CN105203773A - Quantitative detection kit for human Dickkopf-1 protein (DKK-1) - Google Patents
Quantitative detection kit for human Dickkopf-1 protein (DKK-1) Download PDFInfo
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- CN105203773A CN105203773A CN201510626573.XA CN201510626573A CN105203773A CN 105203773 A CN105203773 A CN 105203773A CN 201510626573 A CN201510626573 A CN 201510626573A CN 105203773 A CN105203773 A CN 105203773A
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- 101710099518 Dickkopf-related protein 1 Proteins 0.000 title claims abstract description 138
- 102100030074 Dickkopf-related protein 1 Human genes 0.000 title claims abstract description 136
- 238000001514 detection method Methods 0.000 title claims abstract description 32
- 101000864646 Homo sapiens Dickkopf-related protein 1 Proteins 0.000 title abstract 2
- 102000050762 human DKK1 Human genes 0.000 title abstract 2
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 80
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- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 4
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Classifications
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
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- Urology & Nephrology (AREA)
- Immunology (AREA)
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- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
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- Pathology (AREA)
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- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Hospice & Palliative Care (AREA)
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Abstract
The invention provides a quantitative detection kit for human Dickkopf-1 protein (DKK-1). The quantitative detection kit comprises a calibrator, a quality control product, a DKK-1 monoclonal antibody labeled by fluorescein isothiocyanate and DKK-1 monoclonal antibody labeled by alkaline phosphatase solution, a magnetic separation reagent, cleaning liquid and a substrate solution. An adopted detection method combines magnetic particle separation chemiluminescence immune detection, enzyme labeling technology, magnetic separation technology and chemiluminescence technology, and is convenient and simple to operate, mild in reaction condition, stable in luminescence value and little in influence by outside conditions. Compared with existing detection methods, the quantitative detection kit has the advantages of simple sample treatment process, low detection cost, quickness in detection, accurate testing result and high repeatability.
Description
Technical field
The invention belongs to technical field of immune assay, relate to and be used for antidiastole hepatocellular carcinoma clinically, particularly early hepatocyte cancer, provides a kind of people Dickkopf-1 albumen (DKK-1) immue quantitative detection reagent box, is applicable to human serum DKK-1 and quantitatively detects.
Background technology
Hepatocellular carcinoma (hepatocellularcarcinoma, HCC) is one of modal malignant tumour in world wide, and the incidence of disease occupies the 6th, the world, and mortality ratio occupies third place in the world.Hepatitis b virus infected is the main pathogenic of hepatocellular carcinoma, and cirrhosis is that the most dangerous factor occurs hepatocellular carcinoma, is also the main cause of the death of patient with liver cirrhosis.At present, examination and the most frequently used method of diagnosing hepatocellular carcinoma are iconography (comprising ultrasonic, CT, MRI etc.) and serum alpha-fetoprotein (alphafetoprotein, AFP) detect, but Image detection both expensive, be subject to operator's experience influence, and be difficult to distinguish liver cancer and non-malignant proliferative.AFP is the most widely used hepatocellular carcinoma mark in the current whole world, but also has some limitations, and occurs " false cloudy " or " false positive " result.Therefore, searching one species specificity and the good tumor markers of sensitivity is needed to carry out adjuvant clinical diagnosis.
Dickkopf-1 albumen (DKK-1) is by the secreting glycoprotein of DKK-1 gene code, is one of DKK family member.1998, DKK-1 was found first by Glinka etc. in African toad embryonic cell, and the header area of this protein induced embryo is formed.People DKK-1 gene is positioned at No. 10 chromosomes, and coded product (DKK-1) is containing 266 amino acid, and relative molecular mass is about 29KD, and after glycosylation, relative molecular weight is about 35KD.DKK-1 is the antagonist of Wnt signal transduction pathway, and it is combined by Wnt acceptor and membrane-spanning protein on cell membrane, suppresses the activation of classical wnt signal transduction pathway.Wnt path is one and plays the signal path of important regulative to embryonic development, participates in the process such as cell proliferation, differentiation, apoptosis, in its path the sudden change of signaling molecule or unconventionality expression relevant to various diseases and cancer.
Current many research finds that the expression of DKK-1 in kinds of tumors tissue presents multifarious feature, indicates the generation of itself and kinds of tumors and is in progress closely related.2003, Qin Wen new team Late Cambrian in Shanghai Inst. of Tumor also proves DKK-1 specificity overexpression in mankind's kinds of tumors tissue, and its secretion property high expressed detected in the culture supernatant and serum of mankind's kinds of tumor cells, prompting DKK-1 can be used as the serodiagnosis of tumour serum protein marker for malignant tumours such as liver cancer, cancer of the stomach, lung cancer, breast cancer, cervical carcinomas.2008, by large sample, the research of clinical multiple center trial, the new team of Qin Wen confirms that DKK-1 albumen is for hepatocellular carcinoma, particularly for early liver cancer (BCLC is the 0+A phase by stages) and small cell carcinoma (diameter <2cm), there is good diagnostic value, also can be used as the index of hepatocellular carcinoma curative effect monitoring and Index for diagnosis.DKK-1 can make up the deficiency of alpha-fetoprotein (apha-fetoprotein, AFP) to diagnosis of hepatoma ability, antidiastole hepatocellular carcinoma from the patients with chronic liver of the AFP positive, and DKK-1 and AFP joint inspection can improve hepatocellular carcinoma collective diagnosis rate.
At present, commercially available people DKK-1 immue quantitative detection reagent box mainly adopts Enzyme-multiplied immune technique (only for research), and the method exists the problems such as the range of linearity is narrow, operation steps is complicated, consuming time, and cannot meet clinical practice requirement.Traditional enzyme-linked immunoassay method, the association reaction of antigen, antibody carries out on solid phase (elisa plate reacting hole) surface, reaction time is long, generally can obtain quantitative testing result after 3-4 hour, its sensing range is at 31.2pg/mL-2000pg/mL, and sample needs to carry out dilution process.
Summary of the invention
An object of the present invention is to provide a kind of people Dickkopf-1 protein quantification detection kit.
Kit provided by the invention,
Be 1) or 2):
1) reagent A is comprised;
Described reagent A is made up of the DKK-1 monoclonal antibody solution of marked by fluorescein isothiocyanate and alkali phosphatase enzyme mark DKK-1 monoclonal antibody solution, wherein, the mass ratio of marked by fluorescein isothiocyanate DKK-1 monoclonal antibody and alkali phosphatase enzyme mark DKK-1 monoclonal antibody is 1:8;
The DKK-1 monoclonal antibody solution of described marked by fluorescein isothiocyanate and the solvent of described alkali phosphatase enzyme mark DKK-1 monoclonal antibody solution are anti-reagent buffer;
Described anti-reagent buffer is prepared as follows: the mixing of solution A, sheep blood serum 2, cow's serum, horse serum, antiseptic and blocking agent is obtained anti-reagent buffer;
Described solution A is by Na
+, Mg
2+, Zn
2+, Trizmabase, Sodium azide, sheep blood serum 1, bovine serum albumin(BSA) and water composition;
2) the DKK-1 monoclonal antibody of described marked by fluorescein isothiocyanate, described alkali phosphatase enzyme mark DKK-1 monoclonal antibody and described anti-reagent buffer is comprised.
