CN103588872B - A kind of vitamins D synthetic antigen, its preparation method and application - Google Patents

A kind of vitamins D synthetic antigen, its preparation method and application Download PDF

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CN103588872B
CN103588872B CN201210287219.5A CN201210287219A CN103588872B CN 103588872 B CN103588872 B CN 103588872B CN 201210287219 A CN201210287219 A CN 201210287219A CN 103588872 B CN103588872 B CN 103588872B
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vitamins
synthetic antigen
protein carrier
antigen
solution
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CN103588872A (en
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杨奇
崔建华
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Beijing Bohui Innovation Biotechnology Group Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/77—Ovalbumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors

Abstract

The invention provides a kind of vitamins D synthetic antigen and preparation method thereof, described vitamins D synthetic antigen is the conjugate of vitamins D and protein carrier, described vitamins D is 25-hydroxyvitamin D3 or 1,25-dihydroxyvitamin D3, described protein carrier is one or more in bovine serum albumin, ovalbumin, hemocyanin, human serum albumin.The present invention also provides the application of described vitamins D synthetic antigen in vitamins D immunology detection.The present invention further provides vitamins D detection kit, it is integrated with the advantage of existing clinical vitamins D detection method, be applicable to all microplate reader, Chemiluminescence Apparatus and temporal resolution analyser, detection time shortens greatly, and sensitivity, accuracy, precision all can meet testing requirement.Adopt the immuno sorbent assay kit of highly versatile of the present invention, batch, the rapid detection of vitamins D in serum (or blood plasma) can be realized.

Description

A kind of vitamins D synthetic antigen, its preparation method and application
Technical field
The present invention relates to field of immunology, specifically, relate to a kind of vitamins D synthetic antigen, its preparation method and application.
Background technology
Vitamins D is the very important VITAMIN of a class in body, and Main Function is in vivo the metabolism regulating calcium phosphorus, simultaneously and the state of health of body also have very large relation.
The main circulation form of vitamins D is 25-hydroxy-vitamin D (comprising 25-OH Vintamin D2 and 25-hydroxyvitamin D3), and it is most important vitamins D metabolism product, is widely used.When be deficient in vitamin in body D time can cause a series of disease.Wherein, the biologic activity of 1-25-(OH)2-D3 is maximum, and 1-25-(OH)2-D3 can promote the absorption of calcium, and has other important biological function.
Due to the importance of vitamins D, therefore the content detection of vitamins D in body is also more and more received to the concern of people.
Due to the special molecular structure of vitamins D, all there is no very good detection method for a long time, in general the detection spended time of vitamins D is longer, need special equipment etc., along with the development of Protocols in Molecular Biology, the method at present for measuring 25-hydroxyvitamin D3 mainly contains radio-competitive protein binding method (CPB), radioimmunology (RIA), high performance liquid chromatography (HPLC), the Electrochemiluminescince of Roche and the euzymelinked immunosorbent assay (ELISA) etc. of Britain IDS.Be summarized as follows respectively:
(1) radio-competitive protein binding method
WeiS, TanakaH, KuboT etc., AmultipleassayforvitaminDmetabolteswithouthigh-performan celiquidchromatography.AnalBiochem, 1994, disclose a kind of without the need to HPLC purifying in 222 (2): 359-365 documents, only 0.5ml serum can measure serum 25(OH) method of D3 content.Key step comprises extracts serum with acetonitrile, abstraction and purification on C-18/OH post and silica gel Sep-Pak post, finally measure 25(OH with vitamin D binding protein standard measure) D3, the method has fast easy, sample consumption is few, reproducible, can carry out the advantage such as quantitatively to three kinds of main metabolites of vitamins D simultaneously, but the rate of recovery is lower.
(2) radioimmunology
" Chinese journal of obstetrics and gynecology " the 3rd interim 25-(OH reported in use serum measured by radioimmunoassay in 1993) D3, the method application of radiation rubidium marking tracer, detected result and radioreceptor assay height correlation, there is method easy, highly sensitive, the advantages such as specificity is good, but its shortcoming is that the isotropic substance marked is unstable, very high to Laboratory Request during operation, and the radiation produced has disadvantageous effect to operator ' s health.
(3) high performance liquid chromatography
Namely the detection method to vitamins D in GB and pharmacopeia.Red, orange, green, blue, yellow (ROGBY), due to the plant and instrument of complexity and loaded down with trivial details process, is not suitable for clinical great amount of samples examination.
