BR112016030237B1 - METHOD FOR DETERMINING AN AMOUNT OF VITAMIN D ANALYTE IN A SAMPLE SUSPECTED OF CONTAINING THE 25OH VITAMIN D ANALYTE - Google Patents

Abstract

SPECIFIC BINDING PARTNERS FOR VITAMIN D EPIMERS IN VITAMIN D ASSAYS. The present invention relates to methods that include determining an amount of vitamin D analyte in a sample suspected of containing the vitamin D analyte. provided in an assay medium that includes the sample, a vitamin D epimer antibody that is specific for epimers of the vitamin D analyte, wherein the vitamin D epimer antibody does not bind to any detectable degree to the vitamin D analyte , and a vitamin antibody that is specific to the vitamin D analyte. The assay medium is incubated under conditions for binding of the vitamin D epimer antibody to the epimers of the vitamin D analyte and for binding of the vitamin antibody to the vitamin D analyte. vitamin D to form a vitamin D antibody-binding complex. The amount of vitamin D antibody-binding complex is determined and related to the amount of vitamin D analyte in the sample.

Description

[001] The Subject Application claims the benefit under 35 USC § 119(e) of US Provisional Application 62/018,020, filed June 27, 2014. The complete contents of the above-referenced Patent Application are hereby expressly incorporated herein by reference .

BACKGROUND

[002] This invention relates to compositions, methods and kits for determining the presence and/or quantity of vitamin D analytes, and metabolites thereof, in a sample suspected of containing them.

[003] Most small molecule compounds or haptens such as, for example, drugs and vitamins, exist in isomeric forms, of which only one form is active. In order to obtain an accurate assessment of the active form of an analyte, the presence of the non-active isomer of the analyte must be addressed. Assessments of both forms of an analyte, i.e., active and non-active forms, can lead to inaccuracies that can be harmful to an individual depending on the function of the active form of the analyte. Accurately assessing the level of each of a pair of isomeric analytes in biological samples is important especially where only one of the isomers is active and assessments that include the amount of the non-active isomer distort the level of the analyte in a sample. For example, assessing vitamin D levels in biological samples is important, as vitamin D deficiency is related to several disorders in mammals. In infants, for example, vitamin D assessments that include amounts of 3-epi isomers can lead to inaccurate assessment of vitamin D levels in the baby, which in turn can lead to a lack of appropriate supplementation. It is important to assess the active form of vitamin D so that a baby can receive appropriate vitamin D therapy if necessary.

[004] The term "vitamin D" refers to a group of fat-soluble secosteroids. In humans, vitamin D is unique because it can be ingested as cholecalciferol (vitamin D3) or ergocalciferol (vitamin D2) and because the body can also synthesize it (from cholesterol) when sunlight exposure is adequate. Due to this last property, vitamin D is considered by some to be an essential dietary vitamin, although most consider it an essential nutrient. Vitamin D has an important physiological role in the positive regulation of calcium ion homeostasis. Vitamin D3 is the form of the vitamin synthesized by animals. It is also a common supplement added to dairy products and certain food products as is vitamin D2.

[005] Both dietary and intrinsically synthesized vitamin D3 must undergo metabolic activation to generate bioactive metabolites. In humans, the initial step of vitamin D3 activation occurs primarily in the liver and involves hydroxylation to form the intermediate metabolite 25-hydroxycholecalciferol. Calcidiol is the main form of vitamin D3 in the circulatory system. Vitamin D2 also undergoes metabolic activation similar to 25-hydroxyvitamin D2. Collectively these compounds are called 25-hydroxyvitamin D (abbreviated 25(OH)D) and they are the main metabolites that are measured in serum to determine vitamin D status; 25(OH)D and its epimers are both pre-hormones that need to be converted into 1,25(OH)D to exert biological functions. The comparison of the bioactivity of 1,25(OH)D versus that of 3-epi-1,25(OH)D is complex.

[006] The vitamin D compounds, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 are epimeric at position 3 with the epimers being designated 25-hydroxyvitamin D3 and 3-epi-25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 and 3 -epi-25-hydroxyvitamin D2, respectively. Only one of the epimers of each of these compounds, namely 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2, respectively, are active. The structures for the epimers of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 are mentioned in figure 1.

[007] Assessment of vitamin D levels in biological samples is important, as vitamin D deficiency is related to several disorders in mammals. There is a need for reagents and methods for accurate and sensitive determinations of concentrations of vitamin D, epimeric forms of vitamin D, and vitamin D analogues and metabolites thereof in samples.

SUMMARY

[008] Some examples in accordance with the principles described here are directed to a method of determining an amount of vitamin D analyte in a sample suspected of containing the vitamin D analyte. A combination is provided in an assay medium that includes the sample, a vitamin D epimer binding partner that is specific for epimers of the vitamin D analyte in which the vitamin D epimer binding partner does not bind to any detectable degree to the vitamin D analyte, and a binding partner of vitamin D that is specific for the vitamin D analyte, and the assay medium is incubated under conditions for binding of the vitamin D epimer binding partner to the vitamin D analyte epimers and for binding of the vitamin D binding partner. vitamin D to the vitamin D analyte to form the vitamin D binding-partner complex. The amount of vitamin D binding-partner binding complex is determined and related to the amount of vitamin D analyte in the sample.

[009] Some examples in accordance with the principles described here are directed to a method as mentioned above, in which the vitamin D epimer binding partner, e.g. antibody, is constructed against a compound of formula I: (R1)p -(L)q-Z

[0010] where

[0011] or H, where at least one R1 is not H,

[0012] Y is O, S, CR, or NR4,

[0013]

[0014] -(CH2)w-C(O)-NH(CH2)y-C(O)-, -NR3-C(O)-,

[0015] R is independently H or alkyl,

[0016] R2 is independently H or alkyl,

[0017] R3, R4, R5 and R6 are independently H or alkyl, or R4 and R5 can be considered together to form a bond, or R5 and R6 can be considered together to form a bond,

[0018] R11 is H, alkyl, or acyl,

[0019] n is an integer from 1 to 10,

[0020] w is an integer from 0 to 10,

[0021] x is an integer from 1 to 10,

[0022] y is an integer from 1 to 10,

[0023] p is an integer from 1 to 10,

[0024] L is a linking group,

[0025] q is 0 or 1, and

[0026] Z is an immunogenic poly(amino acid) vehicle or a non-immunogenic poly(amino acid) vehicle, or constructed against two or more of the above compounds.

[0027] Some examples in accordance with the principles described here are directed to a method as mentioned above, in which the vitamin D epimer antibody is constructed against a compound of formula II: NHR1'-(CH2)r'-NR1' '-((CH2)r'-NR1''')s'-(CH2)r'-NR7'-Z'

[0028] in which

[0029] R1', R1'' or R1''' are all independently selected from and H,

[0030] wherein at least one of R1', R1'' or R1''' is not H,

[0031] n' is an integer from 1 to 10,

[0032] r' is independently an integer from 1 to 10,

[0033] s' is an integer from 1 to 10,

[0034] R7' is H or alkyl,

[0035] R11' is H, alkyl, or acyl, and

[0036] Z' is an immunogenic poly(amino acid) vehicle or a non-immunogenic poly(amino acid) vehicle.

[0037] Some examples in accordance with the principles described here are directed to a method of determining an amount of a vitamin D analyte in a sample suspected of containing the vitamin D analyte. The sample, a capture antibody that is a vitamin antibody specific for the vitamin D analyte, and a vitamin D epimer antibody that is specific for epimers of the vitamin D analyte wherein the vitamin D epimer antibody does not bind to any detectable degree to the vitamin D analyte are provided in combination in a test medium. The assay medium is incubated under conditions for binding of the vitamin antibody to the vitamin D analyte to form a vitamin D antibody-binding complex and for binding of the vitamin D epimer antibody to the vitamin D analyte epimers. The vitamin D antibody-binding complex is combined with a detection antibody that binds to the vitamin D analyte in the vitamin D antibody-binding complex wherein the detection antibody comprises a member of a signal production system. The signal produced by the signal production system is measured and related to the amount of vitamin D analyte in the sample.

BRIEF DESCRIPTION OF DRAWINGS

[0038] The drawings provided here are not to scale and are provided for the purposes of facilitating the understanding of certain examples in accordance with the principles described here and are provided by way of illustration and not limitation on the scope of the appended claims.

[0039] Figure 1 is a description of the chemical formulas for the epimeric forms of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2.

[0040] Figure 2 is a schematic diagram of a synthesis of compounds according to examples in accordance with the principles described here.

[0041] Figure 3 is a schematic diagram of a synthesis of compounds according to examples in accordance with the principles described here.

[0042] Figure 4 is a schematic diagram of a synthesis of compounds according to examples in accordance with the principles described here.

[0043] Figure 5 is a schematic diagram of a synthesis of compounds according to examples in accordance with the principles described here.

[0044] Figure 6 is a schematic diagram of a synthesis of compounds according to examples in accordance with the principles described here.

[0045] Figure 7 is a schematic diagram of a synthesis of compounds according to examples in accordance with the principles described here.

[0046] Figure 8 is a schematic diagram of a synthesis of compounds according to examples in accordance with the principles described here.

[0047] Figure 9 illustrates a comparison of standard curves generated using reagents with and without added anti-3-epimer-VD antibody.

[0048] Figure 10 is a standard curve for an immunoassay for determining 3-epi-25(OH)D in samples using anti-3-epimer-VD antibody.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

[0049] Binding partners such as, for example, antibodies, in accordance with the principles described herein can be employed to minimize or eliminate 3-epimer cross-reactivity in assays for non-epimeric forms of vitamin D analytes. Absorption of total non-epimeric vitamin D analyte caused by cross-reactivity of 3-epimer vitamin D with an antibody to vitamin D analyte can be substantially avoided by employing, as blocking agents, antibodies prepared against immunogens that are a compound of formula I in which Z is an immunogenic vehicle.

[0050] Some examples in accordance with the principles described here are directed to a method of determining an amount of vitamin D analyte in a sample suspected of containing the vitamin D analyte. A combination is provided in an assay medium that includes the sample, a vitamin D binding partner that is specific for the vitamin D analyte, and a vitamin D epimer binding partner that is specific for epimers of the vitamin D analyte in which the vitamin D epimer binding partner D does not bind to any detectable degree to the vitamin D analyte. As described in greater detail below, the assay can be homogeneous or heterogeneous, competitive or noncompetitive. In some examples, depending on the nature of the assay, a reagent is included that is a vitamin D analogue and in some examples, a third binding partner that is specific to the binding partner for the vitamin D analyte is employed. The assay medium is incubated under conditions for binding of the vitamin D epimer binding partner to the vitamin D analyte epimers and for binding of the vitamin D binding partner to the vitamin D analyte to form the vitamin D binding partner complex. vitamin D binding. The amount of vitamin D binding-partner complex is determined and related to the amount of vitamin D analyte in the sample.

Overview of assays for Vitamin D

[0051] As mentioned above, some examples in accordance with the principles described here are directed to a method of determining either or both the presence and the amount of a vitamin D analyte in a sample suspected of containing the vitamin analyte. D and may be referred to here as "vitamin D assays." In any of the examples described herein, a binding partner such as, for example, an antibody in accordance with the principles described herein i.e. specific for one or more vitamin D epimers can be employed as a blocking agent to reduce or eliminate interference of vitamin D epimers in an assay for the vitamin D analyte.

[0052] The phrase "vitamin D" refers to one or more of 25-hydroxyvitamin D; calcidiol; 1,25-dihydroxy vitamin D2; 1,25-dihydroxyvitamin D3; 1,25-dihydroxy vitamin D4; 1,25-dihydroxy vitamin D5; and 1,25-dihydroxy vitamin D6; including epimeric forms and metabolites of all of the above. Thus, vitamin D analyte includes vitamin D and vitamin D epimers as defined above. Some examples in accordance with the principles described here are directed to methods of determining either or both the presence and the amount of epimeric forms of vitamin D in a sample suspected of containing epimeric forms of vitamin D (such as, for example, 3- epi-25-hydroxyvitamin D3 or 3-epi-25-hydroxyvitamin D2) and may be referred to herein as "vitamin D epimer assays."

[0053] In one example, by way of illustration and not limitation, of a method for determining a vitamin D analyte, a combination is provided that comprises the sample, a binding partner such as, for example, an antibody to an epimer of vitamin D produced in accordance with the principles described herein, a binding partner such as, for example, an antibody to the vitamin D analyte and a conjugate comprising a vitamin D analogue and a member of a signal production system.

[0054] As mentioned above, the sample and reagents are provided "in combination in the medium." While the order of addition to the medium can be varied to form the combination, there will be certain preferences regarding some embodiments of the assay formats described here. In one example, by way of illustration and not limitation, the order of addition is to add all materials simultaneously and determine the effect that the test medium has on the signal as in a homogeneous test. In another example, by way of illustration and not limitation, each of the reactants, or groups of reactants, may be combined sequentially. In some embodiments, an incubation step may be involved subsequent to each addition as described above. In heterogeneous assays, separation and washing steps can also be employed after one or more incubation steps.

[0055] The phrase "vitamin D analog" refers to a compound that competes with a vitamin D analyte for a receptor such as an antibody to vitamin D or an antibody to an epimer of vitamin D. The vitamin D analog may be a modified vitamin D, where the modification provides a means of linking the vitamin D to another molecule such as, but not limited to, a carrier, a label, a small molecule, or a binding partner for a small molecule, e.g. . The vitamin D analogue can be linked to another molecule directly or indirectly through a linking group. The vitamin D analogue can be, for example, a molecule structurally related to vitamin D or vitamin D conjugated to another molecule through a linking group. In some examples in accordance with the principles described herein, the vitamin D analog may be a compound of formula I wherein Z is a poly(amino acid) label portion or a non-poly(amino acid) label portion or wherein Z is an immunogenic vehicle wherein such a compound of formula I competes with a vitamin D analyte for binding to a vitamin D antibody.

[0056] The sample to be analyzed is one that is suspected of containing a vitamin D analyte. The samples may be biological samples or non-biological samples. Biological samples can be from a mammalian individual or a non-mammalian individual. Mammalian individuals can be, for example, humans or other species of animals. Biological samples include biological fluids such as whole blood, serum, plasma, sputum, lymphatic fluid, semen, vaginal mucus, feces, urine, spinal fluid, saliva, stool, cerebrospinal fluid, tears, mucus, and the like; biological tissue such as hair, skin, cuts or tissue excised from organs or other parts of the body; and so on. In many cases, the sample is whole blood, plasma, or serum. Non-biological samples including, but not limited to, waste streams, for example, can also be analyzed using compound in accordance with the principles described here.

[0057] The sample can be prepared in any convenient medium, which can be, for example, an assay medium, which is described more fully here below. In some cases, a pretreatment may be applied to the sample such as, for example, to lyse blood cells. In some examples, such pretreatment is carried out in a medium that does not subsequently interfere with an assay.

[0058] The combination in the medium is subjected to the conditions for binding the vitamin D analyte and the vitamin D analogue to the vitamin D analyte antibody to form a complex. The amount of the complex is measured, where the amount of the complex is related to one or both of the presence and amount of the vitamin D analyte in the sample.

[0059] An assay for a vitamin D analyte can be carried out either without separation (homogeneous) or with separation (heterogeneous) of any of the test products or components. Heterogeneous assays usually involve one or more separation steps and can be competitive or non-competitive. Immunoassays may involve labeled or unlabeled reagents. Immunoassays involving unlabeled reagents usually comprise the formation of relatively large complexes involving one or more antibodies prepared from immunogenic conjugates in accordance with the principles described here. Such assays include, for example, immunoprecipitin and agglutination methods and corresponding light scattering techniques such as, for example, nephelometry and turbidemetry, for the detection of antibody complexes. Labeled immunoassays include, but are not limited to, chemiluminescence immunoassays, enzyme immunoassays, fluorescence polarization immunoassays, redioimmunoassays, inhibition assays, induced luminescence assays, and fluorescent oxygen channeling assays, for example.

