CN104062440B - A kind of test strips of fast detecting vitamin D - Google Patents
A kind of test strips of fast detecting vitamin D Download PDFInfo
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- CN104062440B CN104062440B CN201410308883.2A CN201410308883A CN104062440B CN 104062440 B CN104062440 B CN 104062440B CN 201410308883 A CN201410308883 A CN 201410308883A CN 104062440 B CN104062440 B CN 104062440B
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- 229930003316 Vitamin D Natural products 0.000 title claims abstract description 117
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 title claims abstract description 117
- 235000019166 vitamin D Nutrition 0.000 title claims abstract description 117
- 239000011710 vitamin D Substances 0.000 title claims abstract description 117
- 229940046008 vitamin d Drugs 0.000 title claims abstract description 117
- 150000003710 vitamin D derivatives Chemical class 0.000 title claims abstract description 116
- 238000012360 testing method Methods 0.000 title claims abstract description 66
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 41
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 41
- 238000001514 detection method Methods 0.000 claims abstract description 36
- 238000003908 quality control method Methods 0.000 claims abstract description 22
- 239000000020 Nitrocellulose Substances 0.000 claims abstract description 3
- 229920001220 nitrocellulos Polymers 0.000 claims abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 36
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 20
- 238000003756 stirring Methods 0.000 claims description 20
- 238000006243 chemical reaction Methods 0.000 claims description 18
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 17
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- 239000010931 gold Substances 0.000 claims description 15
- 229910052737 gold Inorganic materials 0.000 claims description 15
- 239000006228 supernatant Substances 0.000 claims description 15
- 238000002360 preparation method Methods 0.000 claims description 14
- 239000000084 colloidal system Substances 0.000 claims description 13
- 239000003960 organic solvent Substances 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 10
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 8
- 239000002245 particle Substances 0.000 claims description 6
- 101000609762 Gallus gallus Ovalbumin Proteins 0.000 claims description 5
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 5
- 238000000502 dialysis Methods 0.000 claims description 5
- 238000005374 membrane filtration Methods 0.000 claims description 5
- BHTJEPVNHUUIPV-UHFFFAOYSA-N pentanedial;hydrate Chemical compound O.O=CCCCC=O BHTJEPVNHUUIPV-UHFFFAOYSA-N 0.000 claims description 5
- 229920000151 polyglycol Polymers 0.000 claims description 5
- 239000010695 polyglycol Substances 0.000 claims description 5
- 238000001556 precipitation Methods 0.000 claims description 5
- 229940088594 vitamin Drugs 0.000 claims description 5
- 229930003231 vitamin Natural products 0.000 claims description 5
- 235000013343 vitamin Nutrition 0.000 claims description 5
- 239000011782 vitamin Substances 0.000 claims description 5
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 5
- JWUBBDSIWDLEOM-UHFFFAOYSA-N 25-Hydroxycholecalciferol Natural products C1CCC2(C)C(C(CCCC(C)(C)O)C)CCC2C1=CC=C1CC(O)CCC1=C JWUBBDSIWDLEOM-UHFFFAOYSA-N 0.000 claims description 4
- JWUBBDSIWDLEOM-DCHLRESJSA-N 25-Hydroxyvitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@@H](CCCC(C)(C)O)C)=C/C=C1\C[C@@H](O)CCC1=C JWUBBDSIWDLEOM-DCHLRESJSA-N 0.000 claims description 4
- JWUBBDSIWDLEOM-NQZHSCJISA-N 25-hydroxy-3 epi cholecalciferol Chemical compound C1([C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@@H](CCCC(C)(C)O)C)=CC=C1C[C@H](O)CCC1=C JWUBBDSIWDLEOM-NQZHSCJISA-N 0.000 claims description 4
- 239000002202 Polyethylene glycol Substances 0.000 claims description 4
- 229920001223 polyethylene glycol Polymers 0.000 claims description 4
- 239000000376 reactant Substances 0.000 claims description 4
- 108060003951 Immunoglobulin Proteins 0.000 claims description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 3
- 102000018358 immunoglobulin Human genes 0.000 claims description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 2
- 108010058846 Ovalbumin Proteins 0.000 claims description 2
- 229940098773 bovine serum albumin Drugs 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 229940092253 ovalbumin Drugs 0.000 claims description 2
- 239000012460 protein solution Substances 0.000 claims description 2
- 238000010521 absorption reaction Methods 0.000 abstract description 6
- 238000002965 ELISA Methods 0.000 description 12
- 210000002966 serum Anatomy 0.000 description 9
- JWUBBDSIWDLEOM-XHQRYOPUSA-N (3e)-3-[(2e)-2-[1-(6-hydroxy-6-methylheptan-2-yl)-7a-methyl-2,3,3a,5,6,7-hexahydro-1h-inden-4-ylidene]ethylidene]-4-methylidenecyclohexan-1-ol Chemical compound C1CCC2(C)C(C(CCCC(C)(C)O)C)CCC2\C1=C\C=C1/CC(O)CCC1=C JWUBBDSIWDLEOM-XHQRYOPUSA-N 0.000 description 6
- 235000021318 Calcifediol Nutrition 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 238000010790 dilution Methods 0.000 description 5
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- 230000008569 process Effects 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 229930006000 Sucrose Natural products 0.000 description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
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- 238000002474 experimental method Methods 0.000 description 4
- 210000004681 ovum Anatomy 0.000 description 4
- 238000005728 strengthening Methods 0.000 description 4
- 239000005720 sucrose Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 201000004569 Blindness Diseases 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000009739 binding Methods 0.000 description 3
- 229910021538 borax Inorganic materials 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 230000002860 competitive effect Effects 0.000 description 3
