CN109425740A - Abnormal prothrombin (PIVKA- II) magnetic microparticle chemiluminescence immune assay determination kit and preparation method thereof - Google Patents
Abnormal prothrombin (PIVKA- II) magnetic microparticle chemiluminescence immune assay determination kit and preparation method thereof Download PDFInfo
- Publication number
- CN109425740A CN109425740A CN201710768857.1A CN201710768857A CN109425740A CN 109425740 A CN109425740 A CN 109425740A CN 201710768857 A CN201710768857 A CN 201710768857A CN 109425740 A CN109425740 A CN 109425740A
- Authority
- CN
- China
- Prior art keywords
- pivka
- magnetic particle
- added
- sample
- antibody
- 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
- 108010094028 Prothrombin Proteins 0.000 title claims abstract description 12
- 102100027378 Prothrombin Human genes 0.000 title claims abstract description 12
- 230000002159 abnormal effect Effects 0.000 title claims abstract description 12
- 229940039716 prothrombin Drugs 0.000 title claims abstract description 12
- 108010063628 acarboxyprothrombin Proteins 0.000 title claims abstract description 9
- 238000003556 assay Methods 0.000 title claims abstract description 7
- 238000002360 preparation method Methods 0.000 title claims abstract description 6
- 239000011859 microparticle Substances 0.000 title claims abstract description 5
- 238000001514 detection method Methods 0.000 claims abstract description 19
- 239000000758 substrate Substances 0.000 claims abstract description 18
- 239000006249 magnetic particle Substances 0.000 claims abstract description 15
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 claims abstract description 8
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 claims abstract description 8
- 239000003513 alkali Substances 0.000 claims abstract description 8
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000000034 method Methods 0.000 claims abstract description 8
- 102000004190 Enzymes Human genes 0.000 claims abstract description 7
- 108090000790 Enzymes Proteins 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims abstract description 6
- 238000005516 engineering process Methods 0.000 claims abstract description 4
- 238000002038 chemiluminescence detection Methods 0.000 claims abstract description 3
- 238000002372 labelling Methods 0.000 claims abstract description 3
- 239000006210 lotion Substances 0.000 claims abstract description 3
- 238000005406 washing Methods 0.000 claims abstract description 3
- 238000002955 isolation Methods 0.000 claims abstract 2
- 239000006228 supernatant Substances 0.000 claims description 8
- 238000004458 analytical method Methods 0.000 claims description 7
- 239000000047 product Substances 0.000 claims description 7
- 238000012360 testing method Methods 0.000 claims description 7
- 238000004020 luminiscence type Methods 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 4
- 238000003908 quality control method Methods 0.000 claims description 4
- 239000011324 bead Substances 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- -1 polyethylene Polymers 0.000 claims description 3
- 239000003381 stabilizer Substances 0.000 claims description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 2
- 229940098773 bovine serum albumin Drugs 0.000 claims description 2
- 239000011248 coating agent Substances 0.000 claims description 2
- 238000000576 coating method Methods 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims 2
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims 2
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 claims 1
- 239000005977 Ethylene Substances 0.000 claims 1
- 239000004698 Polyethylene Substances 0.000 claims 1
- 239000004743 Polypropylene Substances 0.000 claims 1
- 239000005018 casein Substances 0.000 claims 1
- 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 1
- 235000021240 caseins Nutrition 0.000 claims 1
- 229940056319 ferrosoferric oxide Drugs 0.000 claims 1
- 239000011521 glass Substances 0.000 claims 1
- 229920000573 polyethylene Polymers 0.000 claims 1
- 229920000642 polymer Polymers 0.000 claims 1
- 229920006389 polyphenyl polymer Polymers 0.000 claims 1
- 229920001155 polypropylene Polymers 0.000 claims 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims 1
- 210000002966 serum Anatomy 0.000 abstract description 7
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 238000003018 immunoassay Methods 0.000 abstract description 4
- 210000002381 plasma Anatomy 0.000 abstract description 3
