METHOD OF MEASURING THE ENDOCYTIC VITAMIN D STATUS

FIELD OF THE INVENTION

[001] The present application relates to assays involving biological materials of a specific nature and in particular to an assay for measuring the endocytable vitamin D concentration in a biological sample of a subject, notably blood or serum, and for detection or diagnosis of diseases or conditions related to vitamin D status in blood or serum (G01 N 2333/00, G01 N2800/00)

BACKGROUND OF THE INVENTION

[002] The metabolic pathway leading to the synthesis of active vitamin D involves three reactions that occur in different tissues. In humans the synthesis is initiated in the skin with a UV light-mediated cleavage to produce cholecalciferol (vitamin D _3, VD ₃ ). The other vitamin D isomer“ergocalciferol” (vitamin D ₂ , VD ₂ ) occurs in plants and is taken up with the food. Both vitamin D isomers are metabolized in the liver to 25-hydroxyvitamin D [25(OH)D] which is also the major circulatory form (prohormone). This second step is catalyzed by a cytochrome P450 enzyme, a NAPH-hemoprotein reductase, while the identity of the hepatic 25- hydroxylase still require more elucidation. The 25(OH)D then becomes l ohydroxylated in the kidney to 1 a,25-dihydroxyvitamin D or calcitriol which is the physiologically active form (D- hormone). Calcitriol regulates the absorption of calcium in the intestines, the mineralization of the bones, the differentiation of osteoblasts, the synthesis of bone matrix and neuromuscular functions. It is common medical knowledge that 25(OH)D levels in serum lower than 15 ng per mL serum (37.5 nmol/L) cause a rise of the parathyroid hormone level and leads to an increase in bone resorption (Chapuy MC et al. in J Clin Endocrinol Metab 1996; 81 :1 129-33). A vitamin D deficiency may be caused by gastro-intestinal diseases, liver dysfunction, malabsorption drug- induced heightened metabolism, genetic defects or insufficient exposure to sunlight. Vitamin D deficiency is a known risk factor for senile osteoporosis. Generally, a deficiency of less than 5 ng 25(OH)D per mL serum (12.5 nmol/L) is regarded severe, causing rickets in children and osteomalacia in adults (Scharla et al. Exp Clin Endocrinol. Diabetes, 1996, 104:289-292). Excess of vitamin D due to overdosing causes hypercalcemia. It is not clear whether there is a definitive difference between the effects of VD ₂ and VD ₃ or whether they are equally effective in the raising of serum 25(OH)D, particularly at lower doses of vitamin D (cf: Tripkovic L et al, Am J Clin Nutr. 2012 Jun; 95(6): 1357-1364; Am J Clin Nutr. 2017 Aug;106(2):481-490).

[003] Vitamin D binding protein (DBP) is a member of the albuminoid superfamily. The 56-58 kDa glucoprotein can bind vitamin D metabolites as well as fatty acids and other endotoxins. It is thought that DBP acts as a reservoir in situations of deficiency, increasing the half-life of vitamin D, but also protects against vitamin D intoxication. The concentration of DBP in blood is stably maintained within a relatively narrow range (323-460 mg/L (5.52-7.93 mol/L)) in normal subjects, even in pathological conditions and disorders of the calcium metabolism. Except in pregnancy, no high DBP concentrations have been observed. DBP in serum can be measured by numerous methods, including immunoturbidimetry (Hamashima et al, Clinica Chimica Acta 321 (2002) 23-28). DBP is known to produce chemokinetic effects on neutrophil granulocytes, activate macrophages and sequester actin upon tissue damage.

[004] The DBP level in serum is about 20-fold higher than of vitamin D and therefore 2 to 5% only will be occupied by vitamin D metabolites. For measurement, the vitamin D metabolites are released from DBP by enzymatic digestion, denaturing and/or ligand displacement (EP 2 126 586 B1 , WO 99/6721 1 ; EP 0 753 743, WO 2004/063704). The releasing agents include all types of detergents and surfactants (EP 2 955 516 B1 ) as well as structural analogs of vitamin D such as warfarin, salicylic compounds, certain sulfonic acids, toluene sulfonic acids, naphthalene sulfonic acid, anilinonaphthalene sulfonic acids, etc (WO 03/023391 ). The release of vitamin D from DBP is a decisive step in most laboratory procedures, in particular as the vitamin D metabolites are hydrophobic and cholesterol-like, bound by numerous serum proteins, and so their quantitative determination is technically difficult and results open to interpretation. The confusion as to the vitamin D status gave rise to collaborative initiative led by the Office of Dietary Supplements of the U.S. National institutes of Health and there have recently been proposals for assessing the vitamin D status via a determination of the“free 25(OH)D” in serum, say the 25(OH)D not bound by DBP or albumin (for review: The Importance of 25-Hydroxyvitamin D Assay Standardization and the Vitamin D Standardization Program. J AOAC Int. 2017, 100(5): 1223-1224; and Malmstroem S et al, J AOAC Int. 2017, 100(5): 1323-7).

[005] Calcium and phosphorus are essential minerals required for many critical biologic functions including cell signaling, energy metabolism, skeletal growth and integrity. Calcium and phosphate homeostasis are maintained primarily by regulation of epithelial calcium and phosphate cotransport in the kidney and intestine, processes that are tightly regulated by hormones including calcitriol, fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH). In patients with chronic kidney disease (CKD), as renal function declines, disruption of feedback loops between these hormones have adverse consequences on several organ systems, including the skeleton, heart and vascular system. Complications include vascular calcification, stroke, skeletal fracture and increased risk of death. Increased FGF23 and PTH concentrations, and vitamin D deficiency contributes to the pathogenesis. Therefore, treatment of patients is focused on restoring the feedback loops to maintain normal calcium and phosphate balance to prevent skeletal and cardiovascular complications. Recent evidence is further linking the vitamin D status to disorders such as cancer, diabetes, depression, etc. leading to a higher demand of routine testing for vitamin D levels even in healthy patients. The importance of the vitamin D status in research and health cannot therefore be overstated and clinical laboratories are confronted with the challenge of increasing test numbers and the need to identify persons truly suffering from a low vitamin D status. The state of the art represents a problem.

SUMMARY OF THE INVENTION

[006] The application provides an assay and a method of measuring the effective vitamin D level (including the vitamin D metabolites of the storage form) in a sample of bodily fluid in the presence of vitamin D binding protein (DBP), comprising the steps of a) contacting said sample with megalin/LRP2 ( low-density lipoprotein related protein 2) and/or a soluble fragment thereof under binding conditions to form a complex containing DBP, vitamin D or a metabolite thereof and megalin/LRP2 or a fragment thereof; b) determining the amount of DBP:vitamin D and/or any one of its components; and c) relating the amount of megalin-bound complex of DBP:vitamin D to the effective vitamin D status in said subject.

[007] In a preferred embodiment, the assay and method comprise the use of a fragment of megalin which binds none of the other ligands of megalin/ LRP2, and/or a fusion protein with said fragment of megalin which can bind the DBP:VD complex. A preferred embodiment of said method comprises a differential measurement of hydroxylated chole- calciferol (25-hydroxyvitamin D ₃ ) in the presence of hydroxylated ergocalciferol (25- hydroxyvitamin D ₂ ), 24,25-dihydroxyvitamin D ₂ , and/or 24,25-dihydroxyvitamin D ₃ .

[008] The disclosure encompasses the use of the endogenous DBP present in serum but may include the addition of DBP to obtain a standard concentration of DBP in the test samples. The disclosure may further encompass contacting additionally said sample with cubilin and/or a soluble fragment thereof as cubilin is known to facilitate the endocytic process (Nykjaer A, et al Cubilin dysfunction causes abnormal metabolism of the steroid hormone 25(OH) vitamin D ₃ . PNAS U.S.A. (2001 ) 98(24): 13895-900 [PUBMED:1 1717447]

[009] In one embodiment, the amount of DBP:VD bound by megalin is determined by turbidimetry or nephelometry or, notably, by an immunoassay for DBP. In another embodiment, the megalin and/or said soluble fragment thereof may be bound to particles or beads having diameters ranging from 50 to 200 nm. Alternatively, the disclosure teaches for the sake of completion an immunoassay selected from the group ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), FIA (fluorescence immunoassay), LIA (luminescence immunoassay), or ILMA.

[0010] In another embodiment the method may comprise the steps of (a) providing a defined amount of megalin and/or a fragment thereof coupled to a solid phase; (b) contacting the sample with a solid phase; (c) creating conditions to allow binding of megalin and/or a fragment thereof to the complex formed between DBP and vitamin D metabolite, wherein DBP alone is not bound by megalin and/or a fragment thereof; and washing the solid phase; (e) providing an antibody recognizing the complex of DBP:VD or, facultatively, megalin or a soluble fragment thereof; (f) contacting said megalin and/or fragment thereof bound to DBP:VD with an anti-DBP-antibody, and optionally, immobilizing the complex on the solid phase; and (g) determining the amount of antibody bound to the solid phase, and quantitating the vitamin D status in plasma or serum by correlation with a reference.

[0011] The disclosure also provides a test kit which comprises an antibody specific for DBP and a binding partner comprising megalin or a fragment thereof. The antibody may be one binding to DBP or vitamin D metabolite in said complex. In another embodiment, the test kit may comprise nanoparticles having bound megalin and/or soluble fragments thereof.

[0012] The stated object is achieved because the disclosure provides a method for direct quantitative determination of the effective vitamin D status in serum or plasma. The status is based on the major circulating vitamin D metabolite which is ready for endocytosis and internalization into cells of the kidney, or into cells having an endocytic megalin transport pathway, so that the prohormone (25(OH)D) will be I a-hydroxylated to the physiologically active D-hormone (calcitriol). As ergocalciferol and cholecalciferol are both bound by DBP but the complex of DBP:VD ₃ is predominantly bound by megalin, the disclosed method provides a status of prohormone capable of becoming the active hormone. Vitamin D metabolites such as 24, 25(dihydroxy)vitamin-D _3, while they can still form a complex with DBP, are either much less bound by megalin or their concentration is too low in the circulation to impact the determined effective vitamin D status.

[0013] The DBP:VD3 complex only is endocytosed by the megalin cubulin pathway into cells where the P450 l a-hydroxylase (Cyp27B1 ) is located on the outside of the mitochondrial membrane. Consequently, other vitamin D metabolites will either not become activated or their concentration in the circulation is too low for being relevant with respect to the measured vitamin D status. This applies in particular to 25-hydroxylated ergocalciferol [25(OH)D ₂ ] which complex with DBP is not or much less bound by megalin (see Fig. 8B). The present disclosure therefore contradicts the free hormone hypothesis according to which only the non-protein- bound fraction (the free fraction) of vitamin D metabolites can enter cells and exert biologic effects.

[0014] The determination of the effective vitamin D status can be done in aqueous solution despite of the lipophilic nature of the vitamin D metabolites. There is no need for releasing the vitamin D metabolites from their binding partners (DBP, albumin, proteins of the albuminoid superfamily, etc.) as the method is based on the measurement of the complex of DBP:VD which will be bound or discriminated by megalin or a soluble fragment thereof. A reliable discrimination of vitamin binding protein having bound vitamin D is provided. Megalin or fragments thereof are used to discern the vitamin D status based on the endocytic DBP:VD complex which we submit represents the solely bioactivatable prohormone in the circulation.

