Compounds, products and methods for determining the content or presence of vitamin D
阅读说明：本技术 用于测定维生素d的含量或存在的化合物、产品和方法 (Compounds, products and methods for determining the content or presence of vitamin D ) 是由 章春奇 张向辉 赵卫国 刘宇卉 李临 于 2019-02-01 设计创作，主要内容包括：本发明涉及式I所示的化合物,其可以用于测定含有维生素D分析物(包括维生素D2和维生素D3)及其代谢物的样品中其存在或量。本发明还涉及用于测定样品中维生素D的存在或含量的产品、方法和复合物。<Image he="573" wi="700" file="DDA0001966682800000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The present invention relates to compounds of formula I which can be used to determine the presence or amount of vitamin D analytes (including vitamin D2 and vitamin D3) and metabolites thereof in a sample containing the same. The invention also relates to products, methods and complexes for determining the presence or amount of vitamin D in a sample.)
1. A compound of the formula I, wherein,
wherein Z is selected from C1-C20Alkyl radical, C2-C20Alkenyl and C2-C20Alkynyl, said alkyl, alkenyl and alkynyl being optionally selected from hydroxy, C1-C10Alkoxy radical, C1-C10One or more substituents of ester group and oxime group;
R1selected from hydrogen, hydroxy, C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C2-C20Alkenyloxy and C2-C20An alkynyloxy group;
R2and R3Same or different, independently selected from hydrogen and C1-C20An alkyl group;
r is at least R1Substituted C6-C30Aryl radical, C5-C30Heteroaryl or C9-C30The aromatic group of the condensed aromatic group is,
R1is- (CH)2)pX or- (CH)2)pCOX, wherein X is selected fromA labeling moiety derived from a labeling compound, a biomacromolecule moiety, an N-maleimido group to which the labeling moiety or biomacromolecule moiety is attached, a member of a signal producing system, a small organic molecule, and a binding ligand or carrier for said small organic molecule, p being an integer of 0 to 10.
2. The compound of claim 1, wherein Z is selected from C1-C10Alkyl radical, C2-C10Alkenyl and C2-C10Alkynyl, said alkyl, alkenyl and alkynyl being optionally selected from hydroxy, C1-C10Alkoxy radical, C1-C10Ester groups and oxime groups.
3. A compound according to claim 1 or 2, wherein Z is selected from C1-C10Alkyl radical, C2-C10Alkenyl and C2-C10Alkynyl, said alkyl, alkenyl or alkynyl being optionally selected from hydroxy, C1-C5Alkoxy radical, C1-C5Ester groups and oxime groups.
4. A compound according to any one of claims 1 to 3, wherein Z is C with a branch4-C10Alkyl, C with branched chain4-C10Alkenyl or C with a branch4-C20Alkynyl.
5. The compound of claim 4, wherein C is branched4-C10Alkyl, C with branched chain4-C10Alkenyl or C with a branch4-C20The terminal carbon atom of the alkynyl group is attached to a hydroxyl group or a protected hydroxyl group.
6. A compound according to claim 5, wherein Z is 4, 4-dimethyl-4-hydroxybutyl.
7. A compound according to any one of claims 1 to 6, wherein R is1Selected from hydrogen, hydroxy, C1-C10Alkyl radical, C2-C10Alkenyl radical, C2-C10Alkynyl and C1-C10An alkoxy group.
8. A compound according to any one of claims 1 to 7, wherein R is2And R3Independently selected from hydrogen and C1-C10Alkyl, preferably selected from hydrogen and C1-C5An alkyl group.
9. The compound of any one of claims 1-8, wherein the small organic molecule is selected from the group consisting of biotin, fluorescein, rhodamine, chemiluminescent molecules, dinitrophenol, acridinium esters, alkaline phosphatase, and labeled compound molecules, and wherein the binding partner of the small organic molecule is selected from the group consisting of avidin, an antibody to fluorescein, an antibody to rhodamine, an antibody to chemiluminescent molecules, and an antibody to dinitrophenol.
10. The compound of any one of claims 1-9, wherein the member of the signal producing system is selected from the group consisting of a fluorescent compound, a chemiluminescent compound, a sensitizer, an enzyme, and a radiolabel.
11. A compound according to any one of claims 1 to 10 wherein the member of the signal producing system comprises a particle, preferably the particle is selected from the group consisting of a fluorescent particle, a chemiluminescent particle, a sensitizer particle and a magnetic particle.
12. A compound according to any of claims 1 to 11 wherein the biomacromolecule is selected from the group consisting of a protein molecule, a nucleic acid molecule, a polysaccharide molecule and a lipid molecule.
13. A compound according to any one of claims 1 to 12 wherein the binding ligand of the small organic molecule is selected from antibodies to vitamin D and analogues thereof.
14. A compound according to any one of claims 1 to 13 wherein p is 0, 1,2,3,4 or 5.
15. The compound of any one of claims 1-14, wherein R is at least represented by R1Substituted C6-C20Aryl radical, C5-C20Heteroaryl or C9-C20A fused aryl group.
16. The compound of any one of claims 1-15, wherein R is at least represented by R1Substituted C6-C10Aryl radical, C5-C10Heteroaryl or C9-C10Condensed aryl radicals, e.g. at least by R1Substituted phenyl, at least by R1Substituted pyridyl, at least by R1Substituted quinolyl radicals or substituted by at least R1Substituted isoquinolinyl groups.
17. The compound of any one of claims 1-16, wherein the compound has a structure according to formula II or formula III:
in the formula II, R2-R5The same or different, are independently selected from hydrogen and C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C6-C20Aryl radical, C5-C20Heteroaryl group, C9-C20Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C1-C10Alkyl-substituted amino；
In the formula III, R2-R6The same or different, are independently selected from hydrogen and C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C6-C20Aryl radical, C5-C20Heteroaryl group, C9-C20Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C1-C10Alkyl-substituted amino groups.
18. The compound of claim 17, wherein in formula II, R is2-R5Independently selected from hydrogen, C1-C10Alkyl radical, C1-C10Alkenyl radical, C1-C10Alkynyl, C1-C10Alkoxy, cyano, halogen, nitro, amino and C1-C5An alkyl-substituted amino group; in the formula III, R2-R6Independently selected from hydrogen, C1-C10Alkyl radical, C1-C10Alkenyl radical, C1-C10Alkynyl, C1-C10Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
19. A compound according to claim 17 or 18, wherein in formula II R2-R5Independently selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5An alkyl-substituted amino group; in the formula III, R2-R6Independently selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
20. The compound of any one of claims 1 to 19, selected from the group consisting of:
21. a product for determining the presence and/or amount of vitamin D in a sample comprising the following components:
1) the compound of any one of claims 1-20, and
2) a specific binding member for vitamin D.
