CN109298178A - Cardiac myosin binding protein C(cMyBP-C based on immunomagnetic beads) time-resolved fluoroimmunoassay kit - Google Patents
Cardiac myosin binding protein C(cMyBP-C based on immunomagnetic beads) time-resolved fluoroimmunoassay kit Download PDFInfo
- Publication number
- CN109298178A CN109298178A CN201811310863.3A CN201811310863A CN109298178A CN 109298178 A CN109298178 A CN 109298178A CN 201811310863 A CN201811310863 A CN 201811310863A CN 109298178 A CN109298178 A CN 109298178A
- Authority
- CN
- China
- Prior art keywords
- cmybp
- added
- immunomagnetic beads
- buffer
- kit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000011324 bead Substances 0.000 title claims abstract description 52
- 229960000856 protein c Drugs 0.000 title claims abstract description 11
- 102000013602 Cardiac Myosins Human genes 0.000 title claims abstract description 10
- 108010051609 Cardiac Myosins Proteins 0.000 title claims abstract description 10
- 108091008324 binding proteins Proteins 0.000 title claims abstract description 9
- 102000023732 binding proteins Human genes 0.000 title description 7
- 101000982032 Homo sapiens Myosin-binding protein C, cardiac-type Proteins 0.000 title 1
- 102100026771 Myosin-binding protein C, cardiac-type Human genes 0.000 title 1
- 239000000872 buffer Substances 0.000 claims abstract description 29
- 238000006243 chemical reaction Methods 0.000 claims abstract description 27
- 230000008878 coupling Effects 0.000 claims abstract description 24
- 238000010168 coupling process Methods 0.000 claims abstract description 24
- 238000005859 coupling reaction Methods 0.000 claims abstract description 24
- 238000004458 analytical method Methods 0.000 claims abstract description 21
- 239000000427 antigen Substances 0.000 claims abstract description 21
- 102000036639 antigens Human genes 0.000 claims abstract description 21
- 108091007433 antigens Proteins 0.000 claims abstract description 21
- 238000010166 immunofluorescence Methods 0.000 claims abstract description 17
- 238000004140 cleaning Methods 0.000 claims abstract description 9
- 239000004005 microsphere Substances 0.000 claims abstract description 8
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract 2
- 238000002156 mixing Methods 0.000 claims description 45
- 239000000243 solution Substances 0.000 claims description 38
- 239000007788 liquid Substances 0.000 claims description 30
- 239000006228 supernatant Substances 0.000 claims description 30
- 238000002604 ultrasonography Methods 0.000 claims description 28
- 238000005119 centrifugation Methods 0.000 claims description 27
- 230000004913 activation Effects 0.000 claims description 20
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 18
- 239000003153 chemical reaction reagent Substances 0.000 claims description 17
- 238000002360 preparation method Methods 0.000 claims description 17
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 238000005406 washing Methods 0.000 claims description 13
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 12
- 239000007983 Tris buffer Substances 0.000 claims description 11
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 11
- 239000005018 casein Substances 0.000 claims description 10
- 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 10
- 235000021240 caseins Nutrition 0.000 claims description 10
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 9
- 239000007853 buffer solution Substances 0.000 claims description 7
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 6
- 239000012482 calibration solution Substances 0.000 claims description 6
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 6
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 6
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 6
- 239000002981 blocking agent Substances 0.000 claims description 4
- 239000003755 preservative agent Substances 0.000 claims description 4
- 230000002335 preservative effect Effects 0.000 claims description 4
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 4
- 239000012498 ultrapure water Substances 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 2
- 229940098773 bovine serum albumin Drugs 0.000 claims description 2
- 239000004615 ingredient Substances 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 108010090804 Streptavidin Proteins 0.000 claims 2
- 238000005034 decoration Methods 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 23
- 230000035945 sensitivity Effects 0.000 abstract description 15
- 150000001875 compounds Chemical class 0.000 abstract description 4
- 230000035484 reaction time Effects 0.000 abstract description 3
- 230000009514 concussion Effects 0.000 abstract description 2
- 230000005284 excitation Effects 0.000 abstract description 2
- 238000000034 method Methods 0.000 description 20
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 18
- 239000007995 HEPES buffer Substances 0.000 description 18
- 239000000523 sample Substances 0.000 description 17
- 210000002966 serum Anatomy 0.000 description 13
- 238000012986 modification Methods 0.000 description 12
- 230000004048 modification Effects 0.000 description 12
- 238000011084 recovery Methods 0.000 description 10
- 230000008676 import Effects 0.000 description 9
- 229920000136 polysorbate Polymers 0.000 description 9
- 238000004321 preservation Methods 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 8
- 238000005259 measurement Methods 0.000 description 8
- 239000007787 solid Substances 0.000 description 8
- 239000003643 water by type Substances 0.000 description 8
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 238000003756 stirring Methods 0.000 description 7
- 238000008157 ELISA kit Methods 0.000 description 6
- 206010000891 acute myocardial infarction Diseases 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 230000008901 benefit Effects 0.000 description 5
- 238000003018 immunoassay Methods 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 238000003149 assay kit Methods 0.000 description 4
- 230000007547 defect Effects 0.000 description 4
- 238000003745 diagnosis Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000004090 dissolution Methods 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- -1 rare earth ion Chemical class 0.000 description 4
- 101100537532 Rattus norvegicus Tnni3 gene Proteins 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 238000007885 magnetic separation Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 229910052761 rare earth metal Inorganic materials 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 108090001008 Avidin Proteins 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 101800004937 Protein C Proteins 0.000 description 1
- 102000017975 Protein C Human genes 0.000 description 1
- 101800001700 Saposin-D Proteins 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 102100026893 Troponin T, cardiac muscle Human genes 0.000 description 1
- 101710165323 Troponin T, cardiac muscle Proteins 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 239000011258 core-shell material Substances 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000030609 dephosphorylation Effects 0.000 description 1
- 238000006209 dephosphorylation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000003760 hair shine Effects 0.000 description 1
- 231100001261 hazardous Toxicity 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 1
- 229960001008 heparin sodium Drugs 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000003317 immunochromatography Methods 0.000 description 1
- 102000028557 immunoglobulin binding proteins Human genes 0.000 description 1
- 108091009323 immunoglobulin binding proteins Proteins 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 229910052747 lanthanoid Inorganic materials 0.000 description 1
- 150000002602 lanthanoids Chemical class 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 238000004375 physisorption Methods 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 238000012913 prioritisation Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000003904 radioactive pollution Methods 0.000 description 1
- 238000013102 re-test Methods 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 238000000611 regression analysis Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000013097 stability assessment Methods 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
- G01N33/5434—Magnetic particles using magnetic particle immunoreagent carriers which constitute new materials per se
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- 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
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
-
- 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
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides a kind of cardiac myosin binding protein C (cMyBP-C) time-resolved fluoroimmunoassay kit based on immunomagnetic beads, comprising: calibration object, immunomagnetic beads, immunofluorescence microballoon, analysis buffer, cleaning solution；By by magnetic bead and antibody coupling and by time-resolved fluorescence microballoon and antibody coupling；CMyBP-C antigen forms immunomagnetic beads-cMyBP-C antigen-immunofluorescence microsphere compound after concussion is incubated in reaction tube in the two and sample；Its fluorescence intensity launched under the excitation of ultraviolet light is measured with time identifier device；Reference standard curve is to determine the amount of cMyBP-C antigen in sample.Kit of the present invention substantially reduces the reaction time, improves the efficiency and sensitivity of detection.
Description
Technical field
The invention belongs to bioanalytical chemistry, field of nano biotechnology, in particular to a kind of heart based on immunomagnetic beads
Dirty cardiac myosin binding protein-C (cMyBP-C) time-resolved fluoroimmunoassay kit.
