CN103588872A - Vitamin D synthetic antigen, and preparation method and preparation thereof - Google Patents
Vitamin D synthetic antigen, and preparation method and preparation thereof Download PDFInfo
- Publication number
- CN103588872A CN103588872A CN201210287219.5A CN201210287219A CN103588872A CN 103588872 A CN103588872 A CN 103588872A CN 201210287219 A CN201210287219 A CN 201210287219A CN 103588872 A CN103588872 A CN 103588872A
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- Prior art keywords
- vitamins
- vitamin
- synthetic antigen
- solution
- antigen
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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Abstract
The invention provides a vitamin D synthetic antigen and a preparation method thereof. The vitamin D synthetic antigen is a conjugate of vitamin D and a protein carrier. The vitamin D is 25-hydroxy vitamin D3 or 1,25-dihydroxy vitamin D3; and the protein carrier is one or more selected from bovine serum albumin, ovalbumin, hemocyanin and human serum albumin. The invention also provides application of the vitamin D synthetic antigen to vitamin D immunological detection. The invention further provides a vitamin D detection kit, which integrates the advantages of existing clinical vitamin D detection methods; and the kit can be applied to all enzyme mark instruments, chemiluminescence instruments and time-resolved analyzers, and has greatly shortened detection time, and sensitivity, accuracy and precision met the detection requirements. The immunosorbent assay kit with strong versatility provided by the invention can realize batch and rapid detection on vitamin D in serum (or plasma).
Description
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 that regulates calcium phosphorus, and the state of health of while and body also has 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.While being deficient in vitamin D in body, can cause a series of diseases.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 for vitamins D in body also more and more receives people's concern.
Special molecular structure due to vitamins D, all there is no for a long time very good detection method, in general the detection spended time of vitamins D is longer, need special equipment etc., along with the development of Protocols in Molecular Biology, for measuring the method for 25-hydroxyvitamin D3, mainly contain radiation competitive protein binding assay (CPB), radioimmunology (RIA) at present, high performance liquid chromatography (HPLC), the euzymelinked immunosorbent assay (ELISA) of the Electrochemiluminescince of Roche and Britain IDS etc.Be summarized as follows respectively:
(1) radiation competitive protein binding assay
Wei S, Tanaka H, Kubo T etc., A multiple assay for vitamin D metaboltes without high-performance liquid chromatography.Anal Biochem, 1994,222 (2): in 359-365 document, disclose a kind ofly without HPLC purifying, only 0.5ml serum can be measured serum 25(OH) method of D3 content.Key step comprises with acetonitrile extracts serum, at C-18/OH post and separated and purifying on silica gel Sep-Pak post, finally with vitamin D binding protein standard measure, measure 25(OH) D3, the method has easy to be quick, sample consumption is few, reproducible, can be simultaneously to three of vitamins D kinds of main metabolites, the advantage such as carry out quantitatively, but the rate of recovery is lower.
(2) radioimmunology
< < China journal of obstetrics and gynecology > > the 3rd interim report with the 25-(OH in 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 of mark is unstable, very high to Laboratory Request during operation, and the radiation producing has disadvantageous effect to operator ' s health.
(3) high performance liquid chromatography
It is the detection method to vitamins D in GB and pharmacopeia.Red, orange, green, blue, yellow (ROGBY), due to complicated plant and instrument and loaded down with trivial details process, is not suitable for clinical great amount of samples examination.
(4) Electrochemiluminescince
That Roche Holding Ag adopts at present is the 25-(OH in ElectrochemiluminescDetermination Determination serum) D3, its principle is with electrochemiluminescence agent tris (bipyridine) ruthenium traget antibody, by antigen-antibody reaction and magnetic bead isolation technique, the light intensity of sending on electrode according to tris (bipyridine) ruthenium is carried out quantitative or qualitative to antigen to be determined or antibody.But its technology versatility is poor, need to be with special-purpose equipment, and equipment cost is higher.