22. The product of claim 21, wherein the sample is a biological or non-biological sample.
23. The product of claim 21 or 22, wherein the specific binding member for vitamin D is an antibody to vitamin D and a ligand conjugated thereto.
24. The product according to any one of claims 20 to 22, wherein the product is a composition or a kit.
25. Use of a product according to any of claims 21-24 for detecting the presence and/or amount of vitamin D in a sample.
26. A method for determining the presence and/or amount of vitamin D in a sample comprising the steps of:
1) providing a product according to any one of claims 21-24;
2) allowing said specific binding member for vitamin D in the product to form a complex with said compound;
3) measuring the amount of said complex, which is related to the presence and/or amount of vitamin D in said sample.
27. A complex formed from a compound according to any one of claims 1-20 and a specific binding member for vitamin D.
28. The complex of claim 27, wherein the specific binding member for vitamin D is an antibody to vitamin D and a ligand conjugated thereto.
Technical Field
The present invention is in the field of biotechnology and specifically relates to compounds, products and methods for detecting the presence or amount of vitamin D analytes (including vitamin D2 and vitamin D3) and metabolites thereof in a sample containing the same.
Background
Vitamin D is a lipid-soluble steroid prohormone that has two main forms: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Vitamin D2 is obtained from a nutritional supplement, whereas vitamin D3 is derived from skin exposed to sunlight (ultraviolet radiation) and is obtained mainly from fish, liver oil, egg yolk, and other diets. Vitamins D2 and D3 are metabolized in the liver to 25-OH vitamin D (25- (OH) D) and then converted to 1, 25- (OH) in the kidney2D. 25- (OH) D is the major metabolite in the circulation, and thus, 25- (OH) D values may reflect vitamin D levels in the body. Vitamin D in blood exists in a protein-bound form. The clinical test report shows that the vitamin D in blood refers to the sum of 25- (OH) D, including 25- (OH) D2And 25- (OH) D3. Accurate monitoring of total 25-OH vitamin D levels is critical for clinical use.
Vitamin D plays an important role in maintaining bone mineral density, and it plays a critical role in calcium balance with parathyroid hormone (PTH). Vitamin D deficiency seriously affects the absorption of calcium and phosphorus in human bodies, and can cause rickets, hypocalcemia of newborns, hypothyroidism, osteoporosis of middle-aged and elderly people and other calcium metabolism abnormal diseases. Vitamin D overdose can lead to hypercalcemia and various aging-related diseases.
Vitamin D is present in almost all human tissues and its role is not merely to maintain the balance of calcium and phosphorus. Studies have shown that vitamin D acts by binding to the Vitamin D Receptor (VDR), and recent epidemiological studies have found that vitamin D is associated with a variety of diseases-cancer, heart disease, hypertension, diabetes, autoimmune diseases, infectious diseases and aging.
Currently, the commercially available 25-OH vitamin D detection methods mainly include Radioimmunoassay (RIA), liquid chromatography-tandem mass spectrometry (LC-MS), enzyme-linked immunosorbent assay (ELISA) and chemiluminescence assay (CLIA). Among them, the chemiluminescence method is developed based on its advantages of high sensitivity, wide linear range, convenient operation, no pollution, etc.
Disclosure of Invention
It is an object of the present invention to address the deficiencies of the prior art by providing a composition, product and method for detecting the presence or amount of vitamin D analytes (including vitamin D2 and vitamin D3) and metabolites thereof in a sample containing the same.
In order to achieve the above object, in a first aspect, the present invention provides a compound represented by formula I.
Wherein Z is selected from C1-C20Alkyl radical, C2-C20Alkenyl and C2-C20Alkynyl, said alkyl, alkenyl and alkynyl being optionally selected from hydroxy, C1-C10Alkoxy radical, C1-C10One or more substituents of ester group and oxime group;
R1selected from hydrogen, hydroxy, C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C2-C20Alkenyloxy and C2-C20An alkynyloxy group;
R2and R3Same or different, independently selected from hydrogen and C1-C20An alkyl group;
r is at least R1Substituted C6-C30Aryl radical, C5-C30Heteroaryl or C9-C30The aromatic group of the condensed aromatic group is,
R1is- (CH)2)pX or- (CH)2)pCOX, wherein X is selected from the group consisting of a labeling moiety, a biomacromolecule moiety, linked toN-maleimido group of a labeling moiety or biomacromolecule moiety, a member of a signal generating system, a small organic molecule, and a binding ligand or carrier for said small organic molecule, p being an integer from 0 to 10, wherein said labeling moiety is derived from a labeling compound and said biomacromolecule moiety is derived from a biomacromolecule.
In some embodiments of the invention, Z is selected from C1-C10Alkyl radical, C2-C10Alkenyl and C2-C10Alkynyl, said alkyl, alkenyl and alkynyl being optionally selected from hydroxy, C1-C10Alkoxy radical, C1-C10Ester groups and oxime groups.
In some of the embodiments described above, Z is selected from C1-C10Alkyl radical, C2-C10Alkenyl and C2-C10Alkynyl, said alkyl, alkenyl or alkynyl being optionally selected from hydroxy, C1-C5Alkoxy radical, C1-C5Ester groups and oxime groups.
In some of the above embodiments, Z is C with a branch4-C10Alkyl, C with branched chain4-C10Alkenyl or C with a branch4-C20Alkynyl.
In some of the above embodiments, the C having a branched chain4-C10Alkyl, C with branched chain4-C10Alkenyl or C with a branch4-C20The terminal carbon atom of the alkynyl group is attached to a hydroxyl group or a protected hydroxyl group.
In some of the embodiments described above, Z is 4, 4-dimethyl-4-hydroxybutyl having the following structure:
in some of the embodiments described above, R1Selected from hydrogen, hydroxy, C1-C10Alkyl radical, C2-C10Alkenyl radical, C2-C10Alkynyl radicalAnd C1-C10An alkoxy group.
In some of the embodiments described above, R2And R3Independently selected from hydrogen and C1-C10Alkyl, preferably selected from hydrogen and C1-C5An alkyl group.
In some of the embodiments described above, the small organic molecule is selected from the group consisting of biotin, fluorescein, rhodamine, a chemiluminescent molecule, dinitrophenol, acridinium ester, alkaline phosphatase, and a labeled compound molecule, and the binding partner of the small organic molecule is selected from the group consisting of avidin, an antibody to fluorescein, an antibody to rhodamine, an antibody to a chemiluminescent molecule, and an antibody to dinitrophenol.