[0015] Soluble megalin can be produced in mammalian cells by recombinant methods and purified by affinity chromatography. Megalin fragments soluble in aqueous solution (serum, plasma) are preferred as they allow conditions for the formation of DBP:VD complex close to physiological. [0016] As disclosed, there is no need to release vitamin D from its binding partners as by prior art methods. Thus, there is no need for halogenated solvents, tensids and surfactants (PFOA, CTAB) which makes the method environmentally friendly. The lack of preanalytical purification and preparation steps further allows a direct determination of the effective vitamin D status immediately after sample collection. The concept of determining the formation of said megalin-bound complex can easily be adapted to platforms such as ELISA, turbidimetry and nephelometry. The proteinaceous components of said complex can be reliably quantified.

[0017] The present disclosure is further in conformity with clinical reports that 25- hydroxyvitamin D3 more effective than 25-hydroxyvitamin D ₂ . The disclosure provides an effective status on basis of the endocytable prohormone which will be available for activation. While the 25-hydroxylated vitamin D ₂ and D3 isomers are equally bound by the DBP the provided method makes use of the different binding affinity of megalin to DBP when in interaction with 25-hydroxyvitamin D ₃ . The different binding affinity is striking. It is submitted that a fundamental biological mechanism has been discovered which physiological relevance cannot be overlooked. The present method allows therefore a discrimination of the activatable circulatory vitamin D metabolites and therefore provides a status of the readily activatable vitamin D metabolite (prohormone). With the information provided, vitamin D supplementation therapies can be adapted to the true physiological status of the subject, avoiding toxicity or inefficient therapies.

[0018] The principles of invention will now be described by reference to its advantages, representative examples and drawings which shall, however, not limit the gist of the invention which can be derived from the disclosure contained in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In the accompanying drawings,

Figs. 1A-C are schematic representations showing (A) potential binding sites of human

DBP (dimer) to megalin as predicted by PepSite 2 (Trabuco LG et al, PepSite: prediction of peptide-binding sites from protein surfaces in Nucleic Acids Res. 2012; 40(Web Server issue):W423-426); (B) the interaction between megalin/LRP2, cubulin and their known ligands on the outside of the luminal plasma membrane; and (C) the pathways from vitamin D via the prohormone (25(OH)D to the active hormone (1 ,25(OH) ₂ D - calcitriol) as well as excretion pathways;

Figs. 2A-C are schematic representations of (A) chosen megalin fragments tested for binding of vitamin D binding protein: Cons M1 : M1-K386 (signal peptide + A1-7 + EGF-like ½); M2: (M1-25 signal peptide + E1024-K1429); M3: (M1-25 signal peptide + E2698-R3192; M4: (M1-25 signal peptide + P3510-A4048 - not further examined); (B) the cDNA constructs of megalin fragments (M1 , M2, and M3) cloned into expression vector pcDNA3 (Invitrogen, San Diego, CA); and (C) Western blots of secreted megalin fragments (M1-M3 dimers) and M2 monomer: HEK cell lysates and after Ni-NTA affinity purification from culture supernatants;

Figs. 3A,B show Western blots of (A) megalin fragments from transfected HEK cell lysates

(M1 , M2, M3 + control/Non; ~20 pg) and (B) co-immunoprecipitations of the megalin-bound (M1 , M2, M3) DBP (0,5 pg) in the presence or absence of VD3 (0,2 pg) using anti-His-antibody (WB staining: anti-DBP Ab);

Figs. 4A,B show Western blots (A) of Ni-NTA purified megalin fragments (M1 , M2 and M3 +

Anti-lgG AB as control; ~3 pg) and (B) co-immunoprecipitations of the complex of megalin bound DBP (2 pg) in the presence or absence of VD3 (0,5 pg) using an anti-His-antibody (WB staining: anti-DBP Ab);

Figs. 5A,B show (A) microscale thermophoresis analyses of megalin fragments M1 , M2 and

M3 and DBP (50 nM) in the presence or absence of VD3 (50nM); and (B) the effect of the VD3 concentration on the affinity analysis;

Figs. 6A,B show Western blots (A) of co-immunoprecipitated DBP from different human sera or plasma using purified megalin fragment M1 (3 pg); and a bar diagram (B) showing the results of an ELISA for total DBP and formed DBP/VD3 bound by surface-coated megalin fragment (M1 or M2) ;

Figs. 7A-C are graphs (A) showing the effect of added purified DBP on the formation of M2- megalin-bound DBP:25(OH)D ₃ in a sample of human serum of a subject (RMS); and (B) the correlation between 25(OH)D ₃ serum levels and the binding of DBP:25(OH)D ₃ to megalin M2 fragment in human serum; and (C) the correlation between 25(OH)D ₃ level and ternary complex DBP/25(OH)D ₃ /M2 and the same for the ternary complexes with 25(OH)D ₂ and 24,25(OH)VD, respectively;

Fig. 8A,B show (A) microscale thermophoresis analyses of DBP and various 25(OH)D metabolites: (25(OH)D ₃ , 25(OH)D ₂ , 24,25(OH)D; and (B) microscale thermophoresis analyses of DBP:VD-metabolite when bound by megalin M2 fragment; Fig. 9A,B are graphs showing the linearity range of megalin-bound DBP:25(OH)D ₃ and in the presence of varying concentrations of 24,25(OH)VD and 25(OH)VD ₂ at high concentrations (up to 50 ng/mL)

Fig. 10A,B are graphs showing the linearity range of megalin-bound DBP:25(OH)D ₃ and in the presence of varying concentrations of 24,25(OH)VD and 25(OH)VD ₂ for low concentrations (up to 19 ng/mL).

DETAILED DESCRIPTION OF THE INVENTION

[0020] The instant description provides a method of determining the amount of biologically effective vitamin D in a sample of bodily fluid from a subject. The method comprises the steps of contacting said sample with DBP and megalin or a functional fragment thereof under binding conditions to form a complex of DBP:vitamin D (or a metabolite of vitamin D) which will be specifically bound by megalin, or a functional fragment thereof, to form a ternary complex comprising DBP:vitamin D:megalin. The amount of formed ternary complex in said sample of bodily fluid can then be correlated to the biologically effective amount of vitamin D in said sample of bodily fluid.

[0021] In this context, the term“biologically effective vitamin D” or“effective vitamin D” defines and comprises all structural vitamin D molecules which form a complex with DBP that is recognized and bound by megalin or a functional fragment thereof to form a ternary complex. Those structural vitamin D molecules therefore comprise not only vitamin D (chole- and ergocalciferol) but also the 25-hydroxylated vitamin D metabolites, including the respective epimers. For many years, emphasis has been on measuring total levels of 25-hydroxyvitamin D [25(OH)D] As the measured values were not consistent with physiologies there has recently been hypothesized that“free 25(OH)D” is a potentially better marker of the vitamin D status. The proposed assessment of“free 25(OH)D” however relies on calculations using levels of total 25(OH)D, albumin, and DBP and on the assumption of a constant affinity of DBP for the vitamin D metabolites. This hypothesis works with the assumption that the lipophilic vitamin D metabolites can passively diffuse through cell membranes and that serum DBP and albumin would decrease the readily available amount of vitamin D and the vitamin D status. The determinataion of the“free DBP-unbound fraction of 25(OH)D” shall give the“actual free and available vitamin D status”.

[0022] The vitamin D status is not only essential for normal kidney function and bone health but can also be linked to cancer development and some autoimmune diseases. Given the impact of the vitamin D status on human health, reliable methods are required for clinical practice.

[0023] The concentration of total “25-hydroxyvitamin D” in serum is mostly used in clinical practice but the discussion on the amount or proportion of“free or available” or hidden 25-hydroxy vitamin D” cannot be ignored. However, this hypothesis does not consider that epithelial cells and cells of other tissues (renal tubules, parathyroid gland, placenta etc.) have an endocytic pathways which enable the endocytic internalization of DBP-bound vitamin D metabolites. 25(OH)D is the key form of the prohormone for uptake and innercellular conversion to calcitriol. The vitamin D metabolites are mainly transported and bound in the circulation by the vitamin D binding protein. With prior art measurement methods, the amount of vitamin D metabolites that can become activated is not known and even less known is the amount of prohormone available for endocytic internalization and I a-hydroxylation to the active D-- hormone.

[0024] The present method makes use of that a ternary complex of vitamin D, DBP and megalin must form before the vitamin D metabolite can become internalized by endocytosis, either directly or following interaction with cubilin (cf. Fig. 1A-C). It is well established that vitamin D (cholecalciferol or ergocalciferol) is first hydroxylated in its 25-position to the prohormone in the liver and then present in the circulation. It also well established that the active D-hormone [1 ,25(OH) ₂ D] is synthesized by the enzyme l a-hydroxylase (Cyp27B1 - cytochrome P450) which is located within cells on the outside of the mitochondrial membrane (cf Fig. 1C). The 25-hydroxylated vitamin D metabolite must be first transported from the liver to the respective cells in the kidney. The kidney is the major source of calcitriol (1 ,25(OH) ₂ D) but also extrarenal cells, including lymphocytes, macrophages, keratinocytes, and cells of the parathyroid gland and pancreas can generate calcitriol.

[0025] It is known that 25-hydroxyvitamin D3 is 300% more effective than 25- hydroxyvitamin D2 _. In line therewith, the present disclosure provides a discrimination of endocytable DBP, say DBP having bound a vitamin D3 metabolite. Megalin or fragments thereof are used to discern the physiologically activatable vitamin D3 metabolite. Recombinant megalin fragments can be produced in mammalian cell lines and purified by affinity chromatography. A direct and fast determination of a vitamin D status is provided and there is no need for any additional pre-treatment or sample preparation. The assay ^' s time-scale is, thus, reduced while providing physiological accurate and valid readings. The disclosed approach can easily be adapted to different platforms such as ELISA, turbidimetry and nephelometry.

[0026] . The novel vitamin D status corresponds to the physiologically activated, endocytable vitamin D, in particular, to the 25-hydroxyvitamin D3 in serum or plasma. The concentrations of the other endocytable vitamin D metabolites are much lower. While the 25- hydroxylated vitamin D2 and D3 isomers are equally bound by DBP (Fig. 8A), the higher binding affinity of megalin to DBP:25(OH)VD ₃ compared to DBP:25(OH)VD ₂ and 24, 25-hydroxyvitamin D is striking. A special role seems to be taken by the C3-epimer of 25-hydroxyvitamin D3 [3-epi- 25(OH)D ₃ ], for which there is a near-total lack of data regarding its clinical significance. Although little is known regarding the in vivo importance of 3-epi-25(OH)D ₃ , clinical laboratories face the decision of whether or not to include 3-epi-25(OH)D ₃ in the measurement of the vitamin D status. The data in Figs. 8A,B show that the described method can adequately detect and resolve the C-3 epimeric form of 25(OH)D ₃ . Therefore, the present method allows a distinction for determining the most potent vitamin D metabolites and, in turn, provides a more accurate and physiologically valid vitamin D status which does not include the less active forms. With the information provided by the present disclosure, vitamin D supplementation therapies can therefore be adapted to the physiological status of the subject, avoiding toxicity or inefficient therapies.

[0027] In a preferred embodiment, the method comprises the use of a soluble fragment of megalin and/or a fusion of said soluble megalin fragment which binds the complex of DBP:VD metabolite but none or less of the numerous other ligands of LPR2/megalin. More precisely, a soluble fragment of megalin which has no affinity for albumin or anti-DBP antibody (no Ab cross-reactivity!). Said embodiment may comprise a surface-bound DBP-binding megalin fragment or a fusion protein thereof which contains an epitope comprising :

SEQ ID NO: 01

-D-N-G-N-C-l-H-R-A-W-L-C-D-R-D

or

SEQ ID NO: 2

-G-C-T-H-E-C-V-Q-E-P-F-G-A-K-C- or both epitopes.

