CN106290872A - A kind of golgiosome protein GP 73 quantitative determination reagent kit and detection method thereof - Google Patents
A kind of golgiosome protein GP 73 quantitative determination reagent kit and detection method thereof Download PDFInfo
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- CN106290872A CN106290872A CN201510263625.1A CN201510263625A CN106290872A CN 106290872 A CN106290872 A CN 106290872A CN 201510263625 A CN201510263625 A CN 201510263625A CN 106290872 A CN106290872 A CN 106290872A
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Abstract
The present invention relates to a kind of golgiosome protein GP 73 quantitative determination reagent kit, including GP73 antibody coating plate, GP73 standard substance, concentrate enzyme conjugates, concentration enzyme combination diluent, concentrated cleaning solution, developer A liquid, developer B liquid, stop buffer, shrouding film, golgiosome protein GP 73 quantitative detecting method includes that Preparatory work of experiment process and detecting step, described detecting step include that sample-adding, temperature bathes, wash, added enzyme conjugates, temperature is bathed, washs, develops the color, measured, data process.Detection sensitivity of the present invention is high, and amount of samples is few, simple to operate, and the response time is short, good stability, and its sensitivity and specificity are respectively 39%-64% and 76%-91%.
Description
Technical field
The present invention relates to technical field of biological, particularly relate to a kind of golgiosome protein GP 73 quantitative determination reagent kit and
Its detection method.
Background technology
Hepatocarcinoma (HCC) is one of common malignant tumor of China, and its early symptom is inconspicuous, and one it has been observed that the most belong to
Middle and advanced stage and lose best occasion for the treatment, " early examine, early control " is the key preventing and treating hepatocarcinoma.At present clinically to hepatocarcinoma
Diagnosis mainly iconography and blood serum designated object alpha-fetoprotein (AFP) check, both approaches all can not realize early hepatocarcinoma
Diagnosis, it is therefore necessary to research and development application is in clinical hepatocarcinoma early diagnosis reagent.
Serologic detection is simple to operate, cheap, be suitable for extensive examination, is increasingly paid attention to by scholars.Mesh
Before, GP73 (GP73) is considered as one of best hepatocarcinoma early diagnosis serologic marker thing, and its detection is quick
Perception, more than AFP, has preferable pre-alerting ability to AFP negative patient.According to the literature, the sensitivity of GP73 detection hepatocarcinoma
Being 69%, specificity is 75%.For the diagnosis of early hepatocarcinoma, the sensitivity of GP73 is 62%, and AFP sensitivity is only
25%.AFP level, less than in the liver cancer patient of 20 μ g/L, has crowd's GP73 level of 57% significantly to raise.This shows,
The application of GP73 can be greatly improved the recall rate of the liver cancer patient to AFP negative.Domestic only 1 company's application enzyme connection is exempted from
Epidemic disease method (ELISA) develops clinical GP73 quantitative determination reagent kit.Same kind of products at abroad (ELISA method) is the most only applied
In scientific research, have no the GP73 checkout and diagnosis test kit being applied to clinic.
The Golgi body Glycoprotein G P73 detection method being currently known there is also more defect, as relatively complicated, the most relatively in operation
Long, it is not suitable for using clinically.At present, the Golgi body Glycoprotein G P73 detection kit in China market exists accurately
Spend low, sensitivity is low, the repeated shortcoming such as poor.For these problems, my company and Beijing hepatopathy institute cooperation, from
Source starts, and the raw material such as independent research antigen, antibody, the monoclonal antibody of preparation, multi-resistance titer are all higher than 106, establish matter
The GP73 (GP73) that amount is stable, the sensitiveest measures test kit (euzymelinked immunosorbent assay (ELISA)), greatly reduces
Production cost.
Summary of the invention
The shortcoming of prior art in view of the above, it is an object of the invention to provide Golgi body in a kind of quickly mensuration serum
The test kit of Glycoprotein G P73 content, is used for solving the problems of the prior art.
The technical scheme is that
A kind of golgiosome protein GP 73 quantitative determination reagent kit, it is characterised in that: include GP73 antibody coating plate, GP73
Standard substance, concentrate enzyme conjugates, concentrate enzyme combination diluent, concentrated cleaning solution, developer A liquid, developer B liquid,
Stop buffer, shrouding film.