Background technique
CMyBP-C is the structural proteins of myocardium thick filament, is specifically present in cardiac muscle cell.Work as acute myocardial infarction
(AMI) when occurring, cMyBP-C occurs dephosphorylation and is released into blood, so that the hurried raising of blood cMyBP-C concentration, this
So that serum cMyBP-C has good specificity to AMI Complicated by Heart Failure.It falls ill to the AMI of admission time < 4h
Patient, serum cMyBP-C are significantly increased, and serum cTnI then increases unobvious, and ROC curve is analysis shows that serum cMyBP-
C, the cTnI antidiastole AUC to the AMI patient of admission time < 4h and control group that falls ill is respectively 0.974,0.591, the former
Sensitivity, specificity be respectively 78.9%, 100.0%.Serum cMyBP-C is the sensitive indicator for diagnosing early stage AMI, and special
Property is also preferable, this item value of serum cMyBP-C obtains clinical application.But current clinic there is no the relevant detection of cMyBP-C
Method, clinic generally use enzyme-linked immunosorbent assay (ELISA) to the detection of new albumen index.
The general sensitivity of ELISA kit, detection range are limited, and exist and professional is needed to operate, step
It is cumbersome, the defects of taking a long time.Especially to the detection of New Set, lack the comprehensive of data, essence cannot be provided for clinician
Quasi- quantitative data, is lacking the directive significance of clinical treatment.
Chemiluminescence immune assay (CLIA) is that will have highly sensitive chemical luminescent detecting technology and high specific
Immune response combines, the detection point for various antigens, haptens, antibody, hormone, enzyme, fatty acid, vitamin and drug etc.
Analysis technology.It is an immunoassay for exempting to grow up after analysis, fluoroimmunoassay after radioimmunology analysis, enzyme.
CLIA has the advantage that first compared with other traditional labelling techniques, radiation that detection process is "dead", will not
It is hazardous to the human body；Second, possess higher sensitivity and the wider range of linearity；Third, generally using self-reacting device into
Row operation, is not necessarily to professional operator, excludes manual operation interference, and stability is good；4th, party's science of law application scenarios are wide, can
Antigen, haptens and the antibody of different molecular size are detected, and can be used for the detection of nucleic acid probe.But some drawbacks of CLIA
Limit it in the application development in immunodiagnosis field, such as: the timeliness that shines is short, and single sample can only detect once, some projects
Background is higher and easy by surrounding material interference etc..Most domestic is in the reagent using import producer, such as Roche, refined at present
Training, Beckman etc..
Timed resolved fluoroimmunoassay (TRFIA) is three to run neck and neck at present with chemiluminescence, electrochemical luminescence
One of super quick immunoassay method of kind.Its principle is object of being marked using the rare earth ion of longer fluorescence half-life period, due to this
Marker Stokes is displaced big (> 150nm) and fluorescence lifetime 5~6 orders of magnitude higher than background substance fluorescence lifetime, therefore, surveys
As long as timing delays time of measuring, the signal for measuring marker again after the fluorescence of background substance is sufficiently decayed can effectively disappear
Except the interference of various non-specific fluorescences, very high sensitivity is obtained.
Summary of the invention
Technical problem: in order to solve the defects of prior art, the present invention provides a kind of heart flesh based on immunomagnetic beads
Immunoglobulin binding protein C (cMyBP-C) time-resolved fluoroimmunoassay kit.
Technical solution: the present invention obtains immunomagnetic beads by the way that magnetic bead and antibody coupling are formed compound；Simultaneously by the time
Resolved fluorometric microballoon and antibody coupling obtain immunofluorescence microballoon；The two is passed through in reaction tube with cMyBP-C antigen in sample
Concussion forms immunomagnetic beads-cMyBP-C antigen-immunofluorescence microsphere compound after being incubated for；Excitation of the compound in ultraviolet light
Under launch very strong fluorescence, fluorescence intensity is directly proportional to cMyBP-C antigen concentration therein, measures it with time identifier device
Fluorescence intensity, reference standard curve are the amount that can determine cMyBP-C antigen in sample.
Cardiac myosin binding protein C (cMyBP-C) time-resolved fluorescence provided by the invention based on immunomagnetic beads
Immunoassay kits, comprising: calibration object, immunomagnetic beads, immunofluorescence microballoon, analysis buffer and cleaning solution；And reagent
Buffer used in box includes: buffer system, closed reagent, blocking agent and preservative.
As a kind of prioritization scheme: immunomagnetic beads preparation step are as follows: even with anti-cMyBP-C monoclonal antibody after magnetic bead activation
Connection is closed, and is washed, and is saved.
As advanced optimizing scheme: immunofluorescence microballoon preparation step are as follows: after the activation of time-resolved fluorescence microballoon with it is anti-
The coupling of cMyBP-C monoclonal antibody, is closed, and is washed, and is saved.
As advanced optimizing scheme: immunomagnetic beads partial size is 100nm-5 μm, and surface modification is carboxyl, hydroxyl or strepto-
Any one in Avidin；Immunofluorescence microspherulite diameter is 100nm-500nm, and surface modification is carboxyl, hydroxyl or strepto- parent
With any one in element.
As advanced optimizing scheme: the activation of immunomagnetic beads and coupling concrete operation step are as follows:
Activation: being added the mixing of NHS vortex, adds EDC, vortex mixing；Shaking table 100-500r/min, 30-37 DEG C of reaction
10-30min；Wherein NHS:EDC=25:10；
Coupling: removing supernatant after centrifugation, adds ultrapure water, and ultrasound is redissolved, and repeated centrifugation is redissolved twice, and cMyBP- is added
C monoclonal antibody, vortex mixing, shaking table 100-500r/min, 30-37 DEG C of reaction 1-3h；The label ratio of magnetic bead and antibody is
(50-150):1。
As advanced optimizing scheme: the activation of immunofluorescence microballoon and coupling concrete operation step are as follows:
Activation: being added the mixing of NHS vortex, adds EDC, vortex mixing；Shaking table 100-500r/min, 30-37 DEG C of reaction
10-30min；Wherein NHS:EDC=25:10；
Coupling: removing supernatant after centrifugation, ultrapure water is added, and ultrasound is redissolved, and repeated centrifugation is redissolved twice, and cMyBP-C is added
Monoclonal antibody, vortex mixing, shaking table 100-500r/min, 30-37 DEG C of reaction 1-3h；Time-resolved fluorescence microballoon and antibody
Label ratio is (50-150): 1.
As advanced optimizing scheme: calibration object is that cMyBP-C antigen is configured to 0-500ng/mL series with buffer
The calibration solution of concentration.
As advanced optimizing scheme: analysis buffer formula includes: 10-50mmol/L Tris-HCl, 0.1-5% junket
Albumen, 1--5%BSA, 0.01-1%Proclin300,0.01%-1 Tween 20,0.9%NaCl, buffer tune pH is extremely
7.2-7.4。
As the scheme that advanced optimizes: washing formula of liquid includes: Tris-HCl, 0.01-1% of 10-50mmol/L
The NaCl of Tween 20 and 0.9%, buffer tune pH to 7.2-7.4.
As advanced optimizing scheme: each ingredient in buffer is specific as follows:
Buffer system: any one in PBS, Tris or CBS buffer, and concentration is 10-50mmol/L, pH value is
7.2-7.5；
Closed reagent: any one in bovine serum albumin(BSA) or casein, and mass concentration is 1-5%；
Blocking agent: IgG albumen, and mass concentration is 0.1-1%；
Preservative: any one in Sodium azide or Proclin 300, and mass concentration is 0.01-0.1%.
The measuring method of kit of the present invention are as follows: be added in reaction tube with analysis buffer with volume ratio be 1:70 dilution
100 μ l of immunomagnetic beads, 10 μ l of cMyBP-C calibration object is then added, adding with analysis buffer with volume ratio is 1:70 dilute
The 100 μ l of immunofluorescence microballoon released；Room temperature shakes incubation reaction 15min, with magnetic separation technique by immunomagnetic beads and supernatant
It separates and washed once with cleaning solution, last every hole is added after the shaking of 300 μ l analysis buffers in time-resolved fluorescence detector
Upper measurement.