- 238000007865 diluting Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 10
- 206010028980 Neoplasm Diseases 0.000 description 7
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 6
- 102100023635 Alpha-fetoprotein Human genes 0.000 description 4
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 4
- 238000003745 diagnosis Methods 0.000 description 3
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 3
- 239000011534 wash buffer Substances 0.000 description 3
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 229930003448 Vitamin K Natural products 0.000 description 2
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- XLJMAIOERFSOGZ-UHFFFAOYSA-N cyanic acid Chemical compound OC#N XLJMAIOERFSOGZ-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 208000014018 liver neoplasm Diseases 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- SHUZOJHMOBOZST-UHFFFAOYSA-N phylloquinone Natural products CC(C)CCCCC(C)CCC(C)CCCC(=CCC1=C(C)C(=O)c2ccccc2C1=O)C SHUZOJHMOBOZST-UHFFFAOYSA-N 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 235000019168 vitamin K Nutrition 0.000 description 2
- 239000011712 vitamin K Substances 0.000 description 2
- 150000003721 vitamin K derivatives Chemical class 0.000 description 2
- 229940046010 vitamin k Drugs 0.000 description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 206010073069 Hepatic cancer Diseases 0.000 description 1
- NMOJAXCSURVGEY-UHFFFAOYSA-N N#CC#N.[S] Chemical compound N#CC#N.[S] NMOJAXCSURVGEY-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000004797 therapeutic response Effects 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (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)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention belongs to immunoassay technical field of medical detection; the magnetic microparticle chemiluminescence immune assay determination kit and preparation method thereof of a kind of serum or the abnormal prothrombin (PIVKA- II) in blood plasma is provided in particular, kit specifically includes that II standard items of (1) PIVKA-；(2) the coated magnetic particle of anti-fluorescein isothiocynate (FITC) monoclonal antibody；(3) II sample of PIVKA- of alkali phosphatase enzyme mark and the PIVKA- II of marked by fluorescein isothiocyanate；(4) chemiluminescent substrate of alkali phosphatase enzyme mark；(5) above-mentioned standard product, magnetic particle solution, mixed liquor, sample diluting liquid, washing lotion, chemiluminescent substrate and reaction tube are dispensed.The product that this method enzyme labelling technique, magnetic particle isolation technics and chemiluminescence detection technology combine, provides a kind of close to homogeneous reaction system, has both that simple, sensitivity, specificity is good, the range of linearity is wide, the advantages such as reproducible.
Description
Technical field
The invention belongs to immune detection medical analysis technical fields, and in particular to clinically diagnosis abnormal prothrombin
Content provides a kind of Magneto separate chemical luminous immune detection method of people's abnormal prothrombin, suitable for human serum, blood plasma
Abnormal prothrombin quantitative detection.
Background technique
Tumor markers (Tumor marker, TM) are reflection tumorigenesis, monitor tumour to therapeutic response
One substance can carry out quantitative detection by the methods of immunology, chemistry and molecular biology.The serum levels of tumor markers
It is generally had good correlation with the generation of tumour, development, recession and recurrence, therefore using serum levels measurement result,
The information of forecast etc. is judged and recurred about tumour auxiliary diagnosis, observation of curative effect, prognosis to obtain.
Now, hepatocellular carcinoma (Hepatocellular Carcinoma, HCC) is the sixth-largest most common harm in the whole world
The malignant tumor of digestive tract of human health accounts for the 90% of primary carcinoma of liver, and clinically there are about belonged to when 2/3 liver cancer patient first visit
Middle and advanced stage has lost the killer opportunity of operative treatment.Therefore, highly sensitive detection is the timely and effective early warning that diagnoses the illness
Key.Currently, clinically the most frequently used alpha-fetoprotein (Alphafetoprotein, AFP) sieving and diagnosis HCC, however for AFP
The screening of the patient of low concentration or feminine gender, result are unsatisfactory.
