BACKGROUND INFORMATION

The present invention concerns an in vitro method for measurement of 25-hydroxyvitamin D, wherein the potentially interfering compound 24,25-dihydroxyvitamin D3is blocked by a binding agent specifically binding to 24,25-dihydroxyvitamin D3and not binding to 25-hydroxyvitamin D.

An adequate supply of vitamin D is vital as the term “vitamin” already suggests. A deficiency of vitamin D leads to severe diseases such as rickets or osteoporosis. While vitamin D was still regarded as a single substance at the beginning of the last century, the vitamin D system has changed in the course of the last decades into a complex and manifold network of vitamin D metabolites. Nowadays more than 40 different vitamin D metabolic products are known (Zerwekh, J. E., Ann. Clin. Biochem. 41 (2004) 272-281).

Humans can only produce D3vitamins or calciferols by the action of ultraviolet rays from sunlight on the skin. In the blood Vitamin D3is bound to the so-called vitamin D-binding protein and transported to the liver where it is converted into 25-hydroxyvitamin D3by 25-hydroxylation. A multitude of other tissues are nowadays known to be involved in vitamin D metabolism in addition to the skin and liver, the two organs that have already been mentioned (Schmidt-Gayk, H. et al. (eds.), “Calcium regulating hormones, vitamin D metabolites and cyclic AMP”, Springer Verlag, Heidelberg (1990) pp. 24-47). 25-hydroxyvitamin D and more specifically 25-hydroxyvitamin D2and 25-hydroxyvitamin D3are the central storage form of vitamin D in the human organism with regard to their amounts. When needed these precursors can be converted in the kidneys to form the biologically active 1α,25-dihydroxyvitamin D the so-called D hormone. The biologically active vitamin D regulates among others calcium uptake from the intestine, bone mineralization and it influences a large number of other metabolic pathways such as e.g. the insulin system.

Measuring the vitamin D level itself is of little benefit when determining the vitamin D status of a subject or patient, because concentrations of vitamin D (vitamin D2and vitamin D3) fluctuate greatly depending on food uptake or exposure to sunlight. In addition vitamin D has a relatively short biological half-life in the circulation (24 hours) and it is therefore also for this reason not a suitable parameter for determining the vitamin D status of a patient. The same also applies to physiologically active forms of vitamin D (1,25-dihydroxyvitamin D). These biologically active forms also occur in relatively small and highly fluctuating concentrations compared to 25-hydroxyvitamin D. For all these reasons the quantification of 25-hydroxyvitamin D in particular is a suitable means to globally analyse the total vitamin D status of a subject or patient.

Vitamin D metabolites like 25-hydroxyvitamin D are bound with high affinity by vitamin D-binding protein and to a limited extend also to albumin and some lipoproteins. Methods appropriate to release a vitamin D metabolite from vitamin D-binding protein will under normal circumstances also be more than appropriate to release a vitamin D metabolite also from any other protein.

The binding of 25-hydroxyvitamin D or other vitamin D compounds to the vitamin D-binding protein enormously complicates the determination of vitamin D compounds. All known methods require that the vitamin D compound to be analysed is released or detached from the complex that it forms with the vitamin D-binding protein. In the following this is referred to as the release of a vitamin D compound from vitamin D-binding protein for the sake of simplification although of course it can only be released from a complex of vitamin D compound and vitamin D-binding protein and not from the vitamin D-binding protein alone.

The vitamin D-binding protein is unfolded at acidic pH, but has a high tendency to correctly refold and to re-bind the analyte when the pH is shifted back to neutral conditions. Hence, it is often necessary to firstly release vitamin D compounds from vitamin D-binding protein and then to separate the vitamin D-binding protein from the vitamin D compounds to be analysed.

Due to the high clinical importance of 25-hydroxyvitamin D a large number of methods are known from the literature which allow 25-hydroxyvitamin D to be more or less reliably determined.

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 for example describe the determination of 25-hydroxyvitamin D concentrations in blood samples using high performance liquid chromatography (HPLC).

Other approaches for the determination of 25-hydroxyvitamin D are based among others on the use of vitamin D-binding proteins like those that are present in milk. Thus Holick, M. F. and Ray, R. (U.S. Pat. No. 5,981,779) and DeLuca et al. (EP 0 583 945) describe vitamin D assays for hydroxyvitamin D and dihydroxyvitamin D which are based on the binding of these substances to vitamin D-binding protein where the concentrations of these substances are determined by means of a competitive test procedure. However, a prerequisite of this method is that vitamin D metabolites to be determined firstly have to be isolated from the original blood or serum samples and have to be purified by, for example, chromatography.

Armbruster, F. P. et al. (WO 99/67211) teach that a serum or plasma sample should be prepared for vitamin D determination by ethanol precipitation. In this method the protein precipitate is removed by centrifugation and the ethanolic supernatant contains soluble vitamin D metabolites. These can be measured in a competitive binding assay.