In mentioned reagent box,
Described antiseptic is Proclin-300, and described blocking agent is HBR1 aqueous solution;
In described anti-reagent buffer, the proportioning of described solution A, described sheep blood serum 2, described cow's serum, described horse serum, described Proclin-300 and described HBR1 aqueous solution is 500g-1200g:1mL-10mL:10mL-80mL:3mL-10mL:0.1mL-5mL:1.5mL-5 mL; The concentration of described HBR1 aqueous solution is 20-30mg/mL;
In described solution A, described Na
+come from NaCl, the mass percentage of NaCl is 0.1%-1%; Described Mg
2+come from MgCl
2, described MgCl
2mass percentage be 0.1 ‰-0.5 ‰; Described Zn
2+come from ZnCl
2, described ZnCl
2mass percentage be 0.01 ‰-0.05 ‰; Described sheep blood serum 1 volumn concentration is 0.1%-0.5%; Described Sodium azide mass percentage is 0.1-0.5%; Described Trizmabase concentration is 0.05-0.5mol/L; The mass percentage of described bovine serum albumin(BSA) is 0.4%-2%.
In mentioned reagent box,
In described anti-reagent buffer, the proportioning of described solution A, described sheep blood serum 2, described cow's serum, described horse serum, described Proclin-300, described HBR1 aqueous solution is 1000g:3mL:30mL:5mL; 0.521mL:2.44mL; The concentration of described HBR1 aqueous solution is 20.49mg/mL;
In described solution A, described Na
+come from NaCl, the mass percentage of described NaCl is 0.582%;
Described Mg
2+come from MgCl
2, described MgCl
2mass percentage be 0.203 ‰;
Described Zn
2+come from ZnCl
2, described ZnCl
2mass percentage be 0.0136 ‰;
The volumn concentration of described sheep blood serum 1 is 0.199%;
The mass percentage of described Sodium azide is 0.1%;
The mass percentage of described Trizmabase is 1.205%;
The mass percentage of described bovine serum albumin(BSA) is 0.498%.
In mentioned reagent box,
The DKK-1 monoclonal antibody solution of described marked by fluorescein isothiocyanate is made up of the DKK-1 monoclonal antibody of marked by fluorescein isothiocyanate and described anti-reagent buffer, and the concentration of the DKK-1 monoclonal antibody of described marked by fluorescein isothiocyanate is 0.1-0.5 μ g/mL;
Described alkali phosphatase enzyme mark DKK-1 monoclonal antibody solution is made up of alkali phosphatase enzyme mark DKK-1 monoclonal antibody and described anti-reagent buffer, and the concentration of described alkali phosphatase enzyme mark DKK-1 monoclonal antibody is 0.5-2 μ g/mL;
DKK-1 monoclonal antibody in the DKK-1 monoclonal antibody of described marked by fluorescein isothiocyanate is different with DKK-1 monoclonal antibody in described alkali phosphatase enzyme mark DKK-1 monoclonal antibody.
In mentioned reagent box,
Described kit also comprises Magneto separate reagent;
Described Magneto separate reagent is by covalently bound in magnetic bead surfaces for anti-fluorescein isothiocynate antibody, obtains Magneto separate reagent;
Described anti-fluorescein isothiocynate antibody is polyclonal antibody;
The proportioning of described anti-fluorescein isothiocynate antibody and described magnetic particle is 30 μ g-200 μ g:1mg;
The diameter of described magnetic particle is 0.5-1.5 μm.
Mentioned reagent box also comprises calibration object;
Described calibration object is by the calibration object solution composition of n variable concentrations, and described calibration object solution is made up of DKK-1 antigen and calibration object damping fluid, and described DKK-1 antigen is variable concentrations in described calibration object;
Described calibration object damping fluid is by Na
+, bovine serum albumin(BSA), casein, tetracycline, neomycinsulphate, Sodium azide, Trizmabase, Tween-20 and water composition,
Described Na
+mass percentage be 1%-3%, the mass percentage of described bovine serum albumin(BSA) is 7%-15%, described caseic mass percentage is 0.5-2%, the mass percentage of described tetracycline is 0.01 ‰-0.05 ‰, the mass percentage of described neomycinsulphate is 0.01 ‰-0.05 ‰, the mass percentage of described Sodium azide is 0.05%-0.35%, and described Trizmabase concentration is 0.05-0.5mol/L, and the volumn concentration of described Tween-20 is 0.2-2%.
In mentioned reagent box,
Described Na
+come from NaCl, the mass percentage of described NaCl is 1.75%;
The mass percentage of described bovine serum albumin(BSA) is 10%;
Described caseic mass percentage is 1%;
The mass percentage of described tetracycline is 0.02 ‰;
The mass percentage of described neomycinsulphate is 0.02 ‰;
The mass percentage of described Sodium azide is 0.2%;
The concentration of described Trizmabase is 0.1mol/L;
The volumn concentration of described Tween-20 is 0.4%.
In mentioned reagent box,
Described calibration object is by the calibration object solution composition of n variable concentrations, and n is less than or equal to 6; To be concentration be described calibration object 0,0.7ng/mL, 2.5ng/mL, 7ng/mL, 14ng/mL, 28ng/mLDKK-1 antigenic solution.
Described kit also comprises quality-control product, cleaning fluid and substrate solution;
Described quality-control product is the calibration object of 2 variable concentrations; The calibration object of described quality-control product to be concentration be 0.7 ± 0.14ng/mL and 14 ± 2.8ng/mL.
Described cleaning fluid by Tween-20, sodium chloride and concentration to be 0.15M, pH be 8.0 ± 0.05 Tris-HCl damping fluid form, wherein, the volumn concentration of described Tween-20 is 0.04%, and the concentration of described sodium chloride is 0.25mol/L;
Described substrate solution by acridan and concentration to be 0.25M, pH be 8.0 ± 0.05 Tris-HCl damping fluid form, wherein, described APCL final concentration is 0.25mg/mL.
Another object of the present invention is to provide a kind of method preparing above-mentioned kit.
Method of the present invention, comprises the steps: 6 kinds of equal independent packagings of component in mentioned reagent box; Entire package is kit again.