In some embodiments of the foregoing, the member of the signal producing system is selected from the group consisting of a fluorescent compound, a chemiluminescent compound, a sensitizer, an enzyme, and a radiolabel.
In some embodiments of the foregoing, the member of the signal producing system comprises a particle.
In some embodiments of the foregoing, the particle is selected from the group consisting of a fluorescent particle, a chemiluminescent particle, a sensitizer particle, and a magnetic particle.
In some embodiments of the foregoing, the biomacromolecule is selected from the group consisting of a protein molecule, a nucleic acid molecule, a polysaccharide molecule, and a lipid molecule.
In some embodiments of the foregoing, the binding partner of the small organic molecule is selected from antibodies to vitamin D and analogs thereof.
In some of the embodiments described above, p is 0, 1,2,3,4, or 5.
In some of the above embodiments, R is at least R1Substituted C6-C20Aryl radical, C5-C20Heteroaryl or C9-C20A fused aryl group.
In some of the above embodiments, R is at least R1Substituted C6-C10Aryl radical, C5-C10Heteroaryl or C9-C10Condensed aryl radicals, e.g. at least by R1Substituted phenyl, at least by R1SubstitutionIs at least R1Substituted quinolyl radicals or substituted by at least R1Substituted isoquinolinyl groups.
In some of the embodiments described above, the compound has a structure according to formula II or formula III:
in the formula II, R2-R5The same or different, are independently selected from hydrogen and C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C6-C20Aryl radical, C5-C20Heteroaryl group, C9-C20Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C1-C10An alkyl-substituted amino group;
in the formula III, R2-R6The same or different, are independently selected from hydrogen and C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C6-C20Aryl radical, C5-C20Heteroaryl group, C9-C20Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C1-C10Alkyl-substituted amino groups.
In some of the foregoing embodiments, in formula II, R2-R5Independently selected from hydrogen, C1-C10Alkyl radical, C1-C10Alkenyl radical, C1-C10Alkynyl, C1-C10Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
In some of the embodiments described above, in formula III, R2-R6Independently selected from hydrogen, C1-C10Alkyl radical, C1-C10Alkenyl radical, C1-C10Alkynyl, C1-C10Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
In some of the foregoing embodiments, in formula II, R2-R5Independently selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
In some of the embodiments described above, in formula III, R2-R6Independently selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
The invention provides in a second aspect a product for determining the presence and/or amount of vitamin D in a sample, comprising the following components:
1) a compound of the formula I as described above, and
2) a specific binding member for vitamin D.
According to some embodiments of the foregoing, the product is a composition or a kit.
According to some of the embodiments described above, the sample is a biological or non-biological sample.
According to some of the embodiments described above, the specific binding member for vitamin D is an antibody to vitamin D and a ligand conjugated thereto.
In a third aspect, the present invention provides the use of a product as described above for determining the presence and/or amount of vitamin D in a sample for detecting the presence and/or amount of vitamin D in a sample.
In a fourth aspect, the present invention provides a method for determining the presence and/or amount of vitamin D in a sample, comprising the steps of:
1) providing a product provided by the second aspect of the invention;
2) complexing a specific binding member for vitamin D with a compound of formula I;
3) measuring the amount of said complex, which is related to the presence and/or amount of vitamin D in said sample.
In a fifth aspect, the present invention provides a complex formed between a compound of formula I as provided in the first aspect of the invention and a specific binding member for vitamin D.
Detailed Description
In order that the invention may be readily understood, a detailed description of the invention is provided below. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Where a range of values is provided, it is understood that each intervening value, to the extent that there is no stated or intervening value in that stated range, to the extent that there is no such intervening value, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a specified range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
I. Term(s) for
The term "alkyl" refers to those alkyl groups that are straight, branched, or cyclic, having the indicated number of carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, and the like.
The term "alkenyl" refers to a straight or branched hydrocarbon chain having the indicated number of carbon atoms and at least one carbon-carbon double bond, which may occur at any point along the chain. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, dimethyl pentenyl, and the like.
The term "alkynyl" refers to a straight or branched chain hydrocarbon of the indicated number of carbon atoms containing at least one carbon-carbon triple bond. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and the like.
"aryl" includes groups having aromatic character, including "conjugated" or polycyclic systems containing at least one aromatic ring and not containing any heteroatoms in the ring structure. Examples thereof include phenyl, benzyl, 1,2,3, 4-tetrahydronaphthyl and the like.
"heteroaryl" refers to an aryl group as defined above, but having 1-4 heteroatoms in the ring structure, which may also be referred to as an "aromatic heterocycle" or "heteroaromatic compound". As used herein, the term "heteroaryl" is meant to include a stable 5, 6 or 7 membered monocyclic or 7, 8, 9, 10, 11 or 12 membered bicyclic aromatic heterocyclic ring consisting of carbon atoms and one or more heteroatoms, such as 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, or such as 1,2,3,4, 5 or 6 heteroatoms, independently selected from nitrogen, oxygen and sulfur. The nitrogen atom may be substituted or unsubstituted (i.e., N or NR, where R is hydrogen or other substituent as defined herein). The nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., N → O and S (O))pWherein p is 1 or 2). It should be noted, however, that the total number of sulfur and oxygen atoms in the aromatic heterocycle does not exceed 1.
Examples of heteroaryl groups include pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.
The term "carboxyl" means-COOH or its C1-C6An alkyl ester.
The term "ester group" refers to a compound or fragment containing a carbon or heteroatom bonded to an oxygen atom bonded to a carbon on a carbonyl group. The term "ester" includes alkoxycarbonyl groups such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl and the like.
The term alkoxy includes substituted and unsubstituted alkyl groups covalently bonded to an oxygen atom. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, isopropoxy, propoxy, butoxy, pentoxy, and the like. The term alkenyloxy includes substituted and unsubstituted alkenyl groups covalently bonded to an oxygen atom. Examples of alkenyloxy groups include, but are not limited to, vinyloxy, propenyloxy, butenyloxy, pentenyloxy, and the like. The term "alkynyloxy" includes substituted and unsubstituted alkynyl groups covalently bonded to an oxygen atom. Examples of alkynyloxy include, but are not limited to, ethynyloxy, propynyloxy, butynyloxy, pentynyloxy, and the like.