[0060] A general group of immunoassays includes immunoassays using a limited concentration of an antibody in accordance with the principles described here. Another group of immunoassays involves the use of an excess of one or more of the main reagents such as, for example, an excess of an antibody in accordance with the principles described herein. Another group of immunoassays includes separation-free homogeneous assays in which the signal of a labeled vitamin D analog is modulated upon binding of the labeled vitamin D analog to an antibody produced in accordance with the principles described herein, thereby competing with an analyte of vitamin D that may be present in the sample.

[0061] As mentioned above, tests can be carried out either without separation (homogeneous) or with separation (heterogeneous) of any of the test products or components. Homogeneous immunoassays are exemplified by the EMIT® assay (Siemens Healthcare Diagnostics Inc., Deerfield, IL) described in Rubenstein, et al., United States Patent No. 3,817,837, column 3, line 6 to column 6, line 64; immunofluorescence methods such as those described in Ullman, et al., United States Patent No. 3,996,345, column 17, line 59, column 23, line 25; enzyme channeling immunoassays ("ECIA") such as those described in Maggio, et al., United States Patent No. 4,233,402, column 6, line 25 to column 9, line 63; the fluorescence polarization immunoassay ("FPIA") as described, for example, in, among others, United States Patent No. 5,354,693; and enzyme immunoassays such as enzyme-linked immunosorbent assay ("ELISA"). Exemplary heterogeneous assays are the radioimmunoassay, described in Yalow, et al., J. Clin. Invest. 39:1157 (1960). The relevant portions of the above descriptions are all incorporated herein by reference.

[0062] Other enzyme immunoassays are the enzyme-modulated media immunoassay ("EMMIA") described by Ngo and Lenhoff, FEBS Lett. (1980) 116:285-288; the substrate-labeled fluorescence immunoassay ("SLFIA") described by Oellerich, J. Clin. Chem. Clinic. Biochem. (1984) 22:895-904; the combined enzyme donor immunoassays ("CEDIA") described by Khanna, et al., Clin. Chem. Acta (1989) 185:231-240; homogeneous particle labeled immunoassays such as particle-enhanced turbidimetric inhibition immunoassays ("PETINIA"), particle-enhanced turbidimetric immunoassay ("PE-TIA"); Chromium Affinity Mediated Immunoassay ("ACMIA") assay format, which is described in United States Patent Nos. 7,186,518, 5,147,529, 5,128,103, 5,158,871, 4,661,408, 5,151,348, 5,302,532, 5,422,284, 5,447,870, and 5,434,051, the descriptions of which they are incorporated here in its totality; for example.

[0063] Other tests include acridinium ester label tests such as those described in United States Patent Nos. 6,355,803; 6,673.560; 7,097,995 and 7,319,041, the relevant descriptions of which are incorporated herein by reference. A particular example of an acridinium ester label assay is an acridinium ester label immunoassay using paramagnetic particles as a solid phase ("ADVIA" immunoassay). Other assays include sol particle immunoassay ("SPIA"), disperse dye immunoassay ("DIA"); the metalloimmunoassay ("MIA"); enzyme membrane immunoassays ("EMIA"); and luminoimmunoassays ("LIA"). Other types of assays include immunosensor assays involving monitoring of changes in the optical, acoustic and electrical properties of the present conjugate upon vitamin D analyte binding. Such assays include, for example, optical immunosensor assays, acoustic immunosensor assays, semiconductor immunosensor assays. , electrochemical transducer immunosensor assays, pontentiometric immunosensor assays, amperometric electrode assays.

[0064] Heterogeneous assays usually involve one or more separation steps and can be competitive or non-competitive. A variety of non-competitive competitive heterogeneous assay formats are described in Davalian, et al., United States Patent No. 5,089,390, column 14, line 25 to column 15, line 9, incorporated herein by reference. In an example of a competitive heterogeneous assay, a support having an antibody to a vitamin D analyte bound thereto is contacted with a medium containing the sample suspected of containing the vitamin D analyte and a compound labeled in accordance with the principles described herein. as a labeled vitamin D analogue. The sample suspected of containing the vitamin D analyte was treated with an antibody so that the epimeric form of the analyte binds to all epimeric forms of the vitamin D analyte. The vitamin D analyte in the sample competes for binding to the antibody as to the vitamin D analyte, with the labeled vitamin D analogue. After separation of the support and medium, the label activity of the support or medium is determined by conventional techniques and is related to the amount of vitamin D analyte in the sample. In a variation of the above competitive heterogeneous assay, the support comprises a vitamin D analogue as the labeled reagent and vitamin antibody comprises a label.

[0065] In some examples, a sample to be analyzed is combined in an assay medium with an antibody to the vitamin D analyte and labeled vitamin D analogue. The sample suspected of containing the vitamin D analyte was treated with an antibody to the epimeric form of the analyte binding all epimeric forms of the vitamin D analyte. The medium is examined for either or both of the presence and amount of a complex comprising the labeled vitamin D analog and the antibody to the vitamin D analyte where the presence and/or amount of such complex indicates the presence and/or amount of the vitamin D analyte in the sample.

[0066] In some examples in accordance with the principles described here, the sample to be analyzed is subjected to a pre-treatment to release the vitamin D analyte from endogenous binding substances such as, for example, plasma or serum protein that binds vitamin D. Release of the vitamin D analyte from endogenous binding substances can be carried out, for example, by adding a digesting agent or a releasing agent or a combination of a digesting agent and a releasing agent used sequentially. The digesting agent is one that breaks down endogenous binding substances so that they can no longer bind vitamin D. Such agents include, but are not limited to, proteinase K and proteinase K and protein denaturing agents such as, for example, example, detergents (sodium dodecyl sulfate, for example). Release agents for releasing vitamin D from endogenous binding substrates include, by way of illustration and not limitation, acid denaturing agents such as, for example, salicyclic acid, warfarin, sulfonic acids, toluene sulfonic acids, naphthalene sulfonic acid. -nico, anilinonaphthalene sulfonic acids (ANS) (including, for example, 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS) and 8-anilinonaphthalene-1-sulfonic acid (8-ANS)), salicylic acids and derivatives of the above.

[0067] Conditions such as, for example, duration, temperature, pH and concentration of the releasing agent in the medium for carrying out the digestion or releasing actions are dependent on the nature of the endogenous binding substances, the nature of the sample, and the nature of the release agent, for example. In general, the conditions are sufficient to obtain the desired effect or function. In some examples according to the principles described herein, an effective concentration of release agent is from about 0.01 to about 20 mg/mL, or about 0.01 to about 10 mg/mL, or about 0.01 to about 5 mg/mL, or about 0.1 to about 20 mg/mL, or about 0.1 to about 10 mg/mL, or about 0.1 to about 5 mg /mL, or about 0.1 to about 1 mg/mL. Sample pretreatment to release the vitamin D analyte from endogenous binding substances can be performed as a separate step before conducting an assay or as a first step in an assay. In any case, one or more reagents may be required to stop the action of the digestion agent and/or the releasing agent.

[0068] Conditions for conducting the assays include performing the assay in a buffered aqueous medium at a moderate pH, generally providing optimal assay sensitivity. The aqueous medium may be water alone or may include from 0.1 to about 40 volume percent of a cosolvent. The pH for the medium will be in the range of about 4 to about 11, or in the range of about 5 to about 10, or in the range of about 6.5 to about 9.5, for example. The pH will generally be a compromise between the optimal binding of the binding members of any specific binding pairs, the optimal pH for other assay reagents such as members of the signal-producing system, and so on. Various buffers can be used to obtain the desired pH and maintain the pH during the assay. Illustrative buffers include, by way of illustration and not limitation, borate, phosphate, carbonate, TRIS, barbital, PIPES, HEPES, MES, ACES, MOPS, and BICINE, for example. The particular buffer employed is not critical, but in an individual assay, one or more buffers may be preferred.

[0069] Various auxiliary materials can be used in the test methods. For example, in addition to buffers, the medium may comprise stabilizers for the medium and reagents employed. In some embodiments, in addition to these additives, proteins may be included, such as, for example, albumins; organic solvents such as, for example, formamide; quaternary ammonium salts; polyanions such as, for example, dextran sulfate; binding enhancers, for example polyalkylene glycols; polysaccharides such as, for example, dextran and trehalose. The medium may also comprise agents for preventing the formation of blood clots. Such agents are well known in the art and include, but are not limited to, EDTA, EGTA, citrate, heparin, for example. The medium may also comprise one or more preservatives such as, but limited to, sodium azide, neomycin sulfate, PROCLIN® 300, Streptomycin, for example. The medium may additionally comprise one or more surfactants. Any of the above materials, if employed, are present in a concentration or quantity sufficient to obtain the desired effect or function.

[0070] One or more incubation periods may be applied to the medium at one or more intervals including any intervals between additions of various reagents employed in an assay including those mentioned above. The medium is generally incubated at a temperature and for a time sufficient for binding of various components of the reagents and binding of vitamin D in the sample to occur. Moderate temperatures are normally used to carry out the method and a generally constant temperature, preferably room temperature, during the evaluation period. In some examples, incubation temperatures range from about 5° to about 99°C, or from about 15°C to about 70°C, or from about 20°C to about 45°C, e.g. . The time period for incubation, in some examples, is from about 0.2 seconds to about 24 hours, or about 1 second to about 6 hours, or about 2 seconds to about 1 hour, or about from 1 minute to about 15 minutes, for example. The period of time depends on the temperature of the medium and the binding rate of the various reactants, which is determined by the association rate constant, concentration, dissociation rate constant and binding constant.

[0071] In one example of a method for determining a vitamin D analyte in a sample suspected of containing the vitamin D analyte, a combination is provided in a medium, where the combination includes the sample, a releasing agent (if the sample has not been pretreated to release the vitamin D analyte from endogenous binding substances), an antibody to vitamin D, and a labeled vitamin D analog where the label is a poly(amino acid) label or a not poly(amino acid). Either before, or concomitantly with, the sample suspected of containing the vitamin D analyte is treated with an antibody to the epimeric form of the analyte binding all epimeric forms of the vitamin D analyte. The medium is examined for one or both of the presence and amount of either a complex comprising vitamin D and the antibody to vitamin D or a complex comprising the labeled compound and antibody to vitamin D. The presence and/or amount of one or both complexes indicates the presence and/or amount of the vitamin D analyte in the sample.

[0072] Some known assays use a signal production system (sps) that employs the first and second members of sps. The designation "first" and "second" is completely arbitrary and is not intended to suggest any order or ranking among sps members or any order of addition of sps members in the present methods. Sps members may be related in that activation of one sps member produces a product such as, for example, light or an activated product, which results in activation of another sps member.

[0073] In some assay modalities, the members and sps comprise a sensitizer such as, for example, a photosensitizer, and a chemiluminescent composition where activation of the sensitizer results in a product that activates the chemiluminescent composition. The second sps member generally generates a detectable signal that relates to the amount of bound and/or unbound sps member, that is, the amount of bound or unbound sps member to the vitamin D analyte being detected or to a compound in accordance with the principles described here. In some examples in accordance with the principles described herein, one of the sensitizing reagent or chemiluminescent reagent comprises the present compound reagent.

[0074] In the example tests described below, the sample suspected of containing the vitamin D analyte is treated, before or concomitantly with, with an antibody to the epimeric form of the analyte binding all epimeric forms of the vitamin D analyte.

[0075] As a particular example, an induced luminescence immunoassay can be employed. The induced luminescence immunoassay is referred to in United States Patent No. 5,340,716 (Ullman), the disclosure of which is incorporated herein by reference. In one method, the assay uses a particle having associated with it a photosensitizer where a vitamin D analogue is linked to the particle (particle-compound reagent). The chemiluminescent reagent comprises an antibody to the vitamin D analyte. The vitamin D analyte competes with the particle-compound reagent for binding to the vitamin D antibody. If the vitamin D analyte is present, the lower the number of particle-compound reagent molecules that are in intimate proximity to the chemiluminescent reagent. Therefore, there will be a decrease in the test signal. The photosensitizer generates singlet oxygen and activates the chemiluminescent reagent when the two labels are in close proximity. The activated chemiluminescent reagent subsequently produces light. The amount of light produced is related to the amount of complex formed, which in turn is related to the amount of vitamin D analyte present in the sample.

[0076] In another particular example of an induced luminescence immunoassay, the assay uses a particle having associated with it a chemiluminescent compound where a vitamin D analogue is bound to the particle (particle-compound reagent). The photosensitizing reagent comprises an antibody to vitamin D. The vitamin D analyte competes with the particle-compound reagent for binding to the vitamin D antibody. If the vitamin D analyte is present, the smaller the number of molecules of particle-compound reagent that are in close proximity to the photosensitizing reagent. Therefore, there will be a decrease in the test signal. The photosensitizer generates singlet oxygen and activates the chemiluminescent compound of the particle-compound reagent when the two labels are in close proximity. The activated chemiluminescent compound subsequently produces light. The amount of light produced is related to the amount of complex formed, which in turn is related to the amount of vitamin D analyte present in the sample.

[0077] In another particular example of an induced luminescence assay, a photosensitizing particle is employed that is conjugated to a binding partner for a small molecule such as, for example, avidin or streptavidin (which are binding partners for biotin). . A vitamin D analog comprising biotin (compound-biotin reagent) is also employed. A chemiluminescent reagent comprising an antibody to the vitamin D analyte is employed as part of the detection system. The reaction medium is incubated to allow the avidin or streptavidin of the photosensitizing particles to bind to the compound-biotin reagent by virtue of the bond between the avidin and biotin and to also allow the antibody to the vitamin D analyte that is part of the reagent. chemiluminescent bind to the vitamin D analyte or vitamin D analogue that is now bound to the photosensitizing particles. Then, the medium is irradiated with light to excite the photosensitizer which is capable, in its excited state, of activating oxygen to a singlet state. Because less of the chemiluminescent reagent is now in close proximity to the photosensitizer because of the presence of the vitamin D analyte, there is less activation of the chemiluminescent reagent by singlet oxygen and less luminescence. The medium is then examined for the presence and/or amount of luminescence or light emitted, the presence thereof being related to the presence and/or amount of the vitamin D analyte where a decrease in signal is observed in the presence of the vitamin D analyte. D.

[0078] In another particular example of an induced luminescence assay, a photosensitizing particle is employed that is conjugated to a binding partner for a small molecule such as, for example, avidin or streptavidin (which are binding partners for biotin ). A conjugated reagent comprises an antibody to the vitamin D analyte conjugated to biotin. A vitamin D analogue is employed where the compound is linked to a chemiluminescent particle (chemiluminescent-compound reagent) is also employed. The reaction medium is incubated to allow the avidin or streptavidin of the photosensitizing particles to bind to the antibody-biotin reagent by virtue of the bond between the avidin and biotin and also to allow the compound for the vitamin D analyte to bind to the vitamin D analyte. vitamin D if present in the sample and the vitamin D analogue that is part of the chemiluminescent-compound reagent. Then, the medium is irradiated with light to excite the photosensitizer which is capable, in its excited state, of activating oxygen to a singlet state. Because less of the chemiluminescent reagent compound is now in close proximity to the photosensitizer because of the presence of the vitamin D analyte, there is less activation of the chemiluminescent reagent by singlet oxygen and less luminescence. The medium is then examined for the presence and/or amount of luminescence or light emitted, the presence thereof being related to the presence and/or amount of the vitamin D analyte where a decrease in signal is observed in the presence of the vitamin D analyte. D.