The application of above-mentioned kit in people Dickkopf-1 protein quantification detects also is the scope of protection of the invention;
Or the application of above-mentioned kit in preparation people Dickkopf-1 protein quantification testing product is also the scope of protection of the invention;
Or 6 kinds of component application in preparation people Dickkopf-1 protein quantification testing product are also the scope of protection of the invention in above-mentioned kit;
Present invention also offers people Dickkopf-1 protein quantification detection method in a kind of sample to be tested, comprise the steps:
1) calibration object in above-mentioned kit, quality-control product and sample to be tested are mixed with the described reagent A in arbitrary described kit in claim 1-8 respectively, reaction, obtains immune reaction product;
2) mixed by the Magneto separate reagent in described immune reaction product and above-mentioned kit, reaction, obtain reaction product, by the precipitation magnetic bead cleaning fluid suspendible in described reaction product, collecting precipitation, is Magneto separate product;
Described magnetic bead cleaning fluid is cleaning fluid and water in described kit arbitrary in claim 1-8 are mixed by 1:7, obtains solution;
3) detect the luminous intensity of described Magneto separate product with Chemiluminescence Apparatus, calculate people Dickkopf-1 protein quantification in sample to be tested by luminous intensity.
Or the application of Magneto separate reagent in people Dickkopf-1 protein quantification detects also is the scope of protection of the invention in above-mentioned kit.
The pH value of described anti-reagent buffer is 8.0 ± 0.05.
The pH value of described calibration object damping fluid is 7.5 ± 0.05.
The DKK-1 monoclonal antibody of described marked by fluorescein isothiocyanate is the product obtained with marked by fluorescein isothiocyanate DKK-1 monoclonal antibody A;
Described alkali phosphatase enzyme mark DKK-1 monoclonal antibody is the product obtained with the monoclonal antibody B of alkali phosphatase enzyme mark DKK-1.
The present invention has the following advantages:
The present invention utilizes magnetic microparticles to combine with chemiluminescence, provide the quantitative detecting method that a kind of sensing range is wide, highly sensitive, precision is good, measured value is stable, and be applicable to Full-automatic chemiluminescence apparatus, simple to operate, detect fast, the demand of clinical a large amount of detection serum can be met.
The detection method that this kit adopts is magnetic microparticle separating chemiluminescence immune detection, by enzyme labeling technology, magnetic separation technique and chemiluminescence combine, wherein, the association reaction of antigen, antibody carries out under the condition of approximate liquid phase, thus it is convenient and simple for operation, and reaction conditions is gentle, and luminous value is stablized and affected by external condition less.Compared with prior art, the present invention has sample process process simple (without the need to dilution), and testing cost is low, detects fast (within 30-40 minute, obtaining testing result), the advantages such as test result is accurate, reproducible, can meet clinical practice demand.
Accompanying drawing explanation
Fig. 1 is fundamental diagram of the present invention.
Fig. 2 is that QuantikineELISAHumanDKK-1 measures kit results.
Embodiment
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
All components in detection kit of the present invention all obtains from biological reagent or chemical reagents corporation's purchase by commercial sources.In the present invention, mark fluorescein isothiocynate monoclonal antibody used is that Raygene produces, and article No. is M1656-10; Mark alkaline phosphatase monoclonal antibody is that Raygene produces, and article No. is M6130-10; Antigen is that Raygene produces, and article No. is R1080-05; Goat-anti fluorescein isothiocynate antibody serum serum purchased from animal used as test section of PLA General Hospital first affiliated hospital, article No.: Y11005; Fluorescein isothiocynate (FITC) purchased from SIGMA company, CAT:3326-32-7, article No. is: F7250; Alkaline phosphatase is purchased from BBI company of Britain, and lot number is: 1693AA; SMCC is purchased from thermofisherscientific company, and article No. is: 22360; 2-IT purchased from thermofisherscientific company, CAS:4781-83-3; Article No. is: 26101; Carboxyl magnetic bead colloidal solution is purchased from merk company, and article No. is EM1-100/40; NHS purchased from SIMGA company, CAS:6066-82-6, article No.: 130672; EDC purchased from SIMGA company, CAS:25952-53-8, article No.: E7750.Magnetic particle reagent, substrate solution, cleaning fluid are all from Capitalbio Corporation Co., Ltd.'s finished product kit.
Embodiment 1, people Dickkopf-1 albumen (DKK-1) immue quantitative detection reagent box component and method thereof
One, the preparation of people Dickkopf-1 albumen (DKK-1) immue quantitative detection reagent box
By after following 6 kinds of component independent packagings again entire package be kit, be namely people Dickkopf-1 albumen (DKK-1) immue quantitative detection reagent box: 1, calibration object (containing the DKK-1 of a series of concentration, for Criterion curve); 2, quality-control product (containing certain density DKK-1); 3, reagent A (the DKK-1 monoclonal antibody containing certain density marked by fluorescein isothiocyanate and the DKK-1 monoclonal antibody solution containing certain density alkali phosphatase enzyme mark); 4, Magneto separate reagent (being combined with the magnetic particle suspension of anti-fluorescein isothiocynate antibody); 5, cleaning fluid (for preparing magnetic bead cleaning fluid); 6, substrate solution.In kit, 1,2,3,4,6 is indispensable reagent, and 5 replace using by buying other company's similar products.Mentioned reagent box is placed in the preservation of 4 degree, refrigerator.
Specific as follows:
1, calibration object
1) preparation of calibration object damping fluid
Calibration object damping fluid is by Na
+, protide protective agent, antiseptic, Trizmabase, surfactant and water composition, the final concentration of each component is as follows: Na
+come from NaCl, its concentration is 1.75% (mass percentage); Protide protective agent is bovine serum albumin(BSA) and casein, and concentration is respectively 10% (mass percentage) and 1% (mass percentage); Antiseptic is tetracycline, neomycinsulphate and Sodium azide, and concentration is respectively 0.02 ‰, 0.02 ‰, 0.2% (mass percentage); Trizmabase concentration is 0.1mol/L; Surfactant is Tween-20, and its concentration is 0.4% (volumn concentration).The pH value of calibration object damping fluid is 7.5 ± 0.05.
The collocation method of calibration object damping fluid is specific as follows:
A) get the reagent bottle of a dried and clean, add purified water 500mL;
B) add Trizmabase12.11g, NaCl17.53g, mixing 2 is little of dissolving completely, and adjusted to ph is to 7.5 ± 0.05.