(4) Electrochemiluminescince
The 25-(OH that what current Roche Holding Ag adopted is in ElectrochemiluminescDetermination Determination serum) D3, its principle is with electrochemiluminescence agent tris (bipyridine) ruthenium traget antibody, by antigen-antibody reaction and Beads enrichment technology, carry out quantitative or qualitative according to the light intensity that tris (bipyridine) ruthenium sends on electrode to antigen to be determined or antibody.But its technology poor universality, need with special equipment, and equipment cost is higher.
(5) Enzyme-Linked Immunospot
25(OH in application ELISA method quantitative assay human serum, blood plasma, cell culture supernatant or other related liquid) D3 content.Ultimate principle is 25(OH in application competitive enzyme-linked immune assay sample) D3 level.With polyclonal antibody bag by microwell plate, make insolubilized antibody, added 25(OH to bag successively by the micropore that resists more) D3(sample), biotinylated people 25(OH) D3 antigen, HRP mark avidin, with substrate TMB colour developing after thorough washing.TMB changes into blueness under the catalysis of peroxidase, and changes into final yellow under the action of an acid.25(OH in the depth of color and sample) D3 is proportionate.Under 450nm wavelength, absorbancy (OD value) is measured, calculation sample concentration by microplate reader.The shortcoming of this type of test kit uses biotin labeling 25-hydroxyvitamin D3, marks avidin with HRP, and insolubilized antibody selects polyclonal antibody to detect, and specificity is not strong, and detection time is long.
Summary of the invention
The object of this invention is to provide a kind of novel vitamins D synthetic antigen, its preparation method and application.
In order to realize the object of the invention, a kind of vitamins D synthetic antigen of the present invention, it is the conjugate of vitamins D and protein carrier, described vitamins D is 25-hydroxyvitamin D3 or 1,25-dihydroxyvitamin D3, described protein carrier is one or more in bovine serum albumin, ovalbumin, hemocyanin, human serum albumin.Wherein, the weight ratio of described vitamins D and protein carrier is 1:16 ~ 20.
The present invention also provides the method preparing said vitamin D synthetic antigen, comprises the following steps: 1) be dissolved in the damping fluid of pH8.5 by carrier proteins, obtain solution A; 2) vitamins D is dissolved in the reagent such as ethanol, dimethyl sulfoxide (DMSO) or pyridine, obtains solution B; 3) add activator respectively in solution A and B, obtain protein carrier and the vitamins D of activation; 4) for coupling agent, the protein carrier of above-mentioned activation and vitamins D are carried out coupling with N, N'-bis-succinimidyl carbonate, 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride or N, N'-dicyclohexylcarbodiimide etc.; 5) dialysis, drying, obtain vitamins D synthetic antigen.
Wherein, the damping fluid described in step 1) is phosphate buffered saline buffer, borate buffer solution or Tris damping fluid.
Activator described in step 3) is containing the disodium phosphate soln of quadrol and ethylendiamine dihydrochloride, DMAP-acetone mixture, glutaraldehyde or formaldehyde etc.Activator is preferably the 0.05M disodium phosphate soln pH10.4 containing quadrol and ethylendiamine dihydrochloride, and wherein quadrol concentration is 10%(v/v), ethylendiamine dihydrochloride concentration is 20mg/ml.More preferably activator is the DMAP-acetone mixture of mass ratio 21:80.
The present invention also provides the application of said vitamin D synthetic antigen in vitamins D immunology detection, it is that described vitamins D synthetic antigen and marker are combined as competitive antigen, by with the vitamins D competition binding vitamins D monoclonal antibody in sample, thus detect the vitamin D content in sample.
Wherein, described marker is:
A) horseradish peroxidase or alkaline phosphatase;
B) acridinium ester compounds; Or
C) Eu is chelated with 3+, Sm 3+, Tb 3+or Dy 3+diethylene triamine pentacetic acid (DTPA) (DTPA), 2-[(4-cyano-phenyl) methyl]-Isosorbide-5-Nitrae, 7,10-tetra-azo-cycle dodecane-1,4,7,10-tetraacethyl (DOTA) or 4, the bifunctional chelating agents such as 7-dichloro sulphophenyl-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA).
Described vitamins D monoclonal antibody is purchased from Abcam company of Britain.
The present invention further provides vitamins D immunological detecting kit, it comprises the competitive antigen obtained after marker mark by the enzyme plate of vitamins D monoclonal antibody, said vitamin D synthetic antigen.
Wherein, described vitamins D monoclonal antibody is purchased from Abcam company of Britain.