- UQGFMSUEHSUPRD-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound [Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 UQGFMSUEHSUPRD-UHFFFAOYSA-N 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 239000003547 immunosorbent Substances 0.000 description 3
- 230000009871 nonspecific binding Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 239000004328 sodium tetraborate Substances 0.000 description 3
- 235000010339 sodium tetraborate Nutrition 0.000 description 3
- QYSXJUFSXHHAJI-YRZJJWOYSA-N vitamin D3 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-YRZJJWOYSA-N 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 230000027455 binding Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 239000004816 latex Substances 0.000 description 2
- 229920000126 latex Polymers 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 239000006249 magnetic particle Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- -1 polyethylene Polymers 0.000 description 2
- 239000012857 radioactive material Substances 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 229920002538 Polyethylene Glycol 20000 Polymers 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 206010047626 Vitamin D Deficiency Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 230000003913 calcium metabolism Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
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- 239000000835 fiber Substances 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
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- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 239000004926 polymethyl methacrylate Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
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- 238000012549 training Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of test strips of fast detecting vitamin D, comprise the gripper shoe not absorbing water, in described gripper shoe, be disposed with sample pad, pad, chromatographic film and absorption pad, on described pad, be coated with the vitamin D of label mark and the conjugate of carrier protein, described chromatographic film is nitrocellulose filter, in chromatographic film, be provided with Quality Control C line and detect T line, described detection T line is between pad and Quality Control C line, detect on T line and be coated with vitamin D specific antibody, on Quality Control C line, be coated with the specific antibody that can be combined with carrier protein. Test strips of the present invention is easy to use, simple to operate, detection speed is fast, easily spreading to different medical unit uses, meet the need of market, can fill up domestic and international market blank, the judgement of test strips result easily, symbolically, has broken away from vitamin D and has detected the particular/special requirement to equipment, instrument and personnel.
Description
Technical field
The invention belongs to technical field of biological, be specifically related to a kind of examination of fast detecting vitamin DPaper slip.
Background technology
Vitamin D is regulating calcium metabolism in human body, maintain muscle skeleton and immune health and steadyFixed, prevention part malignant tumour has indispensable effect. The United Nations's health organization statistical numberAccording to the show, about 90% population in the whole world suffers from the disease of vitamin d insufficiency. Due to dividing of vitamin DSub-feature (height lipophilicity) increases its separating-purifying difficulty, and testing conditions requires high, has limitedVitamin D detects in clinical applying, and has also limited us and has obtained more relevant vitamin DInformation. Up to the present, reported and by the human serum vitamin D detection method of clinical acceptanceThere are kit detection and instrument to detect two classes.
One, instrument detects. 1) direct Detection Method (DirectDetectionMethodology). ThisForemost in class methods is liquid phase chromatographic analysis method (HPLC) and the liquid phase look that improved afterwardsSpectrum-mass spectrometry analytic approach (LC/MS). 2) automatic analysis and detection instrument (AutomatedInstrumentationMethodology). It is that accuracy is high that instrument detects its advantage, and shortcoming is instrumentInvestment is large, and sample must extract and purifying by organic solvent, and analyst need to pass through professional training, andAnd in analytic process, organic solvent consumption is large, is unfavorable for environmental protection. Direct Analysis fado limitation at presentUse in Research-oriented Lab.
The NicholsInstituteDiagnostics company of the U.S., DiaSorin company and GermanyRocheDiagnostics company announces that respectively it develops Serum Vitamin D automatic detection instrument. ShouldUse this type of automatic analyzer, blood serum sample was processed and can directly be detected without any need for early stage. ThisThe principle that quasi-instrument detects is chemiluminescence analytical technology. Its advantage is that instrument completes sample automaticallySeparate, extract, detect data preparation, the steps such as result judgement. Approximately one of the sense cycle of sampleHalf an hour. Its shortcoming is that automatic analyzer price is especially expensive, has limited promoting the use of of its.
Two, kit detects, and is divided into again following several by action principle:
1) competitive protein binding assay (CPBA)
2) radio immunoassay (RIA)
3) enzyme linked immunosorbent assay (ELISA)
Current existing kit determination method all uses immune response plate substantially, by directlyOr vitamin D content in indirect enzyme-linked immunosorbent reaction detection sample. In these detection methods, absolutelyThe organic solvent that great majority need to carry out sample extracts, and in certain methods, reactant has tagging,Easily cause environment organic solvent and radioactive material contamination, and detect and must tested by professionalChamber completes by instrumentation. Average detected process is about 100 minutes.