The utility model has the advantages that kit provided by the invention uses magnetic separation technique and binding time resolved fluorometric Microspheres Technique,
Except the high sensitivity, storage time length, "dead" pollution, the standard curve range that possess time-resolved fluorescence Microspheres Technique are wide etc.
Outside plurality of advantages, the reaction time also is greatly shortened by immunomagnetic beads, improves the sensitivity of detection.Magnetic bead and passing through of antibody
Group coupling is learned, pairing antibody dosage is greatly reduced and improves the precision of detection.In addition technology automation easy to accomplish,
The defect that conventional method luminous intensity is weaker and the time is short is overcome, sample is realized and detects immediately.
Detailed description of the invention
Fig. 1 is the measurable concentration range schematic diagram of kit made from 1-4 of the embodiment of the present invention；
Fig. 2 is the linearly interval range schematic diagram of kits concentration made from 1-4 of the embodiment of the present invention；
Fig. 3 is that the fluorescence intensity in heat damage experiment of kit made from the embodiment of the present invention 1 changes over time amplitude and shows
It is intended to；
Fig. 4 is that kit made from the embodiment of the present invention 1 and the clinical blood sample measured value correlation of ELISA kit are illustrated
Figure.
Specific embodiment
In the following with reference to the drawings and specific embodiments, the present invention is furture elucidated, it should be understood that these embodiments are merely to illustrate
It the present invention rather than limits the scope of the invention, after the present invention has been read, those skilled in the art are to of the invention each
The modification of kind equivalent form falls within the application range as defined in the appended claims.
Embodiment 1
A kind of cardiac myosin binding protein C (cMyBP-C) time-resolved fluorescence based on immunomagnetic beads of the present invention is exempted from
The specific preparation process of epidemic disease assay kit:
Step 1: the preparation of immunomagnetic beads
Pretreatment: it takes the 25 μ L of magnetic bead that 1% solid content partial size is the modification of 1 μm of surface carboxyl groups in 2mL import centrifuge tube, adds
Enter the 500 μ L of MES solution of 50mmol/L pH6.0, vortex mixing, 15000rpm, 10min, 4 DEG C of centrifugations, removal supernatant, addition
The 500 μ L of MES solution of 50mmol/L pH6.0, ultrasound are redissolved；
Activation: being added NHS (MES of 50mmol/L pH6.0 is configured) 2 μ L of 25mg/mL, and vortex mixing adds
EDC (MES of 50mmol/L pH6.0 is configured) 2 μ L of 10mg/mL, vortex mixing, shaking table 250r/min, 37 DEG C of reaction 15min；
Coupling: 15000rpm, 10min, 4 DEG C centrifugation, remove supernatant, be added 500 μ L ultrapure waters, ultrasound redissolve, repeat from
The heart redissolves twice, and third time redissolves the 500 μ L of HEPES for using 50mmol/L pH8.0；The anti-cMyBP-C antibody of 25 μ g is added, revolves
Whirlpool mixing, shaking table 250r/min, reacts 2h by 37 DEG C；
Closing: 50 μ L confining liquid (50mmol/L pH8.0Tris+10%BSA+0.1%T-20+0.1%Proclin are added
300), 250 turns of shaking table, react 2h by 37 DEG C；
Washing: supernatant is removed in 15000rpm, 10min, 4 DEG C of centrifugations, and 500 μ L save liquid (50mmol/L pH8.0 HEPES+
5%BSA+0.5%PEG+0.1%T-20+0.1%Proclin 300) it is resuspended, ultrasound；
It saves: 4 DEG C of preservations.
Step 2: the preparation of immunofluorescence microballoon
Pretreatment: take the 25 μ L of time resolution microballoon that 1% solid content partial size is the modification of 300nm surface carboxyl groups in 2mL import
In centrifuge tube, the 500 μ L of MES solution of 50mmol/L pH6.0 is added, vortex mixing, 15000rpm, 10min, 4 DEG C are centrifuged, and go
Except supernatant.The 500 μ L of MES solution of 50mmol/L pH6.0 is added, ultrasound is redissolved；
Activation: being added NHS (MES of 50mmol/L pH6.0 is configured) 2 μ L of 25mg/mL, and vortex mixing adds
EDC (MES of 50mmol/L pH6.0 is configured) 2 μ L of 10mg/mL, vortex mixing, shaking table 250r/min, 37 DEG C of reaction 15min；
Coupling: 15000rpm, 10min, 4 DEG C centrifugation, remove supernatant, be added 500 μ L ultrapure waters, ultrasound redissolve, repeat from
The heart redissolves twice, and third time redissolves the 500 μ L of HEPES for using 50mmol/L pH8.0；The anti-cMyBP-C antibody of 25 μ g is added, revolves
Whirlpool mixing, shaking table 250r/min, reacts 2h by 37 DEG C；
Closing: 50 μ L confining liquid (50mmol/L pH8.0 Tris+10%BSA+0.1%T-20+0.1% are added
Proclin 300), 250 turns of shaking table, 37 DEG C, react 2h；
Washing: supernatant is removed in 15000rpm, 10min, 4 DEG C of centrifugations, and 500 μ L save liquid (50mmol/L pH8.0 HEPES+
5%BSA+0.5%PEG+0.1%T-20+0.1%Proclin 300) it is resuspended, ultrasound；
It saves: 4 DEG C of preservations.
Step 3: the configuration of calibration object
With the 50mmol/L pH7.4 PBS buffer solution containing 2%BSA and 0.01% Sodium azide, by cMyBP-C antigen (Nanjing
The production of Jin Sirui Biotechnology Co., Ltd) it is configured to 0,1,5,10,100, the calibration solution of 500ng/mL series of concentrations, it is standby
With.
Step 4: the configuration of analysis buffer
In pH7.4,20mmol/L Tris-HCl, be added 5% casein, 1%BSA, 0.1%Proclin300,
0.1%Tween 20,0.9%NaCl are stirred and are sufficiently dissolved.
Step 5: the configuration of cleaning solution
In pH7.4,10mmol/L Tris-HCl solution, 0.05%Tween 20,0.9%NaCl is added, stirs and fills
Divide dissolution.
Step 6: saving the configuration of liquid
5%BSA, 0.5%PEG, 0.1%T-20 and 0.1% are added in 50mmol/L pH8.0HEPES solution
Proclin 300 is stirred and is sufficiently dissolved.
Step 7: the configuration of confining liquid
In 50mmol/L pH8.0 Tris solution be added mass concentration be 10% BSA, 0.1%Tween 20,
0.1%Proclin 300 is stirred and is sufficiently dissolved.
Accuracy: according to the method and calculating in " external diagnosis reagent analyzes Performance Evaluation (accuracy-recovery experiment) "
Formula uses the calibration object of 1ng/mL as matrix α, adds the cMyBP-C antigen of 5,10,100ng/mL, detection respectively according to 1: 9
Concentration is denoted as γ, and addition concentration is denoted as β, and each test is repeated 3 times, averages, according to formula: (90 × β+10 × α)/(100 ×
The rate of recovery γ) is calculated, data are shown in Table 1.