Described marker is:
A) horseradish peroxidase or alkaline phosphatase;
B) acridinium ester compounds; Or
C) Eu is chelated with 3+, Sm 3+, Tb 3+or Dy 3+diethylene triamine pentacetic acid (DTPA) (DTPA), 2-[(4-cyano-phenyl) methyl]-Isosorbide-5-Nitrae, 7,10-tetra-azo-cycle dodecane-1,4,7,10-tetraacethyl (DOTA) or 4, the bifunctional chelating agents such as 7-dichloro sulphophenyl-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA).
Also comprise in washings, vitamins D standard substance, substrate nitrite ion, reaction terminating liquid etc. in preferred aforementioned detection kit one or more.
The present invention provides vitamins D synthetic antigen and preparation method thereof first, micromolecular vitamins D is directly connected on carrier proteins, and by connecting the tracer of immunity inspection on protein carrier, such as horseradish peroxidase (or alkaline phosphatase), fluorescein (as lanthanide chelate), chemoluminescence agent (luminol,3-aminophthalic acid cyclic hydrazide or acridinium ester) etc., therefore, most of immunological detection method is applicable to.
Vitamins D detection kit provided by the invention, it is integrated with the advantage of existing clinical vitamins D detection method, be applicable to all microplate reader, Chemiluminescence Apparatus and temporal resolution analyser, achieve a step detection method, compared with two-step approach, detection time shortens greatly, and sensitivity, accuracy, precision all can meet testing requirement.Adopt the immuno sorbent assay kit of highly versatile of the present invention, batch, the rapid detection of vitamins D in serum (or blood plasma) can be realized.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art, is raw materials usedly commercial goods.
The synthesis of embodiment 125-hydroxycholecalciferol antigen and the preparation of enzyme linked immunological kit
The synthesis of 1.125-hydroxycholecalciferol antigen
A, 100mg bovine serum albumin (BSA) is dissolved in the phosphate buffered saline buffer of 10mlpH8.50.05M;
B, 5mg25-hydroxycholecalciferol to be dissolved in 0.5ml ethanol, then to add two succinimidyl carbonate 108mg(and be dissolved in advance in 400 μ l dimethyl formamides), under room temperature, described mixture lucifuge is stirred and spend the night (being generally 16 ~ 22h);
C, in the bovine serum albumin liquid dissolved, adding 2ml, (in Sodium phosphate dibasic damping fluid, quadrol concentration is 10v/v% containing the disodium phosphate soln of the 0.05MpH10.4 of quadrol and ethylendiamine dihydrochloride, ethylendiamine dihydrochloride concentration is 20mg/ml), at room temperature react 3 ~ 4 hours;
D, reacted in backward C gained solution and add 37% formaldehyde solution 1ml, under room temperature, lucifuge stirs;
E, drop in the solution of D by B gained solution, under room temperature, lucifuge stirs and spends the night;
F, end product 0.01MpH7.4 phosphate buffered saline buffer to be dialysed 16 hours, within every 4 hours, change a dialyzate.
1.2 use horseradish peroxidase (HRP) labelled synthesis antigen
G, take 5mgHRP and be dissolved in 1ml distilled water;
H, in above-mentioned solution, add the 0.1MNaIO that 0.2ml newly prepares 4solution, under room temperature, lucifuge stirs 20 minutes;
I, above-mentioned solution loaded in dialysis tubing, dialyse with the sodium-acetate buffer of 1mMpH4.4,4 DEG C are spent the night;
J, to dialysis after solution in add 20 μ l0.2MpH9.5 carbonate buffer solutions, the pH value of above-mentioned hydroformylation HRP is made to be elevated to 9.0 ~ 9.5, then add 10mg25-hydroxycholecalciferol synthetic antigen (being dissolved in advance in 1ml0.01M carbonate buffer solution) immediately, room temperature lucifuge stirs 2 hours gently;
K, add the 4mg/mlNaBH that 0.1ml newly prepares wherein 4solution, mixing, places 2 hours for 4 DEG C;
L, load in dialysis tubing by above-mentioned solution, with the dialysis of 0.15MpH7.4PBS liquid, 4 DEG C are spent the night;
M, by dialysis after solution be transferred in suitable container, dropwise add isopyknic saturated ammonium sulphate solution under stirring, 4 DEG C place 1 hour;
Centrifugal 30 minutes of N, 3000rpm, abandon supernatant; Throw out semi-saturation ammoniumsulphate soln washes secondary, is finally dissolved in by throw out in the PBS liquid of a small amount of 0.15MpH7.4;
O, above-mentioned solution loaded in dialysis tubing, dialyse with the phosphate buffered saline buffer of 0.15MpH7.4, after removing ammonium ion, 10,000rpm removes and precipitates for centrifugal 30 minutes, and supernatant liquor is enzyme conjugates, after packing, and-20 DEG C of stored frozen.