[0028] The sequence for affinity binding of human DBP:VD complex seems to contein SEQ ID NO: 03

-C-V-Q-E-P- or

SEQ ID NO: 04

-l-H-R-A-W-

[0029] Other useful DBP binding epitopes within the megalin M2 region (E1024-K1429) are

SEQ ID NO: 05 (C19 sequence)

-S-D-F-N-G-G-C-T-H-E-C-V-Q-E-P- SEQ ID NO: 06 (D5 sequence)

- C-Y-N-M-R-G-S-F-R-C-S-C-D-T-G

SEQ ID NO: 07 (A2 sequence)

-F-S-F-P-C-K-N-G-R-C-V-P-N-Y-Y

as determined by the interaction of DBP with megalin M2 (E1024-K1429) - SEQ ID NO: 09 - using CelluSpots™ peptide array.

[0030] Preferred embodiment may comprise a determining of the megalin-bound complex of DBP:VD metabolites. This may be done using an antibody recognizing the complex of DBP:VD or after isolation of DBP, vitamin D or its metabolites or megalin or the soluble fragment thereof. In one aspect of the disclosure, the method may comprise providing mixtures of vitamin D metabolites and DBP for establishing standard samples. [0031] In a preferred embodiment, the disclosure may comprise contacting said sample with an amount of added DBP to create standard conditions or a constant excess of DBP for a binding of the prohormone. The disclosure may further encompass contacting the sample with cubilin and/or a soluble fragment thereof.

[0032] Said embodiment may comprise a surface-bound DBP-binding megalin fragment or a fusion protein thereof which contains an epitope having any one or more of the amino acid sequence SEQ ID NO: 01 through SEQ ID NO: 10.

[0033] In one embodiment, the method of the disclosure may relate to a turbidimetric or nephelometric immunoassay. Said megalin and/or a soluble fragment thereof may be bound to nanoparticles having diameters ranging from 50 to 200 nm, so that the complex of DBP:VD is bound to said nanoparticles. The disclosure also provides an immunoassay selected from the group ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), FIA (fluorescence immunoassay), L1A (luminescence immunoassay), or ILMA.

[0034] Consequently, the method may comprise the steps of (a) providing a defined amount of megalin and/or a soluble fragment thereof coupled to a solid phase; (b) contacting the sample with the solid phase having coupled megalin and/or a soluble fragment thereof; (c) creating conditions to allow binding of the complex formed by DBP and vitamin D metabolite, wherein DBP alone does not bind to megalin and/or a soluble fragment thereof; and washing the solid phase; (e) providing an antibody recognizing the ternary complex comprising DBP, vitamin D or its metabolite; (f) contacting said ternary complex with an antibody, optionally an antibody against DBP, and immobilizing the immunocomplex on the solid phase; and (g) determining the amount of antibody bound to the solid phase, and correlating the amount of bound antibody to the endocytic or activatable vitamin D status in blood, plasma, or serum by reference with a standard.

[0035] The instant disclosure further comprises a test kit for use in a method of measuring the effective vitamin D status which comprises an antibody specific for the ternary complex, megalin or a fragment thereof or DBP, or vitamin D and its metabolites. The disclosure also relates to a test kit for use in a method of measuring vitamin D metabolites in a sample of bodily fluid, comprising nanoparticles having bound megalin and/or soluble fragments thereof.

[0036] The achieved object is a simple and reliable method for a direct quantitative determination of the effective status of vitamin D and its metabolites in a sample. When the bodily fluid is blood, serum or plasma, the status describes the endocytable fraction of vitamin D metabolites in the circulation, say the fraction which can be processed innercellularly to the active hormone by respective target cells and tissues as needed. The novel vitamin D status can be determined in aqueous solution despite the highly lipophilic nature of vitamin D.

[0037] Vitamin D ₃ is absorbed and processed by the organism easily and considered more potent than vitamin D2 which has a shorter half life and binds with less affinity to the vitamin D receptor (VDR). 1 a,25(OH)D ₃ and parathyroid hormone (PTH) influence the vitamin D metabolism by positive or negative regulation of the activity of the l a-hydroxylase and 24- hydroxylase (see Fig. 1 C). Again, vitamin D is 25-hydroxylated in the liver to 25(OH)D ₃ or 25(OH)D ₂ , and further hydroxylated in kidney cells to its biologically active form 1 a,25(OH)D. 24,25(OH)D is a further metabolite of 25(OH)D _3, but inactive and destined for excretion. In the circulation, vitamin D metabolites are tightly bound to DBP. Smaller amounts are bound to albumin and lipoproteins. The affinity of 25(OH)D (Ka = 6 x 10 ⁵ M ¹ ) and 1 ,25(OH) ₂ D (Ka = 5.4 x 10 ⁴ M ¹ ) for albumin is substantially lower than the affinity for DBP (25(OH)D (Ka = 7 ^c 10 ⁸ M ¹ ) and 1 ,25(OH) ₂ D (Ka = 4 ^c 10 ⁷ M ¹ )). Because of the abundance of albumin in serum (650 mM) compared to DBP (5 mM), some vitamin D will likely be bound by albumin while not effectively available for endocytosis. Additionally, the vast majority of the DBP in serum is not occupied by any vitamin D metabolite. There has been no disclosure in the prior art defining a parameter which addresses the bioavailabity of the vitamin D metabolites based on their binding to DBP and the following endocytic pathway. As DBP is present in high concentrations in the circulation it was so far not considered in the assessment of the vitamin D status. In the prior art, either total (bound and free) or free circulating vitamin D, separately, were considered sources of information for establishing a vitamin D status.

[0038] Megalin (also known as Low Density Lipoprotein receptor-related Protein 2, LRP2) is a multiligand binding cell receptor with structural similarities to the LDL receptor (LDLR). Megalin can be found in numerous cells and tissues, notably in the plasma membrane of absorptive epithelial cells (Farquhar MG et al, Soc. Nephrol. 6 (1 ): 35-47). LRP2/megalin is known to mediate the endocytosis of its ligands and can form complexes with cubilin which cubulin:megalin complexes are again able to (re)absorb molecules. The cubilin:megalin complex is inter alia responsible for the cellular uptake of lipids, VLDL, certain proteins (albumin, lactoferrin), cobalamin (vitamin B12), and calcidiol. The instant disclosure proposes determining the amount of DBP:VD which is bound by megalin. This can be done for example by an ELISA against human DBP after “isolation or separation” of said ternary complex. This is preferred and the ELISA is already commercially available (Immundiagnostik AG, Bensheim). The term“effective vitamin D” describes formed complex of DBP:VD which has been bound by megalin (or a fragment thereof). The complex with megalin is considered to interact with cubulin so that the “activated vitamin D metabolite” will be endocytosed and subsequently 1 a-hydroxylated to become the active hormone.

[0039] The effective vitamin D status represents an improvement over the prior art since it offers physiological information and a reading of the concentration of circulating vitamin D that will be processed to the D hormone. This status is different to the major circulating storage form because less active forms such as 25(OH)D ₂ will not or much less contribute to the effective vitamin D status. The present disclosure provides a vitamin D status based on the amount DBP:VD, selectively and discriminably bound by megalin and/or cubulin. This endocytic complex therefore corresponds to the status of the effective vitamin D in serum or plasma. The term "bioavailable" as used in the prior art however refers to "total free” vitamin D which is speculative and is based on an assumed diffusion across the plasma membrane of cells.

[0040] There are contradictory views in the prior art with respect to the role of DBP. On the one hand, binding of vitamin D to DBP has been regarded a protection mechanism against excessive amounts of the free vitamin. This view is followed by groups supporting the "free hormone hypothesis". On the other hand, studies in DBP knockout mice showed that these animals have significantly reduced plasma levels of 25(OH)D ₃ and 1 ,25-(OH) ₂ D ₃ . On a vitamin D depleted diet, the animals suffered from vitamin D deficiency and bone formation defects. These results point to the development of alternative pathways which will not be relevant when DBP is present in the circulation.

[0041] Steroid hormones and sterols such as vitamin D are lipophillic and commonly require a carrier protein for effective delivery. There are many ligand-specific serum carriers of steroid hormones and sterols including corticosteroid-binding globulin (CBG) (glucocorticoids, mineralocorticoids), vitamin A (retinol)-binding protein, vitamin D-binding protein (DBP), sex hormone-binding globulin (SHBG) (estrogens, androgens), and thyroid hormone-binding globulin. For example, CBG and SHBG not only act as high affinity serum transporters, but also able bind to cell membranes in their ligand forms, suggesting alternative actions as signal transducers. In a similar fashion, DBP is a macrophage-activating factor (MAF) and actin- binder, which functions seem independent from the binding to vitamin D metabolites. The mechanisms by which ligands are released from binding globulins and acquired by target cells are crucial to steroid hormone signaling pathways.

[0042] This is particularly important for vitamin D where there is increasing evidence for extra-renal, intracrine, conversion of the pro-hormone (25(OH)D) to active D-hormone. The impact of vitamin D is then very much dependent on tissue-specific expression of the l a- hydroxylase and the receptor for 1 ,25(OH) ₂ D, the nuclear vitamin D receptor (VDR). It has been estimated that concentrations of free 1 ,25(OH) ₂ D in serum are approximately 10 ¹³ M, which is much less than the concentrations quoted for binding to the vitamin D receptor (dissociation constant (Kd) = approximately 10 ¹⁰ M). Due to the obvious disparity between the amounts of free hormone available for (supposedly) passive diffusion and the levels required to efficiently occupy intracellular target receptors, the‘free-hormone hypothesis’ raises doubts as a basis for a physiologically relevant (“true”) vitamin D status.

[0043] Total 25(OH)D is currently measured by LC-MS/MS ( Institute of Standards and Technology and the Centers for Disease Control and Prevention). A variety of immunoassays are used to determine concentrations of total 25(OH)D and other vitamin D metabolites. However, all these methods produce highly variable results, likely due to the need for a releasing of the vitamin D metabolites from their carrier proteins. This comprises a risk of a loss of vitamin D through binding to vessel surfaces so that a falsely (low) vitamin D level is determined. Affinity chromatography studies using immobilized DBP and solubilized rabbit kidney membranes have identified the co-receptors for the uptake of 25(OH)D ₃ -DBP complex in kidney tubules, namely a 600-kDa protein (megalin) and a 460-kDa protein (cubilin). These studies show a Ca ² -dependent binding of DBP to cubilin. The authors do not suggest that the binding of DBP to either co-receptor is preceded, influenced or dependent on a formed complex of DBP and 25(OH)D ₃ , nor on the type of vitamin D metabolite bound by DBP (Nykjaer et al, Cubilin dysfunction causes abnormal metabolism of the steroid hormone 25(OH) vitamin D3, Proc Natl Acad Sci U S A. 2001 Nov 20; 98(24): 13895-13900).

[0044] Megalin has three domains: a 4400 amino acid extracellular amino-terminal domain, a 22 amino acid transmembrane domain and a 213 amino acid carboxy- terminal cytoplasmic tail, indicating that it is a type I cell-surface receptor. The extracellular domain contains four cysteine rich clusters of LDLR type A (complement-type) repeats. The complement type repeat consists of approximately 40 amino acids containing six cysteine residues and the SDE (Ser-Asp-Glu) motif responsible for high-affinity binding of positively charged sequences in ligands for LDLR. The four cysteine-rich clusters are flanked by epidermal growth factor (EGF)-type repeats and spacer regions containing YWTD (Tyr-Trp-Thr- Asp) motifs which are responsible for pH-dependent dissociation of ligands in endosomal compartments. The cytoplasmic domain of megalin contains three tetra-amino-acid NPXY motifs, which are essential for endocytosis of the ligand-receptor complex via clathrin-coated pits.