Embodiment 2
A kind of cardiac myosin binding protein C (cMyBP-C) time-resolved fluorescence based on immunomagnetic beads of the present invention is exempted from
The specific preparation process of epidemic disease assay kit:
Step 1: the preparation of immunomagnetic beads
Pretreatment: it takes the 25 μ L of magnetic bead that 1% solid content partial size is the modification of 2 μm of surface carboxyl groups in 2mL import centrifuge tube, adds
Enter the 500 μ L of MES solution of 20mmol/L pH6.0, vortex mixing, 15000rpm, 10min, 4 DEG C of centrifugations, removal supernatant, addition
The 500 μ L of MES solution of 20mmol/L pH6.0, ultrasound are redissolved；
Activation: being added NHS (MES of 20mmol/L pH6.0 is configured) 2 μ L of 15mg/mL, and vortex mixing adds
EDC (MES of 20mmol/L pH6.0 is configured) 2 μ L of 10mg/mL, vortex mixing, shaking table 250r/min, 37 DEG C of reaction 15min；
Coupling: 15000rpm, 10min, 4 DEG C centrifugation, remove supernatant, be added 500 μ L ultrapure waters, ultrasound redissolve, repeat from
The heart redissolves twice, and third time redissolves the 500 μ L of HEPES for using 20mmol/L pH8.0；The anti-cMyBP-C antibody of 50 μ g is added, revolves
Whirlpool mixing, shaking table 250r/min, reacts 2h by 37 DEG C；
Closing: 50 μ L confining liquid (20mmol/L pH8.0 Tris+10%BSA+0.1%T-20+0.1% are added
Proclin 300), 250 turns of shaking table, 37 DEG C, react 2h；
Washing: supernatant is removed in 15000rpm, 10min, 4 DEG C of centrifugations, and 500 μ L save liquid (20mmol/L pH8.0 HEPES+
5%BSA+0.5%PEG+0.1%T-20+0.1%Proclin 300) it is resuspended, ultrasound；
It saves: 4 DEG C of preservations.
Step 2: the preparation of immunofluorescence microballoon
Pretreatment: take the 25 μ L of time resolution microballoon that 1% solid content partial size is the modification of 100nm surface carboxyl groups in 2mL import
In centrifuge tube, the 500 μ L of MES solution of 20mmol/L pH6.0 is added, vortex mixing, 15000rpm, 10min, 4 DEG C are centrifuged, and go
Except supernatant, the 500 μ L of MES solution of 20mmol/L pH6.0 is added, ultrasound is redissolved；
Activation: being added NHS (MES of 20mmol/L pH6.0 is configured) 2 μ L of 15mg/mL, and vortex mixing adds
EDC (MES of 20mmol/L pH6.0 is configured) 2 μ L of 10mg/mL, vortex mixing, shaking table 250r/min, 37 DEG C of reaction 15min；
Coupling: 15000rpm, 10min, 4 DEG C centrifugation, remove supernatant, be added 500 μ L ultrapure waters, ultrasound redissolve, repeat from
The heart redissolves twice, and third time redissolves the 500 μ L of HEPES for using 20mmol/L pH8.0；The anti-cMyBP-C antibody of 50 μ g is added, revolves
Whirlpool mixing, shaking table 250r/min, reacts 2h by 37 DEG C；
Closing: 50 μ L confining liquid (20mmol/L pH8.0Tris+1%BSA+0.1%T-20+0.1%Proclin are added
300), shaking table 250r/min, reacts 2h by 37 DEG C；
Washing: supernatant is removed in 15000rpm, 10min, 4 DEG C of centrifugations, and 500 μ L save liquid (20mmol/L pH8.0HEPES+
1%BSA+0.5%PEG+0.1%T-20+0.1%Proclin 300) it is resuspended, ultrasound；
It saves: 4 DEG C of preservations.
Step 3: the configuration of calibration object
With the 20mmol/L pH7.4Tris buffer containing 2%BSA and 0.01% Sodium azide, by cMyBP-C antigen (Nanjing
The production of Jin Sirui Biotechnology Co., Ltd) it is configured to 0,1,5,10,100, the calibration solution of 500ng/mL series of concentrations, it is standby
With.
Step 4: the configuration of analysis buffer
In pH7.4,20mmol/L Tris-HCl, be added 1% casein, 0.5%BSA, 0.1%Proclin300,
0.1%Tween 20 and 0.9%NaCl is stirred and is sufficiently dissolved.
Step 5: the configuration of cleaning solution
In pH7.4,20mmol/L Tris-HCl solution, 0.05%Tween 20,0.9%NaCl is added, stirs and fills
Divide dissolution.
Step 6: saving the configuration of liquid
1%BSA, 0.5%PEG, 0.1%T-20 and 0.1% are added in 20mmol/L pH8.0 HEPES solution
Proclin 300 is stirred and is sufficiently dissolved.
Step 7: the configuration of confining liquid
1%BSA, 0.1%Tween 20,0.1%Proclin 300 are added in 20mmol/L pH8.0 Tris solution
It stirs and sufficiently dissolves.
Accuracy: according to the method and calculating in " external diagnosis reagent analyzes Performance Evaluation (accuracy-recovery experiment) "
Formula uses the calibration object of 1ng/mL as matrix α, adds the cMyBP-C antigen of 5,10,100ng/mL, detection respectively according to 1: 9
Concentration is denoted as γ, and addition concentration is denoted as β, and each test is repeated 3 times, averages, according to formula: (90 × β+10 × α)/(100 ×
The rate of recovery γ) is calculated, data are shown in Table 1.
Embodiment 3
A kind of cardiac myosin binding protein C (cMyBP-C) time-resolved fluorescence based on immunomagnetic beads of the present invention is exempted from
The specific preparation process of epidemic disease assay kit:
Step 1: the preparation of immunomagnetic beads
Pretreatment: taking the 25 μ L of magnetic bead that 1% solid content partial size is the modification of 500nm surface hydroxyl in 2mL import centrifuge tube,
The 500 μ L of MES solution of 50mmol/L pH6.0 is added, vortex mixing, 15000rpm, 10min, 4 DEG C are centrifuged, and remove supernatant, add
Enter the 500 μ L of MES solution of 50mmol/L pH6.0, ultrasound is redissolved；
Activation: being added NHS (MES of 50mmol/L pH6.0 is configured) 2 μ L of 25mg/mL, and vortex mixing adds
EDC (MES of 50mmol/L pH6.0 is configured) 2 μ L of 10mg/mL, vortex mixing, shaking table 100r/min, 30 DEG C of reaction 30min；
Coupling: 15000rpm, 10min, 4 DEG C centrifugation, remove supernatant, be added 500 μ L ultrapure waters, ultrasound redissolve, repeat from
The heart redissolves twice, and third time redissolves the 500 μ L of HEPES for using 50mmol/L pH8.0；The anti-cMyBP-C antibody of 25 μ g is added, revolves
Whirlpool mixing, shaking table 100r/min, reacts 3h by 30 DEG C；
Closing: 50 μ L confining liquid (50mmol/L pH8.0 Tris+10%BSA+0.1%T-20+0.1% are added
Proclin 300), 250 turns of shaking table, 37 DEG C, react 2h；
Washing: supernatant is removed in 15000rpm, 10min, 4 DEG C of centrifugations, and 500 μ L save liquid (50mmol/L pH8.0 HEPES+
5%BSA+0.5%PEG+0.1%T-20+0.1%Proclin 300) it is resuspended, ultrasound；
It saves: 4 DEG C of preservations.
Step 2: the preparation of immunofluorescence microballoon
Pretreatment: take the 25 μ L of time resolution microballoon that 1% solid content partial size is the modification of 500nm surface hydroxyl in 2mL import
In centrifuge tube, the 500 μ L of MES solution of 50mmol/L pH6.0 is added, vortex mixing, 15000rpm, 10min, 4 DEG C are centrifuged, and go
Except supernatant.The 500 μ L of MES solution of 50mmol/L pH6.0 is added, ultrasound is redissolved；
Activation: being added NHS (MES of 50mmol/L pH6.0 is configured) 2 μ L of 25mg/mL, and vortex mixing adds
EDC (MES of 50mmol/L pH6.0 is configured) 2 μ L of 10mg/mL, vortex mixing, shaking table 100r/min, 30 DEG C of reaction 30min；
Coupling: 15000rpm, 10min, 4 DEG C centrifugation, remove supernatant, be added 500 μ L ultrapure waters, ultrasound redissolve, repeat from
The heart redissolves twice, and third time redissolves the 500 μ L of HEPES for using 50mmol/L pH8.0；The anti-cMyBP-C antibody of 25 μ g is added, revolves
Whirlpool mixing, shaking table 100r/min, reacts 3h by 30 DEG C；
Closing: 50 μ L confining liquid (50mmol/L pH8.0 Tris+10%BSA+0.1%T-20+0.1% are added
Proclin 300), 250 turns of shaking table, 37 DEG C, react 2h；
Washing: supernatant is removed in 15000rpm, 10min, 4 DEG C of centrifugations, and 500 μ L save liquid (10mmol/L pH7.0 HEPES+
5% casein+0.5%PEG+0.1%T-20+0.01% Sodium azide) it is resuspended, ultrasound；
It saves: 4 DEG C of preservations.