The preparation of the enzyme linked immunological kit of 1.325-hydroxycholecalciferol
96 orifice plates wrap by the monoclonal antibody of 25-hydroxyvitamin D3 (purchased from Abcam company of Britain), using labelled antigen obtained above as competitive antigen, preparation enzyme working fluid, preparation calibration serum intermediate value quality controlled serum and high level quality controlled serum (new-born calf serum is containing appropriate 25-hydroxyvitamin D3), preparation substrate solution A(is containing 0.1% hydrogen peroxide, 0.1M sodium-acetate and 10mM citric acid), substrate solution B(is containing 0.05% tetramethyl benzidine, 0.2% citric acid and 10% glycerol), Sample dilution (10mmol/L phosphate buffered saline buffer), stop buffer (2M sulfuric acid) and concentrated cleaning solution (containing 5%Tween20 and 0.2M phosphate buffered saline buffer), obtain 25-hydroxyvitamin D3 detection kit (competition law enzyme linked immunological kit).
The synthesis of embodiment 225-hydroxycholecalciferol antigen and the preparation of chemiluminescence immunoassay kit
The synthesis of 1.125-hydroxycholecalciferol antigen
A, 100mg ovalbumin is dissolved in the borate buffer solution of 8mlpH8.50.05M;
B, 25-hydroxyvitamin D3 5mg is dissolved in 0.5ml anhydrous pyridine;
C, to dissolve ovalbumin solution in add 200 μ l4-Dimethylamino pyridines (42mg) and acetone (200 μ l) mixed solution, under room temperature lucifuge stirring spend the night;
D, in the solution of B, add two succinimidyl carbonate 108mg(be dissolved in 400 μ l dimethyl formamides in advance), under room temperature, described mixture lucifuge is stirred and spend the night;
E, be dissolved in the borate buffer solution of 1mlpH8.50.05M by 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride 10mg, then drop in the mixed solution of D and C, under room temperature, lucifuge stirs and spends the night;
F, end product 0.01MpH7.4 phosphate buffered saline buffer to be dialysed 16 hours, within every 4 hours, change a dialyzate.
1.2 use acridinium ester label synthetic antigen
G, get 25-hydroxyvitamin D3 synthetic antigen 1mg and be dissolved in 800 μ l phosphate buffered saline buffer (0.1mol/L, pH8.0);
H, get acridinium ester 20 μ l and be dissolved in 180 μ lDMF;
I, get acridinium ester solution 160 μ l and divide 4 times to add in antigenic solution, shake up after adding at every turn, room temperature lucifuge reaction 20min;
J, reaction add Methionin (50mg/ml) 400 μ l after terminating, and place 15min with termination reaction;
K, use phosphate buffered saline buffer (10mmol/L, pH6.3) 4 DEG C of lucifuges are dialysed 24 hours, and period changes liquid 6 times;
L, get sephadex G-25 as chromatography column filler purifying, post (0.8cm × 34cm) uses PBS(10mmol/L, pH6.3 in advance) balance, same buffer solution elution is used after loading, distribution is collected, and measurement collection pipe luminous intensity, peak pipe merges 4 DEG C and saves backup.
The preparation of the chemiluminescence immunoassay kit of 1.325-hydroxycholecalciferol
96 orifice plates wrap by the monoclonal antibody of 25-hydroxyvitamin D3 (purchased from Abcam company of Britain), marker solution is prepared as competitive antigen using labelled antigen obtained above, preparation Sample dilution (10mmol/L phosphate buffered saline buffer), substrate solution A(is containing 0.1M nitric acid and 0.1M hydrogen peroxide), substrate solution B(is containing 0.2M sodium hydroxide and 2%TritonX-100), concentrated washing lotion (containing 5%Tween20 and 0.2M phosphate buffered saline buffer), , calibration serum, intermediate value quality controlled serum and high level quality controlled serum (new-born calf serum is containing appropriate 25-hydroxyvitamin D3), obtain 25-hydroxyvitamin D3 detection kit (competing method chemiluminescence immunoassay kit).