[0079] Another example, by way of illustration and not limitation, of an assay format for detecting a vitamin D analyte is the ACMIA assay format. As for the ACIA assay format, chromium particles, which are coated with a vitamin D analogue (chromium particle reagent), are employed as a first component. A second component is an antibody to the vitamin D analyte. This antibody, cross-linked to a reporter enzyme (e.g., β-galactosidase) to form an antibody-enzyme conjugate, is added to a reaction vessel in an amount in excess, that is, an amount greater than that required to bind all of the vitamin D analyte that could be present in a sample. A sample, which is previously subjected to treatment with a releasing agent, is treated with an antibody to the vitamin D analyte, which binds to the vitamin D analyte in the sample. The antibody-enzyme conjugate is mixed with the sample in the medium to allow the vitamin D analyte to bind to the antibody. Next, chromium particle reagent is added to bind any excess antibody-enzyme conjugate. Next, a magnet is applied, which pulls all chromium particles and excess antibody-enzyme out of the suspension, and the supernatant is transferred to a final reaction vessel. The reporter enzyme substrate is added to the final reaction vessel, and enzyme activity is spectrophotometrically measured as a change in absorbance over time. The amount of this signal is related to the amount of vitamin D analyte in the sample.

[0080] Another example of an assay for vitamin D in accordance with the principles described here is an acridinium ester label immunoassay using paramagnetic particles as a solid phase (ADVIA immunoassay). The detection system employed for this example of a vitamin D assay includes a small molecule-labeled vitamin D analog (capture moiety) as the small molecule conjugate or capture conjugate, binding partner for the coated paramagnetic latex particles. by small molecule as a solid phase (SP), and an acridinium ester labeled antibody to the vitamin D analyte (detection antibody). The small molecule may be, for example, biotin or fluorescein and the respective binding partner may be streptavidin or antibody to fluorescein. The vitamin D analogue can be attached to the small molecule directly or through a linking group such as, for example, a protein, for example, bovine serum albumin (BSA). The vitamin D analyte in a patient sample competes with the vitamin D analogue of the capture moiety for binding to the acridinium ester labeled anti-vitamin D detection antibody. The sample suspected of containing vitamin D is subjected to pre-treatment with 1,8-ANS. The assay can be performed on a Centaur®, Centaur® XP or Centaur® CP apparatus (Siemens Healthcare Diagnostics Inc., Newark DE) according to the manufacturer's guidelines provided with it. The amount of this signal is related to the amount of epimeric vitamin D analyte in the sample.

[0081] Another example of an assay for a vitamin D analyte in accordance with the principles described here is an acridinium ester label immunoassay using paramagnetic particles as a solid phase (ADVIA immunoassay). The detection system employed for this example of a vitamin D assay includes a small molecule labeled antibody to the vitamin D analyte (capture antibody) such as biotin conjugate or capture conjugate, streptavidin-coated paramagnetic latex particles such as a solid phase (SP), and a vitamin D analog labeled by acridinium ester (sensing hapten). The acridinium ester label may be directly linked to the vitamin D analogue to form the detection hapten or a linker group may be employed including, for example, a protein such as, for example, BSA. Vitamin D analyte in a patient sample competes with the acridinium ester labeled detection hapten for binding with anti-vitamin D antibody. The sample suspected of containing vitamin D is subjected to pretreatment with 1,8-ANS . The assay can be performed on a Centaur®, Centaur® XP or Centaur® CP apparatus (Siemens Healthcare Diagnostics Inc., Newark DE) according to the manufacturer's guidelines provided with it. In the variations of the acridinium ester assays above, the small molecule may be, for example, biotin or fluorescein. The amount of this signal is related to the amount of epimeric vitamin D analyte in the sample.

[0082] The concentration of the vitamin D analyte in a sample that can be evaluated generally ranges from about 10-5 to about 10-17 M, or from about 10-6 to about 10-14 M, e.g. . Considerations such as whether the assay is qualitative, semiquantitative, or quantitative (with respect to the amount of vitamin D analyte present in the sample), the particular detection technique, and the expected concentration of the vitamin D analyte typically determine the concentrations of the various reagents.

[0083] The concentration of the antibody or antibodies to the epimeric forms of the analyte is dependent on one or more of the suspected concentration of the epimeric forms of the analyte and the expected concentration of the vitamin D (non-epimeric) analyte, for example. In some examples, the concentration of the antibody or antibodies to the epimeric forms of the analyte is sufficient to bind all epimeric forms of the vitamin D analyte in the sample. In some examples, the concentration of the antibody or antibodies to the epimeric forms of the analyte is in excess based on the expected concentration of the epimeric forms of the analyte in the sample.

[0084] The concentrations of the various reagents in the assay medium will generally be determined by the concentration range of interest of the vitamin D analyte, the nature of the assay, and the like. However, the final concentration of each of the reagents is usually determined empirically to optimize the sensitivity of the assay over the range of interest. That is, a variation in vitamin D analyte concentration that is of significance must provide a precisely measurable signal difference. Considerations such as the nature of the signal-producing system and the nature of the analytes typically determine the concentrations of the various reagents.

[0085] As mentioned above, the sample and reagents are provided in combination in the medium. While the order of addition to the medium can be varied, there will be certain preferences regarding some embodiments of the assay formats described here. The simplest order of addition, certainly, is to add all materials simultaneously and determine the effect that the test medium has on the signal as in a homogeneous test. Alternatively, each of the reactants, or groups of reactants, can be combined sequentially. In some embodiments, an incubation step may be involved subsequent to each addition as described above. In heterogeneous assays, washing steps can also be employed after one or more incubation steps.

Compounds

[0086] As mentioned above, some examples according to the principles described here are directed to binding partners such as, for example, aptamers and antibodies, constructed against compounds of formula I: (R1)p-(L)q-Z

[0087] in which

[0088] or H, where at least one R1 is not H,

[0089] Y is O, S, CR, or NR4,

[0090] X is -O-(CH2)n-C(O)-, -(CH2)w-C(O)-, or -NR3-C(O)-; In some examples, O)-, or

[0091] -(CH2)w-C(O)-NH(CH2)y-C(O)- when Y is O or S, for example, or -NR3-C(O)- when Y is CR, for example,

[0092] R is independently H or alkyl,

[0093] R2 is independently H or alkyl,

[0094] R3, R4, R5 and R6 are independently H or alkyl, or R4 and R5 can be considered together to form a bond, or R5 and R6 can be considered together to form a bond,

[0095] R11 is H, alkyl, or acyl,

[0096] n is an integer from 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8 to 9, or 9 to 10, for example,

[0097] w is an integer from 0 to 10, 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8 to 9, or 9 to 10, for example,

[0098] x is an integer from 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8 to 9, or 9 to 10, for example,

[0099] y is an integer from 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8 to 9, or 9 to 10, for example,

[00100] p is an integer from 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8 to 9, or 9 to 10, for example,

[00101] L is a linking group,

[00102] q is 0 or 1, and

[00103] Z is OR8 where R8 is H, alkyl, or NR9R10 where R9 and R10 are independently H or alkyl, a poly(amino acid) portion of immunogenic carrier, a non-immunogenic poly(amino acid) carrier portion, the poly(amino acid) label portion, a non-poly(amino acid) label portion, a poly(amino acid) non-label portion, a non-immunogenic poly(amino acid carrier) portion, or a support; and including mixtures of two or more of the above compounds.

[00104] As used herein, the term "alkyl" includes those alkyl groups of a designated number of carbon atoms of linear, branched or cyclic configuration. Examples of "alkyl" include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec- and tert-butyl, pentyl, hexyl, heptyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbonyl , for example. In some examples, alkyl contains 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2 , or 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10, or 3 to 9 , or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6 , or 4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7 , or 7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8 to 9, or 9 to 10, carbon atoms, which may be unsubstituted or one or more of which may be substituted by one or more hydroxy, alkoxy 1 to 5, or 1 to 4, 1 to 3, or 1 to 2, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 5, or 3 to 4, or 4 to 5 carbon atoms.

[00105] As used here, the term "acyl" means R12C(O)- where R12 is alkyl or aryl.

[00106] As used herein, the term "aryl" means an organic radical derived from an aromatic hydrocarbon by the removal of an atom and containing one or more aromatic rings, generally four aromatic rings, such as, for example, phenyl (from benzene) , naphthyl (from naphthalene), etc., for example, phenyl, naphthyl, fenatrinyl.

[00107] As used herein, the phrase "linking group" refers to a chemical moiety that may comprise about 2 to about 50 atoms, or 4 to about 30 atoms, not containing hydrogen and may comprise a chain of 2 to about 30 atoms, or 3 to about 20 atoms, are all independently selected from the group normally consisting of carbon, oxygen, sulfur, nitrogen, and phosphorus. In some examples, part or all of the linking group may be a portion of the molecule being linked such as, but not limited to, an amino acid residue in a poly(amino acid), for example. The number of heteroatoms in the linking group can be in the range of 0 to about 20, or 1 to about 15, or about 2 to about 10. The linking group can be aliphatic or aromatic. When heteroatoms are present, oxygen is normally present as oxo or oxy, bound to carbon, sulfur, nitrogen or phosphorus; nitrogen is normally present as nitro, nitroso or amino, normally bound to carbon, oxygen, sulfur or phosphorus; sulfur is analogous to oxygen; while phosphorus is bonded to carbon, sulfur, oxygen or nitrogen, usually as mono- or diester of phosphonate and phosphate. Common functionalities in forming a covalent bond between the linking group and the molecule to be conjugated are alkylamine, amidine, thioamide, ether, urea, thiourea, guanidine, azo, thioether and carboxylate, sulfonate, and phosphate esters, amides and thioesters.

[00108] For the most part, when a linking group has a linking functionality (functionality for reacting with a moiety) such as, for example, a non-oxocarbonyl group including nitrogen and sulfur analogues, a phosphate group, a amino, alkylating agent such as halo or tosylalkyl, oxy (hydroxyl or the sulfur analogue, mercapto) oxocarbonyl (e.g. aldehyde or ketone), or active olefin such as a vinyl sulfone or α-, p-unsaturated ester , these functionalities are linked to amine groups, carboxyl groups, active olefins, alkylating agents, for example, bromoacetyl. Where an amine and carboxylic acid or its nitrogen derivative or phosphoric acid are linked, amides, amidines and phosphoramides are formed. Where mercaptan and olefin are linked, thioethers are formed. Where mercaptan and an alkylating agent are linked, thioethers are formed. Where aldehyde and an amine are linked under reducing conditions, an alkylamine is formed. Where the ketone or aldehyde and hydroxylamine (including derivatives thereof, where a substituent is in place of the hydrogen of the hydroxyl group) are bonded, an oxime functionality (=N-O-) is formed. Where a carboxylic acid or phosphate acid and an alcohol are linked, esters are formed.

[00109] As used herein, the term "immunogenic vehicle" means a group or moiety that is conjugated to a hapten. The conjugate of the immunogenic vehicle and the hapten can be injected into an organism capable of eliciting an immune response such as, but not limited to, a mammal, a bird (e.g., chicken or pigeon), an amphibian, or a reptile; or the conjugate may be used to inoculate an in vitro sample (mammal, including human, bird, amphibian or reptile) or may otherwise be employed in a technique for producing a binding partner for the hapten.

[00110] The phrase "binding partner" refers to a molecule that is a member of a specific binding pair, which is one of two different molecules that specifically bind to, and is hereby defined as complementary to, the other molecule . For example, a specific binding pair member may have an area on the surface or in a cavity that specifically binds to a particular spatial and polar organization of the other specific binding pair members. The binding partner may be, by way of illustration and not limitation, an antibody or an aptamer (e.g., nucleic acid aptamer or peptide aptamer), for example. In one example, an immunogenic carrier can be employed as an immunogen to induce an immune response and elicit the production of a binding partner for a hapten. Other techniques include phage display and in vitro selection. Immunogenic vehicles are also sometimes referred to as antigenic vehicles. In some examples in accordance with the principles described herein, immunogens comprising immunogenic carriers, including poly(amino acid) and non-poly(amino acid) immunogenic carriers, are synthesized and used to prepare the antibodies. Haptens are compounds capable of binding especially to the corresponding antibodies, but they themselves do not act as imogens (or antigens) for the preparation of antibodies. Consequently, a hapten is linked to an immunogenic carrier, which can be used, for example, to construct antibodies.

[00111] The molecular weight range (in Daltons) for poly(amino acids) that are immunogenic carriers is from about 5,000 to about 10,000,000, or about 20,000 to about 600,000, or about 25,000 to about of 250,000, for example. "Poly(amino acid) immunogenic carrier moieties" include proteins such as, for example, albumins, serum proteins, for example, globulins, ocular lens proteins and lipoproteins. Illustrative proteins include, but are not limited to, bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), egg ovalbumin, and bovine gamma globulin (BGG), for example. "Non-poly(amino acid) immunogenic carrier portions" include polysaccharides, nucleic acids and particles (biological and synthetic materials). A wide variety of immunogenic vehicles are described in Davalian, et al., United States Patent No. 5,089,390, column 4, line 57 to column 5, line 5, which is incorporated herein by reference.

[00112] As mentioned above, the immunogenic carrier moiety can be a polysaccharide, which is a high molecular weight polymer of monosaccharides that can be prepared naturally or synthetically and generally involves repeated condensations of monosaccharides. Examples of polysaccharides are starches, glycogen, cellulose, carbohydrate gums such as gum arabic, agar and so on. The polysaccharide may also contain poly(amino acid) residues and/or lipid residues.

[00113] As used herein, the term "label" includes poly(amino acid) labels and non-poly(amino acid) labels. The term "poly(amino acid) label moieties" includes labels that are proteins such as, but not limited to, enzymes, antibodies, peptides, and immunogens, for example. With label proteins such as, for example, enzymes, the average weight molecular weight range will be from about 10,000 to about 600,000 or from about 10,000 to about 300,000. There is generally at least one compound in accordance with the principles described herein (analogous group) per about 200,000 molecular weight, or at least about 1 per about 150,000 molecular weight, or at least about 1 per about 100,000 molecular weight. molecular weight, or at least about 1 per about 50,000 molecular weight, or at least about 1 per 40,000, molecular weight, or at least about 1 per 30,000 molecular weight, or at least 1 per 20,000 molecular weight molecular, or at least one per 10,000 molecular, or at least one per 5,000 molecular weight, for example, of the protein. In the case of enzymes, the number of analogous groups is generally from 1 to about 20, about 2 to about 15, about 3 to about 12, or about 6 to about 10.

[00114] Enzymes include, by way of illustration and not limitation, redox enzymes such as, for example, dehydrogenases, for example, glucose-6-phosphate dehydrogenase (G6PDH) and lactate dehydrogenase; enzymes involving the production of hydrogen peroxide and the use of hydrogen peroxide to oxidize a dye precursor such as, for example, horseradish peroxidase, lactoperoxidase and microperoxidase; hydrolases such as, for example, alkaline phosphatase and β-galactosidase; luciferases such as, for example firefly luciferase, and bacterial luciferase; transferases; combinations of enzymes such as, but not limited to, saccharide oxidases, e.g., glucose and galactose oxidase, or heterocyclic oxidases, such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidase a dye precursor, i.e. , peroxidase such as horseradish peroxidase, lactoperoxidase or microperoxidase, for example.

[00115] As used herein, the term "non-poly(amino acid) labels" includes those labels that are not proteins. The non-poly(amino acid) label is capable of being detected directly or is detectable through a reaction that produces a detectable signal. The non-poly(amino acid) label may be isotopic or non-isotopic may be, by way of illustration and not limitation, a radioisotope, a luminescent compound (which includes, but is not limited to, fluorescent compounds and chemiluminescent compounds, for example), a polynucleotide encoding a catalyst, a promoter, a dye, a coenzyme, an enzyme substrate, a radioactive group, a small organic molecule (molecular weight 200 to 2,000), a particle, and an amplifiable polynucleotide sequence, for example.