[0045] Megalin has diverse types of ligands: vitamin-binding proteins and other binding proteins, apolipoproteins, hormones and hormone precursors, drugs and toxins, enzyme and enzyme inhibitors, immune- and stress-response-related proteins, and others including calcium. Megalin knockout mice are unable to recover DBP from the glomerular filtrate, and lose it together with its vitamin D cargo in urine. As a consequence, megalin knockout mice are unable to adequately metabolize 25(OH)D to 1 ,25(OH) ₂ D resulting in a bone phenotype that resembles vitamin D-deficient rickets.

[0046] Cubilin has been identified as a receptor for intrinsic factor-B12 (IF-B) complex in the terminal ileum. It is a 460 kDa receptor with no transmembrane domain and no signals for endocytosis. Cubilin contains 27 CUB domains responsible for the ligand binding and eight EGF-type repeats preceded by a stretch of 1 10 amino acids, where the N-terminal region appears essential for membrane anchoring. A direct association between cubilin and megalin has been demonstrated, whereby molecular cooperation provides the basis for internalization of ligands bound to cubilin. Thus, cubilin binding ligands may undergo megalin-mediated endocytosis, unload its cargo in lysosomes and recycle back to the plasma membrane together with megalin.

[0047] Although megalin-dependent uptake of DBP has a clear role in renal vitamin D endocrinology, it is not yet clear whether a similar mechanism is present in other vitamin D target tissues. Outside the kidney, megalin is expressed by several tissues including the placenta, mammary gland and parathyroid glands, which are known to have l a-hydroxylase activity, suggesting an extra-renal DBP-megalin interaction. [0048] The present disclosure describes a method of determining a vitamin D status based on the detection and quantitation of a complex comprising DBP, vitamin D and either one of megalin and cubulin or both. Different to the prior art, the present method requires no specific release of the vitamin D metabolites from DBP and only the fraction of vitamin D metabolites will be evaluated that is subject to endocytosis and activation. Without wishing to be bound by any theory, the measurement of DBP bound to megalin provides a parameter for endocytable vitamin D metabolites. The provided method can therefore be used to establish a vitamin D status which corresponds to the physiologically active and endocytable vitamin D concentration in serum or plasma.

[0049] With the present method, the various vitamin D metabolites, in particular 25(OH)D ₃ can be discriminated as well as the changes in total serum concentrations of 250HD, 1 ,25(OH) ₂ D and DBP in subjects following supplementation with either cholecalciferol (vitamin D3) or ergocalciferol (vitamin D2). Supplementation will no longer interfere with the measurement of active vitamin D molecules that will be subject to endocytosis and hydroxylation. The fraction of truly bioavailable vitamin D molecules can therefoe easily be discerned from the supplemented vitamin D2 or vitamin D molecules bound by other proteins in the circulation.

[0050] Immunonephelometry can be used to measure the DBP concentration in a sample. Immunonephelometry quantifies the scattering of an incident light source by large soluble antigen-antibody complexes under conditions of a moderate excess of antibody. Under these conditions, the complexes form a stable lattice, and a direct linear relationship is established between an increasing concentration of antigen and the increase in scattered light intensity. Automated nephelometers provide sensitive and precise measurements of DBP concentration in a rapid manner and with minimal requirements for technical skill. These can be used in clinical chemistry laboratories to analyze DBP concentrations. In a preferred embodiment, only the ternary complex formed by DBP, vitamin D and megalin is recognized by the antibody.

[0051] In another aspect, immunoturbidimetry can be used to measure the DBP concentration in a sample. Turbidimetry is the process of measuring the loss of intensity of transmitted light due to the scattering effect of particles suspended in it. Light is passed through a filter creating a light of known wavelength which is then passed through, for example, a cuvette containing a solution. A photoelectric cell collects the light which passes through the cuvette. A measurement is then given for the amount of absorbed light. Immunoturbidimetry is a variant in which an antigen-antibody reaction takes place. The antigen-antibody complexes are particles which can be optically detected by a photometer. In a preferred embodiment, the antibody recognizes a ternary complex formed by DBP, vitamin D and megalin. In another aspect, nanoparticles coated with megalin or fragments thereof are contacted with a sample containing DBP/vitamin D complexes. Increasing concentrations of DBP/vitamin D/megalin ternary complex in the sample result in increased turbidity. EXAMPLES

EXAMPLE 1

[0052] Complex biological functions emerge through intricate protein-protein interaction networks. An important class of protein-protein interaction corresponds to peptide-mediated interactions, in which a short peptide stretch from one partner interacts with a large protein surface from the other partner. Protein-peptide interactions are typically of low affinity and involved in regulatory mechanisms, dynamically reshaping protein interaction networks. Due to the relatively small interaction surface, modulation of protein-peptide interactions has been considered feasible and highly attractive, for example, for therapeutic purposes. Unfortunately, the number of available 3D structures of protein-peptide interfaces is very limited. However, there was limited or no information regarding the interaction of DBP with other proteins, in particular, megalin/LRP2 (UniProtKB/Swiss-Prot, protein accession number P98164, human) in the context of vitamin D transport and metabolism. In order to examine the potential interaction of DBP with megalin at the peptide level, the PepSite2 program (http://pepsite2.russelllab.org) was used to predict peptide-binding spots. The PepSite method relies on preferred peptide binding environments calculated from a set of known protein-peptide 3D structures, combined with distance constraints derived from known peptides. According to the Pepsite prediction, megalin likely interacts with human vitamin D-binding protein through ligand-binding repeats 9, 13-15, 16, 19, 22-25; 27 and 29 located at domains 2 and 3 (see Figs. 1A)

[0053] In agreement with the above prediction, the present inventors selected cysteine- rich complement-type ligand binding repeats (LDLR class A) for analysis of DBP-megalin interaction. EGF-like modules were also included as they are considered important for receptor folding and dissociation of ligands in endosomal compartment. A signal peptide (M1-G25) was introduced at the N-terminis of complement-type repeats to allow sorting to the secretory pathway. A C-terminal 6xHis-tag was added for affinity purification and protein analysis by co- immunoprecipitation and western blotting. The respective cDNA sequences were obtained by PCR and inserted into the mammalian expression vector pcDNA3.1. Since calcium is required for a correct folding of the LDLR-A domain, HEK cells were stable transfected with megalin cDNA sequences (M1 , M2, M3) as they have endogenous calcium channels. Protein purification was done using the His-tag and a commercially available Ni-NTA resin. The cloning strategy for megalin fragments (M1 , M2, M3) into the mammalian expression vector pcDNA3.1 is shown in Figs. 2A-B.

[0054] Megalin M1 cDNA encoded the sequence of amino acids 26 to 386 of human LRP2/megalin (SEQ ID NO:08). The construct included cDNA encoding for an N-terminal 25 amino acid signal peptide and a C-terminal 6x histidine tag. The predicted molecular weight was about 43 kDa for the monomer and about 86 kDa for the dimer. [0055] The HEK-expressed and secreted recombinant megalin M1 fragment had the following amino acid sequence (without signal sequences):

SEQ ID NO: 08

IPADWRCDGT KDCSDDADEI GCAVVTCQQG YFKCQSEGQC IPNSWVCDQD QDCDDGSDER QDCSQSTCSS HQITCSNGQC IPSEYRCDHV RDCPDGADEN DCQYPTCEQL TCDNGACYNT SQKCDWKVDC RDSSDEINCT EICLHNEFSC GNGECIPRAY VCDHDNDCQD GSDEHACNYP TCGGYQFTCP SGRCIYQNWV CDGEDDCKDN

GDEDGCESGP HDVHKCSPRE WSCPESGRCI SIYKVCDGIL DCPGREDENN TSTGKYCSMT LCSALNCQYQ CHETPYGGAC FCPPGYIINH NDSRTCVEFD DCQIWGICDQ KCESRPGRHL CHCEEGYILE RGQYCK

[0056] Megalin M2 cDNA encoded the sequence of amino acids 1024-1429 of human LRP2/megalin (SEQ ID NO:09). The construct included cDNA encoding for an N-terminal 25 amino acid signal peptide and a C-terminal histidine(6x) tag. The predicted molecular weight was about 50 kDa for the monomer and about 100 kDa for the dimer. The HEK-expressed and secreted recombinant protein megalin M2 fragment had the following amino acid sequence (without signal sequences)::

SEQ ID NO: 09

EQCGLFS FPCKNGRCVP NYYLCDGVDD CHDNSDEQLC GTLNNTCSSS AFTCGHGECI

PAHWRCDKRN DCVDGSDEHN CPTHAPASCL DTQYTCDNHQ CISKNWVCDT

DNDCGDGSDE KNCNSTETCQ PSQFNCPNHR CIDLSFVCDG DKDCVDGSDE

VGCVLNCTAS QFKCASGDKC IGVTNRCDGV FDCSDNSDEA GCPTRPPGMC

HSDEFQCQED GICIPNFWEC DGHPDCLYGS DEHNACVPKT CPSSYFHCDN

GNCIHRAWLC DRDNDCGDMS DEKDCPTQPF RCPSWQWQCL GHNICVNLSV

VCDGIFDCPN GTDESPLCNG NSCSDFNGGC THECVQEPFG AKCLCPLGFL LANDSKTCED IDECDILGSC SQHCYNMRGS FRCSCDTGYM LESDGRTCK

[0057] Megalin M3 cDNA encoded the sequence of amino acids 2698 to 3192 of human LRP2/megalin (SEQ ID NO: 10). The cDNA encoded an 25 amino acid signal peptide and a C-terminal 6x histidine tag. The predicted molecular weight was about 60 kDa for the monomer and 120 kDa for the dimer. The construct was expressed in HEK 293 mammalian cells. The secreted megalin M3 fragment. SEQ ID NO. 10 corresponds to following amino acid sequence (without signal sequences): SEQ ID NO: 10

ERC GASSFTCSNG RCISEEWKCD NDNDCGDGSD EMESVCALHT CSPTAFTCAN GRCVQYSYRC DYYNDCGDGS DEAGCLFRDC NATTEFMCNN RRCIPREFIC

NGVDNCHDNN TSDEKNCPDR TCQSGYTKCH NSNICIPRVY LCDGDNDCGD

NSDENPTYCT THTCSSSEFQ CASGRCIPQH WYCDQETDCF DASDEPASCG HSERTCLADE FKCDGGRCIP SEWICDGDND CGDMSDEDKR HQCQNQNCSD SEFLCVNDRP PDRRCIPQSW VCDGDVDCTD GYDENQNCTR RTCSENEFTC GYGLCIPKIF RCDRHNDCGD YSDERGCLYQ TCQQNQFTCQ NGRCISKTFV CDEDNDCGDG

SDELMHLCHT PEPTCPPHEF KCDNGRCIEM MKLCNHLDDC LDNSDEKGCG INECHDPSIS GCDHNCTDTL TSFYCSCRPG YKLMSDKRTC VDIDECTEMP FVCSQKCENV IGSYICKCAP GYLREPDGKT CR

[0058] Protein analysis of megalin M1 , M2, M3 fragments was done by Western blotting employing an antibody against 6x His-tag. Cells transfected with above constructs (M1 , M2, M3) were lysed and the lysate analyzed for the proteins with a His-tag. Megalin fragments were also purified from the cell supernatants using Ni/NTA resin, which bind proteins with a His- tag (right). The results are shown in Fig. 2C.

[0059] The Wester blots show that megalin M1 , M2, M3 fragments were expressed and secreted by HEK cells. The megalin fragments could be kept in solution. No precipitation was observed. The megalin fragments showed a tendency to form dimers but the molecular weights were all consistent with the predicted sizes. 2X Laemmli and 2X Urea sample buffers were used for analysis of megalin monomer and dimer formation but no difference was observed by the use of these buffers. The M2 fragment was the one which could be mostly easily dissociated to the monomer.