C) tetracycline 20mg (producer: SIGMA is added, article No.: 1651009), neomycinsulphate CL10-02220mg (producer: SIGMA, article No.: 33492-100MG-R), Sodium azide CL10-0352.0g (producer: SIGMA, article No.: S8032), polysorbas20 4.0mL (producer: SIGMA, article No.: P1379), bovine serum albumin(BSA) 100g (producer: SIGMA, article No.: V900933), casein 10g (producer: SIGMA, article No.: C7078), mixing 1 is little of dissolving completely, and adjusted to ph is 7.5 ± 0.05;
D) add purified water surely to weigh to 1000g, checking PH most 7.5 ± 0.05;
E) use 0.22 μm of membrane filtration and collect filtrate, post label, 2 ~ 8 DEG C of preservations, obtain calibration object damping fluid.
2) preparation of calibration object
By DKK-1 antigen (company: Raygene; Article No.: R1080-05) with above-mentioned 1) alignment savors damping fluid dilution becomes the calibration object of 28ng/mL, 14ng/mL, 7ng/mL/L, 2.5ng/mL, 0.7ng/mL, equivalent is distributed into the calibration object that concentration is 0ng/mL, 0.7ng/mL, 2.5ng/mL, 7ng/mL, 14ng/mL, 28ng/mL.
2, quality-control product (containing certain density DKK-1)
Using the above-mentioned concentration calibration object that is 0.7ng/mL and 14ng/mL as quality-control product;
3, reagent A (fluorescein isothiocynate (FITC) marks the DKK-1 monoclonal antibody solution of DKK-1 monoclonal antibody and alkali phosphatase enzyme mark)
1), the preparation of anti-reagent buffer
Anti-reagent buffer is by Na
+, Mg
2+, Zn
2+, protide protective agent, animal blood serum, antiseptic, Trizmabase, blocking agent and water composition, the final concentration of each component is as follows:
Na
+come from NaCl, the concentration of NaCl is 0.582% (mass percentage; Mg
2+come from MgCl
2, MgCl
2concentration is 0.203 ‰ (mass percentage), Zn
2+come from ZnCl
2, ZnCl
2concentration be 0.0136 ‰ (mass percentage); Protide protective agent is bovine serum albumin(BSA), and concentration is 0.498% (mass percentage); Animal blood serum is sheep blood serum 1, NBCS and horse serum, and its concentration is respectively 0.499%, 3%, 0.5% (volumn concentration); Antiseptic is Sodium azide and Proclin-300, and concentration is respectively 0.1% (mass percentage) and 0.0521% (volumn concentration); Trizmabase concentration is 0.1mol/L; Blocking agent is its concentration of HBR1 is 0.244 (volumn concentration).The pH value of calibration object damping fluid is 8.0 ± 0.05.
The compound method of anti-reagent buffer is specific as follows:
A) get the reagent bottle of a dried and clean, add purified water 600mL;
B) Trizmabase12.05g, NaCl5.82g, Sodium azide 1.00g is added, mixing 20min
C) 0.203g/mL magnesium chloride solution 1mL (magnesium chloride producer: SIGMA is added, article No.: 449172), 0.0136g/mL liquor zinci chloridi 1mL (zinc chloride producer: SIGMA, article No.: 746355), dissolve mixing 20min, 4MHCl and regulate pH to 10.0 ± 0.05.
D) sheep blood serum (called after 1) 1.992mL (producer: SIGMA is added, article No.: RYA11004), bovine serum albumin(BSA) 4.98g, fully stir and evenly mix, leave standstill 18h, regulate pH to 8.0 ± 0.05 with 4MHCl, add pure water and weigh calmly to 1000g (default volume is 1L); Obtain solution A;
E) stir and evenly mix 1.0h, repetition measurement pH value is 8.0 ± 0.05;
F) sheep blood serum (called after 2) 3.0mL is added, NBCS 30.0mL (producer: Gibco, article No.: 16010-159), horse serum 5.0mL (producer: Gibco, article No.: 16050-114), Proclin-3000.521mL (producer: SIGMA, article No.: 48914-U), abundant mixing 1h, checking pH value is 8.0 ± 0.05;
G) add 20.49mg/mLHBR12.44mL (HBR1 producer: Scantibodies, article No.: 3KC780), mix, use 0.22 μm of membrane filtration and collect filtrate, posting label, 2 ~ 8 DEG C of preservations.
2), marked by fluorescein isothiocyanate DKK-1 monoclonal antibody
Marked by fluorescein isothiocyanate DKK-1 monoclonal antibody is the product obtained by marked by fluorescein isothiocyanate DKK-1 monoclonal antibody, and concrete grammar is as follows:
Get 1mgDKK-1 monoclonal antibody (company: Raygene; Article No.: M1656-10), with concentrate (pH=9.0 ± 0.05, mass percent 1.68% sodium bicarbonate aqueous solution) suspend, and by centrifugal concentrating to 5mg/mL, adding concentration is that (solvent is pH=9.0 ± 0.05 to 0.5mg/mL, 1.68% sodium bicarbonate aqueous solution) 100 μ L (proportioning of DKK-1 monoclonal antibody and fluorescein isothiocynate is 1g:0.18mL), mix.Room temperature lucifuge reacts 20 hours, obtains marked by fluorescein isothiocyanate DKK-1 monoclonal antibody.
Re-use PD10 pillar purifying, with 0.2mol/L carbonate buffer solution PH9.5 wash-out, collect yellow liquid, obtain marked by fluorescein isothiocyanate DKK-1 monoclonal antibody after purifying.
3), the preparation of marked by fluorescein isothiocyanate DKK-1 monoclonal antibody solution
By above-mentioned 2) purifying after marked by fluorescein isothiocyanate DKK-1 monoclonal antibody and above-mentioned 1) in anti-reagent buffer mix, obtain marked by fluorescein isothiocyanate DKK-1 monoclonal antibody solution, wherein the concentration of the anti-human Dickkopf-1 albumen (DKK-1) of marked by fluorescein isothiocyanate is 0.1 μ g/ml.
4) alkali phosphatase enzyme mark DKK-1 monoclonal antibody
Alkali phosphatase enzyme mark DKK-1 monoclonal antibody is the product obtained by the another kind of monoclonal antibody of alkali phosphatase enzyme mark DKK-1, and concrete grammar is as follows:
Get the another kind of monoclonal antibody (company: Raygene of 1mgDKK-1; Article No.: M6130-10), with concentrate (formula: 1.492% triethanolamine, 0.584% sodium chloride, 0.0412%EDTA-Na
22H
2o and water; PH=8.5 ± 0.05) suspend, and by centrifugal concentrating to 2.5mg/mL; Add activator 2-Iminothiolanehydrochloride (2-IT) solution (solvent: N that concentration is 13.76mg/mL, dinethylformamide) 5 μ L, volume ratio 1:80, room temperature adds glycocoll and stops priming reaction after placing 20 minutes, ambient temperatare puts 10 minutes.Re-use SephadexG25 pillar desalination, obtain activating rear antibody.
