CN102796804A - Detection method of specific gamma-glutamyl transferase and application thereof - Google Patents
Detection method of specific gamma-glutamyl transferase and application thereof Download PDFInfo
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- CN102796804A CN102796804A CN2012102614032A CN201210261403A CN102796804A CN 102796804 A CN102796804 A CN 102796804A CN 2012102614032 A CN2012102614032 A CN 2012102614032A CN 201210261403 A CN201210261403 A CN 201210261403A CN 102796804 A CN102796804 A CN 102796804A
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Abstract
The invention provides a detection method of specific gamma-glutamyl transferase, which specifically detects the activity difference of the gamma-glutamyl transferase in serum before and after forming an immunoprecipitation bonder between an anti-apolipoprotein B antibody and low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL); as well as a diagnostic reagent or a diagnostic kit prepared by applying the method. Compared with the prior art, the invention provides a simple and effective diagnostic reagent or diagnostic kit for diagnosis or auxiliary diagnosis of liver cancer. Experiment results show that the specific method for detecting the activity of LDL/VLDL-GGT (gamma-glutamyl transferase) in the serum, provided by the invention, can be used for the diagnostic reagent or the diagnostic kit for detection or auxiliary detection of liver cancer, the average diagnostic specificity can be more than 70%, and the average positive predictive value is 90%. Therefore, the application prospects of the invention are very broad.
Description
Technical field
The present invention relates to a specific specificity γ-Gu Anxianji transferring enzyme method for detecting specificity and an application thereof.
Background technology
Gamma-glutamyl based transferase (GGT in the serum; EC2.3.2.2) exist with isozyme or multiple molecular form; Some GGT can combine with lipoprotein, and as combining with low-density lipoprotein (LDL) and vldl (VLDL), but it forms reason and physiological function it be unclear that.In the prior art; Measuring about serum GGT gross activity is the routine clinical zymetology test item of carrying out; It all has in various degree and increases when acute viral hepatitis, chronic hepatitis, posthepatitic cirrhosis, intrahepatic cholestasis, extrahepatic biliary passages obstruction, primary or secondary liver cancer; But to measure specificity low for the GGT gross activity in the serum, can't be as the specificity inspection of relative disease with it, and especially for the differential diagnosis of liver cancer and other hepatopathys still detection method and the auxiliary products thereof of lack of specific.
Summary of the invention
The problems referred to above to the prior art existence; The purpose of this invention is to provide a specific specificity γ-Gu Anxianji transferring enzyme detection method and an application thereof; Be specificity be directed against with serum in low-density lipoprotein and vldl bonded γ-Gu Anxianji transferase active, to realize coming the differential diagnosis of liver cancer in the test sample and other serious hepatopathys thereof with simple, fast method.
For realizing the foregoing invention purpose, the technical scheme that the present invention adopts is following:
One specific specificity γ-Gu Anxianji transferring enzyme detection method is specially: through detecting the active difference with the gamma-glutamyl based transferase in the serum before and after the immunoprecipitation binding substances that forms between anti-apolipoprotein B antibody (Apo B antibody) and low-density lipoprotein (LDL) and vldl (VLDL).
As a kind of preferred version, the activity of the gamma-glutamyl based transferase in the serum behind the immunoprecipitation binding substances that forms between described anti-apolipoprotein B antibody and low-density lipoprotein (LDL) and vldl (VLDL) is for forming immune conjugate after the activity of the gamma-glutamyl based transferase in the centrifugal supernatant.
As further preferred version, the active method of described detection gamma-glutamyl based transferase is for being substrate and the continuous monitoring method that can on automatic clinical chemistry analyzer, use with L-gamma-glutamyl-3-carboxyl-p-Nitroaniline (3-carboxy-GGPNA).
As preferred version further, above-mentioned detection method comprises the steps:
4) activity of the total gamma-glutamyl based transferase in the working sample serum;
5) add anti-apolipoprotein B antibody liquid and precipitation agent, centrifugal after 5～30 minutes, get supernatant, measure the activity of the gamma-glutamyl based transferase in the supernatant;
6) calculation procedure 1) with step 2) in the active difference of gamma-glutamyl based transferase.
