete Freund's adjuvant every 4 weeks for 10 months. Immune sera were collected in the middle of each immunization interval.

2.2 purification of polyclonal sheep antibodies

In Aerosil(1.5%) with the aid of 25-hydroxyvitamin D₃-3-hemisuccinate-KLH conjugate removes lipid-containing components from the immunized sheep serum. The immunoglobulins were then precipitated with ammonium sulfate (1.7M). The precipitate was dialyzed against 15mM potassium phosphate buffer (pH 7.0) containing 50mM NaCl and purified by chromatography on DEAE-agarose matrix. Obtaining IgG fraction (═ PAB) from column flow-through (flow-through)<25-hydroxy vitamin D₃>S-IgG(DE))。

2.3 purification of 25-hydroxyvitamin D specific antibodies by affinity chromatography.

A preparation of a conjugated 25-hydroxyvitamin D comprising as specificity determinant₂And can be used as an immunoadsorbent for purifying polyclonal antibodies by immunochromatography. This immunoadsorbent is obtained by the following steps:

a) hydroxy vitamin D₃Synthesis of (E) -3-2' -cyanoethyl ether

Under argon, one of the thermometers is arranged inIn a 25ml three-necked round-bottom flask, 20.6mg (50. mu. mol) of 25-hydroxyvitamin D was placed₂(Fluka No.17937) was dissolved in 10ml dry acetonitrile. 1.5ml of tert-butanol/acetonitrile (9: 1) were added to the solution and cooled to 6 ℃ in an ice bath. Then 820. mu.l of acrylonitrile solution (86. mu.l of acrylonitrile in 1.0ml of acetonitrile) was added and stirred for 15 minutes at 6 ℃. Then 205. mu.l of potassium hydride solution (25mg KH in 0.5ml t-butanol/acetonitrile (9: 1)) were added. A clear solution was obtained after a short flocculation. The reaction solution was stirred for a further 45 minutes at 6 ℃ and subsequently 60 minutes at 4 ℃.

Subsequently, the reaction solution was diluted with 10ml of methyl t-butyl ether and washed twice with 10ml of water. The organic phase was dried over about 1G of anhydrous sodium sulfate, filtered through a G3 glass filter and dewatered with a rotary dehydrator. Finally, drying under high vacuum resulted in a clear viscous residue with a mass of about 55 mg.

b) Hydroxy vitamin D₂Synthesis of (E) -3-3' -aminopropyl ether

All the nitriles obtained above were dissolved in 15ml of diethyl ether and mixed with a suspension of 7.5mg of lithium hydride in 7.5ml of diethyl ether with stirring. The reaction mixture was stirred at room temperature for one hour, then a suspension of 38.4mg of lithium aluminium hydride in 6.6ml of diethyl ether was added, giving a very turbid mixture. The reaction mixture was stirred at room temperature for an additional hour, then cooled to 0-5 ℃ in an ice bath and 35ml of water was added carefully. Finally 6.6ml of 10M potassium hydroxide solution are added to make the mixture strongly basic.

Extracted three times with 65ml of methyl tert-butyl ether each time and the combined organic phases are dried over about 5g of anhydrous sodium sulfate, filtered and dehydrated at room temperature with a rotary evaporator. The residue was dried with an oil pump until the mass was constant. The crude product was dissolved in 5ml DMSO and 3.0ml acetonitrile and purified by preparative HPLC.

Eluent a Millipore water + 0.1% trifluoroacetic acid;

eluent B95% acetonitrile + 5% Millipore water + 0.1% TFA;

gradient: from 50% B to 100% B in 100 minutes.

Flow rate: 30 ml/min

Temperature: at room temperature

Column size: the radius is 5.0 cm; the length is 25 cm;

column packing: vydac C18/300/15-20μm

Detection wavelength: 226nm

Analytical HPLC (Vydac C18/300)5 μm; 4.6X 250mm) the fractions with a product content higher than 85% were collected in a round bottom flask and lyophilized. Finally, 13.7mg (yield: 58%) of colorless lyophilized powder was obtained.

c) Synthetic hydroxy vitamin D₂-3-3' -N- (semi-cycloheptyl) aminopropyl ether-N-hydroxysuccinimide ester

11.7mg (25. mu. mol) of the amino derivative are dissolved in 5ml of freshly distilled DMF and 92mg (250. mu. mol) of N-hydroxysuccinimide suberate are added. 3.5. mu.l of triethylamine were added and stirred under argon overnight. The crude product was purified by preparative HPLC method (conditions described above) to obtain 10.1mg (yield: 56%) of N-hydroxysuccinimide ester after lyophilization.