Can be found out by above-mentioned analysis, current existing vitamin D detection method, no matter instrument detectsOr the detection of enzyme linked immunoassay plate, it all exists, and instrument and equipment investment is high, and testing staff requires high,The problem such as organic solvent or radioactive material contamination in testing process, makes vitamin D detect universal being subject toTo restriction, this also causes clinical application to lack test rating support, and medication is with great blindness,Even bring more harm to patient body. For this reason, a kind of easy and simple to handle, quick, price of exploitationThe Serum Vitamin D detection method that low, applicable basic unit promotes the use of is following development trend, is alsoAn urgent demand of clinical position person and extensive patients.
Side direction laminar flow immuno analytical method was risen in the nineties in last century, had had considerable at nearly 10 yearsDevelopment, has been widely used in fast detecting diagnostic field. And the detection of vitamin D is due to samplePurification process complexity, sample is unstable, and reason that marker is rare etc. is failed clinical alwaysExtensively carry out.
Summary of the invention
Technical problem to be solved by this invention is for above-mentioned the deficiencies in the prior art, provides a kind ofThe test strips of fast detecting vitamin D. This test strips is based on competitive immunization combination principle, adoptsLateral chromatography method detects vitamin D concentration, and its detection method is simple, result is easy to judgement, is detected asThis is cheap, is adapted at the units such as basic hospital, clinic and MEC and uses, and supports one's family for reductionElement D testing cost, universal body vitamin D detect, reduce clinical blindness medication has important meaningJustice.
For solving the problems of the technologies described above, the technical solution used in the present invention is: a kind of fast detecting is supported one's familyElement D test strips, comprise the gripper shoe not absorbing water, in described gripper shoe, be disposed with sample pad,Pad, chromatographic film and absorption pad, is characterized in that, is coated with label mark on described padVitamin D and the conjugate of carrier protein, described chromatographic film is nitrocellulose filter, in chromatographic filmBe provided with Quality Control C line and detect T line, described detection T line between pad and Quality Control C line,Detect on T line and be coated with vitamin D specific antibody, on Quality Control C line, be coated with can with carrierProtein bound specific antibody.
The test strips of above-mentioned a kind of fast detecting vitamin D, described label is colloid gold particle.
The test strips of above-mentioned a kind of fast detecting vitamin D, described carrier protein is bovine serum albuminIn vain, the specific antibody that can be combined with carrier protein is BSA immunoglobulin (Ig).
The test strips of above-mentioned a kind of fast detecting vitamin D, described carrier protein is the pure egg of ovum gallinaceumIn vain, the specific antibody that can be combined with carrier protein is the anti-chicken ovalbumin of rabbit.
The test strips of above-mentioned a kind of fast detecting vitamin D, described vitamin D is 25-hydroxyl dimensionRaw plain D3 or 25-OH Vintamin D2.
The test strips of above-mentioned a kind of fast detecting vitamin D, described detection T line and Quality Control C lineBetween distance be 1cm.
The test strips of above-mentioned a kind of fast detecting vitamin D, described vitamin D specific antibodyFor mouse-anti vitamin D monoclonal antibody, the package amount of vitamin D specific antibody is 2 μ L, bagBe 0.25mg/mL～5mg/mL by concentration.
The test strips of above-mentioned a kind of fast detecting vitamin D, described can be combined with carrier proteinThe package amount of specific antibody is 2 μ L, and coated concentration is 0.1mg/mL～1.0mg/mL.
The test strips of above-mentioned a kind of fast detecting vitamin D, the vitamin D of described label markComprise the following steps with the preparation method of the conjugate of carrier protein:
Step 1,1mg～10mg vitamin D is dissolved in 100 μ L organic solvents, then existsIn the carrier protein solution that is 0.1mg/mL～5mg/mL to 10mL concentration under stirring condition, dropwise addVitamin D after entering to dissolve, more dropwise to add concentration of volume percent be 50% glutaraldehyde water solutionConcentration of volume percent to glutaraldehyde in solution is 1%～3%, moves to after stirring 5min～20minStirring reaction 16h～18h at 4 DEG C, reacted reactant liquor is dialysed at 4 DEG C with PBS cushioning liquid8h, changes liquid 2～3 times in dialysis procedure, after dialysate is centrifugal, get supernatant, finally supernatant is used0.45 μ m membrane filtration, obtains the conjugate of vitamin D and carrier protein, and-20 DEG C save backup;
Step 2, to the idol that adds vitamin D described in step 1 and carrier protein in label solutionConnection thing to the final concentration of vitamin D in solution is 10 μ g/mL～1000 μ g/mL, stirs after 5minAdding BSA solution is 0.1%～1% to the quality concentration expressed in percentage by volume of BSA, and at 4 DEG C, lucifuge is stirredAfter mixing reaction 30min, add polyglycol solution to the quality concentration expressed in percentage by volume of polyethylene glycol to be0.1%～1%, centrifugal after continuation reaction 5min, supernatant discarded, will obtain label after precipitation redissolutionThe vitamin D of mark and the conjugate of carrier protein.