[0060] The megalin fragments were further examined for their interaction with DBP in the presence (+) or absence (-) of vitamin D ₃ (VD ₃ ). Co-immunoprecipitations were performed and the results are shown in Figs 3 A,B. In brief, HEK cells were transfected with above cDNA constructs (M1 , M2, M3) and cell lysates used for co-immunoprecipation. Fig. 3A shows a Western blot of cell lysates (20 pg) of HEK cells expressing M1 , M2 and M3. Detection with an antibody against the His-tag. Fig. 3B shows the results of a co-immunoprecipitation with DBP (0,5 pg and VD ₃ (0,2 pg).

[0061] The serum samples included DBP alone or in the presence of added vitamin D ₃ . Each sample was contacted with cell lysate containing megalin fragment. After incubation, the samples were contacted with beads coated with an antibody against His-tag and the DBP pulled down by centrifugation. The bound ligands (DBP) were dissociated from the beads and analyzed by western blot for the expression of DBP using an anti-DBP-antibody. As control, pure DBP alone was analyzed. Fig. 3B confirms that DBP could be pulled down with megalin M1 and M2 fragments. Notably, DBP could only be pulled down in the presence (+) of vitamin D3. This indicates that the megalin fragments M1 and M2 do not interact with DBP alone but only when the DBP is occupied with a vitamin D metabolite. In other words, a ternary complex comprising vitamin D binding protein, vitamin D metabolite and megalin fragment was formed in vitro, and no interaction took place between megalin M1 or M2 fragments and DBP alone.

[0062] Additional co-immunoprecipitations show the in vitro interaction between DBP and Ni-NTA agarose purified soluble megalin M1 , M2, M3 fragments (3 pg) in a similar set-up as above. The results are shown in Figs. 4 A,B. Fig. 4A is a western blot of purified megalin M1 , M2, M3 fragments from culture supernatants (3 pg), secreted by HEK cells transfected with cDNA for megalin M1 , M2 and M3 fragments; detection by anti-His-antibodies. Fig. 4 B shows a western blot with the results of a co-immunoprecipitation with purified megalin M1 , M2, M3 fragments. The serum samples contained DBP alone or in the presence (+) of vitamin D3. After incubation, the samples were contacted with beads coated with an antibody against 6xHis-tag and pulled down by centrifugation. Bound proteins were dissociated from the beads and analyzed by Western blotting using an antibody against vitamin D binding protein. Again, Fig. 4B confirms that DBP could be pull down with purified soluble M1 and M2 fragments. DBP was pulled down only in the presence (+) of vitamin D3. This indicates that soluble purified megalin fragments M1 and M2 do not interact with DBP unless occupied with vitamin D3. Ternary complexes comprising DBP, vitamin D3 and purified megalin M1 or M2 fragments were formed in vitro. No interaction or binding was observed between soluble purified megalin M1 or M2 fragments and DBP alone.

EXAMPLE 2

[0063] For further characterization of the formed complex of purifed soluble megalin M1 or M2 fragments and DBP alone or occupied by vitamin D metabolite, the obtained complexes were further analyzed by microscale thermophoresis and results are shown in Fig. 5A. Microscale thermophoresis (MST) examines the directed movement of particles in a microscopic temperature gradient (thermophoresis). Any change of the hydration shell of biomolecules due to changes in their conformation results in a relative change of the movement along the temperature gradient. This principle can be used to determine the binding affinity of two molecules. This technique allows in particular an examination of interactions in solution without any immobilization on a surface. A spatial temperature difference leads to a depletion of molecule concentration in the region of elevated temperature, which can be then determined. Thermophoresis is usually performed with fluorescently labeled molecules.

[0064] The difference in the molecule's thermophoresis can further be to quantify the binding strength under constant buffer conditions. The thermophoretic movement of the fluorescently labeled molecule is measured by monitoring the fluorescence distribution inside a capillary. The microscopic temperature gradient is generated by an IR-Laser, which is focused into the capillary and absorbed in water. The temperature of the aqueous solution in the laser spot region therefore increases. A homogeneous fluorescence distribution is observed inside the capillary prior the IR-Laser is switched on. When the IR-Laser is switched on, a new fluorescence distribution is established. The thermal relaxation time is fast and induces a binding-dependent drop in the fluorescence of the dye due to its local environmental-dependent response to the temperature step increase. Molecules move then from the locally heated region to the outer cold regions. The local concentration of molecules decreases in the heated region until it reaches a steady-state distribution.

[0065] The normalized fluorescence (Fnorm) measures a concentration ratio, with consideration of the temperature step increase. Due to the linearity of the fluorescence intensity and the thermophoretic depletion, the normalized fluorescence from the unbound molecule and the bound complex superpose linearly. Quantitative binding parameters were obtained using serial dilutions of the binding substrate. By plotting Fnorm against the logarithm of the different concentrations of the dilution series, a sigmoidal binding curve is obtained. This binding curve can directly be fitted with the non-linear solution of the law of mass action, with the dissociation constant Kd as result.

[0066] In brief, purified DBP was labeled with the red fluorescent dye NT-647 using Monolith Protein Labeling Kit Red (NanoTemper Technologies, Munich, Germany). Ni-NTA- purified soluble megalin fragments M1 , M2, and M3 fragments were titrated in the range from 0.488 to 1000 nmol/L. A Monolith NT.1 15 device (NanoTemper Technologies) and the NT Analysis software version 1 .427 (NanoTemper Technologies) were used for measurements.

[0067] Microscale thermophoretic analysis of the binding between His-Tag purified soluble megalin M1 , M2, and M3 fragments and DBP in the absence or in the presence of vitamin D3 were performed. The results are shown in Figure 5A. In line with the above co- immunoprecipitation experiments, M1 and M2 fragments interacted with DBP but M3 did not. The dissociation constants were calculated for each experiment. Both megalin M1 and M2 fragments showed weak binding to DBP in the absence of 25-OH vitamin D3 (M1/DBP: Kd= 442 +/- 44.93 nM; M2/DBP: 134.2 +/- 23.57 nM)). In contrast, high binding affintiy was found for both megalin fragments in the presence of 25(OH)D ₃ as the dissociation constant Kd was markedly lower (M1/DBP/VD ₃ : Kd= 45.65 +/- 4.57 nM; M2/DBP/VD ₃ : 23.7 +/- 2.28 nM). We also observed increased binding of DBP to purified soluble megalin fragments M1 or M2 in the presence of 25(OH)D.

[0068] The result are good evidence of a 25(OH)D ₃ -dependent binding DBP to megalin. Megalin M2 fragment had a higher affinity for the complex of DBP: 25(OH)D ₃ and was used therefore in further studies.

[0069] The impact of the 25(OH)D ₃ concentration on the binding of soluble megalin M1 and M2 fragment to DBP was further assessed using an ELISA which already represents an embodiment of the proposed new complex binding assay for the effective vitamin D status. The results are shown in Fig. 5B. In brief, a microtiter plate was coated with purified soluble megalin M1 or M2 fragment (1 pg/100 pi PBS). Mixtures of DBP and 25(OH)D ₃ were prepared using 1 pg DBP and serial dilutions of 25(OH)D ₃ (VD ₃ ) in PBS (0, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20 pg). Each mixture was applied to the coated wells, incubated at physiological conditions to allow formation of a ternary complex and washed. Detection and quantification of DBP bound by megalin (soluble fragments M1 and M2) was carried out by using a polyclonal rabbit antibody against DBP and HRP-conjugated donkey anti-rabbit antibody. Absorbance was read at 450 nm.

[0070] As shown in Fig. 5B, the binding of soluble megalin M1 and M2 fragments to DBP was dependent on the 25-hydroxy-vitamin D ₃ . More precisely, the absorbance is linear proportional to the vitamin D ₃ concentration as shown in Fig. 9A and Fig. 10A and this relation is not impacted by increasing concentrations of 25(OH)VDBP or 24,25(OH)VD (cf. Fig. 9B, 10B). Importantly, these experiments demonstrate a proof of principle for easy and reliable vitamin D measurements based on the ternary complex.

EXAMPLE 3

[0071] The binding properties of the various megalin soluble fragments were further analyzed by co-immunoprecipitation using samples of serum and plasma from human subjects. The results are shown in Fig. 6A. Purified soluble megalin M1 protein was mixed with two human serum or plasma samples to allow interaction between soluble megalin M1 fragment and endogenous DBP present in the sample. Samples were incubated with Ni-NTA resin to allow interaction of megalin M1/DBP to the resin. After washing the resin, bound complex was eluted from the resin and assayed by western blotting. Fig. 6A (right) shows that DBP was present in both samples and could be detected using a specific antibody. Fig. 6A (left, upper blot) shows that purified soluble megalin M1 fragment interacted in solution with DBP in all samples, as demonstrated by the presence of pulled down DBP. Of note, using a control sample containing serum only but no megalin protein, no DBP was detectable. The presence of purified megalin M1 protein was detected in all samples using an anti-6xHis-tag antibody (Fig. 6A, left, lower blot).

[0072] In summary, the co-immunoprecipitations are proof that the complex of DBP and 25(OH)D ₃ can specifically be bound and isolated from plasma or serum using a suitable soluble megalin fragment. The megalin portion with amino acids 26-386 and 1024-1429 have been shown to be involved in the formation of a ternary complex with DBP and 25(OH)D ₃ . The external megalin region with amino acids 2698-R3192 did not participate in the binding under the described conditions.

[0073] The present application comprises representative amino acid sequences of megalin which can be used in the binding of DBP. Those can further be used for determining the status of endocytic or activatable vitamin D in bodily fluids. The status of endocytable vitamin D overrules any status for“free vitamin D” or“total vitamin D” as there will be no need for distinguishing between "free" or "total" from the "physiological status of vitamin D available for endocytosis and l ohydroxylation”. Conventional methods usually do not determine the physiologically relevant vitamin D status since they cannot analyze the metabolites ready for processing to the active hormone. Thus, the conventional methods are insufficient whereas the present method is directed to the status of circulating prohormone which can and will be hydroxylated in the kidney, and probably in other tissues, giving rise to the active hormone.

EXAMPLE 4

[0074] For detailed characterization of the proportion of the ternary complex of DBP , vitamin D3 and megalin (M1 and M2 fragments) an ELISA was developed using megal in-coated plates. The total amount of DBP in the serum sample was determined using a commercial ELISA for DBP . The results are shown in Figure 6B. The assay shows that a minor proportion (0.0028 %) only of the total amount of DBP in serum has bound hydroxylated vitamin D3 and can be bound by megalin. Using megalin M1 fragment, the assay yielded a concentration value of 9.45 ng complex/mL serum. In the case of megalin M2 fragment, a value of about 12.89 ng complex/mL serum was obtained. This is evidence that the megalin fragment did only DBP when in a complex with the prohormone. Megalin can be thus regarded a means for detection and quantification of the physiologically activated prohormone (bound to DBP ) in a clinical sample.

[0075] We noted that it was sometimes necessary to add an amount of purified DBP (DBP, from about 5 to 25 ng/mL) to induce a ternary complex between megalin, DBP and vitamin D metabolite in human serum. In order to assess the effect of added DBP on the formation of ternary complex we also performed ELISAs with increasing amounts of purified vitamin D binding protein. The results are shown in Fig. 7A. In the serum sample (RMS), addition of purified DBP in a dose range up to 25 ng/mL did not affect the binding of the complex DBP/VD3 to purified soluble megalin M2 fragment. The initial value of 8.65 ng/mL remained unchanged independently of added purified DBP. A slight increase in complex- megalin interaction was observed when adding high amounts of purified DBP up to 40 ng/mL. Above this limit, the ternary complex was formed exponentially. Within limits, the measured levels of ternary complex of DBP-VD3-megalin do therefore not depend on the amount of added purified vitamin D binding protein.