d) Synthetic hydroxy vitamin D₂Immunoadsorbent

20ml of EAH agarose gel were washed with 200ml of 0.5M sodium chloride solution on a G3 glass filter (Amersham Biosciences, No. 17-0569-03). Equilibrated with 200ml of 0.03M potassium phosphate buffer (pH 7.1). After draining off excess liquid through a glass filter, the suspension was added to 200ml of the same buffer and 10ml of DMSO in which 1.7mg (2.3. mu. mol) of N-hydroxysuccinimide ester was dissolved was added. The reaction mixture was stirred with a mixer at room temperature overnight. The mixture was transferred again to a G3 glass filter and drained and washed with 500ml of 0.05M potassium phosphate buffer/0.15M sodium chloride (pH 7.0). After complete discharge, it was resuspended in 25ml of the same buffer and stored by adding 0.15ml of 25% strength sodium azide.

e) Purification of antibodies

10ml of the affinity matrix of d) was packed into a column and equilibrated with a buffer solution (pH 7.5) (PBS) containing 50mM potassium phosphate and 150mM NaCl. 3.6g of PAB<25-hydroxy vitamin D₃>S-IgG (DE) was loaded into the column. Separately in PBS buffer and containing 0.05% TweenThe cartridge was washed with 20 and 30mM sodium chloride in 0.5M NaCl solution. The specifically bound immunoglobulins were separated from the affinity matrix using 3mM HCl solution. The HCl eluate was dialyzed against 1mM ethyl acetate and subsequently lyophilized. Dissolving the lyophilized powder in PBS, and using Superdex 200The aggregates are removed by chromatography and the immunoabsorbed polyclonal antibody obtained by this method is used in the next step. The immunoaffinity matrix was regenerated with 1M propionic acid and stored in PBS solution containing 0.9% sodium azide.

Example 3

Assay for detecting 25-hydroxyvitamin D

Commercial assays were used according to the manufacturer's instructions. The literature describes the determination of vitamin D by HPLC (test 25(OH) vitamin D)₃The determination of 25-hydroxyvitamin D is carried out by the method of "Immunadiagnostik", Bensheim, order No. KC 3400) or by the LC-MS-MS method (Vogeser, M. et al, Clin. chem.50(2004) 1415-.

The general procedure for the preparation of the components and a new immunological test on the basis of the antibodies produced according to the invention is described below:

3.1 Synthesis of hydroxy vitamin D₂-3-3' -N- (semi-cycloheptyl) aminopropyl ether-biotin- (β -Ala) -Glu-Lys (e) conjugate (═ Ag-Bi)

13.7mg (25. mu. mol) of hydroxyvitamin D are added₂-3-3' -aminopropyl ether was dissolved in 3.5ml of DMSO solution, and 28.7mg (30. mu. mol) of biotin- (. beta. -Ala) -Glu-Glu-Lys (. epsilon. -suberic acid-N-hydroxysuccinimide ester (Rocheapplied Science, No.11866656) and 12.5. mu.l of triethylamine were mixed and stirred at room temperature overnight. The reaction solution was diluted with 4.5ml of DMSO solution and filtered off in a 0.45 μm microfiltration Millipore followed by purification by preparative HPLC (conditions see example 2.3 b). Fractions with a product content of over 85% as detected by analytical HPLC were collected and lyophilized. Finally, 9.8mg (yield: 30%) of the purified biotin conjugate was obtained.

3.2 purification of Ruthenized polyclonal antibodies against 25-hydroxyvitamin D (═ PAB-Ru) by affinity chromatography

The affinity-purified antibody was transferred to 100mM potassium phosphate buffer (pH 8.5) and the protein concentration was adjusted to 1mg/ml according to the method of example 2.3 e). The ruthenation reagent (ruthenium (II) tris (bipyridyl) -N-hydroxysuccinimide ester) was dissolved in DMSO and added to the antibody solution in a molar ratio of 7.5: 1. After a reaction time of 60 minutes, the reaction was stopped by addition of L-lysine and the excess labeled reagent was separated by gel permeation chromatography on Sephadex G25.

3.3 detection procedure of immunoassay.

Elecsys by Roche diagnosticsThe system measures the sample. Mu.l of the sample are mixed with 30. mu.l of the release reagent, and simultaneously or successively 15. mu.l of the ruthenium detection antibody are added and the mixture is warmedIncubate for 9 minutes. Next, biotinylated muramyl antigen (50. mu.l) was added and the pH was maintained within a certain desired range by adding a release agent (50. mu.l). After a further 9 minutes of incubation, Streptavidin (SA) (30. mu.l) coated magnetizable polystyrene particles were added and after a further 9 minutes of incubation the amount of bound ruthenated antibody was determined routinely.