Recent studies have shown that abnormal prothrombin (Protein induced by vitamin K absence- II,
PIVKA- II) it is a kind of novel hepatocellular carcinoma marker, specificity with higher is primarily referred to as vitamin K
Lack the liver cell that the albumen that antagonist II induces is caused and be difficult to the phenomenon that being effectively synthesized coagulation factor, can be used as diagnosing cancer of liver
Index, it has also become one of tumour basis and the hot spot of clinical research.
Immunologic assay is determined based on the specific recognition of antibody-antigene, antibody labelling technique and tracer technique
Amount technology has in the body fluid detection of tumor markers and is widely applied.Chemiluminescence immunoassay combines immune response
High specific and chemiluminescence reaction it is highly sensitive small, instrument is simple, low in cost, can reach 10-18Mol detection level, and
It is wider in detection range, it is receive more and more attention in clinical detection assays.
Summary of the invention
The technical problem to be solved in the invention is to provide that a kind of high specificity, high sensitivity, easy to operate, detection is fast
Speed, the analysis assay kit of accurate detection abnormal prothrombin content, while also providing a kind of abnormal prothrombin inspection
The preparation method of test agent box, the kit may be adapted to industrialization production, have a vast market application prospect.
Kit of the invention, it is characterised in that the kit includes: II standard items of (1) PIVKA-；(2) resist different sulphur
The coated magnetic particle of cyanic acid fluorescein (FITC) monoclonal antibody；(3) II sample of PIVKA- of alkali phosphatase enzyme mark, different sulphur cyanogen
The fluorescein-labeled PIVKA- II of acid；(4) chemiluminescent substrate of alkali phosphatase enzyme mark；(5) it is micro- that above-mentioned standard product, magnetic are dispensed
Grain solution, mixed liquor, sample diluting liquid, washing lotion, chemiluminescent substrate and reaction tube.
Technical solution of the invention, steps are as follows for detection and analysis:
(1) it is loaded and is immunoreacted: standard items, quality-control product, the sample to be tested of 15-50 μ L being added in flat based tubes, 45 μ are added
Coated 45 μ L magnetic bead antibody is added in L enzyme labelled antibody, and 300 μ L stabilizers are added, and shakes 30s, and after mixing, 37 DEG C incubate 30 points
Clock；
(2) it washs: flat based tubes being stood 2 minutes on magnet stand, then pour out supernatant, test tube and magnet stand are upside down in together
It is patted dry on blotting paper；300 μ L cleaning solutions are added in every pipe, shakes 30 seconds, flat based tubes is stood 2 minutes on magnet stand, then
Supernatant is poured out, test tube and magnet stand are upside down in together on blotting paper and patted dry；It is repeated 5 times；
(3) luminous substrate solution is added: 200 μ L luminous substrates being added in every pipe；
(4) it reads luminous value: measuring the luminous value of every pipe with chemical luminescence detector.
The beneficial effects of the present invention are:
To there is the present invention highly sensitive chemiluminescence detection technology to combine with the immune response of high specific, operation letter
Single, the used time is short, is not necessarily to long-time colour developing and terminates, and high sensitivity, and specificity is good, and energy is quick, simplicity, is delicately suitable for people
The quantitative detection of abnormal prothrombin in serum, blood plasma.
(1) it can increase coating surface area as solid phase using small magnetic particle, increase effective package amount of antibody,
Antibody is not only saved, and helps to establish the immunologic detection method of wide scope, is particularly suitable for high concentration clinical sample
Measurement, avoids the generation of crotch effect.
(2) in the reaction of liquid phase, using luminescence enhancer, hydrone is arranged from the luminescence sites of luminous substrate, together
When can also shorten luminous peak time.