[0076] For comparison, five serum samples (S3, S6, S8, S9, RMS) from different subjects were analyzed to determine the concentration of total DBP (DBP), 25(OH)D ₃ and ternary complex of DBP:25(OH)VD:M2. DBP was determined by ELISA. The 25(OH)D ₃ content was quantified conventionally by an independent laboratory. The ternary complex DBP:25(OH)VD:M2 megalin was analyzed by a DBP:VD3:megalin ELISA according to the disclosure. Table 1 below summarizes the results for comparison. TABLE 1

[0077] Current standards for healthy control (interquartile) range values are for i) serum DBP 193.5-4345.0 pg/ml (median 423.5 pg/ml; 354.1-586 pg/ml), and for ii) serum 25(OH)D ₃ 30-100 ng/mL (normal range), 20-30 ng/ml (insufficiency), < 20 ng/ml (deficiency). Accordingly, sample S3 was considered to have normal levels of 25(OH)D ₃ ; S9, insufficient; S8 and S6, deficient levels. In other words, from higher to lower levels of 25(OH)D ₃ : S3>S9>S8>S6. From the results it could be concluded that the level of total DBP seemed not to correlate with the level of 25(OH)D ₃ level in human serum samples.

[0078] Although a direct comparison is strictly not possible since different analytes were measured by different methods, it is noteworthy that a correlation in the levels of ternary complex DBP:25(OH)VD:M2 megalin could be found in the samples when compared to 25(OH)D ₃ values; from higher to lower levels of ternary complex S3>S9>S8>S6, identical as determined for 25(OH)D ₃ levels. This further supports the notion that megalin only interacts in vitro with DBP when the prohormone is bound, providing a new status of physiologically active, endocytable vitamin D, which describes the bioavailability of the prohormone in the circulation.

[0079] The correlation between 25(OH)VD ₃ serum levels and ternary complex was furhter analyzed; see graph in Fig. 7B. A linear correlation between serum levels of 25(OH)VD ₃ and DBP:25(OH)VD:M2 megalin was determined (R ² =0.8897) indicating that the method can provide accurate information that can be compared or related to standard values so as to obtain a measurement of the activatable vitamin D status of a subject.

[0080] Moreover, the linearity range of the interaction between megalin and DBP was examined using the claimed method for 25-hydroxyvitamin D ₃ (25(OH)VD ₃ ), 25-hydroxyvitamin D ₂ (25(OH)VDBP) and 24,25-dihydroxyvitamin D (24,25(OH)VD). ELISAs for the complex of DBP:VD:megalin were performed as disclosed. In brief: a microtitre plate coated with purified megalin M2 fragment was incubated with a mixture of DBP and serial dilutions of 25(OH)VD ₂ , 25(OH)VD ₃ or 24,25(OH)VD. For determination of the ternary complex, a polyclonal rabbit anti- DBP antibody and a HRP-conjugated donkey anti-rabbit antibody were used. The results are shown in Fig. 7C. For all vitamin D metabolites, the assay was linear up to 50 ng/ml. The determined sensitivity limit was 2.0 ng/ml.

[0081] The binding properties of DBP and 25(OH)VD ₃ , 25(OH)VD ₂ , 24,25(OH)VD and 3C-epimer of 25(OH)D ₃ on the one hand and megalin on the other was further examined using microscale thermophoresis. Results are shown in Figs. 8 A,B. The purified DBP was labeled with red fluorescent dye NT-647. The vitamin D metabolites were titrated with different concentrations. The dissociation constant Kd was calculated for every interaction partner. The binding affinity was assessed via the obtained dissociation constant Kd. The results show that the interaction affinity of DBP to 25(OH)VD ₃ (Kd = 1.88 +/- 0.28 nM) or 25(OH)VDBP (Kd = 2.81 +/- 0.681 nM) were comparable, whereas the interaction to 24,25(OH)VD appeared to be slightly weaker. The 3C-epi25(OH)VD ₃ bound most strongly.

[0082] The affinity of DBP when interacting with 25(OH)VD ₃ , 25(OH)VD ₂ , or 24,25- 24,25(OH)VD, 3C-epimer of 25(OH)D ₃ and megalin M2 was also analyzed by microscale thermophoresis. The results are shown in Fig. 8B. Purified DBP (50nM) was labeled with the red fluorescent dye NT-647 and mixed with either vitamin D metabolite (37.8 nM). Purified megalin M2 was titrated in different concentrations. The dissociation constant Kd was calculated for every condition. The binding affinity was assessed by the dissociation constant Kd. The analyses show that the binding affinity of megalin M2 fragment to the complex DBP:25(OH)VD ₃ is much stronger (Kd = 33.7 +/- 20.7 nM) than to the complex DBP:25(OH)VD ₂ (Kd = 160 +/- 22.7 nM), or -/DBP-24,25VD (Kd = 171 +/- 24.6 nM). A very high affinity was observed for the 3C epimer which will require further investigation.

[0083] These experiments demonstrate that the binding of megalin to DBP takes only place when bound to the prohormone. This ternary complex can be detected and quantified by any method for determination of proteins. In view of the above, the use of megalin allows for a reliable measurement of the physiologically activated vitamin D, say 25-hydroxyvitamin D ₃ , as this represents the most active form of vitamin D in the circulation. The disclosed method provides for differential measurement of other vitamin D metabolites which is also relevant in view of the vitamin DBP in food supplements.

[0084] Megalin or fragments thereof are used to discern the effective vitamin D status. The described megalin fragments can easily be produced by recombinant methods and even by chemically synthesized. The described megalin fragments remained soluble in aqueous solution so that the conditions close to physiological can be used. There is no longer a need for a release of vitamin D from its binding partners, as required by the prior art methods. Thus, no solvents or surfactants for displacement of the vitamin D from its binding partners are needed. Accordingly, the measurement of the vitamin D status will not be interfered by non-physiological chemicals. No purification steps or time consuming and costly techniques such as LC-MS are necessary. A direct and fast determination of the effective vitamin D status can be done immediately after sample collection. The disclosed principles can further be easily adapted to available platforms and automats. [0085] As other ligands than DBP can also bind to megalin, we have further mapped more closely the binding regions or epitope within the M2 region. The results are contained in the provided sequence for the binding epitopes. Minimum binding epitopes have further been tested.

EXAMPLE 5

[0086] Production of recombinant megalin fragments. Megalin (LRP2) cDNA fragments were amplified by RT-PCR using mRNA from Caco-2 cells (human colon carcinoma epithelial cells). For cDNA transcription synthesis Maxima H Minus First strand cDNA synthesis kit (Thermo Scientific) was used. This kit allows synthess of cDNA up to 20 kb. Oligo dt18 primer and 65C were used. For PCR reactions Platinum PCR Supermix high fidelity PCR kit (Invitrogen) was used. Megalin M1 cDNA: 1 158 bp; Megalin M2 cDNA: 1215 bp; Megalin M3 cDNA: 1482 bp.

[0087] Megalin cDNA fragments were cloned with a C-terminal His-Tag into pcDNA3.1 and the cloned plasmids transfected into mammalian HEK293 cells. The following cloning strategy was applied to clone megalin M2 cDNA encoding for sequence of amino acids 1024- 1429 of human LRP2/megalin (SEQ ID NO:09), and resulted in the construct having: signal peptide (25 amino acids) + E1024-K1429 + 6x His tag. The cloning was done using PCR generated sequences.

[0088] Stable cell line selection was carried out using neomycin (G418, 800 pg/ml). Cell culture supernatants or cell lysates were purified by Ni-NTA resin. Analysis of megalin protein M1 , M2, M3 fragments having a 6xHis-Tag was performed by Western blot with an antibody against 6xHis-tag (Cohesion Biosciences). Cell culture supernantants or cell lysates were purified with Ni-NTA agarose (Thermo Fischer Scientific). Cell lysates or culture supernatant (0.5 - 2 mL) were incubated with 50 pi of pre-equilibrated Ni-NTA resin at 4°C overnight. The resin was washed 3 times with H-buffer + 20mM imidazole. Megalin protein complex was eluted with H-buffer containing 200mM imidazole and buffer exchanged with PBS 1 X. Protein solutions were lyophilized or kept at 4°C prior use.

[0089] The fragment M2 was further mapped for major binding epitopes of Megalin ligands within the M2 region, the region of megalin which binds human DBP. The binding sites of other megalin ligands were removed and the epitopes for binding of human DBP identified. It is imporant that the minimun epitopes do not overlap with the binding epitopes of other ligands off megalin. The results are shown in the Tables below. TABLE 2

Major binding epitopes of Megalin ligands within M2 region (aa E1024-K1429)

TABLE 3

Epitope mapping of ligands binding to the human Megalin M2 region (aa E1024-K1429)

LDL-receptor class A8

EQCGLFS FPCKNGRCVPNYYLCDGVDDCHDNSDEQLCG

(1024-1061)

LDL-receptor class A9 TLNNTCSSSAFTCGHGECIPAHWRCDKRNDCVDGSDEHNCP

(1066-1102) Apo E

LDL-receptor class A10 THAPASCLDTQYTCDNHQCISKNWVCDTDNDCGDGSDEKNCN

(1108-1144) Clq

LDL-receptor class All

STETCQPSQFNCPNHRCIDLSFVCDG DKDCVDGSDEVGCV

(1148-1184)

LDL-receptor class A12

LNCTASQFKCASGDKCIGVTNRCDGVFDCSDNSDEAGCP

(1186-1223)

LDL-receptor class A13 TRPPG/WCHSDEFQCQEDG/C/PA/FI/l/ECDGHPpCLYGSDEHNACV

(1229-1267) RAP Ajbumjn Clg/PTH

LDL-receptor class A14 PKTCPSSYFHCDNGNCIHRAWLCDRDNDCGDMSDEKDCP

(1270-1306) DPB Ig KC

LDL-receptor class A15 rQPFRCPSWQWQCLGHNICVNLSWCDGIFDCPNGTDESPLCN

(1304-1349) Ig KC

GNSCSDFNGGCTHECVQEPFGAKCLCPLGFLLANDSKTCE

Special domain (1350-1389)

DPB Albumin

EGF-likel; calcium-binding PIPE CDILGSCSQHCYNMRGSFRCSDTGYMLESDGRTCK

(1390-1429) Albumen Apo E RAP

[0090] The minimum epitopes within the megalin M2 region for a bindung of human DBP have been listed in Table 3 below. TABLE 4

List of Megalin-M2-peptides cloned and expressed as fusion polypeptides with furin

EXAMPLE 6

[0091] Megalin complex ELISA binding assay. Microtiter plates were coated with soluble purified megalin M1 or M2 fragment (1 pg/100 pi diluted in PBS1X) by incubation at RT for 2 h. Serial dilutions of 25-hydroxvitamin D3 (VD3) 0, 0.3125, 0.625, 1 .25, 2.5, 5, 10, 20 ng in 100 pi PBS were mixed each with 1 pg DBP (DBP). The mixture was incubated at 37°C for 1 h. Unspecific binding sites were blocked with blocking buffer (1 % BSA in PBS1X) at RT for 1 h. The DBP-VD3 mixture or serum samples were added to the megal in-coated wells and incubated at 4°C overnight. After washing with PBST-buffer (0.05% Tween 20 in PBS) 100 pi rabbit-anti-DBP antibody was added (1 : 3500 diluted in blocking buffer) and incubated at R.T for 2 h. After washing with PBST, 100 pi of HRP- conjugated donkey anti-rabbit 2nd antibody (1 : 500 diluted in blocking buffer) was added and incubated at 37°C for 1 h. 100 pi substrate reagent A+B (1 : 1 ) (R&D) was added and incubated at R.T for 30min. Then, 100 pi stop solution was added to the wells. Absorbance was read at 450 nm. The values were compared to standard values of known 25-hydroxvitamin D3 concentration.