[00116] As mentioned above, a "small organic molecule" has a molecular weight of about 200 to about 2,000, or about 200 to about 1,500, or about 200 to about 1,000, or about 200 to about of 500. Such "small organic molecules" include, but are not limited to, biotin molecules, fluorescent molecules (such as fluorescein and rhodamine, for example), chemiluminescent molecules, and dinitrophenol, for example. A binding partner for a small organic molecule is a molecule that specifically recognizes and binds to the small molecule. Binding partners for a small molecule are defined by the nature of the small molecule and include, but are not limited to, avidin, streptavidin, antibody to the small organic molecule (which includes, but is not limited to, antibody to a fluorescent molecule (such as such as antibody to fluorescein and antibody to rhodamine, for example), antibody to a chemiluminescent molecule, and antibody to dinitrophenol, for example.

[00117] As used herein, the terms "poly(amino acid) non-label portion" and "poly(amino acid) non-immunogenic carrier portion" refer to poly(amino acids) that are not normally considered immunogenic labels or carriers although such portions may be labels or immunogenic vehicles in certain cases. For example, an antibody may not be considered a label, but may be a label if the antibody is modified to include a signal-producing portion of a signal-producing system. Furthermore, an antibody may not be considered an immunogenic vehicle, but is still capable of being an immunogenic vehicle in certain cases due to its high molecular weight.

[00118] In some examples, the non-poly(amino acid) label may be selected from the group consisting of supports, magnetic particles, acridinium esters, a combination of magnetic particles and acridinium esters (such as, for example, paramagnet particles). - cas labeled by acridinium ester), chemiluminescent particles and sensitizing particles.

[00119] The term "covalent" refers to the bonding of molecules such as by a direct connection, for example, a chemical bond between molecules. The term "non-covalent" refers to the binding of molecules involving specific bonding between complementary specific binding pair (sbp) members that are attached to the molecules.

[00120] In some examples, compounds according to the principles described here may be associated with a support, for example, by covalent or non-covalent bonding. As mentioned above, in some examples according to the principles described herein, R2 may be a support, which may comprise a water-insoluble, organic or inorganic, solid or fluid material and which may be transparent or partially transparent. The support may have any of a variety of forms, such as, but not limited to, a particle (particulate support) including bead, a film, a membrane, a tube, a cavity, a strip, a rod, a fiber, or a surface. flat such as, for example, a plate or paper, for example. The support may or may not be suspended in the environment in which it is used. Examples of suspendable supports are polymeric materials such as latex, lipid bilayers or liposomes, oil droplets, cells and hydrogels, and magnetic particles, for example. Other carrier compositions include polymers such as, by way of illustration and not limitation, nitrocellulose, cellulose acetate, poly(vinyl chloride), polyacrylamide, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly (ethylene terephthalate), nylon, poly(vinyl butyrate), for example, used alone or in conjunction with other materials. The support may or may not also be labeled with a dye, catalyst or other detectable group, for example.

[00121] In some examples according to the principles described here, the support may be a particle. The particles have an average diameter of at least about 0.02 microns and no more than about 100 microns. In some examples, the particles have an average diameter of about 0.05 microns to about 20 microns, or from about 0.3 microns to about 10 microns. The particle may be organic or inorganic, swellable or non-swellable, porous or non-porous, preferably of a density approaching that of water, generally from about 0.7 g/mL to about 1.5 g/mL, and composed of material which can be transparent, partially transparent, or opaque. The particles may be biological materials such as cells and microorganisms, for example, erythrocytes, leukocytes, lymphocytes, hybridomas, streptococcus, Staphylococcus aureus, and E. coli, viruses, for example. The particles may also be particles comprising organic and inorganic polymers, liposomes, latex particles, magnetic or non-magnetic particles, phospholipid vesicles, chylomicrons, lipoproteins, and the like. In some examples, the particles are chromium dioxide (chromium) particles or latex particles.

[00122] Magnetic particles include paramagnetic particles, ferromagnetic particles and diamagnetic particles. Such particles include, but are not limited to, transition metals from periods 4-7 of the Periodic Table including chromium, copper, cobalt, aluminum, manganese, iron, and nickel, for example.

[00123] Chemiluminescent particles are particles that have a chemiluminescent compound associated with them. The phrase "associated therewith" as used herein means that a compound such as, for example, a chemiluminescent compound and a particle may be associated by direct or indirect binding, adsorption, absorption, incorporation, or solution, for example. Examples of chemiluminescent compounds that may be used are those mentioned in United States Patent Nos. 5,340,716 and 6,251,581, the relevant disclosures of which are incorporated herein by reference. In some examples according to the principles described here, the chemiluminescent compound is a photoactivatable substance that undergoes a chemical reaction under direct or light-sensitized excitation or under reaction with singlet oxygen to form a metastable reaction product that is capable of decomposition. with the simultaneous or subsequent emission of light, generally within the wavelength range of 250 to 1200 nm. The term "photoactivatable" includes "photochemically activatable". In some examples, chemiluminescent compounds are those that react with singlet oxygen to form dioxetanes or dioxetanes. The latter are generally electron-rich olefins are enol ethers, enamines, 9-alkylidene-N-alkylacridans, arylvinylethers, dioxenes, arylimidazoles, 9-alkylidene-xanthenes and lucigenin. Other compounds include luminol and other luminescent compounds and phthalhydrazides that are protected from undergoing a chemiluminescent reaction by virtue of being protected by a photochemically labile protecting group, such compounds including, for example, firefly luciferin, aquaphorin, and luminol. Examples of such chemiluminescent compounds that may be used are those mentioned in United States Patent No. 5,709,994, the relevant disclosure of which is incorporated herein by reference.

[00124] Sensitizing particles are particles that have associated with them a sensitizing compound, which includes, but is not limited to, a photosensitizing compound. Examples of sensitizing compounds that may be used are those mentioned in United States Patent Nos. 5,340,716 and 6,251,581, the relevant disclosures of which are incorporated herein by reference.

[00125] A photosensitizer is a sensitizer for the generation of singlet oxygen generally by excitation with light. In some examples, the photosensitizer absorbs at a longer wavelength than the chemiluminescent compound and has a lower triplet energy than the chemiluminescent compound. The photosensitizer can be photoactivatable (for example, dyes and aromatic compounds). The photosensitizer is generally a compound comprising covalently bonded atoms, usually with multiple conjugated double or triple bonds. The compound must absorb light in the wavelength range of 200-1100 nm, generally 300-1000 nm, preferably 450-950 nm. Typical photosensitizers include, but are not limited to, acetone, benzophenone, 9-thioxanthone, eosin, 9,10-dibromoanthracene, methylene blue, metalloprophyrins (e.g., hematoporphyrin), phthalocyanines, chlorophylls, rose bengal, buckminsterfullerene, etc. example, and derivatives of these compounds. Examples of other sensitizers are enumerated in N.J. Turro, "Molecular Photochemistry", page 132, W. A. Benjamin Inc., N.Y. 1965. The photosensitizer assists photoactivation where activation is by singlet oxygen. Generally, the photosensitizer absorbs light and the excited photosensitizer thus formed activates oxygen to produce singlet oxygen, which reacts with the chemiluminescent compound to provide a metastable luminescent intermediate.

[00126] In the formulas mentioned here, a wavy line through a bond indicates the point of attachment of a moiety in the molecule.

[00127] Some examples according to the principles described here are directed to binding partners such as, for example, antibodies constructed against compounds of formula IV, which are compounds of formula I in which:

[00128] or H, wherein at least one R1 is not H; and including mixtures of two or more of the above compounds.

[00129] Some examples according to the principles described here are directed to binding partners such as, for example, antibodies constructed against derivatives of (7aS,E)-4-((Z)-2-((R)-5 - hydroxy-2-methylene-cyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-one (derived from HMCHEMOIO), or compounds of formula V, which are compounds of formula I in which:

[00130] or H, wherein at least one R1 is not H; and including mixtures of two or more of the above compounds.

[00131] Some examples in accordance with the principles described here are directed to binding partners such as, for example, antibodies constructed against Formula VI compounds, which are compounds of formula I in which: (R1)p-(L)q- is NHR1-(CH2)r-NR1-((CH2)r-NR1)s-(CH2)r-NR7-

[00132] where R1 is independently

[00133] wherein at least one R1 is not H,

[00134] r is independently an integer from 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8 to 9, or 9 to 10, for example,

[00135] s is an integer from 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8 to 9, or 9 to 10, for example, and

[00136] R7 is H or alkyl; and including mixtures of two or more of the above compounds.

[00137] Some examples according to the principles described here are directed to binding partners such as, for example, antibodies constructed against compounds of formula II: NHR1'-(CH2)r'-NR1''-((CH2)r '-NR1''')s'-(CH2)r'-NR7'-Z'

[00138] where

[00139] R1', R1'' or R1''' are all independently selected from the group consisting of

[00140] wherein at least one of R1', R1'' or R1''' is not H,

[00141] r' is independently an integer from 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8 to 9, or 9 to 10, for example,

[00142] s' is an integer from 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8 to 9, or 9 to 10, for example,

[00143] R7' is H or alkyl,

[00144] R11' is H, alkyl, or acyl, and

[00145] Z' is an immunogenic poly(amino acid) carrier or a non-immunogenic poly(amino acid) carrier; and including mixtures of two or more of the above compounds.

[00146] In some examples s' is 1. In some examples R7' is H. In some examples r' is 2. In some examples R1'' and R1''' are H; in some examples R1' and R1''' are H; and in some examples R1' and R1'' are H. In some examples none of R1', R1'' and R1''' are H, that is, R1', R1'' and R1''' are all

[00147] Some examples in accordance with the principles described here are directed to pharmaceutical compositions comprising an effective amount of an antibody constructed against the compound of formula I, or constructed against a compound of formula II, or constructed against a compound of formula V, or constructed against a compound of formula VI, for example.

Compound Preparation

[00148] Examples of methods of preparing compounds that are derived from HMCHEMOIO according to the principles described here are described below, by way of illustration and not limitation. Other methods may be employed to form the above compounds and other compounds consistent with the principles described herein.

[00149] An example of the preparation of a compound of formula VII ((7aS,E)-4-((Z)-2-((R)-5-hydroxy-2-methylenecyclohexylidene)ethylidene)-7a-methylocta -hydro-1H-inden-1-one) is mentioned in figure 2. Referring to figure 2, the compound of formula VIII, wherein R11O is acetyl, is treated to form ketal of formula IX. In one example, the compound of formula VIII is treated with ethylene glycol in an aromatic solvent such as benzene in the presence of a strong organic acid such as, for example, p-toluene sulfonic acid, under conditions (temperature and time) to form a ketal. In some examples, the reaction is conducted at reflux for a period of about 10 hours to about 24 hours, or about 12 to about 20 hours.

[00150] The compound of formula IX is treated to introduce a halide group such as, for example, a chloride group or a bromide group. In one example, the compound of formula IX is treated with N-bromosuccinimide and a free radical initiator such as, for example, azobisisobutyronitrile in an organic solvent such as, for example, an alkane (e.g., hexane), e.g. under conditions for introducing a bromide group into the compound of formula IX to provide a compound of formula X. In some examples, the reaction is conducted at reflux for about 30 minutes.

[00151] The halide of the compound of formula X is removed to provide a compound of formula XI having two double bonds that are conjugated. In one example, the compound of formula for example, under conditions for removing a hydrogen halide to form a double bond (compound of formula XI). In some examples, the temperature during the reaction is about 15°C to about 25°C. The reaction time period is about 2 hours.

[00152] The acetyl group (R11) of the compound of formula XI is removed by treatment with an inorganic base such as, for example, sodium hydroxide or potassium hydroxide, in a polar organic solvent such as, for example, an alkanol ( for example, methanol or ethanol). The reaction components are subjected to the conditions for removing the acetyl group to provide the compound of formula XII. In some examples, the temperature during the reaction is about 15°C to about 25°C, or at room temperature. The reaction time period is about 4 hours to about 8 hours.

[00153] The ketal group of the compound of formula XII is removed, for example, using a strong organic acid such as, for example, p-toluene sulfonic acid, in a mixture of water and a polar organic solvent such as, for example, acetone, under conditions for removing a ketal to produce the compound of formula XIII. In some examples, the temperature during the reaction is about 15°C to about 25°C. The reaction time period is from about 12 hours to about 48 hours.

[00154] The compound of formula VII is formed by treating the compound of formula XIII with ring-opening reagents such as, for example, UV light treatment (photoreaction) in a polar organic solvent such as, for example, an alkanol (e.g., methanol or ethanol), an ether (e.g., ethyl ether), a ketone (e.g., acetone), or a condition of two or more of the above, under conditions for opening a ring of formula XIII . In some examples, the temperature during the photoreaction is from about -20°C to about 0°C. The time period of the photoreaction is about 1 to about 10 minutes, or about 3 to about 5 minutes. The photoreaction is followed by refluxing the intermediate in ethanol for about 3 hours.

[00155] Figure 3 describes, by way of illustration and not limitation, an example of a method of preparing a compound of formula IIa (where Z is BSA, by way of example and not limitation). Referring to Figure 3, the compound of formula VII is reacted with aminooxyacetic acid (XIX) to form the oxime of formula XX (acid 2-((7aS,E)- 4-((Z)-2-( (R)-5-hydroxy-2-methylenecyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-ylideneaminooxy)acetic acid). The reaction is carried out in an organic solvent such as, for example, an alcohol (for example, methanol or ethanol), under conditions for formation of an oxime. In some examples, the temperature during the reaction is from about 10°C to about 30°C, or about 15°C to about 25°C. The reaction time period is about 1 hour to about 30 hours, or about 2 hours to about 24 hours.

[00156] In a separate reaction, an immunogenic poly(amino) acid carrier (in this example, BSA (Z' of formula II is BSA), by way of illustration and not limitation) is combined with a compound of formula XXI to form a compound of formula XXIa. The reaction is carried out in a buffered aqueous medium at a pH of about 5.0 to about 7.0, or about 5.5 to about 6.5, or about 6. An activating agent to facilitate reaction of the carboxylic acid functionality of BSA with one or more of the amine groups of XXI is included in a reaction medium. Such coupling agents include, but are not limited to, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC), N-hydroxysuccinimide (NHS), or N,N,N',N'-tetramethyl-tetrafluoroborate. O-(N-succinimidyl)uronium, or combinations of two or more of the above. The reaction is carried out under conditions for the formation of an amide. In some examples, the temperature during the reaction is about 15°C to about 25°C. The reaction time period is from about 3 hours to about 24 hours, or about 4 hours to about 20 hours, or about 4 hours to about 10 hours, for example.

[00157] The compound of formula XX is treated with an activating agent such as, but not limited to, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), N-hydroxysuccinimide (NHS), or N-Tetrafluoroborate ,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium, or combinations of two or more of the above, for example, to form activated XX. The reaction is carried out in a polar organic solvent such as, for example, an alkanol (e.g. methanol or ethanol), an ether (e.g. ethyl ether or tetrahydrofuran), a ketone (e.g. acetone), dimethyl sulfoxide , acetonitrile, dichloromethane, or dimethylformamide (DMF), or a condition of two or more of the above, which may also contain water. In some examples, the temperature during the reaction is about 15°C to about 30°C, or about 20°C to about 25°C, or about room temperature. The reaction time period is from about 12 hours to about 36 hours, or about 20 to about 30 hours. The activated XX is reacted with the compound of formula XXIa to provide a compound of formula IIa. As shown, R1' is the compound portion of formula IIa and R1'' and R1''' are each H. However, consistent with the principles described here either or both R1'' and R1''' may be the of compound of formula IIa. The reaction is carried out in an organic solvent such as, for example, DMF or DMSO, for example, under conditions for forming an amide. In some examples, the temperature during the reaction is about 15°C to about 30°C, or about 20°C to about 25°C, or about room temperature. The reaction time period is from about 12 hours to about 36 hours, or about 20 to about 30 hours.