(3) present invention is to can detecte luminous substrate production using enzymatic luminous substrate on the basis of EIA enzyme immunoassay
Raw optical signal replaces enzyme to exempt from the chromogenic substrate in analysis, thus its sensitivity greatly improves, and practicability easy to operate is wide,
Can be applied to open semi-automatic chemiluminescence measuring instrument, it can also be used to full automatic measuring system, it can be achieved that high-volume,
Fast detection, use cost is low, is easier to promote and apply.
Detailed description of the invention
Fig. 1 is II chemiluminescence immunoassay examination criteria curve of PIVKA- of the invention, wherein abscissa is PIVKA- II
Concentration, ordinate be relative luminous intensity (RLU).
Specific embodiment
The 1 coated magnetic particle of anti-fluorescein isothiocynate (FITC) monoclonal antibody of embodiment
0.1-5 μm of partial size of magnetic particle is activated with glutaraldehyde, is stirred at room temperature, after mixing 2 hours, adds magnetic field, stands 20-
25min pours out supernatant, is cleaned three times with the 0.01mol/L phosphate buffer that pH value is 7.4, and suspended with the solution,
Concentration is 50-100mg/mL；Anti-FITC monoclonal antibody 60-100 μ g is added in every milliliter of suspension, is stirred overnight at 4 DEG C
Afterwards, add magnetic field, stand 10-15min, pour out supernatant, with containing 0.2%-1.0% bovine serum albumin(BSA), 0.5% ~ 1.0% junket egg
White, 0.02mol/L phosphate buffer (pH 7.2) is closed 3-4 hours in room temperature；It is finally 7.4 with pH value, contains Tween-20
It is cleaned 3-5 times with the phosphate washing buffer of sodium azide preservatives, and is configured to the work of 5 ~ 10mg/mL with the solution
Liquid.Magnetic particle solution is saved at 4 DEG C, should not be frozen, the used time gently shakes up.
2 washing buffer of embodiment
Washing buffer is the phosphate buffer containing 0.2-0.5% Tween-20,300 preservative of 0.5mL/L Proclin,
PH value is 7.4.10 times are diluted with distilled water when use.
The application method of 3 kits of embodiment
One, sample pre-treatments
The empty stomach morning blood serum sample of people is taken, 3000rpm is centrifuged 5min, upper serum is taken to be analyzed.
Two, detection method
Abnormal prothrombin (PIVKA- II) magnetic microparticle chemiluminescence immune assay determination kit.
Before being tested using this kit, magnetic particle solution, FITC label II antibody of PIVKA-, standard need to be first taken out
The experiment reagents such as product, quality-control product, chemiluminescent substrate are placed 15 ~ 30 minutes at room temperature, balance to room temperature；Secondly, will
Water-bath or constant temperature incubator modulate 37 DEG C, with convenient to use；Later, prepare suitable micro sample adding appliance and corresponding suction nozzle, together
When check Chemiluminescence Apparatus whether work normally.
Steps are as follows for detection and analysis:
(1) it is loaded and is immunoreacted: standard items, quality-control product, the sample to be tested of 15-50 μ L being added in flat based tubes, 45 μ are added
Coated 45 μ L magnetic bead antibody is added in L enzyme labelled antibody, and 300 μ L stabilizers are added, and shakes 30s, and after mixing, 37 DEG C incubate 30 points
Clock.
(2) it washs: flat based tubes being stood 2 minutes on magnet stand, then pour out supernatant, test tube and magnet stand are upside down in together
It is patted dry on blotting paper；300 μ L cleaning solutions are added in every pipe, shakes 30 seconds, flat based tubes is stood 2 minutes on magnet stand, then
Supernatant is poured out, test tube and magnet stand are upside down in together on blotting paper and patted dry；It is repeated 5 times；
(3) luminous substrate solution is added: 200 μ L luminous substrates being added in every pipe；
(4) it reads luminous value: measuring the luminous value of every pipe with chemical luminescence detector.