A kind of golgiosome protein GP 73 quantitative determination detection method, it is characterised in that: described detection method includes experiment standard
Standby process and detecting step, described Preparatory work of experiment process is as follows:
(1) test kit and test serum sample are placed room temperature, balance at least 30 minutes；
(2) dilution concentrated cleaning solution: with fresh distilled water or deionized water by thickening and washing in suitable clean container
Liquid dilutes 20 times, mixes standby；If concentrated cleaning solution has crystallization before use, concentrated cleaning solution is positioned over water-bath
Heat in Guo so that it is be completely dissolved；
(3) to sample number into spectrum in GP73 antibody coating plate；
(4) preparation enzyme working solution: draw 120 μ l and concentrate enzyme conjugates, adds to concentrating in enzyme combination diluent bottle,
Mix standby the most up and down；If once do not make to be finished should-20 DEG C of storages, this enzyme working solution at most can freeze thawing 3 times；
The detecting step of a kind of golgiosome protein GP 73 quantitative determination detection method is as follows:
(1) sample-adding: add 50 μ L standard substance or blood serum samples respectively in GP73 antibody coating plate respective aperture, shake gently
Swing；
(2) temperature bath: with shrouding film by rearmounted for GP73 antibody coating plate shrouding 37 DEG C of temperature baths 30 minutes；
(3) washing: take shrouding film off, dries liquid in hole, fully washs with cleaning mixture 5 times, and the most every hole should add
At least 300 μ L cleaning mixture, finally pat dry in clean absorbent paper；
(4) enzyme conjugates is added: every hole adds the enzyme working solution that 100 μ L Preparatory work of experiment process steps (4) obtain, gently
Vibration；
(5) temperature bath: with another shrouding film by rearmounted for GP73 antibody coating plate shrouding 37 DEG C of temperature baths 30 minutes；
(6) washing: take shrouding film off, dries liquid in hole, fully washs with cleaning mixture 5 times, and the most every hole should add
At least 300 μ L cleaning mixture, finally pat dry in clean absorbent paper；
(7) colour developing: every hole adds 50 μ L developer A, adds 50 μ L developer B, mixing of vibrating gently, 37 DEG C
Lucifuge temperature is bathed 10 minutes；
(8) measure: every hole adds 50 μ L stop buffers, mixing of vibrating gently, use microplate reader Double wavelength to survey
Measure each hole absorbance, dominant wavelength 450nm, reference wavelength 630nm, use blank well zeroising；
(9) data process: select data processing software, the Log of the absorbance of each standard substance obtained with detecting step (8)
Value is abscissa, and the Log value of each standard concentration is vertical coordinate, by " the log-concentration logarithm of absorbance " mathematical modulo
Type carries out linear fit, Criterion curve, finds the GP73 of this serum on standard curve with each test serum absorbance
Concentration, wherein abscissa is luminous intensity, and abscissa is GP73 concentration.
The beneficial effects of the present invention is:
This test kit uses double antibody sandwich method that GP73 is carried out detection by quantitative, can be as adjuvant clinical early diagnosis hepatocarcinoma
A kind of mark.Detection sensitivity is high, and amount of samples is few, simple to operate, and the response time, the short client of convenience used.Product has
The effect phase is 9 months, good stability.For 256 example untreated hepatocarcinoma specimen, under the ROC curve of GP73, area is 0.913,
Sensitivity 82.4%, specificity 92.2%.Being significantly larger than AFP, its sensitivity and specificity are respectively 39%-64% and 76%-91%.
Detailed description of the invention
One golgiosome protein GP 73 quantitative determination reagent kit of the present invention, it is characterised in that: include GP73 antibody coating plate,
GP73 standard substance, concentration enzyme conjugates, concentration enzyme combination diluent, concentrated cleaning solution, developer A liquid, developer B
Liquid, stop buffer, shrouding film.