[00158] Figure 4 describes, by way of illustration and not limitation, an alternative example of a method of preparing a compound of formula VIa and converting the compound of formula Vla into a compound of formula IIa (where Z is BSA, by example and not limitation). Referring to Figure 4, the compound of formula VII is reacted with aminooxyacetic acid (XIX) to form the oxime of formula XX. The reaction is carried out in an organic solvent such as, for example, an alcohol (for example, methanol or ethanol), under conditions for formation of an oxime. In some examples, the temperature during the reaction is from about 10°C to about 30°C, or about 15°C to about 25°C. The reaction time period is about 1 hour to about 30 hours, or about 2 hours to about 24 hours.

[00159] The oxime of formula XX is combined with a compound of formula XXI to form compound of formula VIa. The reaction is carried out in an organic solvent such as, for example, an alkane (e.g., hexane or pentane, under conditions for forming an amide. In some examples, the temperature during the reaction is from about 15°C to about of 25° C. The reaction time period is from about 10 hours to about 48 hours.

[00160] The compound of formula VIa is reacted with an immunogenic poly(amino) acid carrier (Z' of formula II is BSA, by way of illustration and not limitation) to provide a compound of formula IIa. As shown, R1' is the compound portion of formula IIa and R1'' and R1''' are each H. However, consistent with the principles described here, the resulting product may be a mixture of compounds, wherein in a compound of the mixture, R1' is the portion of the compound of formula IIa, and in another compound of the mixture R1' and R1'' are both a portion of compound of formula IIa, and in another compound of the mixture R1' and R1''' are both a portion of compound of formula IIa, and in another compound of the mixture all three of R1' and R1'' and R1''' are a portion of compound of formula IIa. The reaction is carried out in an organic solvent such as, for example, an alkane (e.g., hexane or pentane), under conditions for forming an amide. In some examples, the temperature during the reaction is about 15°C to about 25°C. The reaction time period is from about 10 hours to about 48 hours.

[00161] Another example of a method of preparing exemplary compounds in accordance with the principles described herein is described, by way of illustration and not limitation, with reference to figure 5. Other methods may be employed to form compounds consistent with the principles described here. The preparation of a compound of formula XVIII (Y is C in formula I) is mentioned in figure 5. Referring to figure 5, the compound of formula VII (prepared, for example, as described in figure 3) is treated to protect the free hydroxyl group (R13 is a protecting group) to form a compound of formula XIV. The reaction conditions are dependent on the nature of the protecting group, for example.

[00162] In one example, the compound of formula VII is treated with tert-butyldimethylsilyl chloride under conditions to form a silyl ether. The reaction is carried out in a polar organic solvent such as, for example, pyridine, dimethylsulfoxide, an ether (e.g., tetrahydrofuran, ethyl ether or 1,4-dioxane), acetonitrile, dichloromethane, or dimethylformamide (DMF). In some examples, the temperature during the reaction is from about 10°C to about 30°C, or about 15°C to about 25°C. The reaction time period is about 1 hour to about 30 hours, or about 2 hours to about 24 hours.

[00163] Examples of protecting groups, by way of example and not limitation, are silyl groups (such as, but not limited to, trimethylsilyl, trimethylsilyl, tert-butyldimethylsilyl, triisopropylsilyl, tert-butyldiphenylsilyl, for example), t-butoxycarbonyl (t-Boc), fluorenylmethyloxycarbonyl (Fmoc), acetaminomethyl (Acm), triphenyl methyl (Trt), benzyloxycarbonyl, biphenylisopropyloxycarbonyl, 1-amyloxycarbonyl, isobornyloxycarbonyl, alpha-dimethyl-3,5-dimethoxybenzyloxycarbonyl , o-nitrophenylsulfenyl, 2-cyano-1,1-dimentyl-ethoxycarbonyl, bromobenzyloxy, carbamyl, and formyl, for example.

[00164] A compound of formula XV is formed from the compound of formula XIV, for example, by reaction with an alpha-haloester in the presence of zinc (Reformatsky reaction). In this example, the compound of formula XIV is treated with a Reformatsky reagent such as, for example, BrZnCH2COOR14 (Zn and ethyl alpha-bromoacetate in the example shown, w = 1) to provide the compound of formula XV. The reaction is carried out in an organic solvent such as, for example, an ether (for example, tetrahydrofuran or ethyl ether) or an aromatic solvent (for example, benzene or toluene). In some examples, the temperature during the reaction is from about 4°C to about 100°C, or about 10°C to about 90°C. The reaction time period is from about 4 hours to about 24 hours.

[00165] The free hydroxyl group of the compound of formula XV is reduced to form a compound of formula XVI. The compound of formula XV can be treated to reduce the hydroxy group by methods including, but not limited to, tosylate ester formation followed by treatment with a metal hydride such as, for example, LiAlH4 or NaBH4; removal of the hydroxyl to form an alkene (such as, for example, by treatment with a concentrated organic acid, e.g., concentrated sulfuric acid or concentrated hydrochloric acid) followed by hydrogenation in the presence of a catalyst such as, for example, platinum or palladium ; for example. The conditions for the reaction depend on one or both of the nature of the reagents used and the nature of the solvent, for example.

[00166] The resulting compound of formula XVI is treated to hydrolyze the ester group to a carboxylic acid group (deesterification reaction) to provide the compound of formula XVII. Numerous methods are available for deesterification and include, but are not limited to, saponification or treatment with an aqueous base such as, for example, NaOH or KOH with heat; acid hydrolysis or treatment with an aqueous acid such as, for example, an aqueous mineral acid (such as, for example, hydrochloric acid or sulfuric acid), for example. The conditions for the reaction are dependent on one or more of the nature of the reactants and the nature of the ester, for example.

[00167] A compound of formula XVIII is formed by removing protective group R13 from the compound of formula XVII. Various methods can be employed to remove protecting groups and include, but are not limited to, treatment with a dilute mineral acid (such as, e.g., hydrochloric acid or sulfuric acid); treatment with an organic acid (such as, e.g., acetic acid, in a polar organic solvent (such as, for example, an alkanol (e.g., methanol or ethanol), an ether (e.g., ethyl ether or tetrahydrofuran), a ketone (e.g., acetone), dimethylsulfoxy - do, acetonitrile, dichloromethane, or dimethylformamide (DMF), or a condition of two or more of the above, which may also contain water, under conditions for removing the protecting group to produce the compound of formula XVIII. Conditions for the reaction are dependent on one or more of the nature of the reactants and the nature of the protecting group, for example.

[00168] Figure 6 describes, by way of illustration and not limitation, an example of a method of preparing a compound of formula VIb and converting the compound of formula Vlb into a compound of formula IIb (where Z' is KLH, e.g. by way of example and not limitation). Referring to Figure 6, the compound of formula XVIII is treated to activate the carboxylic acid group such as, but not limited to, the reaction to form an N-hydroxysuccinimide (NHS) ester (compound of formula XXII). The reaction is carried out in a polar organic solvent such as, for example, dimethylsulfoxide, an ether (e.g., tetrahydrofuran or ethyl ether), acetonitrile, dichloromethane, or dimethylformamide (DMF), for example, under conditions for forming a NHS ester. In some examples, the temperature during the reaction is about 15°C to about 25°C. The reaction time period is from about 4 hours to about 48 hours.

[00169] The NHS ester of formula XXII is combined with a compound of formula XXI to form compound of formula VIb. The reaction is carried out in an organic solvent such as, for example, an alkane (e.g., hexane or pentane), for example, under conditions for forming an amide. In some examples, the temperature during the reaction is about 15°C to about 25°C. The reaction time period is from about 4 hours to about 48 hours.

[00170] The compound of formula VIb is reacted with an immunogenic poly(amino) acid carrier (Z' of formula II is KLH, for example) to provide a compound of formula IIb. As shown, R1' is the compound portion of formula IIb and R1'' and R1''' are each H. However, consistent with the principles described here, the resulting product may be a mixture of compounds, wherein in a compound of the mixture, R1' is the portion of the compound of formula IIa, and in another compound of the mixture R1' and R1'' are both a portion of compound of formula IIa, and in another compound of the mixture R1' and R1''' are both a portion of compound of formula IIa, and in another compound of the mixture all three of R1' and R1'' and R1''' are a portion of compound of formula IIb. The reaction is carried out in an organic solvent such as, for example, an alkane (e.g., hexane or pentane), under conditions for forming an amide. In some examples, the temperature during the reaction is from about 15°C to about 25°C. The reaction time period is from about 4 hours to about 48 hours.

[00171] In an alternative routine, a compound of formula IIb can be prepared from a compound of formula XVIII in a manner similar to that described above for figure 3 for preparing the compound of formula IIa.

[00172] Another example of a method of preparing exemplary compounds in accordance with the principles described herein is described, by way of illustration and not limitation, with reference to figure 7. Other methods may be employed to form compounds consistent with the principles described here. The preparation of a compound of formula XXVII (Y is O in formula I and w is 0 to 10 or 1 to 10) is mentioned in figure 7. Referring to figure 7, the compound of formula VII (prepared, for example, as described in figure 2) is treated to protect the free hydroxyl group (R15 is a protecting group) to form a compound of formula XXIII. The reaction conditions are dependent on the nature of the protecting group, for example.

[00173] In one example, the compound of formula VII is treated with tert-butyldimethylsilyl chloride (by way of example and not limitation) under conditions to form a silyl ether. The reaction is carried out in a polar organic solvent such as, for example, pyridine, dimethylsulfoxide, an ether (e.g., tetrahydrofuran, ethyl ether, or 1,4-dioxane), acetonitrile, dichloromethane, or dimethylformamide (DMF). ). In some examples, the temperature during the reaction is about 15°C to about 25°C. The reaction time period is from about 4 hours to about 48 hours.

[00174] A compound of formula XXIV is formed from the compound of formula XXIII by reducing the ketone group to a hydroxy group, for example. The compound of formula XXIII can be treated to reduce the hydroxy group by methods including, but not limited to, treatment with a metal hydride such as, for example, LiAlH4 or NaBH4, to provide the compound of formula XXIV. The reaction is carried out in an organic solvent such as, for example, an alkanol (e.g. ethanol). In some examples, the temperature during the reaction is from about 0°C to about 25°C. The reaction time period is about 0.5 hours to about 4 hours.

[00175] A compound of formula XXV is formed from the compound of formula XXIV, for example, by reaction with a halo ester (such as, by way of example and not limitation) an alpha-haloester in the presence of a base. In this example, the compound of formula XXV is treated with, for example, BrCH2COOR16 (ethyl alpha-bromoacetate in the example shown, w is 1 and R16 is ethyl) to provide the compound of formula XXV. The reaction is carried out by forming an alkoxide ion of the hydroxy group of the compound of formula XXIV in the presence of a base such as, for example, KOH, NaOH, Na2CO3, or K2CO3, and reacting the alkoxide with the haloester. The solvent for the reaction is an aqueous solvent, which may contain 1% to 40% of a polar organic solvent as described above. In some examples, the temperature during the reaction is from about 50°C to about 100°C, or about 50°C to about 90°C, for example. The reaction time period is about 0.5 hours to about 24 hours, or about 1 hour to about 8 hours, for example.

[00176] The resulting compound of formula XXV is treated to hydrolyze the ester group to a carboxylic acid group (deesterification reaction) to provide the compound of formula XXVI. Numerous methods are available for deesterification and include, but are not limited to, saponification or treatment with an aqueous base such as, for example, NaOH or KOH, with heat; acid hydrolysis or treatment with an aqueous acid such as, for example, an aqueous mineral acid (such as, for example, hydrochloric acid, sulfuric acid, for example. The conditions for the reaction are dependent on one or more of the nature of the reagents and the nature of the ester, for example.

[00177] A compound of formula XXVII is formed by removing protecting group R15 from the compound of formula XXVI. Various methods can be employed to remove protecting groups and include, but are not limited to, treatment with dilute mineral acid (such as, for example, hydrochloric acid or sulfuric acid); treatment with an organic acid (such as, for example, acetic acid), in a polar organic solvent (such as, for example, an alkanol (for example, methanol or ethanol), an ether (for example, ethyl ether, tetrahydrofuran, or 1,4-dioxane), a ketone (e.g., acetone), dimethyl sulfoxide, acetonitrile, dichloromethane, dimethylformamide (DMF), or a condition of two or more of the above, which may also contain water, under conditions for removing the protecting group to produce the compound of formula XXVII. The conditions for the reaction are dependent on one or more of the nature of the reactants and the nature of the protecting group, for example.

[00178] Figure 8 describes, by way of illustration and not limitation, an example of a method of preparing a compound of formula VIc and converting the compound of formula Vlc into a compound of formula IIc (where Z' is the enzyme G6PDH , by way of example and not limitation). Referring to Figure 8, compound of formula XXVII is treated to activate the carboxylic acid group such as, but not limited to, the reaction to form an N-hydroxysuccinimide (NHS) ester (compound of formula XXVIII). The reaction is carried out in a polar organic solvent such as, for example, dimethylsulfoxide, an ether (e.g., tetrahydrofuran, ethyl ether, or 1,4-dioxane), acetonitrile, dichloromethane, or dimethylformamide (DMF), for example , under conditions for formation of an NHS ester. In some examples, the temperature during the reaction is about 15°C to about 25°C. The reaction time period is from about 1 hour to about 24 hours.

[00179] The NHS ester of formula XXVIII is combined with a compound of formula XXI to form compound of formula VIc. The reaction is carried out in an organic solvent such as, for example, an alkane (e.g., hexane or pentane), for example, under conditions for forming an amide. In some examples, the temperature during the reaction is about 15°C to about 25°C. The reaction time period is from about 1 hour to about 24 hours.

[00180] The compound of formula VIc is reacted with the enzyme, G6PDH (Z' of formula II is G6PDH, for example), to provide a compound of formula IIc. As shown, R1' is the compound portion of formula IIc and R1'' and R1''' are each H. However, consistent with the principles described here, the resulting product may be a mixture of compounds, wherein in a compound of the mixture, R1' is the portion of the compound of formula IIa, and in another compound of the mixture R1' and R1'' are both a portion of compound of formula IIa, and in another compound of the mixture R1' and R1''' are both a portion of compound of formula IIa, and in another compound of the mixture all three of R1' and R1'' and R1''' are a portion of compound of formula IIc. The reaction is carried out in an organic solvent such as, for example, an alkane (e.g., hexane or pentane), under conditions for forming an amide. In some examples, the temperature during the reaction is about 15°C to about 25°C. The reaction time period is from about 1 hour to about 24 hours.

[00181] In an alternative routine, a compound of formula IIc can be prepared from a compound of formula XXVII in a manner similar to that described above for figure 3 for preparing the compound of formula IIa.

[00182] Other compounds according to the principles described here can be prepared in a similar way to those described above.

Preparation of binding partners

[00183] Examples of compounds in accordance with the principles described herein, where Z is an immunogenic poly(amino acid) carrier or a non-immunogenic poly(amino acid) carrier can be employed to prepare binding partners for vitamin D such as, e.g. example, vitamin D aptamers or antibodies to vitamin D, which include, but are not limited to, antibodies specific to vitamin D3, antibodies specific to vitamin D2, antibodies specific to 25-hydroxyvitamin D3, antibodies specific to 25-hydroxyvitamin D2, specific antibodies for 3-epi-25-hydroxyvitamin D3, and antibodies specific for 3-epi-25-hydroxyvitamin D2, for example. Of particular interest are antibodies specific for 3-epi-25-hydroxyvitamin D3, antibodies specific for 3-epi-25-hydroxyvitamin D2, and antibodies specific for epimers of other vitamin D compounds ("antibodies to epimeric vitamin D"). , which can be used in assays for 3-epi-25-hydroxyvitamin D3 and for 3-epi-25-hydroxyvitamin D2, or which can be used in assays for non-epimeric vitamin D to reduce or eliminate interference from epimeric forms of vitamin D such as, for example, 3-epi-25-hydroxyvitamin D3 and 3-epi-25-hydroxyvitamin D2, which may be present in a sample being tested for the presence of vitamin D.