The solution comprising the ruthenated < 25-OH-vitamin D > antibody conjugate contains:

20mM phosphate buffer, pH 6.5

0.1％ oxypyrion

0.1% MIT (N-methylisothiazolone-HCl)

10% DMSO (dimethyl sulfoxide)

1% EtOH (ethanol)

0.1% of surface anesthetic

1% Rabbit immunoglobulin G (DET)

2.0. mu.g/ml PAB-Ru (see example 3.2)

The release agent comprises:

220mM acetate buffer, pH 4.0

0.1％ oxypyrion

0.1％ MIT

10％ DMSO

1％ EtOH

0.1% of surface anesthetic

0.2% rabbit immunoglobulin G

The solution containing biotinylated mural antigens comprises:

20mM phosphate buffer, pH 6.5

0.1％ oxypyrion

10％ DMSO

1％ EtOH

0.1% of surface anesthetic

0.2% rabbit immunoglobulin G

0.18. mu.g/ml Ag-Bi (see example 3.1)

A suspension containing SA-coated latex particles comprises:

streptavidin (SA) -coated magnetizable polystyrene particles with a binding capacity of 470ng/ml, 0.72mg/ml

Example 4

Experimental results and discussion

4.1 use of 25-hydroxyvitamin D as immunogen and immunoadsorbent₃The obtained antibody

Antibodies used in a number of (failed) experiments were prepared using prior art methods, e.g.using 25-hydroxyvitamin D₃Carrying out immunization and immunoadsorption on the vaccine. An example of an antibody that is not suitable for reliable detection as 25-hydroxyvitamin D is shown in figure 4. The absence of correlation between the 25-hydroxyvitamin D values measured by the immunoassay with these antibodies and the reference method (HPLC) is clearly shown in FIG. 4.

4.2 comparative HPLC and LC-MS

The detection of metabolites of vitamin D by the LC-MS-MS method is increasingly used as a reference method for the determination of vitamin D metabolites, as described in Vogeser, M.et al, Clin. chem.50(2004) 1415-1417. Thus, it was investigated whether the results of the earlier HPLC reference method could be compared with the results of the newer LC-MS reference method. As can be seen from fig. 5, both reference methods are comparable. The correlation coefficient was determined to be 0.94 using linear regression.

4.3 use of antibodies against 25-hydroxyvitamin D produced according to the invention for immunoassays

A total of 66 samples were compared for 25-hydroxyvitamin D content using the new immunological test and LC-MS-MS method. As can be seen from fig. 6, the results obtained from the two assays are very relevant. The correlation coefficient obtained by linear regression was 0.85. The results are surprising, since the two analytical methods are based on completely different principles.

Thus, tests for the detection of 25-hydroxyvitamin D can be used to establish a reliable assay for 25-hydroxyvitamin D using antibodies produced according to the present invention.

Claims (9)

1. A method of producing an antibody against 25-hydroxyvitamin D comprising the steps of:

a) with vitamin D containing 25-hydroxy group₃Or 25-hydroxy vitamin D₂As a conjugate of a hapten, is immunized into an experimental animal,

b) separating the serum or plasma of the experimental animal, and

c) by immunoadsorption to a mixture of vitamin D compounds each containing 25-hydroxy vitamin D₂Or 25-hydroxy vitamin D₃On a complementary substrate, thereby binding the antibodies in the serum or plasmaAnd (4) purifying.

2. The method of claim 1, characterized in that 25-hydroxyvitamin D₃The bond of the conjugate is through 25-hydroxy vitamin D₃And 3 bits of (1).

3. The method of claim 1, characterized in that 25-hydroxyvitamin D₂The bond of the conjugate is through 25-hydroxy vitamin D₂And 3 bits of (1).

4. The method of claim 2, characterized in that 25-hydroxyvitamin D₂By 25-hydroxy vitamin D₂And the 3 position of (b) is linked to a substrate used in immunoadsorption.

5. The method of claim 3, characterized in that 25-hydroxyvitamin D₃By 25-hydroxy vitamin D₃And the 3 position of (b) is linked to a substrate used in immunoadsorption.

6. Anti-25-hydroxy vitamin D₃The antibody of (1), the antibody being conjugated with 25-hydroxyvitamin D₂There is a cross-reaction of 10% to 1000%.

7. Anti-25-hydroxy vitamin D₃The antibody of (1), the antibody being conjugated with 25-hydroxyvitamin D₂There is a cross-reaction of 20% to 500%.

8. Use of an antibody according to claim 6 or 7 in the determination of 25-hydroxyvitamin D₃The use in a test of (1).

9. Method for measuring 25-hydroxy vitamin D₃Characterized in that the test kit comprises the antibody of claim 6 or 7.