A kind of golgiosome protein GP 73 quantitative determination detection method, it is characterised in that: described detection method includes experiment standard
Standby process and detecting step, described Preparatory work of experiment process is as follows:
(1) test kit and test serum sample are placed room temperature, balance at least 30 minutes；
(2) dilution concentrated cleaning solution: with fresh distilled water or deionized water by thickening and washing in suitable clean container
Liquid dilutes 20 times, mixes standby；If concentrated cleaning solution has crystallization before use, concentrated cleaning solution is positioned over water-bath
Heat in Guo so that it is be completely dissolved；
(3) to sample number into spectrum in GP73 antibody coating plate；
(5) preparation enzyme working solution: draw 120 μ l and concentrate enzyme conjugates, adds to concentrating in enzyme combination diluent bottle,
Mix standby the most up and down；If once do not make to be finished should-20 DEG C of storages, this enzyme working solution at most can freeze thawing 3 times；
The detecting step of a kind of golgiosome protein GP 73 quantitative determination detection method is as follows:
(1) sample-adding: add 50 μ L standard substance or blood serum samples respectively in GP73 antibody coating plate respective aperture, shake gently
Swing；
(2) temperature bath: with shrouding film by rearmounted for GP73 antibody coating plate shrouding 37 DEG C of temperature baths 30 minutes；
(3) washing: take shrouding film off, dries liquid in hole, fully washs with cleaning mixture 5 times, and the most every hole should add
At least 300 μ L cleaning mixture, finally pat dry in clean absorbent paper；
(4) enzyme conjugates is added: every hole adds the enzyme working solution that 100 μ L Preparatory work of experiment process steps (4) obtain, gently
Vibration；
(5) temperature bath: with another shrouding film by rearmounted for GP73 antibody coating plate shrouding 37 DEG C of temperature baths 30 minutes；
(6) washing: take shrouding film off, dries liquid in hole, fully washs with cleaning mixture 5 times, and the most every hole should add
At least 300 μ L cleaning mixture, finally pat dry in clean absorbent paper；
(7) colour developing: every hole adds 50 μ L developer A, adds 50 μ L developer B, mixing of vibrating gently, 37 DEG C
Lucifuge temperature is bathed 10 minutes；
(8) measure: every hole adds 50 μ L stop buffers, mixing of vibrating gently, use microplate reader Double wavelength to survey
Measure each hole absorbance, dominant wavelength 450nm, reference wavelength 630nm, use blank well zeroising；
(9) data process: select data processing software, the Log of the absorbance of each standard substance obtained with detecting step (8)
Value is abscissa, and the Log value of each standard concentration is vertical coordinate, by " the log-concentration logarithm of absorbance " mathematical modulo
Type carries out linear fit, Criterion curve, finds the GP73 of this serum on standard curve with each test serum absorbance
Concentration, wherein abscissa is luminous intensity, and abscissa is GP73 concentration.
Performance indications:
(1) measurement range: the GP73 using zero standard to add high concentration measures the measurement range of this test kit and is
3-150ng/ml；
(2) standard curve correlation coefficient r: >=0.99；
(3) sensitivity: take test kit alignment reagent and do 20 parallel holes, seek its meansigma methodsAnd deviation SD, from dense
Finding corresponding concentration value on degree-response curve, the sensitivity (minimum detection value) being defined as this test kit is 3ng/ml；
(4) precision: variation within batch coefficient≤15.0%, interassay coefficient of variation≤15.0%
This product compared with the identical product that Beijing Hotgen Biotechnology Co., Ltd. produces, advantage after having:
| Contrast project | This product | Contrast product |
| Sensitivity for analysis | 3ng/ml | ≦25ng/ml |
| Do not treat hepatocarcinoma group-specific | 69.1% | 68% |
| Healthy People specificity | 99.0% | 98.89% |
| Response time | 30min/30min/10min | 60min/30min/15min |
| Operation | 50 μ L sample reaction 30min | Add 50 μ L weak solutions, then add 50 μ L samples |
Clinical study results:
1, in 821 example health examination specimen, specificity is 99.0% (813/821).
2, for 256 example untreated hepatocarcinoma specimen, under the ROC curve of GP73, area is 0.913, sensitivity 82.4%,
Specificity 92.2%, positive rate 69.1% (177/256).
3,4921 example specimen detection by quantitative results (being positive more than 11ng/ml):
4, first visit is non-liver cancer, and GP73 testing result is positive, and the case specimen being diagnosed as hepatocarcinoma subsequently is added up:
Above one embodiment of the present of invention is described in detail, but described content has been only presently preferred embodiments of the present invention,
It is not to be regarded as the practical range for limiting the present invention.All impartial changes made according to the present patent application scope and improvement etc.,
Within all should still belonging to the patent covering scope of the present invention.
Claims (2)
1. a golgiosome protein GP 73 quantitative determination reagent kit, it is characterised in that: include GP73 antibody coating plate, GP73
Standard substance, concentrate enzyme conjugates, concentrate enzyme combination diluent, concentrated cleaning solution, developer A liquid, developer B liquid,
Stop buffer, shrouding film.