[00184] Antibodies may be monoclonal antibodies or polyclonal antibodies and may include an immunoglobulin or fragment thereof, which immunoglobulins include, but are not limited to, various classes and isotypes, such as IgA, IgD, IgE, IgG and IgM, for example. Fragments thereof may include Fab, Fv and F(ab')2, Fab', and the like. Furthermore, aggregates, polymers, and conjugates of immunoglobulins or fragments thereof can be used where appropriate, as long as binding affinity for a particular molecule is maintained.

[00185] Antibodies in accordance with the principles described here can be prepared by techniques including, but not limited to, immunization of a host and collection of sera (polyclonal), continuous preparation of hybrid cell lines and collection of secreted protein (monoclonal) or cloning and expressing nucleotide sequences or mutagenized versions thereof encoding at least the amino acid sequences required for binding natural antibodies, for example.

[00186] Monoclonal antibodies can be prepared by techniques such as continuous preparation of hybrid cell lines and collection of secreted protein (somatic cell hybridization techniques). Monoclonal antibodies can be produced according to techniques of Kohler and Milstein, Nature 265:495-497, 1975. Reviews of monoclonal antibody techniques are found in Lymphocyte Hybridomas, ed. Melchers, et al. Springer-Verlag (New York 1978), Nature 266: 495 (1977), Science 208: 692 (1980), and Methods of Enzymology 73 (Part B): 3-46 (1981).

[00187] In another method for preparing antibodies, the sequence encoding antibody binding sites can be excised from the chromosomal DNA and inserted into a cloning vector, which can be expressed in bacteria to produce recombinant proteins having the binding sites of corresponding antibodies. This method involves cloning and expressing nucleotide sequences or mutagenized versions thereof, encoding at least amino acid sequences required for specific binding of natural antibodies.

[00188] In a method for producing monoclonal antibodies, a first step includes immunizing an antibody-producing animal such as a mouse, a rat, a goat, a sheep, or a cow with an immunogen comprising a compound of formula I in which Z is an immunogenic vehicle, for example. Immunization can be performed with or without an adjuvant such as complete Freund's adjuvant or other adjuvants such as monophosphoryl lipid A and synthetic trehalose dicorynomycolate adjuvant. A next step includes isolating spleen cells from the antibody-producing animal and fusing the antibody-producing spleen cells with an appropriate fusion partner, typically a myeloma cell, such as by use of polyethylene glycol or other techniques. Typically, the myeloma cells used are those that grow normally in hypoxanthine-thymidine (TH) medium, but do not grow in hypoxanthine-aminopterin-thymidine (HAT) medium, used for selection of fused cells. A next step includes selection of the fused cells, typically by selection in HAT medium. A next step includes analysis of the cloned hybrids for production of appropriate antibody using immunoassays such as, for example, an enzyme-linked immunosorbent assay (ELISA) or other appropriate immunoassays for analysis.

[00189] An antibody (prepared from an immunogen in accordance with the principles described herein) with the required specificity can be selected by analytical methodologies, which include, by way of illustration and not limitation, ELISA, dot blots, Western analysis, and Surface Plasmon Resonance, for example. In this way, an antibody is obtained that binds to a vitamin D analyte of interest and does not bind to any detectable degree to a vitamin D molecule that is not of interest in a particular assay. In some examples in accordance with the principles described herein, an antibody that binds to a vitamin D analyte of interest has a binding affinity for the vitamin D analyte of interest of about 107 to about 1014 liters/mole, or about 107 to about 1011 liters/mole, or about 107 to about 1012 liters/mole, or about 108 to about 1014 liters/mole, or about 108 to about 1011 liters/mole, or about 108 to about 1012 liters/mole, for example. In some examples, the antibody that specifically binds the vitamin D analyte of interest should have a binding affinity for the vitamin D molecule that is not of interest of less than about 104 liters/mole, or less than than about 103 liters/mole, or less than about 102 liters/mole, or less than about 10 liters/mole, for example.

[00190] In one example in accordance with the principles described herein, an immunogen prepared from a compound of formula I above, wherein Z is an immunogenic carrier, is employed to prepare antibodies that are specific for 3-epi-25-hydroxyvitamin D3 wherein the antibodies do not bind to any detectable degree with 25-hydroxyvitamin D3 or other variants of vitamin D including non-epivitamin D compounds such as, for example, 25-hydroxyvitamin D; calcidiol; 1,25-dihydroxyvitamin D3; 1,25-dihydroxy vitamin D4; 1,25-dihydroxy vitamin D5; and 1,25-dihydroxy vitamin D6, for example.

[00191] In one example in accordance with the principles described herein, an immunogen prepared from a compound of formula I above, wherein Z is an immunogenic carrier, is employed to prepare antibodies that are specific for 3-epi-25-hydroxyvitamin D3 wherein the antibodies do not bind to any detectable degree with 25-hydroxyvitamin D3 or other variants of vitamin D including non-epivitamin D compounds such as, for example, 25-hydroxyvitamin D; calcidiol; 1,25-dihydroxyvitamin D3; 1,25-dihydroxyvitamin D4; 1,25-dihydroxyvitamin D5; and 1,25-dihydroxyvitamin D6, for example.

[00192] In another example in accordance with the principles described here, an immunogen prepared from a compound of formula I above, wherein Z is an immunogenic vehicle, is employed to prepare antibodies that are specific for 3-epi-25-hydroxyvitamin D2 wherein the antibodies do not bind to any detectable degree with 25-hydroxyvitamin D2 or other variants of vitamin D including non-epivitamin D compounds such as, for example, 25-hydroxyvitamin D; calcidiol; 1,25-dihydroxyvitamin D3; 1,25-dihydroxyvitamin D4; 1,25-dihydroxyvitamin D5; and 1,25-dihydroxyvitamin D6, for example.

[00193] The phrase "any detectable degree" means that the compound that specifically binds a vitamin D analyte of interest (e.g., 3-3pi-25-hydroxyvitamin D) has a binding affinity for a vitamin D molecule that is not of interest (e.g., a non-epivitamin D compound) of less than about 104 liters/mole, or less than about 103 liters/mole, or less than about 102 liters/mole mole, or less than about 10 liters/mole, for example.

[00194] In one example, by way of illustration and not limitation, an immunogen is employed in which Z in the compound of formula I is BSA. This immunogen is used to immunize mice (e.g., BALB/c mice, Swiss Webster mice, or an AJ strain of mice) intraperitoneally. Serum samples from these mice are tested for anti-3-epi-25-hydroxyvitamin D3 antibodies using a conjugate of 3-epi-25-hydroxyvitamin D3 and ovalbumin (ovalbumin conjugate). An ELISA microtiter plate is employed and antibodies are examined for binding to the ovalbumin conjugate and subsequently to 3-epi-25-hydroxyvitamin D3. Mice with high titers are boosted three days before fusion. On the day of fusion, spleen cells are harvested from these mice and are fused with myeloma cell line P3X63Ag8.653 using PEG-assisted fusion protocols. After about ten days, hybridoma supernatants are analyzed for anti-3-epi-25-hydroxyvitamin D3 antibodies using an ELISA plate. Positive clones are also expanded, subcloned and the supernatants are purified using a protein A sepharose column. Purified antibody samples are tested using ELISA for binding to ovalbumin conjugate and free 3-epi-25-hydroxyvitamin D3.

[00195] Particular examples, by way of illustration and not limitation, of antibodies prepared as described above that are specific for 3-epi-25-hydroxyvitamin D3 or specific for 3-epi-25-hydroxyvitamin D2, include antibody 4G8 and antibody 8F10 , for example.

[00196] In some examples, antibodies in accordance with the principles described herein may be employed to purify vitamin D compounds. For example, antibodies such as those described above may be attached to a support and the support employed to purify vitamin D compounds. vitamin D. In one example, antibodies to 3-epi-25-hydroxyvitamin D3 may be bound to an affinity purification chromatography substrate such as, for example, a column, and vitamin D preparations may be contacted with the substrate of chromatography where antibody on the substrate binds 3-epi-25-hydroxyvitamin D3 from the vitamin D preparation, while other vitamin D compounds are eluted from the substrate.

Specific Examples of Assays for Vitamin D Analytes Employing Antibodies According to the Principles Described Here as Blocking Antibodies

[00197] Antibodies in accordance with the principles described here can be employed to minimize or eliminate 3-epimer cross-reactivity in assays for non-epimeric forms of vitamin D analytes. Overestimation of non-epimeric vitamin D analyte Total damage caused by cross-reactivity of 3-epimer vitamin D with an antibody to vitamin D analyte can be substantially avoided by employing, as blocking agents, antibodies prepared against immunogens that are a compound of formula I in which Z is an immunogenic carrier.

[00198] In an example of an assay for non-epimeric vitamin D analyte, the assay uses, as a chemiluminescent reagent, a chemical reagent ("Chemibead") comprising an olefinic dye and 25-hydroxyvitamin D3, a vitamin analog. D. A sample suspected of containing non-epimeric vitamin D analyte is combined in an assay medium with a biotinylated antibody to the non-epimeric vitamin D analyte and a second antibody which is antibody 8F10 and then with a chemical reagent. Chemical reagents bind to the fraction of monoclonal antibody binding sites that is not occupied by non-epimeric vitamin D analyte in the sample. Antibody 8F10 binds to 3-epi-25-hydroxyvitamin D3 which is present in the sample and which interferes with the determination by cross-reaction with the biotinylated antibody. Subsequently, streptavidin-coupled sensitizing beads comprising a photosensitizer (sensitizers) are added to the reaction mixture. This induces the formation of chemical reagent pairs, the concentration of which is inversely related to a non-epimeric vitamin D analyte concentration in the sample. Under illumination at 680 nm, the sensitizing beads generated singlet oxygen that diffuses into the chemical reagents, which are paired with sensibeads, reacts with the olefin dye in the chemiluminescent and activates the chemiluminescent signal at approximately 612 nm which is inversely related to the non-epimeric vitamin D analyte concentration.

Examples of Assays for Vitamin D Analytes Employing a Labeled Compound According to the Principles Described Here

[00199] Assays for vitamin D analytes can be performed using a compound of formula I where Z is a poly(amino acid) label, or a non-poly(amino acid) label or a support.

[00200] In one example, by way of illustration and not limitation, of an assay for detection of vitamin D analyte, an ACMIA assay format is employed. Chromium particles, which are coated with a vitamin D analogue which is a compound of formula I where Z is a non-poly(amino acid) label which is the chromium particle (chromium particle reagent), are employed as a first component. A second component is an antibody to vitamin D analyte. This antibody, cross-linked to a reporter enzyme (e.g., β-galactosidase) to form an antibody-enzyme conjugate, is added to a reaction vessel in an excess amount, that is, an amount greater than that required to bind all of the vitamin D analyte that could be present in a sample. A sample, which is previously subjected to treatment with a releasing agent, is treated with an antibody to vitamin D, which binds to the vitamin D analyte in the sample. The antibody-enzyme conjugate is mixed with the sample in the medium to allow the vitamin D analyte to bind to the antibody. Next, chromium particle reagent is added to bind any excess antibody-enzyme conjugate. Next, a magnet is applied, which pulls all chromium particles and excess antibody-enzyme out of the suspension, and the supernatant is transferred to a final reaction vessel. The reporter enzyme substrate is added to the final reaction vessel, and enzyme activity is spectrophotometrically measured as a change in absorbance over time. The amount of this signal is related to the amount of vitamin D analyte in the sample.

[00201] In the example above, an antibody in accordance with the principles described herein can be employed to block the binding of 3-epi-25-hydroxyvitamin D3, which may be present in the sample, to the antibody to the vitamin D analyte. anti-3-epi-25-hydroxyvitamin D3 antibody may be an antibody constructed against an immunogen that is a compound of formula I in which Z is an immunogenic carrier.

Examination Stage

[00202] In a next step of an assay method, the medium is examined for the presence of a complex comprising the vitamin D analyte and antibody to the vitamin D analyte and/or a complex comprising a vitamin D analogue and antibody for vitamin D. The presence and/or amount of one or both complexes indicates the presence and/or amount of the vitamin D analyte in the sample.

[00203] The phrase "measuring the amount of a vitamin D analyte" refers to the quantitative, semi-quantitative and qualitative determination of vitamin D. Methods that are quantitative, semi-quantitative and qualitative, as well as all other methods for determining the analyte - vitamin D analyte, are considered to be methods of measuring the amount of the vitamin D analyte. For example, a method, which merely detects the presence or absence of the vitamin D analyte in a sample suspected of containing the vitamin D analyte, is considered to be included within the scope of the present invention. The terms "detect" and "determine", as well as other common synonyms for evaluate, are contemplated within the scope of the present invention.

[00204] In many embodiments, examination of the medium involves detecting a signal from the medium. The presence and/or amount of the signal is related to the presence and/or amount of the vitamin D analyte in the sample. The particular mode of detection depends on the nature of the signal producing system. As described above, there are numerous methods by which a label of a signal production system can produce a signal detectable by external means. The activation of a signal production system depends on the nature of the members of the signal system.

[00205] Temperatures during assessments generally range from about 10°C to about 70°C, or from about 20°C to about 45°C, or about 20°C to about 25°C, for example. example. In one method, standard curves are formed using known concentrations of vitamin D analyte. Calibrators and other controls can also be used.

[00206] The luminescence or light produced from any label can be measured visually, photographically, actinometrically, spectrophotometrically, such as using a photomultiplier or photodiode, or by any other convenient method to determine the amount thereof, which is related to the amount of vitamin D analyte in the medium. Examination for the presence and/or quantity of the signal also includes detection of the signal, which is generally merely a step in which the signal is read. The signal is typically read using an instrument, the nature of which depends on the nature of the signal. The instrument may be, but is not limited to, a spectrophotometer, fluorometer, absorption spectrometer, luminometer and chemiluminometer, for example.

Kits comprising reagents for conducting assays

[00207] Kits comprising reagents for conducting assays can be formulated based on the nature of a particular assay. In some examples in accordance with the principles described herein, a kit may comprise a binding partner such as, for example, an antibody constructed against an immunogen that is a compound of formula I wherein Z is an immunogenic carrier. In some examples in accordance with the principles described herein, a kit may comprise a reagent that is a compound of formula I wherein Z is a poly(amino acid) label portion or a non-poly(amino acid) label portion including a support. A kit may also include other reagents for conducting a particular assay for a vitamin D analyte. In some embodiments, a kit comprises in a packaged combination a biotin binding partner such as, for example, avidin or streptavidin, associated with a particle, biotinylated compound of formula I and a compound labeled for the vitamin D analyte. The kit may also include other reagents for carrying out the assay, the nature of which depends on the format of the particular assay.

[00208] The reagents may each be in separate containers or several reagents may be combined in one or more containers depending on the cross-reactivity and stability of the reagents. The kit may also include other separately packaged reagents for conducting an assay such as additional specific binding pair members, signal production system members, and auxiliary reagents, for example.

[00209] The relative quantities of the various reagents in the kits can be varied widely to provide concentrations of the reagents that substantially optimize the reactions that need to occur during the present methods and also substantially optimize the sensitivity of an assay. Under appropriate circumstances, one or more of the reagents in the kit may be supplied as a dry powder, generally lyophilized, including excipients, which upon dissolution will provide a reagent solution having concentrations appropriate for carrying out a method or assay using a compound reagent. according to the principles described here. The kit may also include a written description of a method using reagent that includes a reagent compound in accordance with the principles described herein.

[00210] The phrase "at least" as used herein means that the number of items specified may be equal to or greater than the number reported. The phrase "about" as used here means that the reported number may differ by plus or minus 10%; for example, "about 5" means a range of 4.5 to 5.5.

[00211] The following description is directed to specific examples in accordance with the principles described here by way of illustration and not limitation; The specific examples are not intended to limit the scope of the present invention and the appended claims. Numerous modifications and alternative compositions, methods, and systems can be developed without departing from the spirit and scope of the present invention.