Step 3: the configuration of calibration object
With the 50mmol/L pH7.4 PBS buffer solution containing 2% casein and 0.01% Sodium azide, by cMyBP-C antigen
(Nanjing Genscript Biotechnology Co., Ltd.'s production) be configured to 0,1,5,10,100, the calibration of 500ng/mL series of concentrations it is molten
Liquid, it is spare.
Step 4: the configuration of analysis buffer
In pH7.2,10mmol/L Tris-HCl, be added 0.1% casein, 5%BSA, 0.01%Proclin300,
0.01%Tween 20,0.9%NaCl are stirred and are sufficiently dissolved.
Step 5: the configuration of cleaning solution
In pH7.2,50mmol/L Tris-HCl solution, 0.01%Tween 20,0.9%NaCl is added, stirs and fills
Divide dissolution.
Step 6: saving the configuration of liquid
5%BSA, 0.5%PEG, 0.1%T-20 and 0.1% are added in 50mmol/L pH7.0 HEPES solution
Proclin 300 is stirred and is sufficiently dissolved.
Step 7: the configuration of confining liquid
In 10mmol/L pH7.0 PBS solution be added mass concentration be 15% casein, 0.1%Tween 20,
0.01% Sodium azide is stirred and is sufficiently dissolved.
Accuracy: according to the method and calculating in " external diagnosis reagent analyzes Performance Evaluation (accuracy-recovery experiment) "
Formula uses the calibration object of 1ng/mL as matrix α, adds the cMyBP-C antigen of 5,10,100ng/mL, detection respectively according to 1: 9
Concentration is denoted as γ, and addition concentration is denoted as β, and each test is repeated 3 times, averages, according to formula: (90 × β+10 × α)/(100 ×
The rate of recovery γ) is calculated, data are shown in Table 1.
Embodiment 4
A kind of cardiac myosin binding protein C (cMyBP-C) time-resolved fluorescence based on immunomagnetic beads of the present invention is exempted from
The specific preparation process of epidemic disease assay kit:
Step 1: the preparation of immunomagnetic beads
Pretreatment: taking 1% solid content partial size is that the 25 μ L of magnetic bead of 5 μm of surface Streptavidins modification is centrifuged in 2mL import
The 500 μ L of MES solution of 20mmol/L pH6.0 is added in Guan Zhong, and vortex mixing, 15000rpm, 10min, 4 DEG C are centrifuged, in removal
Clearly, the 500 μ L of MES solution of 20mmol/L pH6.0 is added, ultrasound is redissolved；
Activation: being added NHS (MES of 20mmol/L pH6.0 is configured) 2 μ L of 15mg/mL, and vortex mixing adds
EDC (MES of 20mmol/L pH6.0 is configured) 2 μ L of 10mg/mL, vortex mixing, shaking table 500r/min, 35 DEG C of reaction 10min；
Coupling: 15000rpm, 10min, 4 DEG C centrifugation, remove supernatant, be added 500 μ L ultrapure waters, ultrasound redissolve, repeat from
The heart redissolves twice, and third time redissolves the 500 μ L of HEPES for using 20mmol/L pH8.0；
The anti-cMyBP-C antibody of 50 μ g, vortex mixing is added, shaking table 500r/min, reacts 1h by 35 DEG C；
Closing: 50 μ L confining liquid (20mmol/L pH8.0 Tris+10%BSA+0.1%T-20+0.1% are added
Proclin 300), 250 turns of shaking table, 37 DEG C, react 2h；
Washing: supernatant is removed in 15000rpm, 10min, 4 DEG C of centrifugations, and 500 μ L save liquid (20mmol/L pH8.0 HEPES+
5%BSA+0.5%PEG+0.1%T-20+0.1%Proclin 300) it is resuspended, ultrasound；
It saves: 4 DEG C of preservations.
Step 2: the preparation of immunofluorescence microballoon
Pretreatment: take the 25 μ L of time resolution microballoon that 1% solid content partial size is the modification of 5 μm of surface Streptavidins in 2mL
In import centrifuge tube, be added 20mmol/L pH6.0 500 μ L of MES solution, vortex mixing, 15000rpm, 10min, 4 DEG C from
The heart removes supernatant, and the 500 μ L of MES solution of 20mmol/L pH6.0 is added, and ultrasound is redissolved；
Activation: being added NHS (MES of 20mmol/L pH6.0 is configured) 2 μ L of 15mg/mL, and vortex mixing adds
EDC (MES of 20mmol/L pH6.0 is configured) 2 μ L of 10mg/mL, vortex mixing, shaking table 500r/min, 37 DEG C of reaction 10min；
Coupling: 15000rpm, 10min, 4 DEG C centrifugation, remove supernatant, be added 500 μ L ultrapure waters, ultrasound redissolve, repeat from
The heart redissolves twice, and third time redissolves the 500 μ L of HEPES for using 20mmol/L pH8.0；The anti-cMyBP-C antibody of 50 μ g is added, revolves
Whirlpool mixing, shaking table 500r/min, reacts 1h by 37 DEG C；
Closing: 50 μ L confining liquid (20mmol/L pH8.0Tris+1%BSA+0.1%T-20+0.1%Proclin are added
300), shaking table 250r/min, reacts 2h by 37 DEG C；
Washing: supernatant is removed in 15000rpm, 10min, 4 DEG C of centrifugations, and 500 μ L save liquid (20mmol/L pH8.0 HEPES+
1%BSA+0.5%PEG+0.1%T-20+0.1%Proclin 300) it is resuspended, ultrasound；
It saves: 4 DEG C of preservations.
Step 3: the configuration of calibration object
It is with the 20mmol/L pH7.4 Tris buffer containing 2%BSA and 0.01% Sodium azide, cMyBP-C antigen is (southern
The production of Jing Jinsirui Biotechnology Co., Ltd) it is configured to 0,1,5,10,100, the calibration solution of 500ng/mL series of concentrations, it is standby
With.
Step 4: the configuration of analysis buffer
In pH7.4,20mmol/L Tris-HCl, 1% casein, 0.5%BSA, 1%Proclin300,1% is added
Tween 20 and 0.9%NaCl is stirred and is sufficiently dissolved.
Step 5: the configuration of cleaning solution
In pH7.4,20mmol/L Tris-HCl solution, 1%Tween 20,0.9%NaCl is added, stirs and abundant
Dissolution.
Step 6: saving the configuration of liquid
1%BSA, 0.5%PEG, 1%T-20 and 1%Proclin are added in 20mmol/L pH8.0 HEPES solution
300 stir and sufficiently dissolve.
Step 7: the configuration of confining liquid
1%BSA, 1%Tween 20,1%Proclin 300 are added in 20mmol/L pH9.0 Tris solution to stir
And it sufficiently dissolves.
The kit being prepared in embodiment 1-4 is examined and determine:
Wherein accuracy: according in " external diagnosis reagent analyze Performance Evaluation (accuracy-recovery experiment) " method and
Calculation formula uses the calibration object of 1ng/mL as matrix α, adds the cMyBP-C antigen of 5,10,100ng/mL respectively according to 1: 9,
Detectable concentration is denoted as γ, and addition concentration is denoted as β, and each test is repeated 3 times, averages, according to formula: (90 × β+10 × α)/
(100 × γ) calculates the rate of recovery, and data are shown in Table 1.