The test strips of above-mentioned a kind of fast detecting vitamin D, organic solvent described in step 1 is for justHexane, acetonitrile, dimethyl sulfoxide (DMSO) or Isosorbide-5-Nitrae-dioxane.
The present invention compared with prior art has the following advantages:
1, the test strips of fast detecting vitamin D of the present invention is based on competitive immunization combination principle,Adopt lateral chromatography method to detect vitamin D concentration, its detection method is simple, result is easy to judgement, inspectionSurvey with low costly, be adapted at the unit such as basic hospital, clinic and MEC and use, for reductionVitamin D testing cost, universal body vitamin D detect, reduce clinical blindness medication to be had heavilyWant meaning.
2, test strips of the present invention is easy to use, simple to operate, and detection speed is fast, easily spreads toDifferent medical unit uses, and meets the need of market, and can fill up domestic and international market blank.
3, easily, symbolically, has broken away from vitamin D inspection in test strips result judgement of the present inventionSurvey the particular/special requirement to equipment, instrument and personnel.
4, test strips of the present invention not only can adopt the colloid gold particle thing that serves as a mark, and also can adopt glimmeringPhotochemistry material, coloring agent, latex particle, enzyme or the magnetic-particle thing that serves as a mark, according to routine sideMethod is carried out the mark of the conjugate of vitamin D and carrier protein.
Below in conjunction with drawings and Examples, technical scheme of the present invention is described in further detail.
Brief description of the drawings
Fig. 1 is the structural representation of test strips of the present invention.
Description of reference numerals:
1-gripper shoe; 2-sample pad; 3-pad;
4-chromatographic film; 5-detection T line; 6-Quality Control C line;
7-absorption pad.
Detailed description of the invention
Vitamin D (25-hydroxyvitamin D3) and the carrier protein of embodiment 1 label markThe preparation of conjugate
Step 1, by 1mg vitamin D, (25OHD33-HS is purchased from TorontoResearchChemicals company) be dissolved in 100 μ L organic solvents (n-hexane, acetonitrile, dimethyl sulfoxide (DMSO) orIsosorbide-5-Nitrae-dioxane) in, the carrier egg that is then 0.1mg/mL to 10mL concentration under stirring conditionIn (bovine serum albumin(BSA) BSA, or chicken ovalbumin OVA) solution, dropwise add after dissolving in vainVitamin D, more dropwise to add concentration of volume percent be that 50% glutaraldehyde water solution is to solutionThe concentration of volume percent of glutaraldehyde is 1%, after stirring 5min, moves to stirring reaction 16h at 4 DEG C,The reacted reactant liquor 8h that dialyses at 4 DEG C with PBS cushioning liquid, changes liquid 2～3 in dialysis procedureInferior, after dialysate is centrifugal, get supernatant, finally, by 0.45 μ m membrane filtration for supernatant, tieed upThe conjugate of raw plain D and carrier protein ,-20 DEG C save backup;
Step 2, in 40nm colloidal gold solution (OD1.0 is purchased from the outstanding biotech firm in Shanghai)The conjugate that adds vitamin D described in step 1 and carrier protein is to the end of vitamin D in solutionConcentration is 10 μ g/mL, after stirring 5min, adds the final concentration (mass body of BSA solution to BSALong-pending percentage concentration) be 0.1% (being containing 0.1gBSA in 100mL solution), at 4 DEG C, lucifuge is stirredMixing reaction adds polyglycol solution after 30min (quality volume percentage is dense to the final concentration of polyethylene glycolDegree) be 0.1% (being containing 0.1g polyethylene glycol in 100mL solution), continue after reaction 5min fromThe heart, supernatant discarded, will obtain vitamin D and the carrier egg of label colloid gold label after precipitation redissolutionWhite conjugate (containing neo dohyfral D3 0ng/mL～40ng/mL).
Vitamin D (25-OH Vintamin D2) and the carrier protein of embodiment 2 label marksThe preparation of conjugate
Step 1, by 5mg vitamin D, (25OHD2 is purchased from TorontoResearchChemicalsCompany) be dissolved in 100 μ L organic solvent (n-hexane, acetonitrile, dimethyl sulfoxide (DMSO) or Isosorbide-5-Nitrae-dioxies sixRing) in, the carrier protein (cow's serum that is then 2mg/mL to 10mL concentration under stirring conditionAlbumin BSA, or chicken ovalbumin OVA) dropwise add the vitamin D after dissolving in solution,Dropwise to add concentration of volume percent be 50% glutaraldehyde water solution again to the body of glutaraldehyde in solutionLong-pending percent concentration is 2%, after stirring 10min, moves to stirring reaction 17h at 4 DEG C, reacted anti-Answer the liquid PBS cushioning liquid 8h that dialyses at 4 DEG C, in dialysis procedure, change liquid 2～3 times, dialysateAfter centrifugal, get supernatant, finally by 0.45 μ m membrane filtration for supernatant, obtain vitamin D and carryThe conjugate of body protein ,-20 DEG C save backup;
Step 2, in 40nm colloidal gold solution (OD1.0 is purchased from the outstanding biotech firm in Shanghai)The conjugate that adds vitamin D described in step 1 and carrier protein is to the end of vitamin D in solutionConcentration is 100 μ g/mL, after stirring 5min, adds the final concentration (mass body of BSA solution to BSALong-pending percentage concentration) be 0.5%, at 4 DEG C, after lucifuge stirring reaction 30min, add polyglycol solutionFinal concentration (quality concentration expressed in percentage by volume) to polyethylene glycol is 0.5%, continue reaction 5min after fromThe heart, supernatant discarded, will obtain vitamin D and the carrier egg of label colloid gold label after precipitation redissolutionWhite conjugate (containing neo dohyfral D3 0ng/mL～40ng/mL).