EXAMPLES

[00212] Unless otherwise indicated, materials in the experiments below can be purchased from Sigma-Aldrich Chemical Corporation (St. Louis MO) or Fluka Chemical Corporation (Milwaukee WI). Portions and percentages described here are by weight to volume unless otherwise noted.

Definitions:

[00213] mg = milligram

[00214] g = gram(s)

[00215] ng = nanogram(s)

[00216] mL = milliliter(s)

[00217] μL = microliter(s)

[00218] μmol = micromolar

[00219] °C = degrees centigrade

[00220] min = minute(s)

[00221] sec = second(s)

[00222] hr = hour(s)

[00223] w/v = weight for volume

[00224] TLC = thin layer chromatography

[00225] HPLC = high performance liquid chromatography

[00226] EDA = ethylenediamine

[00227] EtOAc = ethyl acetate

[00228] DMF = dimethylformamide

[00229] DMSO = dimethyl sulfoxide

[00230] MeOP = 1-methoxy-2-propanol

[00231] MES = 2-(N-morpholino)ethanesulfonic acid

[00232] CMO = carboxymatoxyoxime

[00233] TMB = tetramethyl benzidine

[00234] SNHS = sulfo-N-hydroxysuccinimide

[00235] High pH Wash Buffer = 5.5 mM Na3PO4 + 4.4 mM Na2CO3, pH 11

[00236] Haptene Wash Buffer = 50 mM HEPES, 300 mM NaCl, 1 mM EDTA, 0.1%

[00237] TRITON® X405, 0.15% PROCLIN® preservative, and 1 mg/mL neomycin

[00238] DI = deionized

[00239] ELISA = enzyme-linked immunosorbent assay

[00240] UPA = Ultra Particle Analyzer

[00241] LOCI = luminescent oxygen channeling immunoassay

[00242] BSA = bovine serum albumin

[00243] BGG = bovine gamma globulin

[00244] mIgG = mouse immunoglobulin

[00245] MS = mass spectrometry

Example 1 Preparation of compounds from Figure 2 where R11 is acetyl

[00246] 5-Androsten-3α-ol-17-one acetate (VIII) (100 mg) was reacted with ethylene glycol (0.245 mL) and p-toluenesulfonic acid monohydrate (4 mg) in benzene under reflux overnight to provide 5-androsten-3α-ol-17-one acetate ethylene ketal (IX) (112 mg). Ethylene ketal IX (112 mg) was brominated with N-bromosuccinimide (69 mg)/azoisobutyronitrile (3.3 mg) in hexane (13 mL) under reflux for 30 minutes to give compound X, followed by dehydrobromination with tetrabutylammonium fluoride (1M in THF, 1.6 mL) in THF (7.3 mL) at room temperature for 2 hours to give androsta-5,7-dien-3α-ol-17-one ethylene acetate ketal (XI) ( 55mg). Androsta-5,7-dien-3α-ol-17-one acetate ethylene ketal (XI) (55 mg) was reacted with 1N sodium hydroxide (2 mL) in methanol (10 mL) at room temperature for ca. 5 hours to provide androsta-5,7-dien-3α-ol-17-one ethylene ketal (XII) (48 mg). Androsta-5,7-dien-3α-ol-17-one ethylene ketal (XII) (48 mg) was reacted with p-toluenesulfonic acid monohydrate (35 mg) in a mixture of acetone (5 mL) and water ( 0.2 mL) at room temperature overnight to provide androsta-5,7-dien-3α-ol-17-one (XIII) (43.4 mg). Androsta-5,7-dien-3α-ol-17-one (XIII) (43.4 mg) was irradiated under 450 w mercury lamp with a VYCOR® filter (Ace Glass Incorporated, Vineland, NJ) in ether ( 1100 mL) at -20 to 0°C for 5 minutes to provide pre-HMCHEMOIO, which was refluxed in ethanol (15mL) for 3 hours to yield HMCHEMOIO (VII) (13.5 mg).

[00247] HMCHEMOIO (VII) (13.5 mg) from the above was reacted with O-(carboxymethyl)hydroxylamine hemihydrochloride (12 mg) and sodium acetate (24 mg) in 1 mL of methanol overnight at room temperature to provide HMCHEMOIO-CMO (13.2 mg) (compound of formula XX in figure 3 where n = 1).

[00248] Cationized BSA was prepared as follows: Triethylenetetramine (0.5 mL) (compound of formula XXI in figure 3 where each r = 1 and s = 1) 50 mM MES buffer of 4.5 mL pH6 was added , and the pH was adjusted to 6 followed by addition of 20 mg BSA. The combination was mixed to completely dissolve the components. EDAC (5 mg) was added to the above solution every hour for 5 hours. The solution was washed in a 10 mL AMICON® cell (Amicon Inc., Beverly MA) with 10 x 10 mL wash buffer (10 mM phosphate + 300 nM NaCl) at pH 7. Then 100 mg of TWEEN®20 were added to a 50 mL round base bottle. Wash buffer (50 mL) was added and the mixture was stirred for 10 minutes to disperse Tween 20. The cationized BSA (compound of formula XXIa in Figure 3 where each r = 1 and s = 2) was stored in wash buffer with 0 .2% TWEEN® 20 at approximately 5 mg/mL.

[00249] For preparation of HMCHEMOIO-CMO BSA (compound of formula IIa in figure 3), EDAC (25 mg) and 10 mg of NHS were placed in a 10 mL vial with 13.2 mg of HMCHEMOIO-CMO (XX) prepared as described above. DMF (0.5 mL) was added to the vial. The mixture was stirred for 24 hours at room temperature to provide activated HMCHEMOIO-CMO. The solution was clear. TLC (1:1 Ethyl acetate:methanol) indicated no starting material remaining.

[00250] The activated HMCHEMOIO-CMO from above was added dropwise to the cationized BSA solution from above, and the mixture was stirred overnight at room temperature. The mixture was transferred to an Amicon® cell (Amicon Inc., Beverly MA), then washed with 5 x 10 mL of wash buffer and concentrated (30,000 sections) to about 4 mL. The mixture was also separated on an SD-25 column (GE Healthcare Bio-Sciences, Pittsburg PA) using pH 7.0 wash buffer as an eluent to provide a mixture of HMCHEMOIO-BSA CMO compounds (mixture as described above with respect to the compounds of formula IIa in figure 3).

Example 2 Antibody preparation

[00251] AJ line of mice (females, at least eight weeks old) was immunized to generate monoclonal antibodies. The first immunization was 100 μg of immunogen (HMCHE-MOIO-BSA from CMO) of the above in a volume of 100 μl with complete Freund's adjuvant (from Sigma-Aldrich, Cat # F5881). Three weeks later, a booster immunization with the same immunogen was administered at 100 μg in a volume of 100 μL with incomplete Freund's adjuvant (from Sigma-Aldrich Cat # F5506). Subsequently, after another 3 weeks, a second booster immunization with the same immunogen was administered with 100 μg in a volume of 100 μL with Incomplete Freund's Adjuvant. One week after the last booster immunization, the mice were bled and antisera were tested in ELISA for anti-3-epimer antibodies. Subsequently, a prefusion booster was administered on three consecutive days before fusion with the same immunogen (20 μg in a volume of 50 μL in PBS without any adjuvant. On the fourth day, mice were sacrificed and splenectomy was performed. Spleen cells were removed and fusion was performed by standard methods using a non-segregating murine myeloma cell line designated P3-X63Ag8.653 (ATCC CRL-1580™).Cloning was done by standard methods.

[00252] The clones were analyzed by binding and inhibition ELISA. The following ELISA binding immunoassay procedure according to the following protocol. The plates were coated with ovalbumin-conjugated 3-epimer prepared in a similar manner to that described above for the BSA conjugate at 1 μg/mL in PBS at 50 μL per well. Plate coating was carried out for 1 hour or more at room temperature. Plates were then snapped dry and blocked with 200 μL per well of blocking buffer diluent (0.5% casein solution in PBS containing 0.05% TWEEN® 20). Plaque blocking was performed for 1 hour or overnight at 2°C-8°C. The plates were then washed three times and snapped dry. The monoclonal antibody to be analyzed was then added to each well as follows: 25 μL of PBS mixed with 25 μL of culture supernatant transferred from the corresponding well in the melt growth plate. Incubation was for approximately 1 hour at room temperature with shaking of the plate. The plate was washed using a plate washer (Bio-Tek, Winooski VT) with plate stacker with the wash buffer being MILLI-Q® water (Millipore Corporation, Billerica, MA) containing 0.05% TWEEN® 20. An enzyme conjugate (goat anti-mouse IgG coupled to HRP diluted in blocking buffer diluent to 1:3000 was added in 50 μL per well. Incubation was carried out for about 1 hour at room temperature with shaking. The plate it was then washed and a chromogenic solution (TMB from Moss Substrates, Pasadena MD) was added in a volume of 100 μL per well for ten minutes at room temperature.The plates were read at 650 nm using an ELISA plate reader.

[00253] Based on the above analysis technique, hybridomas producing suitable monoclonal antibodies were selected. Such monoclonal antibody was designated 8F10 antibody and is an IgG2a kappa antibody. Another such monoclonal antibody has been designated the 4G8 antibody and is an IgG2a kappa antibody.

[00254] Additionally, the clones were also analyzed using an inhibition ELISA procedure according to the following protocol. Plates were coated with ovalbumin-conjugated 3-epimer at 1 μg/mL in phosphate-buffered saline at 50 μL per well. Plate coating was carried out for 1 hour or more at room temperature or overnight at about 2°C to 8°C. Plates were then snapped dry and blocked with 200 μL per well of blocking buffer diluent (0.5% casein solution in PBS containing 0.05% TWEEN® 20). Blocking of the plate was performed by incubation for 30 minutes or more at room temperature with shaking of the plate. The plates were washed. The monoclonal antibody to be analyzed was then added to each well along with free 3-epimer as follows: 25 μL per well of culture supernatant transferred from the corresponding well in the melt growth plate and added 25 μL of 2μg/mL 3 -epimer-25OH Vitamin D3. Incubation was for approximately 1 hour at room temperature with shaking of the plate. The plate was washed using a plate washer (BioTek, Winooski VT) with plate stacker with the wash buffer being MILLI-Q® water (Millipore Corporation, Billerica, MA) containing 0.05% TWEEN® 20. An enzyme conjugate (goat anti-mouse IgG coupled to HRP diluted in blocking buffer diluent to 1:3000 was added in 50 μL per well. Incubation was carried out for about 1 hour at room temperature with shaking. The plate was in then washed and a chromogenic solution (TMB from Moss Substrates, Pasadena MD) was added in a volume of 100 μL per well. If a desired antibody is present in the hybridoma supernatant, then a decrease in optical density will be observed in comparison. with the cell containing no free 3-epimer. 25OH Vitamin D3 was used in place of 25OH vitamin D3 3-epimer as a control to monitor the specificity of antibodies. Antibodies that bind to the 3-epimer and do not bind to 25OH vitamin D3 were selected.

[00255] Generation of polyclonal antibody: Rabbits were immunized with 500 μg/dose of HMCHEMOIO-CMO BSA. A primary immunization and five booster immunizations were performed two weeks apart and two bleeds and one production bleed were collected and antisera were tested in ELISA as described above.

Example 3 Immunoassay for 25OH Vitamin D Immunoassay Procedure

[00256] The 25OH Vitamin D (25(OH)D) immunoassay format employed was a homogeneous competitive chemiluminescent immunoassay based on LOCI® assay technology. The assay was performed on the automated integrated Siemens Dimension® EXL clinical chemistry system (Siemens Healthcare Diagnostics Inc., Deerfield, IL). The assay measured the total concentration of 25(OH)D2 and/or 25(OH)D3 in serum and plasma samples. LOCI® assay reagents included one release reagent, two synthetic bead reagents, and one biotinylated monoclonal anti-25OH vitamin D antibody reagent. The first bead reagent (designated "Sensibeads") was coated with streptavidin and contained a photosensitive dye. The second chemical reagent (designated "Chemibeads") was coated with a 25(OH)D3 analog and contained chemiluminescent dye. The sample was incubated with the release agent to release 25(OH)D molecules including 3-epimeric compounds of vitamin D binding proteins. A reaction mixture was then incubated with biotinylated antibody to form a 25(OH)D complex. D-biotinylated antibody. Because the biotinylated antibody (a sheep monoclonal antibody) cross-reacted with 3-epi-25(OH)D, 100 μg/mL of 8F10.1 anti-3-epimer-VD antibody was added to minimize the assay signal that comes from the 3-epimer vitamin D compounds. Chemical reagents were added to bind excess free biotinylated antibody. Chemical reagents ("Sensibeads") were then added to bind to the biotin portion of the biotinylated antibody. The chemobead analogue/antibody-biotin/streptavidin-Sensibeads aggregates. Illumination of the reaction mixture with light at 680 nm generates singlet oxygen from Sensibeads, which diffused into the chemical reagents and activated a chemiluminescent reaction. The resulting chemiluminescent signal was measured at 612 nm and is inversely proportional to the total 25(OH)D concentration in the sample.

Preparation of Reagents for Immunoassay

[00257] Synthesis of 25(OH)D3 chemical reagents - The 25(OH)D3 chemical reagents were synthesized by coupling EPRM-EDA with 25(OH)D3 cabarmate. The materials used were EPRM-EDA beads, 1-Ethyl-3-(3-Dimethyl Aminopropyl)Carbodiimide (EDAC) Fluka Sulfo-N-Hydroxysuccinimide (SNHS), 25(OH)D3-3- Carbamate, GAFAC surfactant solution ® at 16%, anhydrous DMSO, MES buffer at 50 mM, pH6 containing 10% MeOP and 1% GAFAC® surfactant.

[00258] Preparation of EPRM-EDA beads - EPRM beads (2000 mg, 20.0 mL) are added to a 40 mL vial. EPRM beads are prepared by a procedure similar to that described in United States Patent No. 7,179,660 and the chemiluminescent compound is 2-(4-(N,N, di-tetradecyl)-anilino-3-phenyl thioxene with europium. EDA (800 mg, 890 μL) is combined with 10 mL of MES buffer, pH 6 (the "buffer") and about 4.2 mL of 6N HCl. The pH of the mixture is, or is adjusted to be, about 6.9. The EDA solution is added to the EPRM beads with vortexing and the mixture is equilibrated at room temperature for 15 minutes. Sodium cyanoborohydride (400 mg) is combined in a vial of 15 mL with 10 mL of DI water and the combination is added to the above bead mixture. The mixture is stirred at 37°C for 18 to 20 hours. The beads are transferred to six 40 mL centrifuge tubes. MES is added to bring the volume to 35 mL and the mixture is centrifuged at 19,000 rpm for 30 minutes. The supernatant is decanted and the beads are resuspended in 2 mL of buffer with a stir stick and additional buffer is added at 35 mL. The mixture is sonicated at 18 Watts of power for 30 seconds, using ice to keep the mixture cold. The washing/sonication step is performed 4 times to remove all activation chemistry. After the last centrifugation of MES buffer, 2 mL of the buffer containing 5% MeOP and 0.1% Tween® 20 (the "second buffer") is added to the tubes for the resuspension step. The second additional buffer is added to 35 mL before sonication. The bead suspension is centrifuged at 19,000 rpm for 30 minutes. The supernatant is discarded. Final sonication used 12 mL of the second buffer in each tube to provide a 25 mg/mL dilution. The particle size is 277 nm as determined on a UPA instrument.