Table 1: the rate of recovery data that different cMyBP-C antigen addition concentration obtain
By above data as it can be seen that kit provided by the invention can effectively be measured cMyBP-C antigen, and it is real
It applies the data in example 1 and shows that the accuracy of the kit can achieve higher level.
The kit assay performance being prepared in embodiment 1-4 is fully assessed:
The assessment of measurable range and linearly interval
With the 50mmol/L pH7.4 PBS buffer solution containing 2%BSA and 0.01% Sodium azide, by cMyBP-C antigen (Nanjing
The production of Jin Sirui Biotechnology Co., Ltd) it is configured to 0,1,5,10,50,100,200,300,400,500ng/mL series of concentrations
Calibration solution.Each concentration retest 3 times when 15min, uses fluorescence analyser reading numerical values；As shown in Figure 1, implementing
The measurable range of kit obtained in example 1 is at (0-400ng/mL)；The measurable range of kit obtained in embodiment 2
At (0-400ng/mL)；The measurable range of kit obtained in embodiment 3 is at (0-300ng/mL)；It is obtained in embodiment 4
Kit measurable range at (0-300ng/mL)；It can be obtained by the above measurement data, kit of the present invention can measure model
It is trapped among 0-400ng/mL；
As shown in Fig. 2, the linearly interval of kit obtained in embodiment 1 is at (0-200ng/mL)；It is obtained in embodiment 2
Kit linearly interval at (0-200ng/mL)；The linearly interval of kit obtained in embodiment 3 is in (0-200ng/
mL)；The linearly interval of kit obtained in embodiment 4 is at (0-200ng/mL)；It can be obtained by the above measurement data, the present invention
Kit linearly interval is in 0-200ng/mL.
The assessment of repeatability
Calibration object each replication 15 of the kit that embodiment 1-4 is obtained respectively under 5,10,100ng/mL concentration
It is secondary, its mean value (M), standard deviation (s) and the coefficient of variation (CV) are calculated, data are as shown in table 2；Calculation formula are as follows: CV=s/M ×
100%；
In formula: CV: the coefficient of variation；The standard deviation of s:15 measurement result；The average value of M:15 measurement result.
Table 2: the mean value (M) that is obtained after the measurement repeatedly of various concentration calibration object, standard deviation (s) and the coefficient of variation (CV)
The assessment of accelerated stability
It chooses embodiment 1 and carries out accelerated stability assessment: the same a collection of reagent that embodiment 1 is obtained in 37 DEG C of environment
Box does the heat damage experiment of lasting 20d (day)；It is detected respectively in 0d, 1d, 3d, 5d, 10d, 15d, 20d, each test weight
It is 3 times multiple, and average；Calculate the fluorescence intensity change amplitude of measurement concentration value, calculation formula △ (0d-nd)/0d.It can by Fig. 3
See, the stabilization of kit is excellent, it is ensured that long-time stability.
The assessment of specificity
The 50ng/mL solution of each index of cTnI, cTnT, cTnC, MYO, CK-MB is taken to be loaded test 1-4 of the embodiment of the present invention
Obtained kit should not generate signal；After tested, the examination that object like above does not obtain 1-4 of the embodiment of the present invention
Agent box itself generates positive reaction, therefore the kit specificity invented is preferably.
The assessment of range of normal value value
With the embodiment of the present invention 1 to 201 Physical Examination persons (male 103, the age 5~75 years old；Female 98, the age 6~77
Year) the horizontal detection of serum cMyBP-C shows that minimum is 0ng/mL, peak 116ng/mL, mean concentration 5ng/
ML, as shown in table 3 below, most suitable CUTOFF value are that (sensitivity 86.36%, specificity is 100%) by > 0.5ng/mL；
With the embodiment of the present invention 2 to 201 Physical Examination persons (male 103, the age 5~75 years old；Female 98, the age 6~77
Year) the horizontal detection of serum cMyBP-C shows that minimum is 0ng/mL, peak is (112) ng/mL, and mean concentration is (5)
Ng/mL, as shown in table 3 below, most suitable CUTOFF value are that (sensitivity (85.23) %, specificity is 100%) by > (0.5) ng/mL；
With the embodiment of the present invention 3 to 201 Physical Examination persons (male 103, the age 5~75 years old；Female 98, the age 6~77
Year) the horizontal detection of serum cMyBP-C shows that minimum is 0ng/mL, peak is (104) ng/mL, and mean concentration is (5)
Ng/mL, as shown in table 3 below, most suitable CUTOFF value are that (sensitivity (80.41) %, specificity is 100%) by > (0.5) ng/mL；
With the embodiment of the present invention 4 to 201 Physical Examination persons (male 103, the age 5~75 years old；Female 98, the age 6~77
Year) the horizontal detection of serum cMyBP-C shows that minimum is 0ng/mL, peak is (106) ng/mL, and mean concentration is (5)
Ng/mL, as shown in table 3 below, most suitable CUTOFF value be > (0.5) ng/mL (sensitivity 81.78%, specificity 100%), it is proposed that
When being detected using this reagent, the normal reference value of serum cMyBP-C level should be set to 0-0.5ng/mL.
Table 3: fixed number evidence (please supplement embodiment 2-4 kit CUTOFF to 1 kit CUTOFF value of the embodiment of the present invention really
Value)
| Critical value | Sensitivity | Specificity | Youden index | |
| Embodiment 1 | > 0.5 | 86.36% | 100.00% | 0.8636 |
| Embodiment 2 | > 0.5 | 85.23% | 100.00% | 0.8523 |
| Embodiment 3 | > 0.5 | 80.41% | 100.00% | 0.8041 |
| Embodiment 4 | > 0.5 | 81.78% | 100.00% | 0.8178 |
The specifically used method of kit of the present invention is as follows:
Step 1: sample prepares
Using heparin sodium heparin tube, venous blood 2ml is adopted, 3000 turns of centrifugation 5min take supernatant to be loaded；Blood serum sample is in 2-8
It DEG C can save 3 days, if necessary to long-term preservation, please be saved at -80 DEG C, avoid multigelation；Sample transport needs to guarantee cold
Cold chain transportation under hiding state.
Step 2: reagent prepares
Reagent is before use, room temperature stores 30min, recovery room temperature；Before reagent pipetting volume, mixing should be shaken.
Step 3: sample-adding detection
Being added in reaction tube with analysis buffer with volume ratio is the diluted 100 μ l of immunomagnetic beads of 1:70, is then added
10 μ l of cMyBP-C calibration object, adding with analysis buffer with volume ratio is 1:70 diluted time-resolved fluorescence microballoon label
100 μ l of antibody；Room temperature shakes incubation reaction 15min, separates immunomagnetic beads with supernatant with magnetic separation technique and uses washing
Liquid washed once, and last every hole measures on time-resolved fluorescence detector after the shaking of 300 μ l analysis buffers is added.
Kit of the present invention is compared with ELISA kit clinic blood sample measured value
It chooses the kit and self-control ELISA kit that embodiment 1 obtains while 100 parts of serum samples is detected.
The cMyBP-C concentration results for measuring blood sample in the process of the present invention are abscissa, to make the cMyBP-C concentration of ELISA kit by oneself
As a result regression analysis, dependent equation are done for ordinate are as follows: Y=1.0178X-0.6763, correlation coefficient r 2=0.9993, such as Fig. 4
Shown, the kit that the method for the present invention obtains and the clinical blood sample measured value of self-control ELISA kit have significant correlation.