The synthesis of embodiment 31,25-dihydroxy vitamin d3 antigen and the preparation of time resolved immuno test kit
The synthesis of 1.11,25-dihydroxy vitamin d3 antigen
A, 80mg hemocyanin is dissolved in the Tris-hydrochloride buffer of 5mlpH8.50.05M;
B, 1-25-(OH)2-D3 5mg is dissolved in 0.6ml dimethyl sulfoxide (DMSO) and dissolves; 3.5mg Methionin and 3mg1-hydroxy benzo triazole are dissolved in 0.25ml N,N-DIMETHYLACETAMIDE, add 16 μ lN, N'-dicyclohexylcarbodiimide, under room temperature, shaking table reacts 1 hour, add 1.5mg4-Dimethylamino pyridine again, mixed solution is added in the dimethyl sulphoxide solution of 1-25-(OH)2-D3, room temperature lucifuge stirring reaction 5 hours, product vacuum is dry;
C, to dissolve hemocyanin solution in dropwise add 2ml glutaraldehyde, fully mix;
D, the product obtained in B is dissolved in 0.5ml dimethyl sulfoxide (DMSO), and drips hemocyanin solution wherein, 37 DEG C of constant-temperature tables reaction 2h;
E, by end product with 0.015MpH7.4 phosphate buffered saline buffer 4 DEG C dialysis 24 hours, every 4 hours change a dialyzate.
1.2 mark 1-25-(OH)2-D3 synthetic antigen with the bifunctional chelating agent being chelated with rare earth ion
F, use Tris-HCl damping fluid (0.05M, pH7.8) dissolve EuCl 3(or any one rare earth ion compound, as Sm 3+, Tb 3+, Dy 3+), make its concentration be 10 -6mol/L;
G, with above-mentioned solution dilution sequestrant diethylene triamine pentacetic acid (DTPA) (DTPA), the mol ratio of rare earth ion and sequestrant is made to be 2:1;
H, above-mentioned solution is placed in 37 DEG C of water bath with thermostatic control reacting by heating 2 hours, prepares difunctional chelating labelled reagent;
I, the above-mentioned chelate products of lyophilize;
J, above-mentioned lyophilize inner complex 1mg is dissolved in 50 μ l dimethyl formamides (DMF);
K, 1-25-(OH)2-D3 synthetic antigen 1mg is dissolved in the carbonate buffer solution of 500 μ lpH9.10.05M;
L, the inner complex of dissolving is divided into 4 parts, every 2 minutes 1 part add in the antigenic solution of dissolving, room temperature lucifuge stirring reaction 30 minutes;
M, get sephadex G-50 as chromatographic stuffing, carry out compression leg flow rate control at 2 ~ 3ml/min with water after dress post, after pillar presses, with 0.1mmol/LNaOH, pillar is processed, flow rate control at 2 ~ 3ml/min, 2 column volumes.Then wash with water flat, balance with column equilibration liquid to purification column, about 1h, column equilibration liquid is the 0.1% high purity BSA aqueous solution, with the 0.05MNH of pH8.0 4hCO 3for elutriant, elutriant also can be that the 0.05MTris-HCl(of pH7.8 is containing 0.9%NaCl), elution flow rate is 1ml/min, is separated end product.Collect the higher several pipes of protein concentration to merge, it is frozen to add equimultiple glycerine-20 DEG C.
The preparation of 1.31,25-dihydroxy vitamin d3 temporal resolution immunoassay kit
96 orifice plates wrap by 1, the monoclonal antibody (purchased from Abcam company of Britain) of 25-dihydroxy vitamin d3, marker solution is prepared as competitive antigen using labelled antigen obtained above, preparation calibration serum, (new-born calf serum is containing appropriate 1 for intermediate value quality controlled serum and high level quality controlled serum, 25-dihydroxy vitamin d3), preparation experiment damping fluid is (containing 1% bovine serum albumin, Tris-HCl damping fluid containing 0.02% disodium ethylene diamine tetraacetate), strengthen liquid (containing 2%TritonX-100, 6% Glacial acetic acid and 0.005% β-NTA) and concentrated washing lotion (containing 5%Tween20 and 0.2MTris-HCl damping fluid), obtain 1, 25-dihydroxy vitamin d3 detection kit (competition law temporal resolution immunoassay kit).
The application of embodiment 425-hydroxycholecalciferol detection kit
Operation steps is as follows:
The 25-hydroxyvitamin D3 detection kit of preparation in A, Example 1, adds calibration serum, quality controlled serum or sample that 50 μ L dilute, then adds the enzyme working fluid of 50 μ L in corresponding enzyme plate micropore.Stick sealing compound, under 37 DEG C of conditions, hatch 1 hour.
B, wash plate 5 times with work washings; Add 300 μ L work washingss to every hole, before carrying out next step operation, enzyme plate is inverted on thieving paper, firmly pats to remove residual work washings.