(5) enzyme linked immunosorbent detection method
Application ELISA standard measure is measured the 25(OH in human serum, blood plasma, cell culture supernatant or other relevant liquid) D3 content.Ultimate principle is 25(OH in application competitive enzyme-linked immune assay sample) D3 level.With the coated microwell plate of polyclonal antibody, make insolubilized antibody, in coated how anti-micropore, add successively 25(OH) D3(sample), biotinylated people 25(OH) avidin of D3 antigen, HRP mark, after thorough washing, with substrate TMB, develop the color.TMB changes into blueness under the catalysis of peroxidase, and changes into final yellow under sour effect.The depth of color and the 25(OH in sample) D3 is proportionate.By microplate reader, under 450nm wavelength, measure absorbancy (OD value), calculation sample concentration.The shortcoming of this type of test kit is to use biotin labeling 25-hydroxyvitamin D3, and with HRP mark avidin, 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 for preparing said vitamin D synthetic antigen, comprises the following steps: 1) carrier proteins is dissolved in the damping fluid of pH8.5, obtains solution A; 2) vitamins D is dissolved in the reagent such as ethanol, dimethyl sulfoxide (DMSO) or pyridine, obtains solution B; 3) in solution A and B, add activator respectively, obtain protein carrier and the vitamins D of activation; 4) with N, N'-bis-succinimidyl carbonates, 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride or N, N'-dicyclohexylcarbodiimide etc. is coupling agent, and the protein carrier of above-mentioned activation and vitamins D are carried out to coupling; 5) dialysis, dry, obtains 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 disodium phosphate soln, DMAP-acetone mixed solution, glutaraldehyde or formaldehyde etc. containing quadrol and quadrol dihydrochloride.Activator is preferably the 0.05M disodium phosphate soln pH10.4 containing quadrol and quadrol dihydrochloride, and wherein quadrol concentration is 10%(v/v), quadrol dihydrochloride concentration is 20mg/ml.More preferably activator is DMAP-acetone mixed solution 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 to competitive antigen, by with sample in vitamins D competition in conjunction with vitamins D monoclonal antibody, thereby detect the vitamin D content in sample.
Wherein, described marker is:
A) horseradish peroxidase or alkaline phosphatase;
B) acridinium ester compounds; Or
C) be chelated with Eu
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 4,7-dichloro sulphophenyl-1,10-phenanthroline-2, the bifunctional chelating agents such as 9-dicarboxylic acid (BCPDA).
Described vitamins D monoclonal antibody is purchased from Britain Abcam company.
The present invention further provides vitamins D immunological detecting kit, it comprises the enzyme plate of coated vitamins D monoclonal antibody, the competitive antigen that said vitamin D synthetic antigen obtains after marker mark.
Wherein, described vitamins D monoclonal antibody is purchased from Britain Abcam company.
Described marker is:
A) horseradish peroxidase or alkaline phosphatase;
B) acridinium ester compounds; Or
C) be chelated with Eu
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 4,7-dichloro sulphophenyl-1,10-phenanthroline-2, the bifunctional chelating agents such as 9-dicarboxylic acid (BCPDA).
In preferred aforementioned detection kit, also comprise one or more in washings, vitamins D standard substance, substrate nitrite ion, reaction terminating liquid etc.
The present invention provides vitamins D synthetic antigen and preparation method thereof first, micromolecular vitamins D is directly connected on carrier proteins, and the tracer of using by connect 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, applicable to most of immunological detection methods.
Vitamins D detection kit provided by the invention, the advantage of existing clinical vitamins D detection method that it is integrated, applicable to all microplate reader, Chemiluminescence Apparatus and temporal resolution analyser, realized a step detection method, compare with two-step approach, greatly shorten detection time, and sensitivity, accuracy, precision all can meet testing requirement.Adopt the immuno sorbent assay kit of highly versatile of the present invention, can realize batch, the rapid detection of vitamins D in serum (or blood plasma).
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art, the raw materials used commercial goods that is.
The preparation of the synthetic and enzyme linked immunological kit of embodiment 1 25-hydroxyvitamin D3 antigen
Synthesizing of 1.1 25-hydroxyvitamin D3 antigens
A, 100mg bovine serum albumin (BSA) is dissolved in the phosphate buffered saline buffer of 10ml pH8.5 0.05M;
B, 5mg 25-hydroxyvitamin D3 is dissolved in 0.5ml ethanol, then adds two succinimidyl carbonate 108mg(to be dissolved in advance in 400 μ l dimethyl formamides), under room temperature, described mixture lucifuge is stirred to spend the night (being generally 16 ~ 22h);
C, to adding in the bovine serum albumin liquid dissolving 2ml to contain the disodium phosphate soln of the 0.05M pH10.4 of quadrol and quadrol dihydrochloride, (in Sodium phosphate dibasic damping fluid, quadrol concentration is 10v/v%, quadrol 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, B gained solution is dropped in the solution of D, under room temperature, lucifuge stirs and spends the night;
F, by 0.01M pH7.4 phosphate buffered saline buffer dialysis 16 hours for end product, within every 4 hours, change a dialyzate.