Time-resolved fluorescence microballoon can wrap up thousands of a fluorescence in each microballoon as a kind of special functional microsphere
Molecule substantially increases the labeling effciency of fluorescence, effectively increases sensitivity for analysis；Fluorescent microsphere surface modification has properly simultaneously
The carboxyl of density or other functional groups improve the stability of marker for the covalent coupling with albumen or antibody.More
Importantly, due to the rare earth ion of microballoon embedding has chelated, without dissociating enhancing step；Time-resolved fluorescence microballoon is used
In the super quick quantitative measurement technology platform of microwell plate/tubular type, fluoremetry can be carried out after need to only washing several times, operating procedure is than solution
It is simple from enhancing lanthanide series fluoroimmunoassay (DELFIA) very much, it is easier to realize automatic operation.
Immunomagnetic beads are the new immunological techniques that developed recently gets up, it by the peculiar advantage of solidified reagents with exempt from
The high degree of specificity of epidemiology reaction is incorporated into one, synthesizes the high score containing superparamagnetism substance with the synthetic method of core-shell structure copolymer
The composite material of sublist face covering, stability is good, can be carried out the substance of later period label, utilizes the function group of these material surfaces
The covalent or non-covalent associations that such as amino, carboxyl, sulfydryl carry out antibody, can be used for combining corresponding antigen or antibody, this
Sample can do displacement under the attraction of externally-applied magnetic field, to reach separation, detect, purified genes, protein, cell, micro- life
The purpose of object etc., immune detection, cell separation, biological macromolecule purifying and in terms of obtained increasingly
It is widely applied.Magnetic bead has the characteristics that following compared with traditional microwell plate: surface area is bigger, can be in conjunction with more albumen point
Son can be connect by covalent bond with probe molecule, and the physisorption than the microwell plate that polystyrene is material is stronger,
It is a kind of small-sized, flowing solid phase carrier, makes reaction that can reach dynamic equilibrium faster, to accelerate reaction speed.Surface
In conjunction with density it is high, concentrate fluorescence signal more, can be combined with different probe molecules, make to detect different in same sample
Determinand is possibly realized.The flexibility of the appearance and coating process of magnetic bead is bigger, can be selected according to different requirement of experiment
It selects.
The These characteristics of magnetic bead in conjunction with time-resolved fluorescence microballoon after can reduce reaction needed for sample size, accelerate anti-
Between seasonable, easily automate.Immunomagnetic beads have been widely used in fields such as chemiluminescence immune assay, nucleic acid extractions at present,
Time-resolved fluorescence microballoon has more research in immunochromatography direction, but is based on immunomagnetic beads binding time resolved fluorometric microballoon
Luminous platform carry out immunoassay detection cardiac marker there is no literature reported on.
The present invention is studied according to above-mentioned principle, obtains stable cMyBP-C calibration object and its dilution, guarantees inspection
The reliability of survey, the perfect antibody marking process of immunomagnetic beads and time-resolved fluorescence microballoon can get high activity, high fluorescence
Intensity, the marker of easily separated cleaning and reaction system, so that detection method is easy, high-efficient, at low cost, reliable in quality.This
The disclosure of the invention various reagents formula obtained based on above-mentioned exploratory experiment, comprising: washing formula of liquid, analysis buffer formula,
Further disclose the preparation process of time resolution microballoon labelled antibody and immunomagnetic beads.Kit of the invention uses Magneto separate
Technology and binding time resolved fluorometric Microspheres Technique, except the high sensitivity, the storage time that possess time-resolved fluorescence microballoon are long, nothing
Outside many advantages, such as radioactive pollution, wide standard curve range, also by the enrichment of immunomagnetic beads and magnetic bead in liquid
In sufficiently diffusion so that combined surface area expands, greatly shorten the reaction time, improve detection sensitivity.Magnetic bead and passing through of antibody
Group coupling is learned, pairing antibody dosage is greatly reduced and improves the precision of detection.In addition technology of the invention is easy to accomplish
Automation, overcomes the defect that conventional method luminous intensity is weaker and the time is short, realizes sample and detect immediately.
Claims (10)
1. cardiac myosin binding protein C (cMyBP-C) time-resolved fluoroimmunoassay kit based on immunomagnetic beads,
It is characterized by comprising: calibration object, immunomagnetic beads, immunofluorescence microballoon, analysis buffer and cleaning solution；And in kit
Buffer used includes: buffer system, closed reagent, blocking agent and preservative.
2. kit according to claim 1, it is characterised in that: the immunomagnetic beads preparation step are as follows: after magnetic bead activation
It is coupled, closes with anti-cMyBP-C monoclonal antibody, wash, save.
3. kit according to claim 1, it is characterised in that: the immunofluorescence microballoon preparation step are as follows: the time point
It is coupled after distinguishing fluorescent microsphere activation with anti-cMyBP-C monoclonal antibody, closes, wash, save.
4. kit according to claim 1, it is characterised in that: the immunomagnetic beads partial size is 100nm-5 μm, and surface is repaired
Decorations are any one in carboxyl, hydroxyl or Streptavidin；The immunofluorescence microspherulite diameter is 100nm-500nm, surface
It is modified to any one in carboxyl, hydroxyl or Streptavidin.
5. kit according to claim 2, it is characterised in that: the activation and coupling concrete operations step of the immunomagnetic beads
Suddenly are as follows:
Activation: being added the mixing of NHS vortex, adds EDC, vortex mixing；Shaking table 100-500r/min, 30-37 DEG C of reaction 10-
30min；Wherein the mass concentration ratio of NHS:EDC is 25:10；
Coupling: removing supernatant after centrifugation, adds ultrapure water, and ultrasound is redissolved, and repeated centrifugation is redissolved twice, and it is mono- that cMyBP-C is added
Clonal antibody, vortex mixing, shaking table 100-500r/min, 30-37 DEG C of reaction 1-3h；The label ratio of magnetic bead and antibody is (50-
150):1。
6. kit according to claim 3, it is characterised in that: the specific behaviour of activation and coupling of the immunofluorescence microballoon
Make step are as follows:
Activation: being added the mixing of NHS vortex, adds EDC, vortex mixing；Shaking table 100-500r/min, 30-37 DEG C of reaction 10-
30min；Wherein the mass concentration ratio of NHS:EDC is 25:10；
Coupling: removing supernatant after centrifugation, ultrapure water is added, and ultrasound is redissolved, and repeated centrifugation is redissolved twice, and cMyBP-C Dan Ke is added
Grand antibody, vortex mixing, shaking table 100-500r/min, 30-37 DEG C of reaction 1-3h；The label of time-resolved fluorescence microballoon and antibody
Ratio is (50-150): 1.
7. kit according to claim 1, it is characterised in that: the calibration object is with the buffer by cMyBP-C
Antigen is configured to the calibration solution of 0-500ng/mL series of concentrations.
8. kit according to claim 1, it is characterised in that: the analysis buffer formula includes: 10-50mmol/L
Tris-HCl, 0.1-5% casein, 1--5%BSA, 0.01-1%Proclin300,0.01%-1Tween20,0.9%
NaCl, buffer tune pH to 7.2-7.4.
9. kit according to claim 1, it is characterised in that: the washing formula of liquid includes: 10-50mmol/L's
The NaCl of the Tween 20 and 0.9% of Tris-HCl, 0.01-1%, buffer tune pH to 7.2-7.4.