EXAMPLE 7

[0092] Co-immu noprecipitation of DBP from human serum with recombinant purified soluble megalin. Ni-NTA purified soluble megalin M1 or M2 protein fragment (3 pg) was first mixed with human serum or plasma samples (30 pi). These samples were incubated with 50 pi of pre-equilibrated Ni-NTA resin at 4°C overnight. The resin was washed 3 times with H-buffer + 20mM imidazole. Bound complex was eluted with H-buffer containing 200mM imidazole and analyzed by Western blot with a polyclonal rabbit antibody against DBP (Abeam). EXAMPLE 8

[0093] Immunoturbidimetry assay for determination of vitamin D status with soluble megalin. Ni-NTA purified soluble megalin M1 or M2 protein fragment (3 pg) is first mixed with human serum or plasma samples (30 pi). Samples are incubated with 50 pi of pre-equilibrated Ni-NTA resin at 4°C overnight. The resin is washed 3 times with H-buffer + 20mM imidazole. Bound complex is eluted with H-buffer containing 200 mM imidazole. The eluted complex is contacted in aqueous solution with an antibody against vitamin D binding protein. The increase in turbidity is measured with a standard turbidimeter and compared with standard values of known 25-hydroxvitamin D3 concentration.

[0094] Alternatively, nanoparticles, for example, latex nanoparticles (aprox. 150 nm), are coated with megalin M1 or M2 fragment and incubated with a serum or plasma sample. The increase of turbidity is then measured and compared to standard values of known 25- hydroxvitamin D3 concentration.

EXAMPLE 9

[0095] ELISA binding assay for linearity range determination. A microtiter plate was coated with purified megalin M1 or M2 protein fragment (1 pg/100 pi diluted in PBS1X) by incubation at RT for 2 h. Mixtures of DBP and 25-hydroxvitamin D3 (VD3), 25-hydroxvitamin D2 (VD2) or 24, 25-hydroxvitamin D (24,25VD) were prepared by mixing 20 pg DBP and serial dilution of VD (0, 0.78, 1 ,56, 3,125, 6.25, 12.5, 25, 50 ng) in 100 pi PBS. The mixture was incubated at 37°C for 1 h. After plate blocking, the above prepared DBP-VD mixture was added to megalin-coated wells and incubated at 4°C, overnight. Then, the plate was incubated with 100 pi diluted rabbit-anti-DBP antibody (1 :1000 diluted in blocking buffer) at RT for 2 h, followed by incubation with 100 pi of HRP-conjugated donkey anti-rabbit antibody (1 : 500 diluted in blocking buffer) at 37°C, 1 h. After substrate reaction, the absorbance was read at 450 nm. The assay was linear up to 50 ng/ml. The sensitivity limit was determined to be 2.0 ng/ml.

EXAMPLE 10

[0096] Microscale Thermophoresis assay. Purified DBP (Merck, 345802) was labeled with the red fluorescent dye NT-647 by using a Monolith Protein Labeling Kit Red (NanoTemper Technologies, Munich, DE). Ni-NTA-purified megalin fragments (M1 , M2) were titrated in the range of 0.488 to 1000 nmol/L. Purified DBP (Merck, 345802) was likewas labeled with the red fluorescent dye NT-647 by using the Monolith Protein Labeling Kit Red. 25-hydroxvitamin D3 (VD3) was titrated in concentrations in the range of 0.0488 to 100 nmol/L. 25-hydroxvitamin D2 (VD2), 24, 25-hydroxvitamin D (24,25VD) and 3epi25(OH)VD ₃ were titrated in concentrations in the range of 0.0163 to 37 nmol/L. 37.8 nM of VD3, VD2 and 24,25VD was respectively added to 50nM of NT-647 labeled DBP. A Monolith NT.1 15 device (NanoTemper Technologies) was used for measurements. NT Analysis software version 1.427 (NanoTemper Technologies) was used for analysis. Parameters: laser power, 100%; LED, 80; laser on-time, 30 seconds; laser off-time, 5 seconds; temperature, 25°C. FNorm: normalized fluorescence; Kd: dissociation constant.

[0097] The dissociation constants Kd of the vitamin D metabolites to DBP gave the following ranking of the binding affinities. 3epi25(OH) ₂ VD3 > 25(OH) ₂ VD3 > 25(OH) ₂ VD ₂ > 24,25(OH) ₂ VD3 > 1 ,25(OH) ₂ VD3. The binding affinity of DBP-VD ₂ is comparable to Kd of DBP- VD3. The binding of 24,25(OH)VD to DBP is marginally lower. The high binding affinity of the 3C epimer of 25(OH)D ₃ is surprising and will require further investigation as this epimer seems therefore particularly useful as food supplement, if not toxic for other reasons.

SEQUENCE LISTING

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<120> Method of measuring the endocytic vitamin D status

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<151> 2018-01-03

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Phe Arg Cys Gly Ser Gly His Cys lie Pro Ala Asp Trp Arg Cys Asp

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Gly Thr Lys Asp Cys Ser Asp Asp Ala Asp Glu lie Gly Cys Ala Trp 50 55 60

Thr Cys Gin Gin Gly Tyr Phe Lys Cys Gin Ser Glu Gly Gin Cys lie 65 70 75 80

Pro Asn Ser Val Trp Cys Asp Gin Asp Gin Asp Cys Asp Asp Gly Ser

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Asp Glu Arg Gin Asp Cys Ser Gin Ser Thr Cys Ser Ser His Gin lie

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Thr Cys Ser Asn Gly Gin Cys lie Pro Ser Glu Tyr Arg Cys Asp His

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Val Arg Asp Cys Pro Asp Gly Ala Asp Glu Asn Asp Cys Gin Tyr Pro 130 135 140

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Glu Cys lie Pro Arg Ala Tyr Val Cys Asp His Asp Asn Asp Cys Gin

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Ser Gly Pro His Asp Val His Lys Cys Ser Pro Arg Glu Trp Ser Cys

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Pro Glu Ser Gly Arg Cys lie Ser lie Tyr Lys Val Cys Asp Gly lie

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Tyr Cys Ser Met Thr Leu Cys Ser Ala Leu Asn Cys Gin Tyr Gin Cys 305 310 315 320

His Glu Thr Pro Tyr Gly Gly Ala Cys Phe Cys Pro Pro Gly Tyr lie 325 330 335 lie Asn His Asn Asp Ser Arg Thr Cys Val Glu Phe Asp Asp Cys Gin

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He Trp Gly He Cys Asp Gin Lys Cys Glu Ser Arg Pro Gly Arg His

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Val Ala Cys Leu Ala Pro Ala Ser Gly Gin Glu Cys Asp Ser Ala His

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Pro Cys Lys Asn Gly Arg Cys Val Pro Asn Tyr Tyr Leu Cys Asp Gly 50 55 60

Val Asp Asp Cys His Asp Asn Ser Asp Glu Gin Leu Cys Gly Thr Leu 65 70 75 80

Asn Asn Thr Cys Ser Ser Ser Ala Phe Thr Cys Gly His Gly Glu Cys

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Ser Asp Glu His Asn Cys Pro Thr His Ala Pro Ala Ser Cys Leu Asp

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Thr Gin Tyr Thr Cys Asp Asn His Gin Cys He Ser Lys Asn Val Trp 130 135 140

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Asn Ser Thr Glu Thr Cys Gin Pro Ser Gin Phe Asn Cys Pro Asn His

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Arg Cys lie Asp Leu Ser Phe Val Cys Asp Gly Asp Lys Asp Cys Val

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Asp Gly Ser Asp Glu Val Gly Cys Val Leu Asn Cys Thr Ala Ser Gin

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Phe Lys Cys Ala Ser Gly Asp Lys Cys lie Gly Val Thr Asn Arg Cys 210 215 220

Asp Gly Val Phe Asp Cys Ser Asp Asn Ser Asp Glu Ala Gly Cys Pro 225 230 235 240

Thr Arg Pro Pro Gly Met Cys His Ser Asp Glu Phe Gin Cys Gin Glu

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Asp Gly lie Cys lie Pro Asn Phe Trp Glu Cys Asp Gly His Pro Asp

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Cys Leu Tyr Gly Ser Asp Glu His Asn Ala Cys Val Pro Lys Thr Cys

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Pro Ser Ser Tyr Phe His Cys Asp Asn Gly Asn Cys lie His Arg Ala 290 295 300

Trp Leu Cys Asp Arg Asp Asn Asp Cys Gly Asp Met Ser Asp Glu Lys 305 310 315 320

Asp Cys Pro Thr Gin Pro Phe Arg Cys Pro Ser Trp Gin Trp Gin Cys

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Leu Gly His Asn lie Cys Val Asn Leu Ser Val Val Cys Asp Gly lie

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Phe Asp Cys Pro Asn Gly Thr Asp Glu Ser Pro Leu Cys Asn Gly Asn

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Ser Cys Ser Asp Phe Asn Gly Gly Cys Thr His Glu Cys Val Gin Glu 370 375 380 Pro Phe Gly Ala Lys Cys Leu Cys Pro Leu Gly Phe Leu Leu Ala Asn 385 390 395 400

Asp Ser Lys Thr Cys Glu Asp lie Asp Glu Cys Asp lie Leu Gly Ser

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Cys Ser Gin His Cys Tyr Asn Met Arg Gly Ser Phe Arg Cys Ser Cys

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Asp Thr Gly Tyr Met Leu Glu Ser Asp Gly Arg Thr Cys Lys His His

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His His His His

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Val Ala Cys Leu Ala Pro Ala Ser Gly Gin Glu Cys Asp Ser Ala His

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Phe Arg Cys Gly Ser Gly His Cys Glu Arg Cys Gly Ala Ser Ser Phe

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Thr Cys Ser Asn Gly Arg Cys lie Ser Glu Glu Trp Lys Cys Asp Asn 50 55 60

Asp Asn Asp Cys Gly Asp Gly Ser Asp Glu Met Glu Ser Val Cys Ala 65 70 75 80

Leu His Thr Cys Ser Pro Thr Ala Phe Thr Cys Ala Asn Gly Arg Cys

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Val Gin Tyr Ser Tyr Arg Cys Asp Tyr Tyr Asn Asp Cys Gly Asp Gly

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Ser Asp Glu Ala Gly Cys Leu Phe Arg Asp Cys Asn Ala Thr Thr Glu

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6 Phe Met Cys Asn Asn Arg Arg Cys lie Pro Arg Glu Phe lie Cys Asn 130 135 140

Gly Val Asp Asn Cys His Asp Asn Asn Thr Ser Asp Glu Lys Asn Cys 145 150 155 160

Pro Asp Arg Thr Cys Gin Ser Gly Tyr Thr Lys Cys His Asn Ser Asn

165 170 175 lie Cys lie Pro Arg Val Tyr Leu Cys Asp Gly Asp Asn Asp Cys Gly

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Asp Asn Ser Asp Glu Asn Pro Thr Tyr Cys Thr Thr His Thr Cys Ser

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Ser Ser Glu Phe Gin Cys Ala Ser Gly Arg Cys lie Pro Gin His Trp 210 215 220

Tyr Cys Asp Gin Glu Thr Asp Cys Phe Asp Ala Ser Asp Glu Pro Ala 225 230 235 240

Ser Cys Gly His Ser Glu Arg Thr Cys Leu Ala Asp Glu Phe Lys Cys

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Asp Gly Gly Arg Cys lie Pro Ser Glu Trp lie Cys Asp Gly Asp Asn

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Asp Cys Gly Asp Met Ser Asp Glu Asp Lys Arg His Gin Cys Gin Asn