[00259] The EPRM chemical reagent is prepared in a manner similar to the method described in United States Patent No. 6,153,442 and United States Patent Application Publication No. 20050118727A, the relevant descriptions of which are incorporated herein by reference. The EPRM chemical reagent comprises an inner aminodextran layer and an outer aldehyde dextran layer having free aldehyde functionalities. See, for example, United States Patent Nos. 5,929,049, 7,179,6 and 7,172,906, the relevant disclosures of which are incorporated herein by reference. The reaction is carried out at a temperature of about 0 to about 40°C over a period of about 16 to about 64 hours at a pH of about 5.5 to about 7.0, or about 6. in a buffered aqueous medium employing a suitable buffer such as, for example, MES. The reaction is quenched by addition of a suitable quenching agent such as, for example, carboxymatoxamine hemihydrochloride (CMO), and subsequent washing of the particles.

[00260] Aldehyde groups in the outer dextran aldehyde layer are reacted with ethylene diamine under reductive amination conditions to form the EPRM-EDA reagent having pendant moieties comprising an ethylene chain and a terminal amine group. Reductive amination conditions include the use of a reducing agent such as, for example, a metal hydride. The reaction is carried out in an aqueous medium at a reaction temperature of about 20°C to about 100°C over a period of about 1 hour to about 48 hours.

[00261] Synthesis of 25(OH)D3-3-Carbamate (25(OH)D3-3-Carbamate) - a mixture of 22 mg (55 μmol) 25(OH)D3 purchased from ChemReagents.com, Sugarland TX, 100 mg (420 μmol) of disuccinimidyl carbonate (DSC), 100 μL of triethylamine in 1 mL of anhydrous acetonitrile in a 5 mL flask (lined with foil) were stirred at room temperature for 18 hours under nitrogen to prepare 25 (OH)D3 activated. TLC (EtOAc:Hexane = 2:1) showed no starting material left. A suspension was prepared by adding 150 mg of carboxymatoxylamine hemihydrochloride (CMO), 0.3 mL of triethylamine, and 1 mL of DMF to a 10 mL vial. A solution containing activated 25(OH)D3 was added dropwise to the CMO suspension with stirring, which was continued for another 18 hours. Vacuum was applied to remove solvents as much as possible (keeping the heating bath temperature below 50°C). EtOAc (25 mL) was added to the residue, which was washed three times with 2 mL of brine. The organic phase was dried with anhydrous Na2SO4 and filtered; the solvent was removed using a Rotavap. The crude product (42 mg) was obtained after drying and was purified by HPLC. The crude product (24 mg) was obtained after drying under high vacuum. The product was dissolved in 1.2 mL of anhydrous DMSO. The aliquots were transferred into vials, which were kept at -70°C.

[00262] Coupling of EPRM-EDA with the hapten 25(OH)D3-Carbamate - 1.2 mg of hapten was added to a 2 mL vial. 11.2 mg of EDAC and 15.5 mg of SNHS plus 3.73 mL of dry DMSO were added to a 5 mL vial. The EDAC/SNHS solution was swirled to dissolve the contents. 1.14 mL of EDAC/SNHS solution was added to the vial containing hapten. The mixture was rotated for 22 hours. EPRM-EDA (200 mg) was washed once with High pH Wash Buffer and then with MES pH6 buffer. To a 5 mL vial was added 1.08 mL (100 mg) of wash buffer followed by 143 mL of 16% GAFAC® surfactant. To a small tube was added 256 of dry DMSO followed by 49 μL of EDAC/SNHS/hapten. The DMSO/hapten solution was added dropwise to the bead mixture (vortexed during addition). The bead/hapten mixture was rotated overnight at room temperature.

[00263] The bead/hapten mixture was transferred to a 50 mL centrifuge tube and diluted to 35 mL with 10% 1-methoxy-2-propanol/1% GAFAC® surfactant/MES buffer, pH6. The tube was centrifuged at 18,500 rpm at 10 °C for 30 minutes. The supernatant was discarded and replaced with 1 mL of the same buffer. The pellet was resuspended with a stir stick. The bottle was filled to 35 mL with the same buffer. The tube was sonicated at 18-21 Watts for 1 minute using ice to keep the tube cool. Centrifugation/washing was repeated six times. After the sixth wash, the buffer was changed to Haptene Wash Buffer pH7.2 and two more washes were performed. After the last wash and resuspending with 1 mL of Haptene Wash Buffer, 4 mL of Haptene Wash Buffer was added. The bead mixture was sonicated at 50% power in a beaker sonicator. The particle size was measured by UPA as 298 nm. Percent solid assay was performed and the buffer mixture was diluted to 10 mg/mL. This chemical reagent was formulated in 50 mM MES buffer.

[00264] Biotinylation of anti-25(OH)D antibody - NHS-PEO4-biotin (Pierce Chemical Company, Rockford IL) was coupled with the anti-25(OH)D antibody (a sheep monoclonal antibody from Bioventix, Farnham, Surrey, United Kingdom). 3 mg of anti-25(OH)D antibody was buffer exchanged twice with 10 mL each of Antibody Dialysis Buffer (10 mM NaH2PO4 pH 7.0/300 mM NaCl) in 10 mL of Amicon and then concentrated to 3.0 mg/mL. 1 mg of NHS-PEO4-biotin was dissolved in 100 μL of Antibody Dialysis Buffer to prepare 10 mg/mL of biotin reagent solution, which was added (35 μL) to the anti-25(OH)D antibody solution. The reaction mixture was equilibrated at room temperature overnight. The mixture was washed three times with 10 mL each of Antibody Dialysis Buffer in 10 mL of Amicon, and then concentrated to about 1 mL. Concentration was measured in UV A280. The biotinylated anti-25(OH)D antibody reagent was formulated in an aqueous buffer containing 25 mM citric acid buffer, 300 mM NaCl, 1 mM EDTA, blocking protein and preservatives, pH 5.0.

[00265] Sensibeads - Streptavidin sensitizing bead was prepared using a method analogous to that described in United States Patent Nos. 6,153,442. 7,022,529, 7,229,842 and United States Patent Application Publication No. 20050118727A. The photosensitizer was bis-(tri-hexyl)silicon-t-butyl-phthalocyanine. The Sensibead reagent concentration was 200 μg/mL in HEPES buffer, pH 8.0, containing 150 mM NaCl.

[00266] Release Reagent - sodium salicylate in 5 mM HEPES buffer. Immunoassay Results

[00267] The effect of adding anti-3-epimer-VD antibody 8F10.1 (anti-VD 3-epimer antibody) on 3-epimer cross-reactivity to the assay is shown in table 1. Sera from ten patients containing 25(OH)D2 or 25(OH)D3 compounds were boosted with 100ng/mL of vitamin D3 3-epimer compound purchased from Sigma (Stock No. 751324). 3-epimer cross-reactivity was calculated as the difference between the values of 25(OH)D2 or 25(OH)D3 with and without the reinforced 3-epimer compound divided by the amount of 3-epimer compound added. With the addition of 100 μg/mL of the anti-3-epimer-VD antibody 8F10.1, the average 3-epimer cross-reactivity was reduced from 13.7% to less than 2%. Table 1

[00268] Figure 9 illustrates a comparison of inherited standard curves using reagents with and without addition of anti-3-epimer-VD antibody 8F10.1 (3-epimer Ab). The complete overlay of the curves shows that the 8F10.1 anti-3-epimer-VD antibody does not affect the evaluation of 25(OH)D3 (25OH Vit D3 in figure 9), indicating that the antibody specific to the 3-epimer compound and it has no observable cross-reactivity with 25(OH)D3.

Example 4 Immunoassay for 3-epi-25OH Vitamin D Immunoassay Procedure

[00269] The measurement of 3-epi-25OH Vitamin 3 (3-epi-25(OH)D) employed a homogeneous competitive chemiluminescent immunoassay based on LOCI® assay technology similar to that described above in example 3. The assay was performed on the automated integrated Siemens Dimension® EXL clinical chemistry system (Siemens Healthcare Diagnostics Inc. LOCI® assay reagents used for the evaluation of 3-epi-25(OH)D included a release agent, two synthetic bead reagents, and a of biotinylated monoclonal anti-3-epimer antibody. The first bead reagent (Sensibeads) was coated with streptavidin and contained photosensitive dye. The second chemical reagent (Chemibeads) was coated with a 3-epimer analog (compound of formula IIa above, wherein Z' is a non-poly(amino acid) label, namely, EPRM-EDA chemical reagent) prepared in a manner similar to that described above by example 1. The sample was incubated with the release agent to release 25(OH)D molecules including 3-epimeric compounds of vitamin D binding proteins. A reaction mixture was then incubated with biotinylated antibody to form a 3-epi-25(OH)D/biotinylated antibody complex. Chemical reagents were added to recover excess free biotinylated antibody. Sensibeads are then added and bind to the biotin portion of the biotinylated antibody. The chemobead analogue/antibody-biotin/streptavidin-Sensibeads aggregates. Illumination of the reaction mixture by light at 680 nm generated singlet oxygen from the Sensibeads, which diffused into the chemical reagents and activated a chemiluminescent reaction. The resulting chemiluminescent signal is measured at 612 nm and is inversely proportional to the total 3-epi-25(OH)D concentration in the sample.

Preparation of Reagents for Immunoassay

[00270] Synthesis of 3-Epimer Analog chemical reagents - Chemibeads for use in this assay for the detection of 3-epi-25(OH)D were prepared in a similar manner to that described above in Example 3 for synthesis of 25 Chemibeads (OH)D3 with the hapten being a compound of formula IIa where Z' is the Chemibead.

[00271] Chemibead chemical reagent - analogous 3-epimer chemical reagents in MES buffer at 50 nM.

[00272] Biotinylation of 8F10.1 anti-3-epimer-VD antibody - Biotinylation of the 8F10.1 anti-3-epimer-VD antibody was carried out in a manner similar to that described above in example 3 for antibody biotinylation anti-25(OH)D.

[00273] Biotinylated Antibody Reagent - Biotinylated anti-3-epimer-VD 8F10.1 antibody established above in 25 mM citrus buffer.

[00274] Sensibead Reagent - Sensibead Reagent was the same as in Example 3.

Immunoassay Results

[00275] Figure 10 illustrates a standard curve for the immunoassay for the determination of 3-epi-25(OH)D in samples using anti-3-epimer-VD antibody 8F10.1. Chemiluminescent kilobeads represent the chemiluminescent signal measured as described above.

[00276] All publications and patent applications cited in this specification are incorporated herein by reference as if each publication or patent application were specifically and individually indicated to be incorporated by reference.

[00277] It should be understood that the examples described above are merely illustrative of some of the many specific examples that represent the principles described here. Clearly, those skilled in the art can readily develop numerous other arrangements without departing from the compound as defined by the following claims.

Claims (12)

1. Method for determining an amount of vitamin D analyte in a sample suspected of containing the 25OH vitamin D analyte, comprising: (a) providing in combination in a test medium: (i) the sample , (ii) a vitamin D epimer binding partner that is specific for epimeric vitamin D, and (iii) a 25-hydroxyvitamin D (25OH vitamin D) binding partner that is specific for the 25OH vitamin D analyte ; (b) incubating the assay medium under conditions for binding of the vitamin D epimer binding partner to the epimeric vitamin D and for binding of the 25OH vitamin D binding partner to the 25OH vitamin D analyte to form a vitamin D binding partner binding complex; and (c) determining the amount of the complex bound to the vitamin D binding partner and relating the amount of the complex bound to the vitamin D binding partner to the amount of 25OH vitamin D in the sample; said vitamin D binding partner being an antibody that specific for the 25OH vitamin D analyte, but with cross-reactivity with epimeric vitamin D; said epimeric vitamin D is 3-epi-25-hydroxyvitamin D, and said vitamin D epimer binding partner is an antibody that specifically binds said 3-epi-25-hydroxyvitamin D, but not binds to any detectable degree with 25OH vitamin D. 2. Method according to claim 1, characterized by the fact that the vitamin D binding partner comprises a member of a signal production system or a solid support. 3. Method according to claim 2, characterized by the fact that the vitamin D binding partner comprises a member of a signal production system that is a label. 4. Method according to claim 1, characterized by the fact that the combination further comprises a vitamin D analogue, wherein the vitamin D analogue is a compound of the formula: (R1)p-(L)qZ , in which in which represents a bond to L, Y is O, S, CR, or NR4, X is -O-(CH2)nC(O)-, -(CH2)wC(O)-, -(CH2)wC(O)- (CH2)x- C(O)-, -(CH2)wC(O)-NH(CH2)yC(O)-, or -NR3-C(O)-, R is independently H or alkyl, R2 is H , R3 and R4 are independently H or alkyl, R5 and R6 are H, R11 is H, n is an integer from 0 to 10, w is an integer from 1 to 10, x is an integer from 1 to 10, y is an integer from 1 to 10, p is 1, L is a linking group, q is 0 or 1, and Z is a label. 5. Method according to claim 1, characterized by the fact that the vitamin D epimer binding partner is constructed against a compound of the formula: (R1)p-(L)qZ, in which in which represents a bond to L, Y is O, S, CR, or NR4, X is -O-(CH2)nC(O)-, -(CH2)wC(O)-, -(CH2)wC(O)- (CH2)x- C(O)-, -(CH2)wC(O)-NH(CH2)yC(O)-, or -NR3-C(O)-, R is independently H or alkyl, R2 is independently H, R3 and R4 are independently H or alkyl, R5 and R6 are H, R11 is H, n is an integer from 0 to 10, w is an integer from 1 to 10, x is an integer from 1 to 10 , y is an integer from 1 to 10, p is 1, L is a linking group, q is 0 or 1, and Z is an immunogenic carrier. 6. Method according to claim 5, characterized by the fact that in the compound of formula (R1)p-(L)qZ, R1 is 7. Method of determining an amount of a 25OH vitamin D analyte in a sample suspected of containing the 25OH vitamin D analyte, characterized by the fact that it comprises: (a) providing in combination in a test medium: ( i) the sample, (ii) a capture antibody that is a 25OH vitamin antibody specific for the 25OH vitamin D analyte, and (iii) an epimeric vitamin D antibody that is specific for epimeric vitamin D; (b) incubating the assay medium under conditions for binding of the vitamin D epimer binding partner to the epimeric vitamin D and for binding of the 25OH vitamin D binding partner to the 25OH vitamin D analyte to form a binding complex -vitamin D antibody; (c) combining the vitamin D antibody-binding complex with a detection antibody that binds to the vitamin D analyte in the vitamin D antibody-binding complex, wherein the detection antibody comprises a member of a production system of signal, and (d) measuring a signal produced by the signal production system and relating the amount of the signal to the amount of the vitamin D analyte in the sample; said vitamin D antibody being specific for the 25OH vitamin D analyte, but having cross-reactivity with epimeric vitamin D; said epimeric vitamin D is 3-epi-25-hydroxyvitamin D, and said vitamin D epimer binding partner is an antibody that specifically binds said 3-epi-25-hydroxyvitamin D, but not binds to any detectable degree with 25-hydroxyvitamin D. 8. Method according to claim 7, characterized in that it also comprises separating the binding-antibody complex from the medium. 9. Method according to claim 7, characterized in that the capture antibody comprises a solid support. 10. The method of claim 7, wherein the capture antibody comprises a particle, wherein the particle is a magnetic particle or the particle comprises one of a photosensitizer or a chemiluminescent compound. 11. Method according to claim 7, characterized by the fact that the vitamin D epimer antibody is constructed against a compound of the formula: (R1)p-(L)qZ in which represents a bond to L, Y is O, S, CR, or NR4, X is -O-(CH2)nC(O)-, -(CH2)wC(O)-, -(CH2)wC(O)- (CH2)x- C(O)-, -(CH2)wC(O)-NH(CH2)yC(O)-, or -NR3-C(O)-, R is independently H or alkyl, R2 is independently H, R3 and R4 are independently H or alkyl, R5 and R6 are H, R11 is H, n is an integer from 0 to 10, w is an integer from 1 to 10, x is an integer from 1 to 10 , y is an integer from 1 to 10, p is 1, L is a linking group, q is 0 or 1, and Z is an immunogenic carrier. 12. Method according to claim 11, characterized by the fact that in the compound of formula (R1)p-(L)qZ, R1 is