Alternatively EP 0 753 743 teaches that the proteins can be separated from blood or serum samples using a periodate salt. In this case vitamin D compounds are determined in the protein-free supernatant from the samples treated with periodate. In some commercial tests acetonitrile is recommended for the extraction of serum or plasma sample (e.g. in the radioimmunoassay from DiaSorin or in the vitamin D test from the “Immundiagnostik” Company).

In recent years a number of different release reagents were proposed which should in principle be suitable for releasing vitamin D compounds from any binding protein present in the sample. However, this release or detachment should be carried out under relatively mild conditions thus enabling a direct use of the sample treated with the release reagent in a binding test (see for example WO 02/57797 and US 2004/0132104). Despite immense efforts in recent years, all available methods for determining vitamin D have disadvantages such as laborious sample preparation, poor standardization, poor agreement between test procedures or bad recovery of spiked vitamin D (see for this in particular Zerwekh, J. E., supra).

In U.S. Pat. No. 7,087,395 metal hydroxides as well as cyclodextrin and derivatives thereof, and metal salicylates have been used to release vitamin D compounds from vitamin D-binding protein, which result in an irreversible denaturation of vitamin D-binding protein or other serum proteins. Surfactants like Triton X100 or Tween-20 have been used to prevent the vitamin D compound from being non-specifically attached to lipids and proteins in the sample after denaturation.

It is particularly difficult to automate a test for a vitamin D compound. The automation requires solving a very difficult problem i.e. surviving a tightrope walk: On the one hand it is necessary to release the vitamin D compounds from vitamin D-binding protein with the aid of a suitable release reagent, on the other hand, the conditions have to be selected such that the sample can be directly analysed further. A prerequisite of this direct further analysis is that, on the one hand, the endogenous vitamin D-binding protein does not bind or no longer to a significant extent binds to the vitamin D compounds during this analysis and thus does not interfere with this analysis and, on the other hand, that the release reagent used does not interfere with the binding of detection reagents such as antibodies, or vitamin D-binding protein. In addition it is known that different alleles of the vitamin D-binding protein are present in the human population which behave biochemically differently. The release and measurement of vitamin D compounds should be comparable for various alleles/phenotypes.

Recently (WO2011/144661) a vitamin D assays that uses an appropriate sample treatment, can be performed online and without precipitation/separation of any vitamin D-binding protein that may be present in the sample. This method is based on the use of vitamin D releasing reagent containing a hydrogen carbonate salt and/or a substance capable of releasing hydrogen carbonate ions (HCO3−) upon hydrolysis in a total concentration of 0.1 M to 2.0 M, a reducing agent, and an alkalinising agent. By using this releasing reagent any vitamin D compound is released from vitamin D-binding protein, while at the same time vitamin D-binding protein comprised in the sample is inactivated and no longer binding to vitamin D. The released vitamin D compound can be measured by appropriate means.

In biological samples many vitamin D related compounds that are structurally closely related to each other are present. 24,25-dihydroxyvitamin D3is present in quite significant quantities in almost all biological samples and does interfere with the measurement of 25-hydroyvitamin D. Its concentration depends on the total amount of 25-hydroxyvitamin D in a sample as well as on the clinical background of an individual leading to variations between patient cohorts with different medicinal background. It could be shown that it is possible to avoid the interference caused by 24,25-dihydroxyvitamin D3by incubating a sample with a binding agent that binds 24,25-dihydroxyvitamin D3and does not bind to 25-hydroxyvitamin D.

SUMMARY OF THE INVENTION

In one embodiment the present invention concerns an in vitro method for determining the concentration of 25-hydroxyvitamin D without interference by 24,25-dihydroxyvitamin D3, the method comprising the steps of:a) providing a sample obtained from a subject,b) mixing the sample(ba) with a first binding agent binding to 24,25-dihydroxyvitamin D3, thereby forming a complex between the first binding agent and 24,25-dihydroxyvitamin D3;(bb) with a second binding agent binding to 25-hydroxyvitamin D, thereby forming a complex between the second binding agent and 25-hydroxyvitamin D;c) measuring the complex formed in (bb), thereby determining the concentration of 25-hydroxyvitamin D without interference by 24,25-dihydroxyvitamin D3.

In a further embodiment the present invention concerns to the use of the method(s) according to the present invention for determining the concentration of 25-hydroxyvitamin D without interference by 24,25-dihydroxyvitamin D3.

The present invention further is related in an embodiment to the use of a first binding agent binding to 24,25-dihydroxyvitamin D3and a second binding agent binding to 25-hydroxyvitamin D in in vitro method(s) according to the present invention for determining the concentration of 25-hydroxyvitamin D without interference by 24,25-dihydroxyvitamin D3in a sample obtained from a subject.

According to a further embodiment the present invention is related to a kit to perform the method(s) according to the present invention comprising at leasta) a first binding agent binding to 24,25-dihydroxyvitamin D3, wherein the first binding agent is a monoclonal antibody or a functionally active part of the monoclonal antibody, andb) a second binding agent binding to 25-hydroxyvitamin D, wherein the second binding agent is a vitamin D-binding protein.