C, add the substrate solution A of 50 μ L and the substrate solution B of 50 μ L to every hole, stick sealing compound, hatch at 37 DEG C, reaction 15 minutes in constant incubator or constant temperature blast drying oven.
D, add 50 μ L stop buffers to every hole.Absorbancy is read in 450nm, 630nm wavelength place immediately after adding stop buffer.
E, drawing standard curve: with OD value for ordinate zou, 25-OH-D3 concentration is X-coordinate, carries out exponential curve fitting.According to the OD value of typical curve and each pattern detection, typical curve finds corresponding 25-OH-D3 concentration value (μ g/L or nmol/L).
Test kit performance analysis:
(1) the comparing of 25-hydroxyvitamin D3 detection kit and high effective liquid chromatography for measuring result
Select 10 samples of the larger 25-hydroxyvitamin D3 scope of representative (0-100 μ g/L), adopt above-mentioned two kinds of methods to detect respectively, the enterprising line correlation analysis on the basis of comparative data: relation conefficient (r) >=0.90, deviation≤15%.
(2) sensitivity technique
Carry out concentration gradient dilution to sample, using diluent as detection sample, replicate(determination) 10 times, the mean light absorbency of detected result deducts the concentration corresponding to twice standard deviation S D value, and result is not higher than 5 μ g/L.
(3) withinrun precision detects
Use intermediate value quality controlled serum, replicate(determination) 10 times, CV value≤10%.
(4) linearity range
In kit assay scope (5-100 μ g/L), linearly dependent coefficient (r) >=0.990.
(5) specificity analyses
In sample during the μ g/L of Vitamin D3 500,000 I.U/GM concentration≤100, detected result is not affected, namely compared with same this detected result of increment not adding Vitamin D3 500,000 I.U/GM, its result error≤10%.
The application of embodiment 525-hydroxycholecalciferol detection kit
Operation steps is as follows:
The 25-hydroxyvitamin D3 detection kit of preparation in A, Example 2, to corresponding bag by the calibration serum, quality controlled serum or the sample that add 50 μ L in plate micropore and diluted, then adds the marker working fluid of 50 μ L.Stick sealing compound, under 37 DEG C of conditions, hatch 1 hour.
B, wash plate 5 times with work washings; Add 300 μ L work washingss to every hole, before carrying out next step operation, enzyme plate is inverted on thieving paper, firmly pats to remove residual work washings.
C, add the substrate solution A of 50 μ L and the substrate solution B of 50 μ L to every hole.
D, add substrate solution after on chemical illumination immunity analysis instrument, read result immediately.
E, drawing standard curve: take light intensity value as ordinate zou, 25-OH-D3 concentration is X-coordinate, carries out exponential curve fitting.According to the light intensity value of typical curve and each pattern detection, typical curve finds corresponding 25-OH-D3 concentration value (μ g/L or nmol/L).Test kit performance analysis:
(1) the comparing of measurement result of 25-hydroxyvitamin D3 detection kit and high performance liquid chromatography
Select 10 samples of the larger 25-hydroxyvitamin D3 scope of representative (0-100 μ g/L), adopt above-mentioned two kinds of methods to detect respectively, the enterprising line correlation analysis on the basis of comparative data: relation conefficient (r) >=0.90, deviation≤15%.
(2) sensitivity technique
Carry out concentration gradient dilution to sample, using diluent as detection sample, replicate(determination) 10 times, the mean light absorbency of detected result deducts the concentration corresponding to twice standard deviation S D value, and result is not higher than 5 μ g/L.
(3) withinrun precision detects
Use intermediate value quality controlled serum, replicate(determination) 10 times, CV value≤10%.
(4) linearity range
In kit assay scope (5-100 μ g/L), linearly dependent coefficient (r) >=0.990.
(5) specificity analyses
In sample during the μ g/L of Vitamin D3 500,000 I.U/GM concentration≤100, detected result is not affected, namely compared with same this detected result of increment not adding Vitamin D3 500,000 I.U/GM, its result error≤10%.
Embodiment 61, the application of 25-hydroxyvitamin D3 detection kit
Operation steps is as follows:
In A, Example 3 preparation 1,25-hydroxyvitamin D3 detection kit, to corresponding bag by the calibration serum, quality controlled serum or the sample that add 50 μ L in plate micropore and diluted, then adds the Europium label working fluid of 50 μ L.Stick sealing compound, vibrator slowly shakes 5 minutes, hatches 1 hour under 37 DEG C of conditions.