10. kit according to claim 1, it is characterised in that: each ingredient in the buffer is specific as follows:
The buffer system: any one in PBS, Tris or CBS buffer, and concentration is 10-50mmol/L, pH value is
7.0-9.0；
The closed reagent: any one in bovine serum albumin(BSA) or casein, and mass concentration is 1-15%；
The blocking agent: IgG albumen, and mass concentration is 0.1-1%；
The preservative: any one in Sodium azide or Proclin 300, and mass concentration is 0.01-0.1%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811310863.3A CN109298178A (en) | 2018-11-06 | 2018-11-06 | Cardiac myosin binding protein C(cMyBP-C based on immunomagnetic beads) time-resolved fluoroimmunoassay kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811310863.3A CN109298178A (en) | 2018-11-06 | 2018-11-06 | Cardiac myosin binding protein C(cMyBP-C based on immunomagnetic beads) time-resolved fluoroimmunoassay kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109298178A true CN109298178A (en) | 2019-02-01 |
Family
ID=65146782
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811310863.3A Pending CN109298178A (en) | 2018-11-06 | 2018-11-06 | Cardiac myosin binding protein C(cMyBP-C based on immunomagnetic beads) time-resolved fluoroimmunoassay kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109298178A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110988331A (en) * | 2019-11-22 | 2020-04-10 | 四川朴澜医疗科技有限公司 | Microfluidic chip detection method based on magnetic bead technology and reagent freeze-drying technology and microfluidic chip |
| CN111024946A (en) * | 2019-11-19 | 2020-04-17 | 江苏美克医学技术有限公司 | Trichomonas vaginalis fluorescence immunochromatography assay kit and preparation method thereof |
| CN114778817A (en) * | 2022-06-17 | 2022-07-22 | 山东中鸿特检生物科技有限公司 | Kit for detecting interferon in serum and plasma, detection method and application |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507947A (en) * | 2011-11-11 | 2012-06-20 | 南方医科大学 | CEA TRFIA (time-resolved fluoroimmunoassay) kit based on IMB (immunomagnetic beads) |
| US20120156702A1 (en) * | 2009-05-11 | 2012-06-21 | King's College London | Cmybp-c and mlc2 as diagnostic markers of cardiac injury |
| CN105891463A (en) * | 2014-12-31 | 2016-08-24 | 川北医学院 | Beta-HCG quantitative detection kit based on nanometer magnetic particle time resolution fluorescence |
| CN105911288A (en) * | 2016-04-06 | 2016-08-31 | 上海奥普生物医药有限公司 | Cup-type time-resolved fluorescent immunoassay kit for high-sensitivity C-reactive protein based on microspheres, and preparation method and application thereof |
| CN106226513A (en) * | 2016-08-02 | 2016-12-14 | 北京乐普医疗科技有限责任公司 | A kind of method of immunomagnetic beads detection by quantitative related antigen and application thereof |
| CN107884586A (en) * | 2017-10-31 | 2018-04-06 | 吴灿军 | A kind of method of the homogeneous immune detection target protein of Magneto separate |
| CN108181287A (en) * | 2018-02-09 | 2018-06-19 | 广州源起健康科技有限公司 | A kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects H-FABP kits |
-
2018
- 2018-11-06 CN CN201811310863.3A patent/CN109298178A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120156702A1 (en) * | 2009-05-11 | 2012-06-21 | King's College London | Cmybp-c and mlc2 as diagnostic markers of cardiac injury |
| CN102507947A (en) * | 2011-11-11 | 2012-06-20 | 南方医科大学 | CEA TRFIA (time-resolved fluoroimmunoassay) kit based on IMB (immunomagnetic beads) |
| CN105891463A (en) * | 2014-12-31 | 2016-08-24 | 川北医学院 | Beta-HCG quantitative detection kit based on nanometer magnetic particle time resolution fluorescence |
| CN105911288A (en) * | 2016-04-06 | 2016-08-31 | 上海奥普生物医药有限公司 | Cup-type time-resolved fluorescent immunoassay kit for high-sensitivity C-reactive protein based on microspheres, and preparation method and application thereof |
| CN106226513A (en) * | 2016-08-02 | 2016-12-14 | 北京乐普医疗科技有限责任公司 | A kind of method of immunomagnetic beads detection by quantitative related antigen and application thereof |
| CN107884586A (en) * | 2017-10-31 | 2018-04-06 | 吴灿军 | A kind of method of the homogeneous immune detection target protein of Magneto separate |
| CN108181287A (en) * | 2018-02-09 | 2018-06-19 | 广州源起健康科技有限公司 | A kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects H-FABP kits |
Non-Patent Citations (2)
| Title |
|---|
| LIN,GF等: "A Magnetic Nanoparticle-Based Time-Resolved Fluoroimmunoassay for Determination of the Cytokeratin 19 Fragment in Human Serum", 《JOURNAL OF FLUORESCENCE》 * |
| 孙传学等: "心肌肌球蛋白结合蛋白C在急性心肌梗死中的表达及其临床意义", 《医学综述》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111024946A (en) * | 2019-11-19 | 2020-04-17 | 江苏美克医学技术有限公司 | Trichomonas vaginalis fluorescence immunochromatography assay kit and preparation method thereof |
| CN110988331A (en) * | 2019-11-22 | 2020-04-10 | 四川朴澜医疗科技有限公司 | Microfluidic chip detection method based on magnetic bead technology and reagent freeze-drying technology and microfluidic chip |
| CN110988331B (en) * | 2019-11-22 | 2024-02-23 | 四川微康朴澜医疗科技有限责任公司 | Microfluidic chip detection method and microfluidic chip based on magnetic bead technology and reagent freeze-drying technology |
| CN114778817A (en) * | 2022-06-17 | 2022-07-22 | 山东中鸿特检生物科技有限公司 | Kit for detecting interferon in serum and plasma, detection method and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109298177A (en) | Time-resolved fluorescence immunoassay method based on Magneto separate | |
| CN104634980B (en) | The super quick detection kit of cardiac muscle troponin I and super quick detection method | |
| WO2016127318A1 (en) | Cardiac troponin i ultra-sensitive detection reagent kit, and ultra-sensitive detection method therefor | |
| CN103364568B (en) | Laminin nano-magnetic particle chemiluminescent immunity quantitative detection kit and preparation method thereof | |
| CN102998467B (en) | β human chorionic gonadotrophin magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof | |
| JPH0654317B2 (en) | Immunoassay device | |
| CN104215770A (en) | Two-particle-based retinol binding protein detection kit | |
| CN108333357A (en) | A kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects CK-MB kits | |
| JPH07506185A (en) | Immunoassay using non-specific anti-hapten antibodies and materials for use in the immunoassay | |
| CN109211867A (en) | A kind of micro-fluidic fluorescence immunoassay chip of rapid quantitative detection BNP | |
| CN102998466A (en) | Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for growth hormone (GH), and preparation method of kit | |
| CN109298178A (en) | Cardiac myosin binding protein C(cMyBP-C based on immunomagnetic beads) time-resolved fluoroimmunoassay kit | |
| CN102998465A (en) | Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for angiotensin (Ang) I, and preparation method of kit | |
| CN109187971A (en) | Neuronspecific enolase chemiluminescence immune detection reagent kit and preparation method thereof | |
| CN101949945B (en) | Kit for detecting free thyroxin by using magnetic particles as solid-phase carriers and preparation method thereof | |
| CN101377509A (en) | III type precollagen N end peptide chemiluminescence immune analysis quantitative determination reagent kit and preparing method thereof | |
| CN110426515A (en) | A kind of time-resolved fluoroimmunoassay chromatographic technique detects kit and its application of dirty underwater trace drugs | |
| CN104677889B (en) | Method for detecting alpha fetoprotein by using magnetic immune probe based on luminol functionalization | |
| CN106771199B (en) | A kind of colorimetric immunoassay analysis method of detection tumor markers for non-diagnostic purpose | |
| CN109142753A (en) | Squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit and preparation method thereof | |
| CN108181287A (en) | A kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects H-FABP kits | |
| CN108872595A (en) | A kind of carcinomebryonic antigen detection kit and preparation method thereof | |
| CN104950111A (en) | Liquid chip kit for quantitatively detecting concentration of myeloperoxidase (MPO) in sample and preparation method of liquid chip kit | |
| CN110082537A (en) | Vascular endothelial growth factor chemiluminescence immune detection reagent kit and preparation method thereof | |
| CN109187972A (en) | A kind of magnetic microparticle chemiluminescence kit of quantitative detection plasma renin content and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190201 |