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Gin Asn Cys Ser Asp Ser Glu Phe Leu Cys Val Asn Asp Arg Pro Pro 290 295 300

Asp Arg Arg Cys lie Pro Gin Ser Trp Val Cys Asp Gly Asp Val Asp 305 310 315 320

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Ser Glu Asn Glu Phe Thr Cys Gly Tyr Gly Leu Cys lie Pro Lys lie

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Phe Arg Cys Asp Arg His Asn Asp Cys Gly Asp Tyr Ser Asp Glu Arg

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7 Gly Cys Leu Tyr Gin Thr Cys Gin Gin Asn Gin Phe Thr Cys Gin Asn 370 375 380

Gly Arg Cys lie Ser Lys Thr Phe Val Cys Asp Glu Asp Asn Asp Cys 385 390 395 400

Gly Asp Gly Ser Asp Glu Leu Met His Leu Cys His Thr Pro Glu Pro

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Thr Cys Pro Pro His Glu Phe Lys Cys Asp Asn Gly Arg Cys lie Glu

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Met Met Lys Leu Cys Asn His Leu Asp Asp Cys Leu Asp Asn Ser Asp

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Glu Lys Gly Cys Gly lie Asn Glu Cys His Asp Pro Ser lie Ser Gly 450 455 460

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Arg Pro Gly Tyr Lys Leu Met Ser Asp Lys Arg Thr Cys Val Asp lie

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Asp Glu Cys Thr Glu Met Pro Phe Val Cys Ser Gin Lys Cys Glu Asn

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Val lie Gly Ser Tyr lie Cys Lys Cys Ala Pro Gly Tyr Leu Arg Glu

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Pro Asp Gly Lys Thr Cys Arg His His His His His His

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Ala Asp Glu lie Gly Cys Ala Val Val Thr Cys Gin Gin Gly Tyr Phe

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8 Lys Cys Gin Ser Glu Gly Gin Cys lie Pro Asn Ser Trp Val Cys Asp 35 40 45

Gin Asp Gin Asp Cys Asp Asp Gly Ser Asp Glu Arg Gin Asp Cys Ser

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Gin Ser Thr Cys Ser Ser His Gin lie Thr Cys Ser Asn Gly Gin Cys 65 70 75 80 lie Pro Ser Glu Tyr Arg Cys Asp His Val Arg Asp Cys Pro Asp Gly

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Ala Asp Glu Asn Asp Cys Gin Tyr Pro Thr Cys Glu Gin Leu Thr Cys

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Asp Asn Gly Ala Cys Tyr Asn Thr Ser Gin Lys Cys Asp Trp Lys Val

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Asp Cys Arg Asp Ser Ser Asp Glu lie Asn Cys Thr Glu lie Cys Leu

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His Asn Glu Phe Ser Cys Gly Asn Gly Glu Cys lie Pro Arg Ala Tyr 145 150 155 160

Val Cys Asp His Asp Asn Asp Cys Gin Asp Gly Ser Asp Glu His Ala

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Cys Asn Tyr Pro Thr Cys Gly Gly Tyr Gin Phe Thr Cys Pro Ser Gly

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Arg Cys lie Tyr Gin Asn Trp Val Cys Asp Gly Glu Asp Asp Cys Lys

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Asp Asn Gly Asp Glu Asp Gly Cys Glu Ser Gly Pro His Asp Val His

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Lys Cys Ser Pro Arg Glu Trp Ser Cys Pro Glu Ser Gly Arg Cys lie 225 230 235 240

Ser lie Tyr Lys Val Cys Asp Gly lie Leu Asp Cys Pro Gly Arg Glu

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Asp Glu Asn Asn Thr Ser Thr Gly Lys Tyr Cys Ser Met Thr Leu Cys

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9 Ser Ala Leu Asn Cys Gin Tyr Gin Cys His Glu Thr Pro Tyr Gly Gly 275 280 285

Ala Cys Phe Cys Pro Pro Gly Tyr lie lie Asn His Asn Asp Ser Arg 290 295 300

Thr Cys Val Glu Phe Asp Asp Cys Gin lie Trp Gly lie Cys Asp Gin 305 310 315 320

Lys Cys Glu Ser Arg Pro Gly Arg His Leu Cys His Cys Glu Glu Gly

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Tyr lie Leu Glu Arg Gly Gin Tyr Cys Lys

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Glu Gin Cys Gly Leu Phe Ser Phe Pro Cys Lys Asn Gly Arg Cys Val 1 5 10 15

Pro Asn Tyr Tyr Leu Cys Asp Gly Val Asp Asp Cys His Asp Asn Ser

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Asp Glu Gin Leu Cys Gly Thr Leu Asn Asn Thr Cys Ser Ser Ser Ala

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Phe Thr Cys Gly His Gly Glu Cys lie Pro Ala His Trp Arg Cys Asp 50 55 60

Lys Arg Asn Asp Cys Val Asp Gly Ser Asp Glu His Asn Cys Pro Thr 65 70 75 80

His Ala Pro Ala Ser Cys Leu Asp Thr Gin Tyr Thr Cys Asp Asn His

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Gin Cys lie Ser Lys Asn Trp Val Cys Asp Thr Asp Asn Asp Cys Gly

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Asp Gly Ser Asp Glu Lys Asn Cys Asn Ser Thr Glu Thr Cys Gin Pro

115 120 125

Ser Gin Phe Asn Cys Pro Asn His Arg Cys lie Asp Leu Ser Phe Val

10 130 135 140

Cys Asp Gly Asp Lys Asp Cys Val Asp Gly Ser Asp Glu Val Gly Cys 145 150 155 160

Val Leu Asn Cys Thr Ala Ser Gin Phe Lys Cys Ala Ser Gly Asp Lys

165 170 175

Cys lie Gly Val Thr Asn Arg Cys Asp Gly Val Phe Asp Cys Ser Asp

180 185 190

Asn Ser Asp Glu Ala Gly Cys Pro Thr Arg Pro Pro Gly Met Cys His

195 200 205

Ser Asp Glu Phe Gin Cys Gin Glu Asp Gly lie Cys lie Pro Asn Phe 210 215 220

Trp Glu Cys Asp Gly His Pro Asp Cys Leu Tyr Gly Ser Asp Glu His 225 230 235 240

Asn Ala Cys Val Pro Lys Thr Cys Pro Ser Ser Tyr Phe His Cys Asp

245 250 255

Asn Gly Asn Cys lie His Arg Ala Trp Leu Cys Asp Arg Asp Asn Asp

260 265 270

Cys Gly Asp Met Ser Asp Glu Lys Asp Cys Pro Thr Gin Pro Phe Arg

275 280 285

Cys Pro Ser Trp Gin Trp Gin Cys Leu Gly His Asn lie Cys Val Asn 290 295 300

Leu Ser Val Val Cys Asp Gly lie Phe Asp Cys Pro Asn Gly Thr Asp 305 310 315 320

Glu Ser Pro Leu Cys Asn Gly Asn Ser Cys Ser Asp Phe Asn Gly Gly

325 330 335

Cys Thr His Glu Cys Val Gin Glu Pro Phe Gly Ala Lys Cys Leu Cys

340 345 350

Pro Leu Gly Phe Leu Leu Ala Asn Asp Ser Lys Thr Cys Glu Asp lie

355 360 365

Asp Glu Cys Asp lie Leu Gly Ser Cys Ser Gin His Cys Tyr Asn Met

11 370 375 380

Arg Gly Ser Phe Arg Cys Ser Cys Asp Thr Gly Tyr Met Leu Glu Ser 385 390 395 400

Asp Gly Arg Thr Cys Lys

405

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Glu Arg Cys Gly Ala Ser Ser Phe Thr Cys Ser Asn Gly Arg Cys lie 1 5 10 15

Ser Glu Glu Trp Lys Cys Asp Asn Asp Asn Asp Cys Gly Asp Gly Ser

20 25 30

Asp Glu Met Glu Ser Val Cys Ala Leu His Thr Cys Ser Pro Thr Ala

35 40 45

Phe Thr Cys Ala Asn Gly Arg Cys Val Gin Tyr Ser Tyr Arg Cys Asp 50 55 60

Tyr Tyr Asn Asp Cys Gly Asp Gly Ser Asp Glu Ala Gly Cys Leu Phe 65 70 75 80

Arg Asp Cys Asn Ala Thr Thr Glu Phe Met Cys Asn Asn Arg Arg Cys

85 90 95 lie Pro Arg Glu Phe lie Cys Asn Gly Val Asp Asn Cys His Asp Asn

100 105 110

Asn Thr Ser Asp Glu Lys Asn Cys Pro Asp Arg Thr Cys Gin Ser Gly

115 120 125

Tyr Thr Lys Cys His Asn Ser Asn lie Cys lie Pro Arg Val Tyr Leu 130 135 140

Cys Asp Gly Asp Asn Asp Cys Gly Asp Asn Ser Asp Glu Asn Pro Thr 145 150 155 160

Tyr Cys Thr Thr His Thr Cys Ser Ser Ser Glu Phe Gin Cys Ala Ser

165 170 175

12 Gly Arg Cys lie Pro Gin His Trp Tyr Cys Asp Gin Glu Thr Asp Cys 180 185 190

Phe Asp Ala Ser Asp Glu Pro Ala Ser Cys Gly His Ser Glu Arg Thr

195 200 205

Cys Leu Ala Asp Glu Phe Lys Cys Asp Gly Gly Arg Cys lie Pro Ser 210 215 220

Glu Trp lie Cys Asp Gly Asp Asn Asp Cys Gly Asp Met Ser Asp Glu 225 230 235 240

Asp Lys Arg His Gin Cys Gin Asn Gin Asn Cys Ser Asp Ser Glu Phe

245 250 255

Leu Cys Val Asn Asp Arg Pro Pro Asp Arg Arg Cys lie Pro Gin Ser

260 265 270

Trp Val Cys Asp Gly Asp Val Asp Cys Thr Asp Gly Tyr Asp Glu Asn

275 280 285

Gin Asn Cys Thr Arg Arg Thr Cys Ser Glu Asn Glu Phe Thr Cys Gly 290 295 300

Tyr Gly Leu Cys lie Pro Lys lie Phe Arg Cys Asp Arg His Asn Asp 305 310 315 320

Cys Gly Asp Tyr Ser Asp Glu Arg Gly Cys Leu Tyr Gin Thr Cys Gin

325 330 335

Gin Asn Gin Phe Thr Cys Gin Asn Gly Arg Cys lie Ser Lys Thr Phe

340 345 350

Val Cys Asp Glu Asp Asn Asp Cys Gly Asp Gly Ser Asp Glu Leu Met

355 360 365

His Leu Cys His Thr Pro Glu Pro Thr Cys Pro Pro His Glu Phe Lys 370 375 380

Cys Asp Asn Gly Arg Cys lie Glu Met Met Lys Leu Cys Asn His Leu 385 390 395 400

Asp Asp Cys Leu Asp Asn Ser Asp Glu Lys Gly Cys Gly lie Asn Glu

405 410 415

13 Cys His Asp Pro Ser lie Ser Gly Cys Asp His Asn Cys Thr Asp Thr 420 425 430

Leu Thr Ser Phe Tyr Cys Ser Cys Arg Pro Gly Tyr Lys Leu Met Ser

435 440 445

Asp Lys Arg Thr Cys Val Asp lie Asp Glu Cys Thr Glu Met Pro Phe 450 455 460

Val Cys Ser Gin Lys Cys Glu Asn Val lie Gly Ser Tyr lie Cys Lys 465 470 475 480

Cys Ala Pro Gly Tyr Leu Arg Glu Pro Asp Gly Lys Thr Cys Arg

485 490 495

14