Get 1.5mg alkaline phosphatase (company: BBI; Article No.: 1693AA), with concentrate (formula: 0.448% triethanolamine, 17.532% sodium chloride, 0.02% magnesium chloride, 0.001% zinc chloride; PH=7.6 ± 0.05) suspend, and by centrifugal concentrating to 5mg/mL; Add Succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) solution (solvent: N that concentration is 6.69mg/mL, dinethylformamide) 12 μ L, volume ratio 1:40, room temperature places 20 minutes, add glycocoll and stop priming reaction, room temperature places 10 minutes.Use SephadexG25 pillar desalination, obtain activating rear alkaline phosphatase.
The ratio that DKK-1 antibody (being as a solution) after activation and alkaline phosphatase (being as a solution) after activation add 1mg alkaline phosphatase in 1mgDKK-1 antibody is mixed, add 5 μ L1M magnesium chloride brine mixings simultaneously, place 4 DEG C of reactions 20 hours.
Use Supperdex200 gel chromatography column separating purification, remove the DKK-1 antibody and alkaline phosphatase that do not connect, with 0.05mol/L Triethanolamine buffer PH7.0 wash-out, elution flow rate is 0.75ml/min, pillar diameter/length is 1.6/60cm, observe out peak position and comparison standard molecule collection of illustrative plates collection connector, connector is stored in 4 DEG C, obtains alkali phosphatase enzyme mark DKK-1 monoclonal antibody.
5), the preparation of alkali phosphatase enzyme mark DKK-1 monoclonal antibody solution
By above-mentioned 4) alkali phosphatase enzyme mark DKK-1 monoclonal antibody and above-mentioned 1) in anti-reagent buffer mix, obtain alkali phosphatase enzyme mark DKK-1 monoclonal antibody solution, wherein the concentration of alkali phosphatase enzyme mark DKK-1 monoclonal antibody is 0.8 μ g/mL.
6) reagent A
Marked by fluorescein isothiocyanate DKK-1 monoclonal antibody solution is mixed with alkali phosphatase enzyme mark DKK-1 monoclonal antibody solution equal-volume, obtain reagent A, wherein, the mass ratio 1:8 of marked by fluorescein isothiocyanate DKK-1 monoclonal antibody and alkali phosphatase enzyme mark DKK-1 monoclonal antibody; 2-8 DEG C of preservation after abundant mixing.
4, Magneto separate reagent (being combined with the magnetic particle suspension of anti-fluorescein isothiocynate antibody)
Magneto separate reagent is by covalently bound for goat-anti fluorescein isothiocynate antibody serum (polyclonal antibody, producer: animal used as test section of PLA General Hospital first affiliated hospital article No.: Y11005) surperficial at magnetic particle, obtains Magneto separate reagent; Wherein, the proportioning of anti-fluorescein isothiocynate antibody and magnetic particle is 150 μ g:1mg.
The diameter (producer: merck company, article No.: EM1-100/40) between 0.5-1.5 μm of magnetic particle, has superparamagnetism, and surface is containing carboxyl (COOH-) reactive group.
The concrete preparation method of Magneto separate reagent:
(1) by magnetic bead and activator EDC (purchased from SIMGA company, CAS:25952-53-8, article No.: E7750) and NHS (purchased from SIMGA company, CAS:6066-82-6, article No.: 130672) mix with certain proportion, room temperature reaction 120 minutes, obtains activated magnetic beads; Above-mentioned magnetic bead and activator mix ratio are: add 5mgEDC and 5mgNHS in 1000mg magnetic bead; (2) mixed with certain proportion with goat-anti fluorescein isothiocynate antibody by magnetic bead after activation, blending ratio is: add 150mg goat-anti fluorescein isothiocynate antibody in 1000mg magnetic bead, and 4 degree of oscillating reactionss 10 hours, obtain the magnetic bead of connection albumen; (3) processed by the magnetic bead confining liquid after connection albumen, room temperature closes 1 hour, obtains magnetic bead solution; Confining liquid is the MES damping fluid of pH7.0,0.01M containing 1g/100mLBSA; (4) the magnetic bead solution (3) obtained sedimentation under magnetic fields abandoned supernatant after 20 minutes; The stainless steel sift that 1500 order materials are 304 steel is crossed after magnetic bead being suspended in the MES damping fluid of 100mlpH7.0,0.01M, sieve in process, continuous vibration is also rinsed repeatedly with the MES damping fluid of pH7.0,0.01M, until compass screen surface remains the agglomerated particle that can not sieve, magnetic bead after sieving answers particle homogeneous, without aggegation after suspendible again; (5) the magnetic bead conserving liquid after sieving is diluted to 5mg/ml, room temperature suspendible is 4 DEG C of preservations after 2 hours, obtain the magnetic bead being combined with anti-fluorescein isothiocynate antibody; Conserving liquid is the trizmabase damping fluid containing 1% bovine serum albumin(BSA); Again the magnetic bead being combined with anti-fluorescein isothiocynate antibody is suspended in the trizmabase damping fluid containing 1% bovine serum albumin(BSA), obtains Magneto separate reagent;
Trizmabase damping fluid containing 1% bovine serum albumin(BSA) is bovine serum albumin(BSA), trizmabase and water are mixed, obtain the trizmabase damping fluid containing bovine serum albumin(BSA), wherein, the concentration of bovine serum albumin(BSA) is 1% (for mass percentage), the concentration of trizmabase is 0.1mol/L.
5, cleaning fluid
Cleaning fluid: be 8.0 ± 0.05 concentration at pH be add Tween-20 and sodium chloride in the Tris-HCl damping fluid of 0.15M, wherein the concentration of Tween-20 is 0.04% (volumn concentration), and the concentration of sodium chloride is 0.25mol/L.
6, substrate solution
Substrate solution: to be 0.25M, pH by dioxane compound (APCL) and concentration be 8.0 ± 0.05 Tris-HCl damping fluid mix, dissolving, makes APCL final concentration be 0.25mg/mL, the solution obtained.
Two, magnetic microparticles chemiluminescence immunoassay kit is utilized to carry out quantitative measurement to people Dickkopf-1 albumen (DKK-1) content in sample
1, principle
Fig. 1 is fundamental diagram of the present invention.
This kit reaction principle is double antibody sandwich method, and the DKK-1 namely in the same sample of DKK-1 monoclonal antibody, calibration object or the quality-control product that mark of the DKK-1 monoclonal antibody that marks of fluorescein isothiocynate (FITC) and alkaline phosphatase (AP) is combined the compound forming " sandwich " structure.Add the magnetic particle being connected with anti-FITC antibody subsequently, antigen antibody complex is made to be connected on magnetic particle by the specific binding of anti-FITC antibody and FITC, Direct precipitation in externally-applied magnetic field, the compound that immune response is formed and other separating substances unconjugated.Remove the compound of washing and precipitating after supernatant, add enzymatic chemical substrate solution.Substrate is catalyzed cracking under enzyme effect, forms unstable excited state intermediate, just sends photon when excited state intermediate gets back to ground state, forms luminescence-producing reaction, can use the luminous intensity of light-emitting appearance detection reaction.Luminous intensity is directly proportional to the concentration of DKK-1 in sample.Use four parameter Logistic equation models of improvement can calculate DKK-1 protein concentration in sample.