2. a golgiosome protein GP 73 quantitative determination detection method, it is characterised in that: described detection method includes Preparatory work of experiment
Process and detecting step, described Preparatory work of experiment process is as follows:
(1) test kit and test serum sample are placed room temperature, balance at least 30 minutes；
(2) dilution concentrated cleaning solution: with fresh distilled water or deionized water by thickening and washing in suitable clean container
Liquid dilutes 20 times, mixes standby；If concentrated cleaning solution has crystallization before use, concentrated cleaning solution is positioned over water-bath
Heat in Guo so that it is be completely dissolved；
(3) to sample number into spectrum in GP73 antibody coating plate；
(4) preparation enzyme working solution: draw 120 μ l and concentrate enzyme conjugates, adds to concentrating in enzyme combination diluent bottle, gently
Gently mix standby up and down；If once do not make to be finished should-20 DEG C of storages, this enzyme working solution at most can freeze thawing 3 times；
The detecting step of a kind of golgiosome protein GP 73 quantitative determination detection method is as follows:
(1) sample-adding: add 50 μ L standard substance or blood serum samples respectively in GP73 antibody coating plate respective aperture, shake gently
Swing；
(2) temperature bath: with shrouding film by rearmounted for GP73 antibody coating plate shrouding 37 DEG C of temperature baths 30 minutes；
(3) washing: take shrouding film off, dries liquid in hole, fully washs with cleaning mixture 5 times, and the most every hole should add
At least 300 μ L cleaning mixture, finally pat dry in clean absorbent paper；
(4) enzyme conjugates is added: every hole adds the enzyme working solution that 100 μ L Preparatory work of experiment process steps (4) obtain, gently
Vibration；
(5) temperature bath: with another shrouding film by rearmounted for GP73 antibody coating plate shrouding 37 DEG C of temperature baths 30 minutes；
(6) washing: take shrouding film off, dries liquid in hole, fully washs with cleaning mixture 5 times, and the most every hole should add
At least 300 μ L cleaning mixture, finally pat dry in clean absorbent paper；
(7) colour developing: every hole adds 50 μ L developer A, adds 50 μ L developer B, mixing of vibrating gently, 37 DEG C
Lucifuge temperature is bathed 10 minutes；
(8) measure: every hole adds 50 μ L stop buffers, mixing of vibrating gently, use microplate reader Double wavelength to survey
Measure each hole absorbance, dominant wavelength 450nm, reference wavelength 630nm, use blank well zeroising；
(9) data process: select data processing software, the Log of the absorbance of each standard substance obtained with detecting step (8)
Value is abscissa, and the Log value of each standard concentration is vertical coordinate, by " the log-concentration logarithm of absorbance " mathematical modulo
Type carries out linear fit, Criterion curve, finds the GP73 of this serum on standard curve with each test serum absorbance
Concentration, wherein abscissa is luminous intensity, and abscissa is GP73 concentration.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018227643A1 (en) * | 2017-06-13 | 2018-12-20 | 首都医科大学附属北京地坛医院 | Target marker gp73 for detecting steatohepatitis and detection application method |
| CN110824172A (en) * | 2019-11-26 | 2020-02-21 | 南通大学 | Method for measuring LEP/APN ratio in venous blood |
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| US20050287630A1 (en) * | 1995-04-27 | 2005-12-29 | Abgenix, Inc. | Human antibodies derived from immunized xenomice |
| CN101062939A (en) * | 2007-05-17 | 2007-10-31 | 北京热景生物技术有限公司 | Device and reagent case for detecting hepatic carcinoma fucosido-fucosyl gorky protein GP73 |
| CN101328214A (en) * | 2007-06-20 | 2008-12-24 | 中国科学院广州生物医药与健康研究院 | Liver disease blood serum specific protein and use thereof |
| CN101988926A (en) * | 2009-08-03 | 2011-03-23 | 中国科学院广州生物医药与健康研究院 | Sandwich ELISA quantitative detection method of Golgi protein GP73 and detection kit thereof |
| CN202794175U (en) * | 2012-06-19 | 2013-03-13 | 北京勤邦生物技术有限公司 | Beta-stimulant ELISA (Enzyme-Linked Immuno-Sorbent Assay) detection kit |
-
2015
- 2015-05-21 CN CN201510263625.1A patent/CN106290872A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050287630A1 (en) * | 1995-04-27 | 2005-12-29 | Abgenix, Inc. | Human antibodies derived from immunized xenomice |
| CN101062939A (en) * | 2007-05-17 | 2007-10-31 | 北京热景生物技术有限公司 | Device and reagent case for detecting hepatic carcinoma fucosido-fucosyl gorky protein GP73 |
| CN101328214A (en) * | 2007-06-20 | 2008-12-24 | 中国科学院广州生物医药与健康研究院 | Liver disease blood serum specific protein and use thereof |
| CN101988926A (en) * | 2009-08-03 | 2011-03-23 | 中国科学院广州生物医药与健康研究院 | Sandwich ELISA quantitative detection method of Golgi protein GP73 and detection kit thereof |
| CN202794175U (en) * | 2012-06-19 | 2013-03-13 | 北京勤邦生物技术有限公司 | Beta-stimulant ELISA (Enzyme-Linked Immuno-Sorbent Assay) detection kit |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018227643A1 (en) * | 2017-06-13 | 2018-12-20 | 首都医科大学附属北京地坛医院 | Target marker gp73 for detecting steatohepatitis and detection application method |
| JP2020525763A (en) * | 2017-06-13 | 2020-08-27 | 首都医科大学附属北京地壇医院 | Target marker GP73 used for detection of steatohepatitis and detection method |
| CN110824172A (en) * | 2019-11-26 | 2020-02-21 | 南通大学 | Method for measuring LEP/APN ratio in venous blood |
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Application publication date: 20170104 |