Kit performance indicator of the present invention
Sensitivity for analysis: 1.53 mAU//mL
Precision: variation within batch CV%≤10%, interassay coefficient of variation CV%≤15；
Linear coefficient: r >=0.9900；
The range of linearity: 8.5 mAU/mL-63000 mAU/mL；
Specificity: measuring the cross reacting material of high concentration, as a result as follows:
| Potential cross reaction object | Maximum detectable concentration |
| Human anti-mouse antibody | 109.5 ng/mL |
| Bilirubin C | 19.9 mg/dL |
| Bilirubin F | 18.8 mg/dL |
| Rheumatoid factor | 950 IU/mL |
| Hemoglobin | 513 mg/dL |
Claims (6)
1. abnormal prothrombin magnetic microparticle chemiluminescence immune assay determination kit and preparation method thereof, it is characterised in that be enzyme
Labelling technique, magnetic particle isolation technics and chemiluminescence detection technology etc. combine, and generate detection kit；
It is characterized in that the kit includes: II standard items of (1) PIVKA-；(2) anti-fluorescein isothiocynate (FITC) Dan Ke
The grand coated magnetic particle of antibody；(3) PIVKA- of II sample of PIVKA- of alkali phosphatase enzyme mark, marked by fluorescein isothiocyanate
Ⅱ；(4) chemiluminescent substrate of alkali phosphatase enzyme mark；(5) it is dilute that above-mentioned standard product, magnetic particle solution, mixed liquor, sample are dispensed
Release liquid, washing lotion, chemiluminescent substrate and reaction tube.
2. according to claim 1, it is characterised in that: the chemiluminescent substrate of the alkali phosphatase enzyme mark, chemistry hair
Light substrate is APS-5.
3. according to claim 1, it is characterised in that: the concentration of II sample of PIVKA- of alkali phosphatase enzyme mark is 1-3mg/
ML, marked by fluorescein isothiocyanate PIVKA- II sample concentration be 2-8 mg/mL.
4. according to claim 1, it is characterised in that: the material of the reaction tube is polyethylene, polypropylene, transparent polyphenyl
Ethylene or glass.
5. according to claim 1, which is characterized in that in the coated magnetic particle step of preparation anti-FITC monoclonal antibody
(2) in: for the antibody coating magnetic particle diameter between 0.01-2 μm, ferroso-ferric oxide is kernel, has superparamagnetism, table
Face is that package has amino (NH2) or carboxyl (COOH) active group polymer, use concentration are as follows: 1-8 mg/mL；
Close coated magnetic particle with confining liquid, the confining liquid be containing 0.2% ~ 1.0% bovine serum albumin(BSA), 0.5% ~
1.0% casein, the 0.02mol/L phosphate buffer that pH value is 7.2.
6. according to claim 1, which is characterized in that steps are as follows for detection and analysis:
(1) it is loaded and is immunoreacted: standard items, quality-control product, the sample to be tested of 15-50 μ L being added in flat based tubes, 45 μ are added
Coated 45 μ L magnetic bead antibody is added in L enzyme labelled antibody, and 300 μ L stabilizers are added, and shakes 30s, and after mixing, 37 DEG C incubate 30 points
Clock；
(2) it washs: flat based tubes being stood 2 minutes on magnet stand, then pour out supernatant, test tube and magnet stand are upside down in together
It is patted dry on blotting paper；300 μ L cleaning solutions are added in every pipe, shakes 30 seconds, flat based tubes is stood 2 minutes on magnet stand, then
Supernatant is poured out, test tube and magnet stand are upside down in together on blotting paper and patted dry；It is repeated 5 times；
(3) luminous substrate solution is added: 200 μ L luminous substrates being added in every pipe；