The application of one specific specificity γ-Gu Anxianji transferring enzyme detection method is meant with method for preparing to be used to diagnose or the diagnostic reagent or the diagnostic kit of auxiliary diagnosis liver cancer.
As a kind of preferred version, above-mentioned diagnostic reagent or diagnostic kit comprise anti-apolipoprotein B antibody liquid, damping fluid, precipitation agent.
As further preferred version, described buffer reagent is pH7.0,30mmol/L MOPS damping fluid.
As further preferred version, described precipitation agent is 30% polyethylene glycol 6000.
Compared with prior art; The invention provides the diagnostic reagent or the diagnostic kit of a kind of simple and effective diagnosis or auxiliary diagnosis liver cancer; Experimental result shows; Specific detection serum LDL provided by the invention/VLDL-GGT activity methods can be used for detecting or the diagnostic reagent or the diagnostic kit of auxiliary detection liver cancer, and average specificity can reach more than 70%, and average positive predictive value is 90%.Therefore, its application prospect is very wide.
Description of drawings
Fig. 1 is the structural representation of specificity gamma-glutamyl based transferase detection kit provided by the invention.
Embodiment
Below in conjunction with embodiment and Comparative Examples to the present invention do further in detail, intactly explanation.
Embodiment
1, preparation specificity gamma-glutamyl based transferase detection kit (10 person-portion) is specifically as shown in Figure 1, comprising three pipe reagent, is respectively:
Antibody liquid (1): anti-people Apo B antibody+damping fluid;
Damping fluid (2) pH7.0,30mmol/L MOPS;
Precipitation agent (3): 30% polyethylene glycol 6000.
A. reagent is formed:
1) control group (gross activity group): pH7.0,30mmol/L MOPS damping fluid 850 μ L.
2) antibody group (to be determined group): contain anti-apolipoprotein B antibody 850 μ L, 30% polyethylene glycol 6000 precipitation agent, 420 μ L.
B. operation steps:
(1) antibody group: in centrifuge tube, add patients serum's sample 100 μ L, antibody liquid 80 μ L, precipitation agent 20 μ L place 20min for 37 ℃, centrifugal (5500g * 5min), get supernatant and on Biochemical Analyzer, measure the GGT activity.
(2) control group: in centrifuge tube, add patients serum's sample 100 μ L, damping fluid 80 μ L, precipitation agent 20 μ L place 20min for 37 ℃, centrifugal (5500g * 5min), get supernatant and on Biochemical Analyzer, measure the GGT activity.
(3) result calculates: active (U/L)=(control group pipe-antibody group pipe) * 2 of LDL/VLDL-GGT.
Annotate: the method that the GGT determination of activity can adopt each laboratory on Biochemical Analyzer, to use, reagent is provided for oneself.Concrete detection method is following:
A) detect principle: this law is a substrate with the bigger L-gamma-glutamyl-3-carboxyl-p-Nitroaniline of solubleness, and glycylglycine is the acceptor of γ-Gu Anxianji.Under the catalysis of GGT, glutamyl is transferred on the glycylglycine molecule, discharges xanchromatic 2-nitro-5-benzaminic acid simultaneously, causes increasing of 405nm～410nm wavelength absorbancy.Absorbancy increases speed and the active proportional relation of GGT.
B) detection reagent: reagent composition and the end level in reaction solution are following: pH7.7,37 ℃, 150mmol/L glycylglycine damping fluid, L-gamma-glutamyl-3-carboxyl-p-Nitroaniline of 6mmol/L, sample volume mark 1: 11.
C) detecting instrument parameter: 37 ℃ of temperature, wavelength 410nm, bandwidth≤2nm, cuvette optical path 1.0cm, incubation time 180s, time of lag 60s, monitoring time 180s, reading point>=6, factor 1159.
D) method of calculation: GGT (U/L)=Δ A/min * (106 * 2.75)/(9490 * 0.25)=Δ A/min * 1159 (molar absorptivity of 2-nitro-5-benzaminic acid at the 405nm place is 9490)
2, detect performance verification
1) ApoB antibody sedimentation effect:
Get 16 parts of serum samples, the LDL-C concentration range of 1～No. 8 sample is at 1.5～10.3mmol/L, and the GGT field of activity of 9～No. 16 samples is used the antibody liquid post precipitation respectively at 30～1950U/L, and supernatant carries out LEP.Electrophoresis result shows that all samples has only RHDL (HDL) 1 band, and does not have LDL and VLDL district band, explains that this ApoB antibody capable precipitates LDL and VLDL in the serum sample well.