As used herein, "amine" or "amino" refers to unsubstituted or substituted-NH2. "alkylamino" includes the group-NH-thereof2And a group of compounds in which the nitrogen atom of (a) is bonded to at least one alkyl group. Examples of alkylamino include benzylamino, methylamino, ethylamino, phenethylamino and the like. "dialkylamino" includes wherein-NH2And a group in which the nitrogen atom of (a) is bonded to at least two alkyl groups. Examples of dialkylamino groups include, but are not limited to, dimethylamino and diethylamino. "arylamino" and "diarylamino" include groups in which a nitrogen atom is bonded to at least one or two aryl groups, respectively. "aminoaryl" and "aminoaryloxy" refer to amino-substituted aryl, aryloxy groups. "Alkylarylamino," "alkylaminoaryl," or "arylaminoalkyl" refers to an amino group bonded to at least one alkyl group and at least one aryl group. "alkylaminoalkyl" refers to an alkyl, alkenyl, or alkynyl group bonded to a nitrogen atom that is also bonded to an alkyl group. "amido" includes groups in which a nitrogen atom is bonded to an acyl group. Examples of amido include, but are not limited to, alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido.
"aryl" includes groups having aromatic character, including "conjugated" or polycyclic systems containing at least one aromatic ring and not containing any heteroatoms in the ring structure. Examples thereof include phenyl, benzyl, 1,2,3, 4-tetrahydronaphthyl and the like.
"heteroaryl" refers to an aryl group as defined above, but having 1-4 heteroatoms in the ring structure, which may also be referred to as an "aromatic heterocycle" or "heteroaromatic compound". As used herein, the term "heteroaryl" is meant to include a stable 5, 6 or 7 membered monocyclic or 7, 8, 9, 10, 11 or 12 membered bicyclic aromatic heterocyclic ring consisting of carbon atoms and one or more heteroatoms, such as 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, or such as 1,2,3,4, 5 or 6 heteroatoms, independently selected from nitrogen, oxygen and sulfur. The nitrogen atom may be substituted or unsubstituted (i.e., N or NR, where R is hydrogen or other substituent as defined herein). The nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., N → O and S (O))pWherein p is 1 or 2). It should be noted, however, that the total number of sulfur and oxygen atoms in the aromatic heterocycle does not exceed 1.
Examples of heteroaryl groups include pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.
Furthermore, the terms "aryl" and "heteroaryl" include polycyclic aryl and heteroaryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzimidazole, benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline, naphthyridinyl, indole, benzofuran, purine, benzofuran, deazapurine, indolizine.
The cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring may be substituted at one or more ring positions (e.g., ring carbon atoms or heteroatoms, such as nitrogen atoms) with substituents as described above, e.g., alkyl, alkenyl, alkynyl, halogen, hydroxy, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, arylalkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, arylalkyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonate, phosphinate, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), amido (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), Amidino, imino, mercapto, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonic, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or aromatic or heteroaromatic fragment. The aryl and heteroaryl groups can also be fused or bridged to non-aromatic alicyclic or heterocyclic rings to form a polycyclic ring system (e.g., tetralin, methylenedioxyphenyl).
The term "oximino" refers to-C ═ N-OH.
Detailed description of the preferred embodiments
The present invention will be described in more detail below.
In order to achieve the above object, in a first aspect, the present invention provides a compound represented by formula I.
Wherein Z is selected from C1-C20Alkyl radical, C2-C20Alkenyl and C2-C20Alkynyl, said alkyl, alkenyl and alkynyl being optionally selected from hydroxy, C1-C10Alkoxy radical, C1-C10One or more substituents of ester group and oxime group;
R1selected from hydrogen, hydroxy, C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C2-C20Alkenyloxy and C2-C20An alkynyloxy group;
R2and R3Same or different, independently selected from hydrogen and C1-C20An alkyl group;
r is at least R1Substituted C6-C30Aryl radical, C5-C30Heteroaryl or C9-C30The aromatic group of the condensed aromatic group is,
R1is- (CH)2)pX or- (CH)2)pCOX, wherein X is selected from a labeling moiety derived from a labeling compound, a biomacromolecule moiety, an N-maleimido group to which the labeling moiety or biomacromolecule moiety is attached, a member of a signal generating system, a small organic molecule, and a binding ligand or carrier for said small organic molecule, and p is an integer from 0 to 10, such as an integer from 1 to 5.
In some embodiments of the invention, Z is selected from C1-C10Alkyl radical, C2-C10Alkenyl and C2-C10Alkynyl, said alkyl, alkenyl and alkynyl being optionally selected from hydroxy, C1-C10Alkoxy radical, C1-C10Ester groups and oxime groups. For example, Z is hydroxy-substituted C1-C10An alkyl group. For example Z is C1-C10Alkoxy-substituted C1-C10An alkyl group. For example, Z is hydroxy-substituted C2-C10An alkenyl group. For example Z is C1-C10Alkoxy-substituted C2-C10An alkenyl group.
In some of the embodiments described above, Z is selected from C1-C10Alkyl radical, C2-C10Alkenyl and C2-C10Alkynyl, said alkyl, alkenyl or alkynyl being optionally selected from hydroxy, C1-C5Alkoxy radical, C1-C5Ester groups and oxime groups. For example, Z is hydroxy-substituted C1-C5An alkyl group. For example Z is C1-C5Alkoxy-substituted C1-C5An alkyl group. For example, Z is hydroxy-substituted C2-C5An alkenyl group. For example Z is C1-C5Alkoxy-substituted C2-C5An alkenyl group.
In some of the above embodiments, Z is C with a branch4-C10Alkyl, C with branched chain4-C10Alkenyl or C with a branch4-C20Alkynyl. For example, the branches are selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl. The number of the branched chain may be one or more.
In some of the above embodiments, the C having a branched chain4-C10Alkyl, C with branched chain4-C10Alkenyl or C with a branch4-C20The terminal carbon atom of the alkynyl group is attached to a hydroxyl group or a protected hydroxyl group. For example, the branches are selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl. The number of the branched chain may be one or more.
In some of the embodiments described above, Z is 4, 4-dimethyl-4-hydroxybutyl, having the following structure:
in some of the embodiments described above, R1Selected from hydrogen, hydroxy, C1-C10Alkyl radical, C2-C10Alkenyl radical, C2-C10Alkynyl and C1-C10An alkoxy group. Preferably, R1Can be hydrogen, hydroxy, C1-C5Alkyl radical, C2-C5Alkenyl radical, C2-C5Alkynyl or C1-C5Alkoxy, and the like. For example R1Is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.
In some of the embodiments described above, R2And R3Independently selected from hydrogen and C1-C10Alkyl, preferably selected from hydrogen and C1-C5An alkyl group. For example, R2And R3Are all hydrogen. For example, R2Is C1-C5Alkyl radical, R3Is hydrogen. For example, R2And R3Are all methyl. For example R2Is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butylIsobutyl or tert-butyl. For example R3Is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.