2, people Dickkopf-1 albumen (DKK-1) magnetic microparticle separating chemiluminescence immunologic detection method
(1) immune response: respectively by 30 μ LDKK-1 calibration objects in the kit of above-mentioned, 30 μ L quality-control products and 30 μ L samples to be tested in differential responses pipe, again respectively to adding 60 μ L reagent A in each reaction tube, be placed on oscillator and mix, 37 DEG C of incubation 15min;
(2) Magneto separate: add 30 μ L Magneto separate reagent in the kit of above-mentioned in each reaction tube processed through (1) again, be placed on oscillator and mix, 37 DEG C of incubation 5min, make magnetic particle sedimentation in externally-applied magnetic field, remove supernatant, add 200 μ L magnetic bead cleaning fluids (cleaning fluid in the kit of above-mentioned one and purified water diluted by 1:7 and be mixed with magnetic bead cleaning fluid), remove magnetic field, concussion makes the abundant suspendible of magnetic particle 30 seconds, and then make magnetic particle sedimentation in magnetic field, remove supernatant; Repetition like this 2-4 time;
(3) read value: to each reaction tube processed through (2) often pipe add 200 μ L substrate solutions, detect luminous intensity with Chemiluminescence Apparatus.
Specific as follows:
Each reagent, sample cup and reaction tube are placed in instrument, and the running program in strict accordance with Full-automatic chemiluminescence immunoassay analysis meter is carried out experimental implementation and calculates concentration of specimens.Instrument has possessed the temperature of reaction 37 DEG C ± 1 DEG C required for experiment, the reaction time of this project and operation steps have been input in full automatic running program, each walks the time needed for testing, instrument spectral measurement range 300nm ~ 650nm can to obtain this this project after selected this project (DKK-1).
DKK-1 kit is in measurement range, and luminous intensity is directly proportional to DKK-1 concentration in sample to be tested, uses four parameter Logistic equation models of improvement to calculate DKK-1 concentration in sample.
y＝(A1-A0)/(1+(x/X0)^P)+A0
Y: luminous intensity
X: concentration value
A1: asymptotic line valuation on curve
A0: asymptotic line valuation under curve
X0: the concentration value that knee point is corresponding
P: the parameter that rate of curve is relevant
Embodiment 2, kit performance evaluation
According to the feature of external diagnosis reagent, detect the range of linearity, minimum detectability, accuracy, the precision of kit prepared by embodiment 1 as usual.Concrete operation step is as follows:
One, reagent prepares:
Before experiment, first take out reagent A, calibration object, Magneto separate reagent, substrate solution, cleaning fluid, the quality-control product in the kit of of embodiment 1, balance to room temperature.
Two, instrument prepares:
This kit adopts the ChemLite of Capitalbio Corporation Co., Ltd.
tM1200 Full-automatic chemiluminescence immunoassay analysis meters.
Specific as follows:
In sample cup, add calibration object, sample or quality-control product, sample single tube reaction application of sample amount is 30 μ L, and according to repeating addition in pipe number calculation sample cup, minimum application of sample amount is not less than 50 μ L, adds the reagent of sufficient quantity.
Each reagent, sample cup and reaction tube are placed in instrument, and the running program in strict accordance with Full-automatic chemiluminescence immunoassay analysis meter is carried out experimental implementation and calculates concentration of specimens.Instrument has possessed the temperature of reaction 37 DEG C ± 1 DEG C required for experiment, the reaction time of this project and operation steps have been input in full automatic running program, each walks the time needed for testing, instrument spectral measurement range 300nm ~ 650nm can to obtain this this project after selected this project (DKK-1).
Four, performance index testing result:
1, the range of linearity:
Dose-response curve is linear: the four parameter Logistic equation models using improvement, within the scope of 0.2-28ng/mL, and dose-response curve correlation coefficient r >=0.99.
By close to the range of linearity upper limit high level sample calibration object damping fluid (be damping fluid in example 1,10%BSA; 0.1MTrizmabase concentration; PH=7.5 ± 0.05) dilute 5 concentration as sample to be tested (human serum) by 4 times, specifically as shown in table 1, wherein dilution ratio is the sample to be tested concentration of 1 is 28ng/mL.
With the kit of a preparation in embodiment 1 and the detection method of two, sample to be tested is detected, measure the range of linearity.Each dilute concentration tests 3 times.
Obtain the average (Yi) of measurement result respectively, with dilute concentration (Xi) for independent variable, with measurement result average (Yi) for dependent variable, obtain equation of linear regression, calculate linear regression coeffficient (r) by formula (1).
Use four parameter Logistic equation models of improvement, in 0.2ng/mL-28ng/mL sensing range, dose-response curve correlation coefficient r=0.999.
Table 1 range of linearity experimental result
2, accuracy:
The DKK-1 high level sample (A) concentration being about 14 ± 2.8ng/mL joins concentration and is about in the low value sample B of 0.7 ± 0.14ng/mL, add A liquid volume be not more than 10% of cumulative volume, specifically in table 2.
With the kit of a preparation in embodiment 1 and the detection method of two, sample to be tested is detected, accuracy of measurement.Recovery R is calculated according to formula (2).
In formula:
R---the recovery;
V---add the volume of A liquid;
V
0---the volume of B liquid;
C---B liquid adds the detectable concentration after A liquid;
C
0---the detectable concentration of B liquid;
Cs---the detectable concentration of A liquid.
Reclaimed by application of sample and evaluate its accuracy, the acquired results dilution recovery is between 90%-110%, and concrete data are in table 2.
Table 2 accuracy experimental result (ng/mL)
3, lowest detectable limit (sensitivity):
With 0ng/mL calibration object in kit as sample to be tested, replication 20 times, draw the RLU value (relative light unit) of 20 measurement results, calculate its mean value (M) and standard deviation (SD), draw RLU value corresponding to M+2SD, carry out 2 regression fits according to the concentration-RLU value result between zero-dose calibration object and adjacent calibration object and draw linear function, the RLU value of M+2SD is brought in above-mentioned equation, obtain corresponding concentration value.
By lowest detectable limit detection method in detection scheme, repeat 5 experiments, concrete data are in table 3.