2) ST influence:
Get 3 parts of basic, normal, high LDL/VLDL-GGT active sample, add behind antibody liquid and the precipitation agent respectively at 37 ℃ of placements 5,10,15,20,25,30min, the result shows, in 5～30min, and the sedimentation effect basically identical.It is the ST that this experiment box is selected 20min.
3) precision experiment:
Get the active 3 parts of fresh mix serum of different GGT, the GGT activity is respectively 52,264,875U/L, and active 15 times of every duplicate samples replication LDL/VLDL-GGT calculates withinrun precision.3 parts of active averages of serum LDL/VLDL-GGT are respectively 8.4,29,123U/L, and s is 0.69,1.78,4.38, and CV is 8.21,6.14,3.56% in batch.The every interval 1h of every duplicate samples measures, and every batch of repetition is averaged for 3 times, measures 5 batches altogether, calculates betweenrun precision.3 parts of active averages of serum LDL/VLDL-GGT are respectively 8.6,31,128U/L, and s is 0.84,2.24,6.98, and CV is 9.77,7.23,5.45% between batch.
4) linear experiment:
Get the active hepatocarcinoma patient serum of a high GGT; Through detecting the LDL/VLDL-GGT activity is 587U/L; Get a normal human serum through detecting the active 0U/L of being of LDL/VLDL-GGT, the two is pressed different ratios mix (1: 9～9: 1), the LDL-VLDL-GGT that measures each biased sample respectively is active.The result shows that the LDL-VLDL-GGT activity is good in 0～587U/L scope internal linear.
5) term of reference is by knowing that the LDL/VLDL-GGT normal reference value is≤10U/L in the serum in the above-mentioned linear experimental result.
6) test kit clinical application result
A) sample source: 150 parts of patients serums, wherein liver cancer is 65 parts, 53 parts of liver cirrhosis, 32 parts of chronic hepatitiss.All clinical diseases are per capita through pathologic finding, B ultrasonic and clinical definite.75 parts of health check-up healthy person serum specimens, wherein the man is 39,36 of woman, the range of age 20～54 years old, no haemolysis, piarhemia.After above serum is collected, put-30 ℃ of refrigerators and preserve.
B) the active detected result of each hepatic diseases group serum LDL/VLDL-GGT: 150 parts of hepatic diseases patients serum LDL/VLDL-GGT are detected, and concrete outcome is seen table 1.
The active result of each hepatic diseases group serum LDL of table 1/VLDL-GGT
Annotate: * *: compare p＜0.01, *: compare p＜0.05 with normal group with normal group;
##: compare p＜0.01 with the chronic hepatitis group;
◆ ◆: compare p＜0.01 with liver cirrhosis group;
△ △: compare p＜0.01 with slow liver+liver cirrhosis group.
The result who compares between group shows that the active median of liver cancer group LDL/VLDL-GGT is significantly higher than normal group and other hepatopathy groups.Serum LDL/VLDL-GGT activity reaches 86.15% to the diagnosis susceptibility of liver cancer.If liver cancer group and chronic hepatitis group are compared, its differential diagnosis specificity reaches 81.25%, and positive predictive value is 90.32%; Liver cancer group and liver cirrhosis group are compared, and its differential diagnosis specificity reaches 67.92%, and positive predictive value is 76.71%; Liver cancer group and chronic hepatitis are added liver cirrhosis group relatively, and its differential diagnosis specificity can reach 72.94%, and positive predictive value is 70.89%.
Experimental result shows, specific detection serum LDL provided by the invention/VLDL-GGT activity methods can be used for detecting or the diagnostic reagent or the diagnostic kit of auxiliary detection liver cancer.
Be necessary at last in this explanation to be: above instance only is used for technical scheme of the present invention is done explanation in further detail; Can not be interpreted as the restriction to protection domain of the present invention, some nonessential improvement that those skilled in the art's foregoing according to the present invention is made and adjustment all belong to protection scope of the present invention.