1.2 use horseradish peroxidase (HRP) mark synthetic antigens
G, take 5mg HRP and be dissolved in 1ml distilled water;
H, to the 0.1M NaIO that adds the new preparation of 0.2ml in above-mentioned solution
4solution, under room temperature, lucifuge stirs 20 minutes;
I, above-mentioned solution is packed in dialysis tubing, with the sodium-acetate buffer dialysis of 1mM pH4.4,4 ℃ are spent the night;
J, to dialysis after solution in add 20 μ l 0.2M pH9.5 carbonate buffer solutions, make the pH value of above-mentioned hydroformylation HRP be elevated to 9.0～9.5, then add immediately 10mg 25-hydroxyvitamin D3 synthetic antigen (being dissolved in advance in 1ml 0.01M carbonate buffer solution), room temperature lucifuge stirs 2 hours gently;
K, the 4mg/ml NaBH that adds 0.1ml newly to prepare wherein
4solution, mixes, and places 2 hours for 4 ℃;
L, above-mentioned solution is packed in dialysis tubing, with the dialysis of 0.15M pH7.4 PBS liquid, 4 ℃ are spent the night;
M, the solution after dialysis is transferred in suitable container, under stirring, dropwise adds isopyknic saturated ammonium sulphate solution, place 1 hour for 4 ℃;
Centrifugal 30 minutes of N, 3000rpm, abandon supernatant; Throw out is washed secondary with semi-saturation ammoniumsulphate soln, finally throw out is dissolved in the PBS liquid of a small amount of 0.15M pH7.4;
O, above-mentioned solution is packed in dialysis tubing, with the phosphate buffered saline buffer dialysis of 0.15M pH7.4, remove after ammonium ion, 10,000rpm removes precipitation in centrifugal 30 minutes, and supernatant liquor is enzyme conjugates, after packing, and-20 ℃ of stored frozen.
The preparation of the enzyme linked immunological kit of 1.325-hydroxycholecalciferol
The monoclonal antibody (purchased from Britain Abcam company) of coated 25-hydroxyvitamin D3 on 96 orifice plates, using labelled antigen obtained above as competitive antigen, preparation enzyme working fluid, preparation calibration serum intermediate value quality controlled serum and high value 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 diluent (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 preparation of the synthetic and chemiluminescence immunoassay kit of embodiment 2 25-hydroxyvitamin D3 antigens
Synthesizing of 1.1 25-hydroxyvitamin D3 antigens
A, 100mg ovalbumin is dissolved in the borate buffer solution of 8ml pH8.5 0.05M;
B, 25-hydroxyvitamin D3 5mg is dissolved in 0.5ml anhydrous pyridine;
C, in the ovalbumin solution dissolving, add 200 μ l DMAPs (42mg) and acetone (200 μ l) mixed solution, under room temperature, lucifuge stirring is spent the night;
D, in the solution of B, add two succinimidyl carbonate 108mg(to be dissolved in advance 400 μ l dimethyl formamides), under room temperature, described mixture lucifuge is stirred and spent the night;
E, 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride 10mg is dissolved in the borate buffer solution of 1mlpH8.5 0.05M, then drops in the mixed solution of D and C, under room temperature, lucifuge stirs and spends the night;
F, by 0.01M pH7.4 phosphate buffered saline buffer dialysis 16 hours for end product, within every 4 hours, change a dialyzate.