B, wash plate 5 times with work washings; Add 300 μ L work washingss to every hole, before carrying out next step operation, enzyme plate is inverted on thieving paper, firmly pats to remove residual work washings.
C, add the enhancing liquid of 100 μ L to every hole, slowly concussion 5 minutes on vibrator.
D, use time resolved immuno fluorometric analyser detect.
E, drawing standard curve: take fluorescent value as ordinate zou, 1,25-(OH) 2-D3 concentration is X-coordinate, carries out exponential curve fitting.According to the fluorescent value of typical curve and each pattern detection, typical curve finds corresponding 1,25-(OH) 2-D3 concentration value (ng/L or pmol/L).Test kit performance analysis:
(1) 1, comparing of the measurement result of 25-hydroxyvitamin D3 detection kit and high performance liquid chromatography
10 samples of the larger 1-25-(OH)2-D3 scope (0-240ng/L) of representative are selected to be detected by two kinds of methods, the enterprising line correlation analysis on the basis of comparative data: relation conefficient (r) >=0.90, deviation≤15%.
(2) sensitivity technique
Carry out concentration gradient dilution to sample, using diluent as detection sample, replicate(determination) 10 times, the mean light absorbency of detected result deducts the concentration corresponding to twice standard deviation S D value, and result is not higher than 3ng/L.
(3) withinrun precision detects
Use intermediate value quality controlled serum, replicate(determination) 10 times, CV value≤10%.
(4) linearity range
In kit assay scope (3-240ng/L), linearly dependent coefficient (r) >=0.990.
(5) specificity analyses
In sample during the μ g/L of Vitamin D3 500,000 I.U/GM concentration≤10, detected result is not affected, namely compared with same this detected result of increment not adding Vitamin D3 500,000 I.U/GM, its result error≤10%.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.

Claims (9)

1. a vitamins D synthetic antigen, it is characterized in that, it is the conjugate of vitamins D and protein carrier, and described vitamins D is 25-hydroxyvitamin D3 or 1,25-dihydroxyvitamin D3, described protein carrier is one or more in bovine serum albumin, ovalbumin, human serum albumin;
The weight ratio of described vitamins D and protein carrier is 1:16 ~ 20;
The preparation method of vitamins D synthetic antigen comprises the steps:
1) protein carrier is dissolved in the damping fluid of pH8.5, obtains solution A;
2) vitamins D is dissolved in ethanol, dimethyl sulfoxide (DMSO) or pyridine, obtains solution B;
3) add activator respectively in solution A and B, obtain protein carrier and the vitamins D of activation;
4) with N, N'-bis-succinimidyl carbonate, 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride or N, N'-dicyclohexylcarbodiimide for coupling agent, the protein carrier of above-mentioned activation and vitamins D are carried out coupling;
5) dialysis, drying, obtain vitamins D synthetic antigen;
Wherein, step 3) described in activator be containing the disodium phosphate soln of quadrol and ethylendiamine dihydrochloride, DMAP-acetone mixture, glutaraldehyde or formaldehyde.
2. prepare the method for vitamins D synthetic antigen described in claim 1, it is characterized in that, comprise step:
1) protein carrier is dissolved in the damping fluid of pH8.5, obtains solution A;
2) vitamins D is dissolved in ethanol, dimethyl sulfoxide (DMSO) or pyridine, obtains solution B;
3) add activator respectively in solution A and B, obtain protein carrier and the vitamins D of activation;
4) with N, N'-bis-succinimidyl carbonate, 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride or N, N'-dicyclohexylcarbodiimide for coupling agent, the protein carrier of above-mentioned activation and vitamins D are carried out coupling;
5) dialysis, drying, obtain vitamins D synthetic antigen;
Wherein, step 3) described in activator be containing the disodium phosphate soln of quadrol and ethylendiamine dihydrochloride, DMAP-acetone mixture, glutaraldehyde or formaldehyde.
3. method according to claim 2, is characterized in that, step 1) described in damping fluid be phosphate buffered saline buffer, borate buffer solution or Tris damping fluid.
4. the application of vitamins D synthetic antigen in vitamins D immunology detection described in claim 1.
5. application according to claim 4, it is characterized in that, it is that described vitamins D synthetic antigen and marker are combined as competitive antigen, by with the vitamins D competition binding vitamins D monoclonal antibody in sample, thus detect the vitamin D content in sample.