(4) it reads luminous value: measuring the luminous value of every pipe with chemical luminescence detector.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710768857.1A CN109425740A (en) | 2017-08-31 | 2017-08-31 | Abnormal prothrombin (PIVKA- II) magnetic microparticle chemiluminescence immune assay determination kit and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710768857.1A CN109425740A (en) | 2017-08-31 | 2017-08-31 | Abnormal prothrombin (PIVKA- II) magnetic microparticle chemiluminescence immune assay determination kit and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109425740A true CN109425740A (en) | 2019-03-05 |
Family
ID=65505106
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710768857.1A Pending CN109425740A (en) | 2017-08-31 | 2017-08-31 | Abnormal prothrombin (PIVKA- II) magnetic microparticle chemiluminescence immune assay determination kit and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109425740A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110850085A (en) * | 2019-11-06 | 2020-02-28 | 迪瑞医疗科技股份有限公司 | Abnormal prothrombin chemiluminescence immunoassay kit and preparation method thereof |
| CN113372447A (en) * | 2021-05-26 | 2021-09-10 | 重庆中元汇吉生物技术有限公司 | anti-PIVKA-II monoclonal antibody and application thereof |
| CN115932250A (en) * | 2023-01-05 | 2023-04-07 | 苏州惠中生物科技有限公司 | Magnetic particle chemiluminescence reagent for quantitative detection of anti-aPS/PT antibody and chemiluminescence detection kit |
| US12105093B2 (en) | 2020-07-28 | 2024-10-01 | University Of Washington | Quantitation of GLA proteins by mass spectrometric analysis |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103592445A (en) * | 2013-10-16 | 2014-02-19 | 北京利德曼生化股份有限公司 | Kit for detecting procalcitonin |
| CN104807997A (en) * | 2015-05-05 | 2015-07-29 | 南京格耀生物科技有限公司 | Kit for detecting CA125 and SP17 contents based on chemiluminiscence method as well as method and application thereof |
| CN104820102A (en) * | 2015-05-05 | 2015-08-05 | 南京格耀生物科技有限公司 | Kit of detecting the content of Lp-PLA2 and CRP on the basis of chemiluminescence, and method and application thereof |
| CN104880564A (en) * | 2015-04-30 | 2015-09-02 | 南京格耀生物科技有限公司 | Kit for detecting resistin as well as preparation method and detection method of kit |
| CN106119340A (en) * | 2016-07-04 | 2016-11-16 | 福建广生堂药业股份有限公司 | Detection method, detectable and the detection kit of a kind of de-γ carboxyl thrombinogen |
| CN106468711A (en) * | 2016-09-07 | 2017-03-01 | 北京热景生物技术股份有限公司 | DCP sharp separation detection kit |
| CN106610430A (en) * | 2016-05-23 | 2017-05-03 | 天津亿美诺生物科技有限公司 | Kit for detecting MUC-16 content based on chemiluminescence |
-
2017
- 2017-08-31 CN CN201710768857.1A patent/CN109425740A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103592445A (en) * | 2013-10-16 | 2014-02-19 | 北京利德曼生化股份有限公司 | Kit for detecting procalcitonin |
| CN104880564A (en) * | 2015-04-30 | 2015-09-02 | 南京格耀生物科技有限公司 | Kit for detecting resistin as well as preparation method and detection method of kit |
| CN104807997A (en) * | 2015-05-05 | 2015-07-29 | 南京格耀生物科技有限公司 | Kit for detecting CA125 and SP17 contents based on chemiluminiscence method as well as method and application thereof |
| CN104820102A (en) * | 2015-05-05 | 2015-08-05 | 南京格耀生物科技有限公司 | Kit of detecting the content of Lp-PLA2 and CRP on the basis of chemiluminescence, and method and application thereof |
| CN106610430A (en) * | 2016-05-23 | 2017-05-03 | 天津亿美诺生物科技有限公司 | Kit for detecting MUC-16 content based on chemiluminescence |
| CN106119340A (en) * | 2016-07-04 | 2016-11-16 | 福建广生堂药业股份有限公司 | Detection method, detectable and the detection kit of a kind of de-γ carboxyl thrombinogen |