Claims (8)
1. a specific specificity γ-Gu Anxianji transferring enzyme detection method is characterized in that: through detecting the active difference with the gamma-glutamyl based transferase in the serum before and after the immunoprecipitation binding substances that forms between anti-apolipoprotein B antibody and low-density lipoprotein (LDL) and vldl (VLDL).
2. specificity gamma-glutamyl based transferase detection method according to claim 1 is characterized in that: the activity of the gamma-glutamyl based transferase in the serum behind the immunoprecipitation binding substances that forms between described anti-apolipoprotein B antibody and low-density lipoprotein (LDL) and vldl (VLDL) is for forming immune conjugate after the activity of the gamma-glutamyl based transferase in the centrifugal supernatant.
3. specificity gamma-glutamyl based transferase detection method according to claim 1 is characterized in that: the active method of described detection gamma-glutamyl based transferase is to be substrate and the continuous monitoring method that can on automatic clinical chemistry analyzer, use with L-gamma-glutamyl-3-carboxyl-p-Nitroaniline (3-carboxy-GGPNA).
4. specificity gamma-glutamyl based transferase detection method according to claim 1 is characterized in that: said specificity gamma-glutamyl based transferase detection method comprises the steps:
1) activity of the total gamma-glutamyl based transferase in the working sample serum;
2) add anti-apolipoprotein B antibody liquid and precipitation agent, centrifugal after 5～30 minutes, get supernatant, measure the activity of the gamma-glutamyl based transferase in the supernatant;
3) calculation procedure 1) with step 2) in the active difference of gamma-glutamyl based transferase.
5. the application of the said specificity gamma-glutamyl of claim 1 a based transferase detection method is characterized in that: be meant with method for preparing to be used to diagnose or the diagnostic reagent or the diagnostic kit of auxiliary diagnosis liver cancer.
6. according to the application of the said specificity gamma-glutamyl of claim 5 based transferase detection method, it is characterized in that: above-mentioned diagnostic reagent or diagnostic kit comprise anti-apolipoprotein B antibody liquid, damping fluid, precipitation agent.
7. according to the application of the said specificity gamma-glutamyl of claim 6 based transferase detection method, it is characterized in that: described buffer reagent is pH7.0,30mmol/L MOPS damping fluid.
8. according to the application of the said specificity gamma-glutamyl of claim 6 based transferase detection method, it is characterized in that: described precipitation agent is 30% polyethylene glycol 6000.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103290097A (en) * | 2013-05-24 | 2013-09-11 | 宁波美康生物科技股份有限公司 | Gamma-glutamoyl transferase detection reagent |
| CN113164110A (en) * | 2019-09-17 | 2021-07-23 | 诺尔生物医药有限公司 | System and method for measuring liver enzyme levels in blood |
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| CN100427609C (en) * | 2001-10-24 | 2008-10-22 | 加利福尼亚大学董事会 | Identification of 5-lipoxygenase as a major gene contributing to atherosclerosis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100427609C (en) * | 2001-10-24 | 2008-10-22 | 加利福尼亚大学董事会 | Identification of 5-lipoxygenase as a major gene contributing to atherosclerosis |
Non-Patent Citations (3)
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| ALDO PAOLICCHI ET AL: "-Lipoprotein- and LDL-associated serum -glutamyltransferase in patients with coronary atherosclerosis", 《ATHEROSCLEROSIS》, 31 December 2006 (2006-12-31) * |
| 于嘉屏 等: "免疫沉淀法测定血清中与两种载脂蛋白结合的γ-谷氨酰基转移酶活性及其诊断肝癌的价值", 《中华检验医学杂志》, vol. 26, no. 6, 30 June 2003 (2003-06-30) * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103290097A (en) * | 2013-05-24 | 2013-09-11 | 宁波美康生物科技股份有限公司 | Gamma-glutamoyl transferase detection reagent |
| CN103290097B (en) * | 2013-05-24 | 2014-12-24 | 宁波美康生物科技股份有限公司 | Gamma-glutamoyl transferase detection reagent |
| CN113164110A (en) * | 2019-09-17 | 2021-07-23 | 诺尔生物医药有限公司 | System and method for measuring liver enzyme levels in blood |
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Application publication date: 20121128 |