6. application according to claim 5, is characterized in that, described marker is:
A) horseradish peroxidase or alkaline phosphatase;
B) acridinium ester compounds; Or
C) Eu is chelated with 3+, Sm 3+, Tb 3+or Dy 3+diethylene triamine pentacetic acid (DTPA), 2-[(4-cyano-phenyl) methyl]-Isosorbide-5-Nitrae, 7,10-tetra-azo-cycle dodecane-Isosorbide-5-Nitrae, 7,10-tetraacethyl or 4,7-dichloro sulphophenyl-1,10-phenanthroline-2,9-dicarboxylic acid.
7. application according to claim 5, is characterized in that, described vitamins D monoclonal antibody is the vitamins D monoclonal antibody product of Abcam company.
8. vitamins D immunological detecting kit, is characterized in that, it comprises the competitive antigen obtained after marker mark by the enzyme plate of vitamins D monoclonal antibody, vitamins D synthetic antigen according to claim 1;
Wherein, described vitamins D monoclonal antibody is the vitamins D monoclonal antibody product of Abcam company; Described marker is: a) horseradish peroxidase or alkaline phosphatase; B) luminol,3-aminophthalic acid cyclic hydrazide or acridinium ester compounds; Or c) be chelated with Eu 3+, Sm 3+, Tb 3+or Dy 3+diethylene triamine pentacetic acid (DTPA), 2-[(4-cyano-phenyl) methyl]-Isosorbide-5-Nitrae, 7,10-tetra-azo-cycle dodecane-Isosorbide-5-Nitrae, 7,10-tetraacethyl or 4,7-dichloro sulphophenyl-1,10-phenanthroline-2,9-dicarboxylic acid.
9. test kit according to claim 8, is characterized in that, described test kit also comprise in washings, vitamins D standard substance, substrate nitrite ion, reaction terminating liquid one or more.
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* Cited by examiner, † Cited by third party
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CN104062440B (en) * 2014-06-30 2016-05-11 韩雅君 A kind of test strips of fast detecting vitamin D
US10114030B2 (en) 2014-07-10 2018-10-30 Shenzhen New Industries Biomedical Engineering Co., Ltd Detection agent for detecting 25-hydroxy vitamin D, preparation method and use
CN107325173A (en) * 2016-04-28 2017-11-07 上海惠斯生物科技有限公司 A kind of artificial antigen of 25(OH)VD 3, preparation method and application
CN106199016A (en) * 2016-06-30 2016-12-07 深圳市亚辉龙生物科技股份有限公司 25 hydroxy-vitamine D chemiluminescence immune detection reagent kits and preparation method thereof
EP3483172B1 (en) * 2016-07-05 2022-01-26 Shenzhen Yhlo Biotech Co., Ltd Acridine-marker conjugate and preparation method thereof, and chemiluminescence kit
WO2018006269A1 (en) * 2016-07-05 2018-01-11 深圳市亚辉龙生物科技股份有限公司 Acridine-marker conjugate and preparation method thereof, and chemiluminescence immunoassay kit
US20190309030A1 (en) * 2016-07-05 2019-10-10 Shenzhen Yhlo Biotech Co., Ltd. Acridine labelled conjugates and preparation methods therefor and chemiluminescent kits
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CN108362688B (en) * 2018-01-02 2021-08-24 北京利德曼生化股份有限公司 Detection kit for chemiluminescence of 25-hydroxy vitamin D magnetic particles
CN110713533B (en) * 2019-11-26 2021-03-16 杭州隆基生物技术有限公司 25-hydroxy vitamin D3Antigen and method for producing the same
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CN113495158A (en) * 2020-03-20 2021-10-12 郑州达诺生物技术有限公司 Sample pretreatment agent, vitamin B12 quantitative detection kit and use method

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101273062A (en) * 2005-09-29 2008-09-24 霍夫曼-拉罗奇有限公司 Antibodies against 25-hydroxyvitamin D
WO2012162165A2 (en) * 2011-05-20 2012-11-29 Siemens Healthcare Diagnostics, Inc. Antibodies to 25-hydroxyvitamin d2 and d3 and uses thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101273062A (en) * 2005-09-29 2008-09-24 霍夫曼-拉罗奇有限公司 Antibodies against 25-hydroxyvitamin D
WO2012162165A2 (en) * 2011-05-20 2012-11-29 Siemens Healthcare Diagnostics, Inc. Antibodies to 25-hydroxyvitamin d2 and d3 and uses thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
25-羟基维生素D3人工完全抗原的合成与鉴定;万巍巍等;《细胞与分子免疫学杂志》;20111231;第27卷(第11期);第1173页摘要第2-5行,第1174页1.2节第1-10行,图1 *

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