1.2 use acridinium ester mark synthetic antigens
G, get 25-hydroxyvitamin D3 synthetic antigen 1mg and be dissolved in 800 μ l phosphate buffered saline buffers (0.1mol/L, pH8.0);
H, get acridinium ester 20 μ l and be dissolved in 180 μ l DMF;
I, get acridinium ester solution 160 μ l and divide 4 times to add in antigenic solution, after adding, shake up at every turn, room temperature lucifuge reaction 20min;
J, reaction add Methionin (50mg/ml) 400 μ l after finishing, and place 15min with termination reaction;
K, with 4 ℃ of lucifuges of phosphate buffered saline buffer (10mmol/L, pH6.3) dialysis 24 hours, during change liquid 6 times;
L, get sephadex G-25 as chromatography column filler purifying, post (0.8cm * 34cm) is used PBS(10mmol/L, pH6.3 in advance) balance, after loading, use same buffer solution elution, distribute and collect, measurement collection pipe luminous intensity, peak pipe merges 4 ℃ and saves backup.
The preparation of the chemiluminescence immunoassay kit of 1.3 25-hydroxyvitamin D3s
The monoclonal antibody (purchased from Britain Abcam company) of coated 25-hydroxyvitamin D3 on 96 orifice plates, the labelled antigen obtained above of usining is prepared marker solution as competitive antigen, preparation sample diluent (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 value 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 preparation of the synthetic and temporal resolution immunoassay kit of embodiment 3 1-25-(OH)2-D3 antigens
Synthesizing of 1.1 1-25-(OH)2-D3 antigens
A, 80mg hemocyanin is dissolved in the Tris-hydrochloride buffer of 5ml pH8.50.05M;
B, 1-25-(OH)2-D3 5mg is dissolved in 0.6ml dimethyl sulfoxide (DMSO) and is dissolved; 3.5mg Methionin and 3mg 1-hydroxy benzo triazole are dissolved in 0.25ml N,N-DIMETHYLACETAMIDE, add 16 μ l N, N'-dicyclohexylcarbodiimide, under room temperature, shaking table reaction is 1 hour, add again 1.5mg DMAP, mixed solution is added in the dimethyl sulphoxide solution of 1-25-(OH)2-D3, room temperature lucifuge stirring reaction 5 hours, product vacuum-drying;
C, in the hemocyanin solution dissolving, dropwise add 2ml glutaraldehyde, fully mix;
D, the product obtaining in B is dissolved in to 0.5ml dimethyl sulfoxide (DMSO), and drips wherein hemocyanin solution, 37 ℃ of constant-temperature tables reaction 2h;
E, by 4 ℃ of dialysis of 0.015M pH7.4 phosphate buffered saline buffer 24 hours for end product, within every 4 hours, change a dialyzate.
1.2 use are chelated with the bifunctional chelating agent mark 1-25-(OH)2-D3 synthetic antigen of rare earth ion
F, use Tris-HCl damping fluid (0.05M, pH7.8) dissolve EuCl
3(or any rare earth ion compound, as Sm
3+, Tb
3+, Dy
3+), making its concentration is 10
-6mol/L;
G, with above-mentioned solution dilution sequestrant diethylene triamine pentacetic acid (DTPA) (DTPA), the mol ratio that makes rare earth ion and sequestrant is 2:1;
H, above-mentioned solution is placed in to 37 ℃ of water bath with thermostatic control reacting by heating 2 hours, prepares difunctional chelating labelled reagent;
I, the above-mentioned chelating product of lyophilize;
J, above-mentioned lyophilize inner complex 1mg is dissolved in to 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 l pH9.1 0.05M;
L, the inner complex of dissolving is divided into 4 parts, in every 2 minutes 1 part of antigenic solutions that add dissolving, room temperature lucifuge stirring reaction 30 minutes;
M, get sephadex G-50 as chromatographic stuffing, after dress post, water carries out compression leg flow rate control at 2 ~ 3ml/min, after pillar presses, with 0.1mmol/LNaOH, pillar is processed, and flow rate control is at 2 ~ 3ml/min, 2 column volumes.Then wash with water flatly, with column equilibration liquid, purification column is carried out to balance, about 1h, column equilibration liquid is the 0.1% high purity BSA aqueous solution, with the 0.05M NH of pH8.0
4hCO
3for elutriant, elutriant can be also that the 0.05MTris-HCl(of pH7.8 contains 0.9%NaCl), elution flow rate is 1ml/min, separated end product.Collect several pipes that protein concentration is higher and merge, times glycerine-20 such as add ℃ frozen.