Vitamin D (25-hydroxyvitamin D3) and the carrier protein of embodiment 3 label marksThe preparation of conjugate
Step 1, by 10mg vitamin D, (25OHD3 is purchased from TorontoResearchChemicalsCompany) be dissolved in 100 μ L organic solvent (n-hexane, acetonitrile, dimethyl sulfoxide (DMSO) or Isosorbide-5-Nitrae-dioxies sixRing) in, the carrier protein (cow's serum that is then 5mg/mL to 10mL concentration under stirring conditionAlbumin BSA, or chicken ovalbumin OVA) dropwise add the vitamin D after dissolving in solution,Dropwise to add concentration of volume percent be 50% glutaraldehyde water solution again to the body of glutaraldehyde in solutionLong-pending percent concentration is 3%, after stirring 20min, moves to stirring reaction 18h at 4 DEG C, reacted anti-Answer the liquid PBS cushioning liquid 8h that dialyses at 4 DEG C, in dialysis procedure, change liquid 2～3 times, dialysateAfter centrifugal, get supernatant, finally by 0.45 μ m membrane filtration for supernatant, obtain vitamin D and carryThe conjugate of body protein ,-20 DEG C save backup;
Step 2, in 40nm colloidal gold solution (be purchased from Shanghai outstanding a biotech firm), add stepThe conjugate of vitamin D described in one and carrier protein to the final concentration of vitamin D in solution is1000 μ g/mL, add final concentration (the quality volume percentage of BSA solution to BSA after stirring 5minConcentration) be 1.0%, at 4 DEG C, after lucifuge stirring reaction 30min, add polyglycol solution to poly-secondThe final concentration (quality concentration expressed in percentage by volume) of glycol is 1.0%, centrifugal after continuation reaction 5min, abandonsRemove supernatant, after precipitation is redissolved, obtain the vitamin D of colloid gold label and the conjugate of carrier protein(containing neo dohyfral D3 0ng/mL～40ng/mL).
The preparation of embodiment 4 vitamin D monoclonal antibodies
Vitamin D and the carrier in step 1 with embodiment 1, embodiment 2 or embodiment 3, preparedThe conjugate of albumen is as immunogene, by 50 μ g/ only～200 μ g/ dosage immunity Balb/c mouse only,Make it produce antiserum; After about 6-8 week, getting mouse boosting cell and SP2/0 myeloma cell merges and obtainsHybridoma; The immune response plate that gained hybridoma application 25-hydroxyvitamin D3 is coated,Through enzyme linked immunoassay, screening obtains the monoclonal antibody of the anti-25-hydroxyvitamin D3 of stably excretingCell line, the cell line that obtains of screening is through clone's final election, final election adopts 25-OH Vintamin D2Coated immune response plate, obtains monoclonal antibody thin of the anti-25-hydroxy-vitamin D of stably excretingBorn of the same parents' strain; Amplification cultivation cell in DMEM nutrient solution, cell culture supernatant is through centrifugal, denseContracting, through G albumen (GEHealthcareLifeSciences) chromatographic column purifying, obtains after purifyingMouse-anti vitamin D monoclonal antibody ,-20 DEG C save backup.