Table 3 lowest detectable limit experimental result
| Sensitivity (ng/mL) | |
| Experiment 1 | 0.01 |
| Experiment 2 | 0.05 |
| Experiment 3 | 0.02 |
| Experiment 4 | 0.01 |
| Experiment 5 | 0.03 |
4, precision:
In batch: use DKK-1 (0.7 ± 0.14) ng/mL and the sample in (14 ± 2.8) ng/mL two concentration ranges as sample to be tested.The method of operating of by specification, carries out 10 replications to sample to be tested respectively with chemical luminescence detector device.Repeatability (CV) is calculated by formula (3).
measure the mean value of concentration for 10 times
X
i: measure concentration value at every turn
Between batch: use DKK-1 concentration to be that (14 ± 2.8) ng/mL is as sample to be tested.Use the kit of 3 different batches, the method for operating of by specification, with chemical luminescence detector device, 30 replications (every batch kit measurement 10 times) are carried out to sample to be tested.Difference between batch (CV) is calculated by formula (4).
In formula:
measure the mean value of concentration for 30 times
Measure the standard deviation of concentration for SD:30 time
Detect precision by precision detection method in detection scheme, concrete data are in table 4.
Table 4 Precision Experiment result
Embodiment 3, kit provided by the invention compare with external kit
1, kit provided by the invention and the comparison of external kit performance index as follows:
Same sample is detected with existing kit, as follows with kit testing result of the present invention comparison:
Table 5 performance index comparative result
2, kit provided by the invention and external kit carry out the comparison of clinical sample measured value
The QuantikineELISAHumanDKK-1 produced with kit provided by the invention and R & DSystems company measures kit (research with) and detects 100 parts of human serum samples (patient is in the know) respectively simultaneously, the results are shown in Figure 2, the serum DKK-1 concentration adopting kit provided by the invention to record is ordinate, with the result of the QuantikineELISAHumanDKK-1 kit measurement of R & DSystems company production for horizontal ordinate does regretional analysis, dependent equation is: y=0.9877x – 0.0871, correlation coefficient r is: 0.993.Show through statistical procedures result, the clinical sample measured value correlativity that kit provided by the invention and R & D company kit record is good.
Claims (10)
1. a people Dickkopf-1 protein quantification detection kit is 1) or 2):
1) reagent A is comprised;
Described reagent A is made up of the DKK-1 monoclonal antibody solution of marked by fluorescein isothiocyanate and alkali phosphatase enzyme mark DKK-1 monoclonal antibody solution, wherein, the mass ratio of marked by fluorescein isothiocyanate DKK-1 monoclonal antibody and alkali phosphatase enzyme mark DKK-1 monoclonal antibody is 1:8;
The DKK-1 monoclonal antibody solution of described marked by fluorescein isothiocyanate and the solvent of described alkali phosphatase enzyme mark DKK-1 monoclonal antibody solution are anti-reagent buffer;
Described anti-reagent buffer is prepared as follows: the mixing of solution A, sheep blood serum 2, cow's serum, horse serum, antiseptic and blocking agent is obtained anti-reagent buffer;
Described solution A is by Na
+, Mg
2+, Zn
2+, Trizmabase, Sodium azide, sheep blood serum 1, bovine serum albumin(BSA) and water composition;
2) the DKK-1 monoclonal antibody of described marked by fluorescein isothiocyanate, described alkali phosphatase enzyme mark DKK-1 monoclonal antibody and described anti-reagent buffer is comprised.
2. kit according to claim 1, is characterized in that:
Described antiseptic is Proclin-300, and described blocking agent is HBR1 aqueous solution;
In described anti-reagent buffer, the proportioning of described solution A, described sheep blood serum 2, described cow's serum, described horse serum, described Proclin-300 and described HBR1 aqueous solution is 500g-1200g:1mL-10mL:10mL-80mL:3mL-10mL:0.1mL-5mL:1.5mL-5 mL; The concentration of described HBR1 aqueous solution is 20-30mg/mL;
In described solution A, described Na
+mass percentage is 0.1%-1%; Described Mg
2+mass percentage is 0.1 ‰-0.5 ‰; Described Zn
2+mass percentage is 0.01 ‰-0.05 ‰; Described sheep blood serum 1 volumn concentration is 0.1%-0.5%; Described Sodium azide mass percentage is 0.1-0.5%; Described Trizmabase concentration is 0.05-0.5mol/L; The mass percentage of described bovine serum albumin(BSA) is 0.4%-2%.
3. kit according to claim 1 or 2, is characterized in that:
In described anti-reagent buffer, the proportioning of described solution A, described sheep blood serum 2, described cow's serum, described horse serum, described Proclin-300, described HBR1 aqueous solution is 1000g:3mL:30mL:5mL; 0.521mL:2.44mL; The concentration of described HBR1 aqueous solution is 20.49mg/mL;
In described solution A, described Na
+come from NaCl, the mass percentage of described NaCl is 0.582%;
Described Mg
2+come from MgCl
2, described MgCl
2mass percentage be 0.203 ‰;
Described Zn
2+come from ZnCl
2, described ZnCl
2mass percentage be 0.0136 ‰;
The volumn concentration of described sheep blood serum 1 is 0.199%;
The mass percentage of described Sodium azide is 0.1%;
The mass percentage of described Trizmabase is 1.205%;
The mass percentage of described bovine serum albumin(BSA) is 0.498%.
4., according to described kit arbitrary in claim 1-3, it is characterized in that:
The DKK-1 monoclonal antibody solution of described marked by fluorescein isothiocyanate is made up of the DKK-1 monoclonal antibody of marked by fluorescein isothiocyanate and described anti-reagent buffer, and the concentration of the DKK-1 monoclonal antibody of described marked by fluorescein isothiocyanate is 0.1-0.5 μ g/mL;
Described alkali phosphatase enzyme mark DKK-1 monoclonal antibody solution is made up of alkali phosphatase enzyme mark DKK-1 monoclonal antibody and described anti-reagent buffer, and the concentration of described alkali phosphatase enzyme mark DKK-1 monoclonal antibody is 0.5-2 μ g/mL;
DKK-1 monoclonal antibody in the DKK-1 monoclonal antibody of described marked by fluorescein isothiocyanate is different with DKK-1 monoclonal antibody in described alkali phosphatase enzyme mark DKK-1 monoclonal antibody.
5., according to described kit arbitrary in claim 1-4, it is characterized in that:
Described kit also comprises Magneto separate reagent;
Described Magneto separate reagent is by covalently bound in magnetic bead surfaces for anti-fluorescein isothiocynate antibody, obtains Magneto separate reagent;
Described anti-fluorescein isothiocynate antibody is polyclonal antibody;
The proportioning of described anti-fluorescein isothiocynate antibody and described magnetic particle is 30 μ g-200 μ g:1mg;
The diameter of described magnetic particle is 0.5-1.5 μm.
