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 PDF

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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
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pivka
magnetic particle
added
sample
antibody
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张波
郗日沫
张学记
王鹏
雷达
刘向阳
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Jiangsu Maideke Biological Technology Co Ltd
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Jiangsu Maideke Biological Technology Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—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

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
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  • Engineering & Computer Science (AREA)
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  • Urology & Nephrology (AREA)
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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

Abnormal prothrombin (PIVKA- II) magnetic microparticle chemiluminescence immune assay determination reagent Box and preparation method thereof
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.
CN201710768857.1A 2017-08-31 2017-08-31 Abnormal prothrombin (PIVKA- II) magnetic microparticle chemiluminescence immune assay determination kit and preparation method thereof Pending CN109425740A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
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

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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

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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
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Cited By (3)

* Cited by examiner, † Cited by third party
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

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Application publication date: 20190305