The preparation of embodiment 5 colloid gold label vitamin D pads
Glass film (Ahlstrom8964) is cut into wide rectangular of 5mm, the glass film shearing is putOn smooth clean glass plate, collaurum prepared by embodiment 1, embodiment 2 or embodiment 31.5 times of diluted for the vitamin D of mark and the conjugate of carrier protein, then by every test paperThe amount of bar 20 μ L～50 μ L is added on glass film equably, and glass film is evenly soaked into, 37 DEG C of bakingsDry (60 minutes), protection against the tide is kept in Dark Place for subsequent use, and described dilution is for containing sucrose, Tween-20With the sodium tetraborate solution of PEG20000, in dilution, the concentration of sodium tetraborate is 2mM～20mM,In every 100mL dilution, contain 0.5g～10g sucrose, 0.1mL～1mLTween-20 and 0.5g～5gPEG20000。
The preparation of embodiment 6 chromatographic film
Chromatographic film is selected CAM (milliporeHF13502), is cut into the wide bar shaped of 30mm;Detect coated mouse-anti vitamin D monoclonal antibody on T line 5, coated concentration 0.25mg/mL～5mg/mL (preferably 0.25mg/mL, 0.5mg/mL, 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mLOr 5mg/mL), by 2 μ L/ test strips antibody amount preparations, when the carrier protein adopting is that ox blood is pureWhen albumen, on Quality Control C line 6, (be purchased from the auspicious neat biotechnology in Shanghai has coated BSA immunoglobulin (Ig)Limit company), coated concentration be 0.1mg/mL～1.0mg/mL (preferably 0.1mg/mL, 0.3mg/mL,0.5mg/mL, 0.8mg/mL or 1.0mg/mL), by the antibody amount preparation of 2 μ L/ test strips, when adoptingWith carrier protein while being chicken ovalbumin, on Quality Control C line 6, the coated anti-OVA of rabbit, coated denseDegree for 0.1mg/mL～1.0mg/mL (preferably 0.1mg/mL, 0.3mg/mL, 0.5mg/mL,0.8mg/mL or 1.0mg/mL), by the antibody amount preparation of 2 μ L/ test strips, by the acetic acid fibre cutting outTie up plain film and be fixed on clean poly (methyl methacrylate) plate, film instrument (the outstanding biological scientific & technical corporation in Shanghai) is drawn in applicationPreparation detects T line 5 and Quality Control C line 6, detects T line 5 and Quality Control C line 6 spacing 1cm; WillAt room temperature vacuum drying of the chromatographic film preparing 1 hour, sealing lucifuge is moistureproof preserves.
Embodiment 7 assembles test strips
Test strips gripper shoe 1 is purchased from the outstanding biological scientific & technical corporation in Shanghai, and the long 7.8cm of gripper shoe 1 is wide5mm; Chromatographic film prepared by embodiment 6 is cut into wide 5mm, and the chromatographic film 4 of long 3cm makes to detectT line is positioned at the central authorities of chromatographic film 4; Sample pad 2 size 5mm × 2.3cm, absorption pad 7 sizes5mm × 2.0cm; Press shown in Fig. 1, by sample pad 2, pad 3, chromatographic film 4 and absorption pad 7Be pasted on successively in gripper shoe 1, between sample pad 2 and pad 3, pad 3 and chromatographic film 4Between and between chromatographic film 4 and absorption pad 7, all have the overlapping of 1mm～2mm, examined fastSurvey the test strips of vitamin D, in the environment of 2 DEG C～8 DEG C, lucifuge is moistureproof stores, the term of validity 12 months.
The application of embodiment 8 test strips
Normal concentration sample preparation: according to step 1 in embodiment 1, embodiment 2 or embodiment 3Method is prepared the conjugate of 25-hydroxy-vitamin D and BSA; HPLC detects 25-hydroxyl in conjugateThe concentration of base vitamin D; By centrifugal concentrating (MilliporeAmiconcentrifugalfilter)Get standard samples, and use the PBS solution dilution standard sample that BSA concentration is 1mg/mL,Obtaining vitamin D concentration is respectively: 0ng/mL, 10ng/mL, 20ng/mL, 25ng/mL, 30ng/mL,The normal concentration sample of 35ng/mL, 40ng/mL, 60ng/mL and 80ng/mL, wherein 0ng/mLSample is the PBS solution of BSA concentration 1mg/mL.
Normal concentration sample detection: the normal concentration sample of getting 20 μ L adds sample pad near padRegion; 150 μ L detection agents are dropwise added to the position of sample pad near test strips end, 10～15Observed result after minute; Described detection reagent is to contain four of sucrose, Tween-20 and PEG20000Dobell's solution, the concentration that detects sodium tetraborate in reagent is 2mM～20mM, every 100mL inspectionIn test agent, contain 0.5g～10g sucrose, 0.1mL～1mLTween-20 and 0.5g～5gPEG20000。
The detection principle of test strips of the present invention is: detected sample and detection reagent mixed liquor are by capillaryGravitation guiding is spread to reaction film along test strips, when mixed liquor process pad, what pad was coated withThe vitamin D of colloid gold label and the conjugate of carrier protein is released and with mixed liquor along reaction filmDiffusion; The vitamin D of the colloid gold label that the vitamin D in detected sample and pad dischargeBe combined with the vitamin D monoclonal antibody that the conjugate competition of carrier protein is coated with detecting T line; IfIn sample to be checked, the content of vitamin D, higher than the detection threshold setting, detects coated the resisting of T lineThe binding site of body is sealed completely by sample vitamin D, stop colloid gold label vitamin D withThe combination with it of the conjugate of carrier protein, detects T line and does not develop the color, and the vitamin D of colloid gold labelThe specificity that can be combined with carrier protein that can be coated with Quality Control C line with the conjugate of carrier proteinAntibody combines, the colour developing of Quality Control C line; If vitamin D content is lower than set in detected sample liquidDetection threshold, the binding site that detects the coated antibody of T line can not be sealed completely, and then colloidThe gold vitamin D of mark and the conjugate of carrier protein vitamin D antibody in chromatographic film are combined,Detect the colour developing of T line, the C of Quality Control simultaneously line antibody also can with the vitamin D of colloid gold label and carrierThe conjugate combination of albumen, the colour developing of Quality Control C line; If Quality Control district does not show, test strips inefficacy.