| CN106468711A (en) * | 2016-09-07 | 2017-03-01 | 北京热景生物技术股份有限公司 | DCP sharp separation detection kit |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110850085A (en) * | 2019-11-06 | 2020-02-28 | 迪瑞医疗科技股份有限公司 | Abnormal prothrombin chemiluminescence immunoassay kit and preparation method thereof |
| US12105093B2 (en) | 2020-07-28 | 2024-10-01 | University Of Washington | Quantitation of GLA proteins by mass spectrometric analysis |
| CN113372447A (en) * | 2021-05-26 | 2021-09-10 | 重庆中元汇吉生物技术有限公司 | anti-PIVKA-II monoclonal antibody and application thereof |
| CN115932250A (en) * | 2023-01-05 | 2023-04-07 | 苏州惠中生物科技有限公司 | Magnetic particle chemiluminescence reagent for quantitative detection of anti-aPS/PT antibody and chemiluminescence detection kit |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102759631B (en) | The latex enhancing immune of a kind of quantitative detection Procalcitonin PCT is than turbid kit | |
| CN106885908B (en) | The detection kit and its detection method of blood-serum P SMD4 albumen and application | |
| CN103604931B (en) | A kind of people's S100 protein assay reagent and preparation method | |
| CN105785043B (en) | For quantitatively detecting AFP L3% kit | |
| TWI698639B (en) | Prostate antigen standards and uses thereof | |
| CN102901812A (en) | Magnetic particle chemiluminescence immunoassay kit and assay method for human thyroglobulin antibodies (TGAb) | |
| CN104034892A (en) | Magnetic particle chemiluminescence immune assay kit of tumor marker AFP (alpha fetal protein) and detection method thereof | |
| CN107167595A (en) | A kind of immunochromatography reagent bar of fluorogenic quantitative detection INHB and preparation method thereof | |
| CN109425740A (en) | Abnormal prothrombin (PIVKA- II) magnetic microparticle chemiluminescence immune assay determination kit and preparation method thereof | |
| CN103364558A (en) | Human tumor marker carcinoembryonic antigen (CEA) magnetic particle chemiluminiscence immunoassay kit and detection method | |
| US20180364249A1 (en) | Method of quantitative determination of sarcosine in a biological sample | |
| CN101231288B (en) | Novel method for analyzing human thymidine kinase fluorescence immune based on magnetic nanometer particular | |
| CN102135535B (en) | Immune colloidal metal detection technology capable of directly performing semi-quantitative analysis, preparation method and application | |
| CN107328942A (en) | A kind of fluorogenic quantitative detection PAPP A immunochromatography reagent bar and preparation method thereof | |
| CN103344634A (en) | Chemiluminescence immune assay kit and method for magnetic particle of human alpha fetoprotein (AFP) | |
| CN109307765A (en) | Type pepsinogen II detection kit | |
| CN102135498B (en) | Semi-quantitative colloidal metal detection technology taking multi-capture property as characteristic and preparation method and use thereof | |
| CN109307766A (en) | Pepsinogen I detection kit | |
| CN102656457A (en) | Kit for diagnosing prostate cancer and diagnosis method | |
| CN107121548A (en) | Quantitatively detect test paper, preparation method and the detection method of tumor markers | |
| CN106645756A (en) | Kit for detecting NMP22 (Nuclear Matrix Protein 22) and preparation method thereof | |
| CN102226808A (en) | Trypsinogen-2 chemiluminescent immunoassay kit and preparation method thereof | |
| CN202916286U (en) | Latex enhanced turbidimetric immunoassay kit for quantitatively detecting procalcitonin (PCT) | |
| CN103969447A (en) | Vascular endothelial growth factor quantitative determination kit and light-emitting substrate solution thereof | |
| CN109001471A (en) | Free beta-human chorionic gonadotropin chemiluminescence detection kit and preparation method thereof and application method |
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: 20190305 |