In some of the above embodiments, R is at least R1Substituted C6-C20Aryl radical, C5-C20Heteroaryl or C9-C20A fused aryl group.
In some of the above embodiments, R is at least R1Substituted C6-C10Aryl radical, C5-C10Heteroaryl or C9-C10A fused aryl group.
In some of the above embodiments, R is at least R1Substituted phenyl, pyridine, quinoline or isoquinoline.
In some of the other embodiments described above, X is a member of a signal producing system.
In some other embodiments described above, X is a small organic molecule.
In some other embodiments of the foregoing, X is a binding ligand or a carrier for the small organic molecule.
In some of the embodiments described above, the small organic molecule is selected from the group consisting of biotin, fluorescein, rhodamine, a chemiluminescent molecule, dinitrophenol, acridinium ester, alkaline phosphatase, and a labeled compound molecule, and the binding partner of the small organic molecule is selected from the group consisting of avidin, an antibody to fluorescein, an antibody to rhodamine, an antibody to a chemiluminescent molecule, and an antibody to dinitrophenol.
In some embodiments of the foregoing, the member of the signal producing system is selected from the group consisting of a fluorescent compound, a chemiluminescent compound, a sensitizer, an enzyme, and a radiolabel.
In some embodiments of the foregoing, the member of the signal producing system comprises a particle. Preferably, the particle is selected from the group consisting of a fluorescent particle, a chemiluminescent particle, a sensitizer particle, and a magnetic particle.
In some embodiments of the foregoing, the binding partner of the small organic molecule is selected from antibodies to vitamin D and analogs thereof.
In some embodiments of the foregoing, the biomacromolecule is selected from the group consisting of a protein molecule, a nucleic acid molecule, a polysaccharide molecule, and a lipid molecule.
In some other embodiments of the foregoing, R1Is- (CH)2)pX, e.g. - (CH)2) And (4) X. X may be a biomacromolecule moiety or an N-maleimido group to which a biomacromolecule moiety is attached. The biomacromolecule is for example biotin polyethylene glycol amino (which may have a molecular weight of more than 1 dalton, for example between 2 and 100 dalton, for example between 2 and 80 dalton) or Bovine Serum Albumin (BSA).
In some other embodiments of the foregoing, R1Is- (CH)2)pCOX, e.g., -CH2COX. X may be a biomacromolecule moiety or an N-maleimido group to which a biomacromolecule moiety is attached. The biomacromolecule is for example biotin polyethylene glycol amino (which may have a molecular weight of more than 1 dalton, for example between 2 and 100 dalton, for example between 2 and 80 dalton) or Bovine Serum Albumin (BSA).
In some of the embodiments described above, p is 0, 1,2,3,4, or 5.
E.g. at least by R1Substituted phenyl, at least by R1Substituted pyridyl, at least by R1Substituted quinolyl radicals or substituted by at least R1Substituted isoquinolinyl groups.
In some of the embodiments described above, the compound has a structure according to formula II or formula III:
in the formula II, R2-R5The same or different, are independently selected from hydrogen and C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C6-C20Aryl radical, C5-C20Heteroaryl group, C9-C20Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C1-C10An alkyl-substituted amino group;
in the formula III, R2-R6The same or different, are independently selected from hydrogen and C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C6-C20Aryl radical, C5-C20Heteroaryl group, C9-C20Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C1-C10Alkyl-substituted amino groups.
In some of the foregoing embodiments, in formula II, R2-R5Independently selected from hydrogen, C1-C10Alkyl radical, C1-C10Alkenyl radical, C1-C10Alkynyl, C1-C10Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
In some of the embodiments described above, in formula III, R2-R6Independently selected from hydrogen, C1-C10Alkyl radical, C1-C10Alkenyl radical, C1-C10Alkynyl, C1-C10Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
In some of the foregoing embodiments, in formula II, R2-R5Independently selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
In some of the embodiments described above, in formula III, R2-R6Independently selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
In some of the embodiments described aboveIn the formula, in the formula II and/or in the formula III, R2Selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups. For example, R2Is hydrogen. For example, R2Hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.
In some of the embodiments described above, R in formula II and/or formula III3Selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups. For example, R3Hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.
In some of the embodiments described above, R in formula II and/or formula III4Selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups. For example, R4Hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.
In some of the embodiments described above, R in formula II and/or formula III5Selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups. For example, R5Hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.
In some of the embodiments described above, in formula III, R6Selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups. For example, R5Hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.
In some of the embodiments described above, the,
specific examples of the compounds of formula I of the present invention include, but are not limited to, the following compounds:
the invention provides in a second aspect a product for determining the presence and/or amount of vitamin D in a sample, comprising the following components:
1) a compound according to formula I above, and
2) a specific binding member for vitamin D.
According to some embodiments of the foregoing, the product is a composition or a kit.
According to some of the embodiments described above, the sample is a biological or non-biological sample.
According to some of the embodiments described above, the specific binding member for vitamin D is an antibody to vitamin D and a ligand conjugated thereto.
In a third aspect, the present invention provides the use of a product as described above for determining the presence and/or amount of vitamin D in a sample for detecting the presence and/or amount of vitamin D in a sample.
In a fourth aspect, the present invention provides a method for determining the presence and/or amount of vitamin D in a sample, comprising the steps of:
1) providing a compound represented by formula I above;
2) complexing a specific binding member for vitamin D with a compound of formula I;
3) measuring the amount of said complex, the amount of said conjugate being related to the presence and/or amount of vitamin D in said sample.
In a fifth aspect, the present invention provides a complex formed between a compound of formula I as described above and a specific binding member for vitamin D.
Example III
In order that the present invention may be more readily understood, the following detailed description will proceed with reference being made to examples, which are intended to be illustrative only and are not intended to limit the scope of the invention. The starting materials or components used in the present invention may be commercially or conventionally prepared unless otherwise specified.