The preparation of 1.31,25-dihydroxy vitamin d3 temporal resolution immunoassay kit
On 96 orifice plates, be coated with 1, the monoclonal antibody of 25-dihydroxy vitamin d3 (purchased from Britain Abcam company), the labelled antigen obtained above of usining is prepared marker solution as competitive antigen, preparation calibration serum, (new-born calf serum is containing appropriate 1 for intermediate value quality controlled serum and high value 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 4 25-hydroxyvitamin D3 detection kit
Operation steps is as follows:
A, get in embodiment 1 the 25-hydroxyvitamin D3 detection kit of preparation, in corresponding enzyme plate micropore, add calibration serum, quality controlled serum or the sample of 50 μ L dilutions, then add the enzyme working fluid of 50 μ L.Stick sealing compound, under 37 ℃ of conditions, hatch 1 hour.
B, with work washings, wash plate 5 times; To every hole, add 300 μ L work washingss, before carrying out next step operation, enzyme plate is inverted on thieving paper, firmly pat to remove residual work washings.
C, to every hole, add the substrate solution A of 50 μ L and the substrate solution B of 50 μ L, stick sealing compound, at 37 ℃, hatch, in constant incubator or constant temperature blast drying oven, react 15 minutes.
D, to every hole, add 50 μ L stop buffers.After adding stop buffer, in 450nm, 630nm wavelength place, read absorbancy immediately.
E, drawing standard curve: take OD value as ordinate zou, 25-OH-D3 concentration is X-coordinate, carries out exponential curve fitting.OD value according to typical curve and each pattern detection finds corresponding 25-OH-D3 concentration value (μ g/L or nmol/L) on typical curve.
Test kit performance analysis:
(1) comparison of 25-hydroxyvitamin D3 detection kit and high effective liquid chromatography for measuring result
10 samples selecting the larger 25-hydroxyvitamin D3 scope of representative (0-100 μ g/L), adopt respectively above-mentioned two kinds of methods to detect, in the enterprising line correlation analysis in the basis of comparative data: relation conefficient (r) >=0.90, deviation≤15%.
(2) sensitivity detects
Sample is carried out to concentration gradient dilution, using diluent as detecting sample, replicate(determination) 10 times, the mean light absorbency of detected result deducts the corresponding concentration of 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 test kit analyst coverage (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, on not impact of detected result, do not compare its result error≤10% with adding same this detected result of increment of Vitamin D3 500,000 I.U/GM.
The application of embodiment 5 25-hydroxyvitamin D3 detection kit
Operation steps is as follows:
A, get in embodiment 2 the 25-hydroxyvitamin D3 detection kit of preparation, to the calibration serum, quality controlled serum or the sample that add 50 μ L to dilute in corresponding coated plate micropore, then add the marker working fluid of 50 μ L.Stick sealing compound, under 37 ℃ of conditions, hatch 1 hour.
B, with work washings, wash plate 5 times; To every hole, add 300 μ L work washingss, before carrying out next step operation, enzyme plate is inverted on thieving paper, firmly pat to remove residual work washings.
C, to every hole, add the substrate solution A of 50 μ L and the substrate solution B of 50 μ L.
D, add after substrate solution reading result on chemical illumination immunity analysis instrument immediately.
E, drawing standard curve: take light intensity value as ordinate zou, 25-OH-D3 concentration is X-coordinate, carries out exponential curve fitting.Light intensity value according to typical curve and each pattern detection finds corresponding 25-OH-D3 concentration value (μ g/L or nmol/L) on typical curve.Test kit performance analysis:
(1) comparison of the measurement result of 25-hydroxyvitamin D3 detection kit and high performance liquid chromatography
10 samples selecting the larger 25-hydroxyvitamin D3 scope of representative (0-100 μ g/L), adopt respectively above-mentioned two kinds of methods to detect, in the enterprising line correlation analysis in the basis of comparative data: relation conefficient (r) >=0.90, deviation≤15%.
(2) sensitivity detects
Sample is carried out to concentration gradient dilution, using diluent as detecting sample, replicate(determination) 10 times, the mean light absorbency of detected result deducts the corresponding concentration of 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 test kit analyst coverage (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, on not impact of detected result, do not compare its result error≤10% with adding same this detected result of increment of Vitamin D3 500,000 I.U/GM.