6., according to described kit arbitrary in claim 1-5, it is characterized in that: described kit also comprises calibration object;
Described calibration object is by the calibration object solution composition of n variable concentrations, and described calibration object solution is made up of DKK-1 antigen and calibration object damping fluid, and described DKK-1 antigen is variable concentrations in described calibration object;
Described calibration object damping fluid is by Na
+, bovine serum albumin(BSA), casein, tetracycline, neomycinsulphate, Sodium azide, Trizmabase, Tween-20 and water composition,
Described Na
+mass percentage be 1%-3%, the mass percentage of described bovine serum albumin(BSA) is 7%-15%, described caseic mass percentage is 0.5-2%, the mass percentage of described tetracycline is 0.01 ‰-0.05 ‰, the mass percentage of described neomycinsulphate is 0.01 ‰-0.05 ‰, the mass percentage of described Sodium azide is 0.05%-0.35%, and described Trizmabase concentration is 0.05-0.5mol/L, and the volumn concentration of described Tween-20 is 0.2-2%.
7. kit according to claim 6, is characterized in that:
Described Na
+come from NaCl, the mass percentage of described NaCl is 1.75%;
The mass percentage of described bovine serum albumin(BSA) is 10%;
Described caseic mass percentage is 1%;
The mass percentage of described tetracycline is 0.02 ‰;
The mass percentage of described neomycinsulphate is 0.02 ‰;
The mass percentage of described Sodium azide is 0.2%;
The concentration of described Trizmabase is 0.1mol/L;
The volumn concentration of described Tween-20 is 0.4%.
8. the kit according to claim 6 or 7, is characterized in that:
Described calibration object is by the calibration object solution composition of n variable concentrations, and n is less than or equal to 6;
Described kit also comprises quality-control product, cleaning fluid and substrate solution;
Described quality-control product is the calibration object of 2 variable concentrations;
Described cleaning fluid by Tween-20, sodium chloride and concentration to be 0.15M, pH be 8.0 ± 0.05 Tris-HCl damping fluid form, wherein, the volumn concentration of described Tween-20 is 0.04%, and the concentration of described sodium chloride is 0.25mol/L;
Described substrate solution by acridan and concentration to be 0.25M, pH be 8.0 ± 0.05 Tris-HCl damping fluid form, wherein, described APCL final concentration is 0.25mg/mL.
9. prepare a method for arbitrary described kit in claim 1-8, comprise the steps: 6 kinds of equal independent packagings of component in described kit arbitrary in claim 1-8; Entire package is kit again.
10. the application of arbitrary described kit in people Dickkopf-1 protein quantification detects in claim 1-8;
Or the application of arbitrary described kit in preparation people Dickkopf-1 protein quantification testing product in claim 1-8;
Or 6 kinds of component application in preparation people Dickkopf-1 protein quantification testing product in arbitrary described kit in claim 1-8;
Or people Dickkopf-1 protein quantification detection method in a kind of sample to be tested, comprise the steps:
1) mixed with the described reagent A in arbitrary described kit in claim 1-8 respectively by calibration object, quality-control product and the sample to be tested in described kit arbitrary in claim 1-8, reaction, obtains immune reaction product;
2) mixed by the Magneto separate reagent in described kit arbitrary in described immune reaction product and claim 1-8, reaction, obtain reaction product, by the precipitation magnetic bead cleaning fluid suspendible in described reaction product, collecting precipitation, is Magneto separate product;
Described magnetic bead cleaning fluid is cleaning fluid and water in described kit arbitrary in claim 1-8 are mixed by 1:7, obtains solution;
3) detect the luminous intensity of described Magneto separate product with Chemiluminescence Apparatus, calculate people Dickkopf-1 protein quantification in sample to be tested by luminous intensity;
Or the application of Magneto separate reagent in people Dickkopf-1 protein quantification detects in arbitrary described kit in claim 1-8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510626573.XA CN105203773A (en) | 2015-09-28 | 2015-09-28 | Quantitative detection kit for human Dickkopf-1 protein (DKK-1) |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510626573.XA CN105203773A (en) | 2015-09-28 | 2015-09-28 | Quantitative detection kit for human Dickkopf-1 protein (DKK-1) |
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|---|---|
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510626573.XA Pending CN105203773A (en) | 2015-09-28 | 2015-09-28 | Quantitative detection kit for human Dickkopf-1 protein (DKK-1) |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110531085A (en) * | 2019-08-30 | 2019-12-03 | 北京利德曼生化股份有限公司 | A kind of magnetic microparticle chemiluminescence detection kit and preparation method thereof measuring human nerve silk light chain protein content |
| CN111044724A (en) * | 2019-12-22 | 2020-04-21 | 上海复星长征医学科学有限公司 | Thymidine kinase 1 magnetic particle chemiluminescence assay kit and preparation method thereof |
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|---|---|---|---|---|
| CN101400406A (en) * | 2006-01-13 | 2009-04-01 | 诺瓦提斯公司 | Compositions and methods of use for antibodies of Dickkopf-1 and/or -4 |
| CN102507951A (en) * | 2011-11-25 | 2012-06-20 | 广东药学院 | Enzyme-linked immuno sorbent assay (ELISA) kit for performing joint detection on tumor marker |
| CN104880564A (en) * | 2015-04-30 | 2015-09-02 | 南京格耀生物科技有限公司 | Kit for detecting resistin as well as preparation method and detection method of kit |
-
2015
- 2015-09-28 CN CN201510626573.XA patent/CN105203773A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101400406A (en) * | 2006-01-13 | 2009-04-01 | 诺瓦提斯公司 | Compositions and methods of use for antibodies of Dickkopf-1 and/or -4 |
| CN102507951A (en) * | 2011-11-25 | 2012-06-20 | 广东药学院 | Enzyme-linked immuno sorbent assay (ELISA) kit for performing joint detection on tumor marker |
| CN104880564A (en) * | 2015-04-30 | 2015-09-02 | 南京格耀生物科技有限公司 | Kit for detecting resistin as well as preparation method and detection method of kit |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110531085A (en) * | 2019-08-30 | 2019-12-03 | 北京利德曼生化股份有限公司 | A kind of magnetic microparticle chemiluminescence detection kit and preparation method thereof measuring human nerve silk light chain protein content |
| CN111044724A (en) * | 2019-12-22 | 2020-04-21 | 上海复星长征医学科学有限公司 | Thymidine kinase 1 magnetic particle chemiluminescence assay kit and preparation method thereof |
| CN111044724B (en) * | 2019-12-22 | 2023-11-10 | 复星诊断科技(上海)有限公司 | Thymidine kinase 1 magnetic particle chemiluminescence assay kit and preparation method thereof |
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