Reagents and instrumentation:
synthesis example 1
Synthesis of derivatives containing N-maleimidoaryl substituted vitamin D
Weighing 125- (OH) VD350mg of the aqueous solution was dissolved in 2.5mL of anhydrous DMSO to prepare a 20mg/mL solution. A4 mL centrifuge tube was charged with 1.25mL of 25- (OH) VD3(62.4umol) in a molar ratio of 1.2:1 (PMPI: 25- (OH) VD)3) 16.04mg of PMPI (p-maleimidophenyl isocyanate) (74.88umol) was added thereto, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was added with saturated aqueous sodium chloride solution, extracted with ethyl acetate, and extracted with anhydrous Na2SO4Drying, spin-drying, and purification by thin layer chromatography gave 15mg of compound 2 in 39% yield.1HNMR(300MHz,CDCl3):0.56(s,3H,Me),0.90(d,3H,Me,J＝5.8Hz),0.79–2.46(several m,19H),1.25(s,6H,Me),2.62(dd,1H,J＝13.5,3.2Hz),2.75(d,1H,J＝11.2Hz),3.35(dd,2H,J＝11.5,6.1Hz),3.65(s,1H,OH),3.67 (t,2H,J＝5.7Hz),4.85–5.01(m,1H,H3),5.06(dd,2H,CH2),6.04and 6.22(2d,2H, CH,J＝11.2Hz),6.95(d,1H,CH),7.00(d,1H,CH)，7.25–7.62(m,4H,ArH)， 9.0(s,1H,NH).
Synthesis example 2
Synthesis of N-maleimidoaryl substituted vitamin D containing labels
10mg of SHPEGnBiotin (5kD) (2umol) was weighed out and dissolved in 0.02M PBS (0.15M NaCl, 25mM EDTA) pH7.2 buffer solution, 2.4mg (4umol) of Compound 2 was added at a molar ratio of 2:1 and stirred at room temperature for 2 h. Purifying by desalting column, and lyophilizing to obtain compound 3 lyophilized powder 12 mg.
Synthesis example 3
Synthesis of chloromethyl aryl substituted vitamin D derivatives
Weighing 125- (OH) VD350mg of the aqueous solution was dissolved in 2.5mL of anhydrous DMSO to prepare a 20mg/mL solution. A2 mL centrifuge tube was charged with 1.25mL of 25- (OH) VD3(62.4. mu. mol) of DMSO solution, 31.37mg of 4- (chloromethyl) phenyl isocyanate (187.2. mu. mol) was added in a molar ratio of 3:1, the mixture was stirred at room temperature for 3 hours, a saturated aqueous solution of sodium chloride was added to the reaction mixture, the mixture was extracted with ethyl acetate, and the mixture was extracted with anhydrous Na2SO4Drying, spin-drying, and purification by column chromatography gave 12.5mg of compound 4, 35% yield.
1HNMR(300MHz,CDCl3):0.54(s,3H,Me),0.88(d,3H,Me,J＝5.8Hz), 0.76–2.51(several m,19H),1.30(s,6H,Me),2.55(dd,1H,J＝12.6,3.3Hz),2.64(d,1H, J＝10.9Hz),3.23(dd,2H,J＝10.5,5.8Hz),3.43(s,1H,OH),3.54(t,2H,J＝4.6Hz), 4.65(s,2H,CH2),4.74–4.97(m,1H,H3),5.03(dd,2H,CH2),5.95and 6.11(2d,2H,CH, J＝11.2Hz)，7.41–7.68(m,4H,ArH)，8.5(s,1H,NH).
Synthesis example 4
Marker for synthesizing chloromethyl aryl substituted vitamin D
Weighing 10mg NH2PEGnBiotin (5kD) (2umol), dissolved in 0.02M PBS (0.15M N)aCl, 25mM EDTA) pH7.2, 2.27mg (4. mu. mol) of Compound 4 was added in a molar ratio of 2:1 and stirred at 37 ℃ for 16 h. Purifying by desalting column, and lyophilizing to obtain compound 5 lyophilized powder 14 mg.
Synthesis example 5
Synthesis of chloromethyl heteroaryl substituted vitamin D derivatives
Weighing 125- (OH) VD350mg of the aqueous solution was dissolved in 2.5mL of anhydrous DMSO to prepare a 20mg/mL solution. A4 mL centrifuge tube was charged with 1.25mL of 25- (OH) VD3(62.4. mu. mol) of DMSO solution, 40.92mg of 8- (chloromethyl) -5-isocyanatoquinoline (187.2. mu. mol) was added in a molar ratio of 3:1, the mixture was stirred at room temperature for 3 hours, a saturated aqueous solution of sodium chloride was added to the reaction mixture, the mixture was extracted with ethyl acetate, and the mixture was extracted with anhydrous Na2SO4Drying, spin-drying and purification by column chromatography gave 20mg of compound 6 in 52% yield.
1HNMR(300MHz,CDCl3):0.57(s,3H,Me),0.89(d,3H,Me,J＝6.3Hz), 0.73–2.49(several m,19H),1.33(s,6H,Me),2.57(dd,1H,J＝12.4,3.6Hz),2.67(d,1H, J＝11.3Hz),3.25(dd,2H,J＝10.1,6.2Hz),3.46(s,1H,OH),3.58(t,2H,J＝4.7Hz), 4.64(s,2H,CH2),4.70–4.94(m,1H,H3),5.05(dd,2H,CH2),5.97and 6.15(2d,2H, CH,J＝10.3Hz)，7.62–9.01(m,5H,ArH)，8.7(s,1H,NH).
Synthesis example 6
Marker for synthesizing chloromethyl heteroaryl substituted vitamin D
Weighing 10mg NH2PEGnBiotin (5kD) (2umol) was dissolved in 0.02M PBS (0.15M NaCl, 25mM EDTA) pH7.2 buffer, 2.48mg (4umol) of Compound 6 was added at a molar ratio of 2:1, and stirred at 37 ℃ for 16 h. Purifying by desalting column, and lyophilizing to obtain compound 7 lyophilized powder 13 mg.
Synthesis example 7
Synthetic succinate-substituted vitamin D-containing markers
2mg of the compound 8_ 25-hydroxyvitamin D is weighed in a 2mL centrifuge tube3Hemisuccinate was dissolved in 1mL of anhydrous DMSO, and 1.9mg of EDAC and 2.3mg of NHS were added thereto, followed by stirring at room temperature for 1 hour.
Weighing 10mg NH2PEGnBiotin (5kD) (2umol), dissolved in 0.02MPBS (0.15M NaCl, 25mM EDTA) pH7.2 buffer solution, activated 25-hydroxyvitamin D was added in a molar ratio of 2:13Succinate, stirred at room temperature for 2 h. Purifying by a desalting column to obtain a compound 9.
Synthesis example 8
Synthetic BSA conjugates with and aryl substituted vitamin D
A2 mL centrifuge tube was added with 1mL of 10mg/mL BSA (0.02M PBS, pH7.2, 25mM EDTA) buffer, 1M DTT was added to a final concentration of 10mM, vortexed, mixed, and allowed to stand at room temperature for 2 h. Desalting with desalting column to remove excessive DTT. Then, 150uL of compound 2 (20 mg/mL) was added at a molar ratio of 10:1 (Compound 2: BSA). Stir at room temperature for 2h and dialyze the protein into a buffer solution of 0.02MPBS, pH 7.2. Purification by dialysis yielded conjugate 10.