Result judgement:
Negative: detect T line and do not develop the color, Quality Control C line shows band, is judged as feminine gender, with "-"Represent, represent to exceed set detection threshold by institute's sample product vitamin D content (vitamin D content fillsFoot);
Positive: to detect T line and Quality Control C line and all demonstrate band, be judged as the positive, with "+" tableShow, (vitamin D content is not lower than set detection threshold to represent in institute's sample product vitamin D contentFoot);
Invalid: when Quality Control C line does not develop the color, to be judged as invalid test strips.
Normal concentration sample detection the results are shown in Table 1.
Table 1 normal concentration vitamin D sample detection result
| 25-hydroxy-vitamin D concentration (ng/mL) | Detection line T | Nature controlling line C |
| 0 | + | + |
| 10 | + | + |
| 20 | + | + |
| 25 | + | + |
| 30 | - | + |
| 35 | - | + |
| 40 | - | + |
| 60 | - | + |
| 80 | - | + |
Table 1 result shows: 1) sample vitamin D: 0,10ng/mL, 20ng/mL and 25ng/mLT line and C line are all positive; 2) T line is in the time that sample vitamin D concentration is more than or equal to 30ng/mLDisappear. This experimental result is through duplicate test between different batches.
Human serum vitamin D concentration detects:
(1) people's whole blood sample 5 examples, through traditional enzyme-linked immunoassay method (25-hydroxy-vitamin D enzyme connectionImmune reagent kit, IDS company of Britain) detect the concentration of 25-hydroxy-vitamin D in people's whole blood sample;
(2) get each sample 20 μ L, detect people's whole blood sample by above-mentioned normal concentration sample detection methodIn 25-hydroxy-vitamin D, the results are shown in Table 2.
The traditional enzyme linked immunosorbent detection of table 2 and ELISA test strip result of the present invention contrast
As can be seen from Table 2, the normal concentration scope of traditional enzyme linked immunosorbent detection (75nmol/L～Sample 150nmol/L) detects T line and does not develop the color in ELISA test strip, and lower than normal concentration modelThe colour developing of its ELISA test strip of the sample enclosing T line.
Embodiment 9 ELISA test strip restrictive conditions
1, specificity experiment
1) non-specific binding experiment
The PBS solution of BSA of preparation variable concentrations, concentration range is by 1mg/mL to 20mg/mL,Get the BSA solution of the each concentration of 20 μ L, by method detectable antigens antibody described in embodiment 6 and general eggThe non-specific binding reaction of white molecule. The BSA solution of testing result demonstration variable concentrations all can notMake vitamin D test strip of the present invention detect T heading line off. Prove vitamin D of the present inventionColloidal gold strip reacts without non-specific binding with common protein molecular.
2) specific binding experiment
The vitamin D blood serum sample of choosing concentration known, adds respectively vitamin D protein conjugate(strengthening) or 1mg/mLBSA solution (dilution), then apply vitamin D of the present invention and detect examinationPaper slip is observed and is detected the variation of T line. Testing result is as shown in table 3.
ELISA test strip result after table 3 concentration known vitamin D serum samples diluted or strengthening
| Sample 25-hydroxy-vitamin D concentration | Vitamin D actual concentrations | T line | C line |
| 17ng/mL | 17ng/mL | + | + |
| 17ng/mL dilutes (50%v/v) | 8.5ng/mL | + | + |
| 17ng/mL strengthening | 25ng/mL | + | + |
| 17ng/mL strengthening | 35ng/mL | - | + |
| 38ng/mL | 38ng/mL | - | + |
| 38ng/mL dilutes (50%v/v) | 19ng/mL | + | + |
| 38ng/mL dilutes (66%v/v) | 25ng/mL | + | + |
| 38ng/mL dilutes (80%v/v) | 30ng/mL | - | + |
As can be seen from Table 3, in ELISA test strip result, detect vitamin in the variation of T line and sampleThe variation of D concentration is relevant, changes and there is no obvious relation with other protein concentrations.
2, vitamin D test strips stability experiment
By after dry test strips sealing, be placed in 37 DEG C of incubators 7 days, then to take out, balance is to room temperature;After the dry sealing of test strips of same product batch number, be kept at 4 DEG C, before test, take out, balance extremelyRoom temperature; Then compare two test strips immunoassay experimental results. Result show 37 DEG C store one week rightThis ELISA test strip result is without impact.
Test strips of the present invention not only can adopt the colloid gold particle thing that serves as a mark, and also can adopt fluorescenceLearn material, coloring agent, latex particle, enzyme or the magnetic-particle thing that serves as a mark, enter according to conventional methodThe mark of the conjugate of row vitamin D and carrier protein.
The above, be only preferred embodiment of the present invention, not the present invention done to any restriction, allAny simple modification, change and the equivalence knot of above embodiment being done according to invention technical spiritStructure changes, and all still belongs in the protection domain of technical solution of the present invention.