Embodiment 61, the application of 25-hydroxyvitamin D3 detection kit
Operation steps is as follows:
A, get in embodiment 31 of preparation, 25-hydroxyvitamin D3 detection kit, to the calibration serum, quality controlled serum or the sample that add 50 μ L to dilute in corresponding coated plate micropore, then adds the Europium label working fluid of 50 μ L.Stick sealing compound, vibrator slowly shakes 5 minutes, under 37 ℃ of conditions, hatches 1 hour.
B, with work washings, wash plate 5 times; To every hole, add 300 μ L work washingss, before carrying out next step operation, enzyme plate is inverted on thieving paper, firmly pat to remove residual work washings.
C, to every hole, add the enhancing liquid of 100 μ L, slowly concussion 5 minutes on vibrator.
D, use temporal resolution immunofluorescence analysis instrument 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, on typical curve, find 1 of correspondence, 25-(OH)
2-D3 concentration value (ng/L or pmol/L).Test kit performance analysis:
(1) 1, the comparison of the measurement result of 25-hydroxyvitamin D3 detection kit and high performance liquid chromatography
Select 10 samples of the larger 1-25-(OH)2-D3 scope of representative (0-240ng/L) to detect by two kinds of methods, in the enterprising line correlation analysis in the basis of comparative data: relation conefficient (r) >=0.90, deviation≤15%.
(2) sensitivity detects
Sample is carried out to concentration gradient dilution, using diluent as detecting sample, replicate(determination) 10 times, the mean light absorbency of detected result deducts the corresponding concentration of 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 test kit analyst coverage (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, on not impact of detected result, do not compare its result error≤10% with adding same this detected result of increment of Vitamin D3 500,000 I.U/GM.
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, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a vitamins D synthetic antigen, it is characterized in that, 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.
2. synthetic antigen according to claim 1, is characterized in that, the weight ratio of described vitamins D and protein carrier is 1:16 ~ 20.
3. the method for preparing vitamins D synthetic antigen described in claim 1 or 2, is characterized in that, comprises 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) in solution A and B, add activator respectively, obtain protein carrier and the vitamins D of activation;
4) with N, N'-bis-succinimidyl carbonates, 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride or N, N'-dicyclohexylcarbodiimide is coupling agent, and the protein carrier of above-mentioned activation and vitamins D are carried out to coupling;
5) dialysis, dry, obtains vitamins D synthetic antigen;
Wherein, activator described in step 3) is disodium phosphate soln, DMAP-acetone mixed solution, glutaraldehyde or formaldehyde containing quadrol and quadrol dihydrochloride.
4. method according to claim 3, is characterized in that, the damping fluid described in step 1) is phosphate buffered saline buffer, borate buffer solution or Tris damping fluid.
5. the application of vitamins D synthetic antigen in vitamins D immunology detection described in claim 1 or 2.
6. application according to claim 5, it is characterized in that, it is that described vitamins D synthetic antigen and marker are combined as to competitive antigen, by with sample in vitamins D competition in conjunction with vitamins D monoclonal antibody, thereby detect the vitamin D content in sample.
7. application according to claim 6, is characterized in that, described marker is:
A) horseradish peroxidase or alkaline phosphatase;
B) 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-Nitraes, 7,10-tetraacethyl or 4,7-dichloro sulphophenyl-1,10-phenanthroline-2,9-dicarboxylic acid.
8. application according to claim 6, is characterized in that, the vitamins D monoclonal antibody product that described vitamins D monoclonal antibody is Abcam company.
9. vitamins D immunological detecting kit, is characterized in that, it comprises the enzyme plate of coated vitamins D monoclonal antibody, the competitive antigen that the vitamins D synthetic antigen described in claim 1 or 2 obtains after marker mark;
Wherein, the vitamins D monoclonal antibody product that described vitamins D monoclonal antibody is 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-Nitraes, 7,10-tetraacethyl or 4,7-dichloro sulphophenyl-1,10-phenanthroline-2,9-dicarboxylic acid.
10. test kit according to claim 9, is characterized in that, described test kit also comprises one or more in washings, vitamins D standard substance, substrate nitrite ion, reaction terminating liquid.
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| CN104062440A (en) * | 2014-06-30 | 2014-09-24 | 韩雅君 | Test strip for rapidly detecting vitamin D |
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