A2 mL centrifuge tube was charged with 1mL of 10mg/mL BSA (0.02M PBS, pH7.2, 25mM EDTA) buffer, followed by 150uL of 20mg/mL Compound 4 at a molar ratio of 10:1 (Compound 4: BSA). After stirring at room temperature for 24h, the protein was dialyzed into 0.02M PBS, pH7.2 buffer, and purified by dialysis to give conjugate 11.
The name of the experiment: development of kit for quantitative detection of 25-hydroxyvitamin D by chemiluminescence method
Purpose of the experiment: research and development of kit for quantitatively detecting 25-hydroxyvitamin D
Experiment design: detection by competitive chemiluminescence
Reagents and instrumentation:
resisting 25-OH VD3Antibody (Bioventix), biotinylated-25-OH VD3Compound 3 (self-produced), biotinylated-25-OH VD3Compound 5 (Secury), biotinylated-25-OH VD3Compound 7 (Secury), biotinylated-25-OH VD3Compound 9 (Secury), carboxyl microspheres (JSR), phosphate buffer (0.02M PBS, pH 7.2), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDAC (thermo fisher), Tween-20, 0.1M MES buffer (pH 6.0).
LiCA HT (Shanghai Boyang Biotechnology Co., Ltd.), Hitachi high-speed refrigerated centrifuge
The experimental steps are as follows:
coating for resisting 25-OH VD3Preparation of microspheres of antibodies
First, 10mg of carboxyl functionalized microspheres were taken in a 2mL centrifuge tube and washed once with 0.1M MES (pH 6.0) buffer at 4 ℃ at 10000rpm for 15 min.
Secondly, 200uL of 0.1M MES (pH 6.0) buffer solution is added for even ultrasonic dispersion, 8uL of 1mg/mL anti-25-OH vitamin D goat monoclonal antibody is added, then 100uL of 5mg/mL EDAC (0.1M MES) solution is added, and stirring is carried out at room temperature for 4 hours.
And thirdly, adding 50uL of 200mg/mL BSA blocked carboxyl microspheres.
And thirdly, centrifuging and washing the microspheres for three times by using PBS (phosphate buffer solution) containing 0.5% Tween-20, and finally fixing the volume to 10mg/mL by using the PBS.
Detection experiment
Experiment one:
the name of the experiment: different biotinylated aryl-substituted-25-OH VD3Detection of derivatives
Purpose of the experiment: screening for optimal biotinylated aryl substituted 25-OH VD3Derivatives of the same
Experiment design: the same reaction scheme, N-maleimide aryl substituted Bio-25-OH-VD was chosen33 and chloromethyl aryl substituted Bio-25-OH-VD35 Performance comparison
The experimental steps are as follows:
1. diluting the luminescent microspheres to 30 mu g/mL, biotinylating to-25-OH VD3Derivatives 3 and 5 were diluted in a gradient of 5ng/mL, 0.5ng/mL and 0.05ng/mL, respectively.
2. Adding horse serum into a 25-hydroxy vitamin D solution with the concentration of 1mg/mL to prepare calibrators 1-6, and assigning values to the calibrators, wherein the values are respectively 0, 4.07, 8.15, 17.75, 33.13 and 65.12.
3. Adding a sample solution according to a reaction mode, and then sequentially adding the coated luminescent microspheres and the biotinylated 25-OHV3Derivatives, 25uL each.
4. A first stage incubation was performed: incubate at 37 ℃ for 17 min.
5. Add 175ul of universal solution.
6. Performing a second stage incubation: incubate at 37 ℃ for 15 min.
7. And (6) reading.
And (3) division of the calibration product:
Cal1/Cal2
1.58
1.77
1.19
1.80
2.17
1.74
Cal2/Cal3
1.37
1.36
1.19
1.10
1.37
1.24
Cal3/Cal4
1.59
1.32
1.10
1.50
1.43
1.25
Cal4/Cal5
1.31
1.18
1.08
1.65
1.41
1.11
Cal5/Cal6
1.97
1.14
0.95
3.01
1.51
1.18
Cal1/Cal6
8.94
4.28
1.59
14.81
8.99
3.52
and (3) data analysis:
from the viewpoint of the distinction between the signal amount and the calibrator, Bio-25-OH-VD33 raising reagent signal amount and distinguishability ratio Bio-25-OH-VD35 high.
The overall discrimination was best at a biotin reagent concentration of 5 ng/mL.
And (4) experimental conclusion:
from the calibrator signal quantity and discrimination, N-maleimidoaryl substituted Bio-25-OH VD33 reagent performance is superior to that of chloromethyl aryl substituted Bio-25-OH VD35。
Experiment two:
the name of the experiment: different biotinylated aryl and heteroaryl substituted-25-OH VD3Detection of derivatives
Purpose of the experiment: heteroaryl substituted 25-OH VD3Derivatives and aryl substituted 25-OH VD3Comparison of the Properties of the markers of the derivatives
Experiment design: same reaction scheme, choosing aryl substituted Bio-25-OH-VD35 and aryl substituted Bio-25-OH-VD37 Performance comparison
The experimental steps are as follows:
1. diluting the luminescent microspheres to 30 mu g/mL, biotinylating to-25-OH VD3Derivatives 5, 7 were diluted in a gradient of 5ng/mL, 0.5ng/mL, 0.05ng/mL, respectively.
2. Adding horse serum into a 25-hydroxy vitamin D solution with the concentration of 1mg/mL to prepare calibrators 1-6, and assigning values to the calibrators, wherein the values are respectively 0, 4.07, 8.15, 17.75, 33.13 and 65.12.
3. Adding a sample solution according to a reaction mode, and then sequentially adding the coated luminescent microspheres and the biotinylated 25-OHV3Derivatives, 25uL each.