Claims (7)
1. a test strips for fast detecting vitamin D, comprises the gripper shoe (1) not absorbing water, instituteState in gripper shoe (1), be disposed with sample pad (2), pad (3), chromatographic film (4) andAbsorption pad (7), is coated with vitamin D and the carrier egg of label mark on described pad (3)White conjugate, described chromatographic film (4) is nitrocellulose filter, chromatographic film is provided with matter on (4)Control C line (6) and detection T line (5), described detection T line (5) be positioned at pad (3) andBetween Quality Control C line (6), detect on T line (5) and be coated with vitamin D specific antibody, matterOn control C line (6), be coated with the specific antibody that can be combined with carrier protein; Described vitamin DSpecific antibody is mouse-anti vitamin D monoclonal antibody, the package amount of vitamin D specific antibodyBe 2 μ L, coated concentration is 0.25mg/mL～5mg/mL; The described spy that can be combined with carrier proteinThe package amount of heterogenetic antibody is 2 μ L, and coated concentration is 0.1mg/mL～1.0mg/mL, and its feature existsIn, the preparation method of the vitamin D of described label mark and the conjugate of carrier protein comprises followingStep:
Step 1,1mg～10mg vitamin D is dissolved in 100 μ L organic solvents, then existsIn the carrier protein solution that is 0.1mg/mL～5mg/mL to 10mL concentration under stirring condition, dropwise addVitamin D after entering to dissolve, more dropwise to add concentration of volume percent be 50% glutaraldehyde water solutionConcentration of volume percent to glutaraldehyde in solution is 1%～3%, moves to after stirring 5min～20minStirring reaction 16h～18h at 4 DEG C, reacted reactant liquor is dialysed at 4 DEG C with PBS cushioning liquid8h, changes liquid 2～3 times in dialysis procedure, after dialysate is centrifugal, get supernatant, finally supernatant is used0.45 μ m membrane filtration, obtains the conjugate of vitamin D and carrier protein, and-20 DEG C save backup;
Step 2, to the idol that adds vitamin D described in step 1 and carrier protein in label solutionConnection thing to the final concentration of vitamin D in solution is 10 μ g/mL～1000 μ g/mL, stirs after 5minAdding BSA solution is 0.1%～1% to the quality concentration expressed in percentage by volume of BSA, and at 4 DEG C, lucifuge is stirredAfter mixing reaction 30min, add polyglycol solution to the quality concentration expressed in percentage by volume of polyethylene glycol to be0.1%～1%, centrifugal after continuation reaction 5min, supernatant discarded, will obtain label after precipitation redissolutionThe vitamin D of mark and the conjugate of carrier protein.
2. the test strips of a kind of fast detecting vitamin D according to claim 1, its featureBe, described label is colloid gold particle.
3. the test strips of a kind of fast detecting vitamin D according to claim 1, its featureBe, described carrier protein is bovine serum albumin(BSA), the specific antibody that can be combined with carrier proteinFor BSA immunoglobulin (Ig).
4. the test strips of a kind of fast detecting vitamin D according to claim 1, its featureBe, described carrier protein is chicken ovalbumin, the specific antibody that can be combined with carrier proteinFor the anti-chicken ovalbumin of rabbit.
5. the test strips of a kind of fast detecting vitamin D according to claim 1, its featureBe, described vitamin D is 25-hydroxyvitamin D3 or 25-OH Vintamin D2.
6. the test strips of a kind of fast detecting vitamin D according to claim 1, its featureBe, the distance between described detection T line (5) and Quality Control C line (6) is 1cm.
7. the test strips of a kind of fast detecting vitamin D according to claim 1, its featureBe, organic solvent described in step 1 is n-hexane, acetonitrile, dimethyl sulfoxide (DMSO) or Isosorbide-5-Nitrae-dioxy sixRing.
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| CN104749385A (en) * | 2015-02-06 | 2015-07-01 | 普迈德（北京）科技有限公司 | Immunochromatography reagent strip for quantitative detection of 25-hydroxyvitamin D and preparation method of immunochromatography reagent strip |
| AU2017209523A1 (en) * | 2016-01-22 | 2018-08-09 | Affimedix, Inc. | Device for detection of vitamin D metabolites |
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| CN106405072B (en) * | 2016-09-06 | 2018-01-02 | 北京华科泰生物技术有限公司 | 25 hydroxy-vitamine D fluorescence immune chromatographies activation fluorescent latex microballoon |
| CN106405071B (en) * | 2016-09-06 | 2018-01-05 | 北京华科泰生物技术有限公司 | 25 hydroxy-vitamine Ds3Fluorescence immune chromatography activation fluorescent latex microballoon |
| CN106381289A (en) * | 2016-11-14 | 2017-02-08 | 刘玲 | Monoclonal antibody of resisting 25-hydroxyvitamin D3 as well as preparation cell lines and method of monoclonal antibody |
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| CN111458525A (en) * | 2020-01-16 | 2020-07-28 | 卢氏实验室公司 | Test strip and kit for detecting human body physiological substance concentration and preparation method |
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| CN112129956B (en) * | 2020-09-02 | 2022-05-27 | 武汉生之源生物科技股份有限公司 | Colloidal gold labeling solution for colloidal gold immunochromatography, and preparation method and application thereof |
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