4. A first stage incubation was performed: incubate at 37 ℃ for 17 min.
5. Add 175ul of universal solution.
6. Performing a second stage incubation: incubate at 37 ℃ for 15 min.
7. And (6) reading.
And (3) division of the calibration product:
Cal1/Cal2
1.58
1.78
1.19
1.55
1.67
1.15
Cal2/Cal3
1.38
1.37
1.20
1.34
1.30
1.14
Cal3/Cal4
1.60
1.33
1.10
1.52
1.25
1.07
Cal4/Cal5
1.32
1.18
1.09
1.26
1.13
1.06
Cal5/Cal6
1.99
1.14
0.95
1.71
1.10
0.96
Cal1/Cal6
9.12
4.36
1.61
6.82
3.36
1.43
and (3) data analysis:
from the viewpoint of the discrimination between the signal amount and the calibrator, Bio-25-OH-VD is used at the same biotin reagent concentration35 and Bio-25-OH-VD37 lower end scores were closer, but Bio-25-OH-VD35 overall reagent signal and discrimination was high. The overall discrimination was best at a biotin reagent concentration of 5 ng/mL.
And (4) experimental conclusion:
aryl substituted Bio-25-OH-VD from calibrator signal and discrimination35 reagent Performance superior to heteroaryl substituted Bio-25-OH-VD37。
Experiment three:
the name of the experiment: different biotinylated 25-OH VD3Detection of derivatives
Purpose of the experiment: aryl substituted 25-OH VD3Derivatives and succinic acid substituted 25-OH VD3Comparison of the Properties of the markers of the derivatives
Experiment design: same reaction scheme, choosing aryl substituted Bio-25-OH-VD33 and succinic acid substituted Bio-25-OH-VD39 Performance comparison
The experimental steps are as follows:
1. diluting the luminescent microspheres to 30 mu g/mL, biotinylating to-25-OH VD3Derivatives 3 and 9 were diluted in a gradient of 5ng/mL, 0.5ng/mL and 0.05ng/mL, respectively.
2. Adding horse serum into a 25-hydroxy vitamin D solution with the concentration of 1mg/mL to prepare calibrators 1-6, and assigning values to the calibrators, wherein the values are respectively 0, 4.07, 8.15, 17.75, 33.13 and 65.12.
3. Adding a sample solution according to a reaction mode, and then sequentially adding the coated luminescent microspheres and the biotinylated 25-OHV3Derivatives, 25uL each.
4. A first stage incubation was performed: incubate at 37 ℃ for 17 min.
5. Add 175ul of universal solution.
6. Performing a second stage incubation: incubate at 37 ℃ for 15 min.
7. And (6) reading.
And (3) division of the calibration product:
Cal1/Cal2
1.80
2.17
1.75
1.76
2.03
1.56
Cal2/Cal3
1.10
1.37
1.25
1.10
1.31
1.17
Cal3/Cal4
1.50
1.44
1.25
1.46
1.34
1.16
Cal4/Cal5
1.66
1.41
1.11
1.57
1.29
1.07
Cal5/Cal6
3.05
1.52
1.19
2.40
1.32
1.11
Cal1/Cal6
15.12
9.23
3.61
10.64
6.09
2.50
and (3) data analysis:
from the viewpoint of the distinction between the signal amount and the calibrator, Bio-25-OH-VD33 raising reagent signal amount and distinguishability ratio Bio-25-OH-VD3And 9 high.
The overall discrimination was best at a biotin reagent concentration of 5 ng/mL.
And (4) experimental conclusion:
from the calibrator signal quantity and discrimination, N-maleimidoaryl substituted Bio-25-OH VD33 the performance of the reagent is superior to that of succinic acid-substituted Bio-25-OH VD39。
Experiment four:
the name of the experiment: different biotinylated 25-OH VD3Detection of derivatives
Purpose of the experiment: exploration of different biotinylated 25-OH VD3Comparison of the Performance of the derivatives on plate-type chemiluminescence
Experiment design: same reaction mode, detection by plate chemiluminescence
The experimental steps are as follows:
1. avidin 2ug/ml coated, 100 ul/well, 4 ℃ overnight
2. Washing the plate: washing the microporous plate with diluted washing solution for 5 times, adding no less than 400 μ L of washing solution into each hole, soaking for 10 s each time, and drying on clean absorbent paper.
3. CB diluted Biotin-25-OH VD3 compound (1/10000)3, 5, 9, 100 ul/well was added.
4. Washing the plate: washing the microporous plate with diluted washing solution for 5 times, adding no less than 400 μ L of washing solution into each hole, soaking for 10 s each time, and drying on clean absorbent paper.
5. Adding a sample: 50 μ L of sample was added to each well. Adding an antibody: add 100. mu.L of antibody per well
6. And (3) incubation: mix well for 5 seconds with a micro-shaker, seal the plate with a sealing membrane, incubate for 2 hours at 37 ℃.
7. Washing the plate: washing the microporous plate with diluted washing solution for 5 times, adding no less than 400 μ L of washing solution into each hole, soaking for 10 s each time, and drying on clean absorbent paper.
8. Adding an enzyme marker: in addition to the blank control wells, 100. mu.L of enzyme label was added to each well
9. And (3) incubation: mix well for 5 seconds with a micro-shaker, seal the plate with a sealing membrane, incubate for 1 hour at 37 ℃.
10. Washing the plate: washing the microporous plate with diluted washing solution for 5 times, adding no less than 400 μ L of washing solution into each hole, soaking for 10 s each time, and drying on clean absorbent paper.
11. Adding a substrate solution: add 100 μ L of the prepared chemiluminescent substrate working solution into each well, and use 8-channel pipette, mix for 5 seconds with shaking by micro-shaker.
12. And (3) detection: and (3) adding the luminescent substrate solution, standing for 5 minutes at room temperature (20-27 ℃) in a dark place, immediately and sequentially measuring the luminescent value (RLU) of each hole on a microplate luminescence analyzer for 0.1-1.0 second/hole. And (3) detection results:
Bio-25-OH VD33
Bio-25-OH VD35
Bio-25-OH VD39
S0
5970
1958
2655
S1
4325
1855
2409
S2
3640
1640
1999
S3
2057
1527
1702
S4
1854
1477
1364
S5
1536
1395
1520
S6
1230
1287
1273
S7
705
1063
922
S0/S1
1.38
1.06
1.10
S1/S2
1.19
1.13
1.21
S2/S3
1.77
1.07
1.18
S3/S4
1.11
1.03
1.25
S4/S5
1.21
1.06
0.90
S5/S6
1.25
1.08
1.19
S6/S7
1.74
1.21
1.38
and (3) data analysis:
Bio-25OH VD33 and Bio-25-OH VD39 integral discrimination average ratio Bio-25-OH VD of detection sample35 high. Bio-25-OH VD33 ratio Bio-25-OH VD39 the integral discrimination of the detection sample is slightly higher, and the sensitivity of the detection of the low-end sample is higher.
And (4) experimental conclusion:
from the discrimination of the test samples, N-maleimidoaryl substituted Bio-25-OH VD33 the best performance of the reagent.
It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.
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