CN107345969A - Purposes of the serum markers comprising AFP, GP73 and CEACAM1 in diagnosing hepatic diseases - Google Patents
Purposes of the serum markers comprising AFP, GP73 and CEACAM1 in diagnosing hepatic diseases Download PDFInfo
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- CN107345969A CN107345969A CN201610292960.9A CN201610292960A CN107345969A CN 107345969 A CN107345969 A CN 107345969A CN 201610292960 A CN201610292960 A CN 201610292960A CN 107345969 A CN107345969 A CN 107345969A
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
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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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA
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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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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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
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
Abstract
The present invention relates to purposes of the serum markers comprising AFP, GP73 and CEACAM1 in diagnosing hepatic diseases.Specifically, the invention provides include the purposes at least one bound fraction for specifically binding AFP (alpha-fetoprotein) protein, the bound fraction of at least one specific binding GP73 (GP73) protein with the reagent of at least one bound fraction for specifically binding CEACAM1 (carcinomebryonic antigen relevant cell adhesion molecule 1) protein in preparation is for the kit of diagnosis and/or prognosis liver diseases.
Description
Technical field
The present invention relates to AFP (alpha-fetoprotein), GP73 (GP73) and CEACAM1
(carcinomebryonic antigen relevant cell adhesion molecule 1) is as the purposes in diagnosis for liver disease label.
In particular it relates to specifically exist with reference to AFP, GP73 and CEACAM1 antibody
Prepare the purposes in the kit for diagnosing liver cancer.
Background technology
WHO data shows within 2008, and the incidence of disease of the liver cancer in all tumours is located at the 5th,
The death rate be located at the 3rd, its be number five respectively in men and women's common cancer position and the 7th,
The same year new cases 74.83 ten thousand, death 69.59 ten thousand, and have the trend risen year by year.And
Hepatocellular carcinoma (HCC) accounts for overall 70% to 85% as the main parting of liver cancer,
Referring to Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM:
Estimates of worldwide burden of cancer in 2008:GLOBOCAN 2008.
International journal of cancer Journal international du cancer 2010,
127(12):2893-2917.Some early liver cancers can pass through ablation, ablation, embolism
Art successful treatment, and the effective treatment method of late stage treatment is few, referring to Global Cancer
Facts&Figures 3rd Edition；However, due to early stage HCC symptom unobvious and have
The shortage of the screening strategy of effect, it has been late period during HCC diagnosis more than 80%, and prognosis mala,
Referring to Sun, Y., et al. (2013) " Annexin A2 is a discriminative serological
candidate in early hepatocellular carcinoma."Carcinogenesis 34(3):
595-604；And Zhu, W.W.et al. (2013) " Evaluation of midkine as a
diagnostic serum biomarker in hepatocellular carcinoma."Clin Cancer Res
19(14):3944-3954.Survival rate is only 3%-5% within 5 years.
China is hepatitis B big country, and newly hair or death have accounted for world's entirety to liver cancer
50%, referring to Song P1, Gao J, Inagaki Y, et al.Biomarkers:evaluation of
screening for and early diagnosis of hepatocellular carcinoma in Japan and
china[J].Liver Cancer,2013,2(1):31-39.Great doctor is caused to China therefrom
Burden is treated, therefore, the prognosis early diagnosed to improving liver cancer patient is most important, for improving
Health of our people level is significant.
It is clinical at present that iconography progress hepatocarcinoma early diagnosis is mainly combined by alpha-fetoprotein (AFP).
AFP expressions in HCC patient substantially increase, and are clinical the most frequently used liver cancer markers.
MARRERO etc. (referring to Marrero JA, Feng Z, Wang Y, et al.Alpha-fetoprotein,
des-gamma carboxyprothrombin,and lectin-bound alpha-fetoprotein in
early hepatocellular carcinoma[J].Gastroenterology,2009,137(1):
110-118) 419 HCC patients and 208 early stage HCC patients are detected, defined
When 20NG/ML is critical value, AFP sensitivity and specificity are respectively 59% and 90%, 53%
With 90%.LOK etc. is (referring to Lok AS, Sterling RK, Everhart JE, et al.
Des-gamma-carboxy prothrombin and alpha-fetoprotein as biomarkers for
the early detection of hepatocellular carcinoma[J].Gastroenterology,2010,
138(2):493-502) result of study of 1031 parts of samples is shown, in HCC initial stage (0 month),
When critical value takes 20NG/ML and 200NG/ML, sensitivity is respectively 61% and 22%, special
Different degree is respectively 81% and 100%.AFP is still clinically very important HCC in China at present
Diagnosis index.
But AFP has higher false negative rate in hepatocarcinoma early diagnosis, on the other hand, iconography
Detection price is higher and is easily influenceed by operator's experience, it is difficult to liver cancer and non-malignant proliferative are distinguished,
Therefore it is badly in need of finding that new Specific marker is used for hepatocarcinoma early diagnosis.
At present it has been reported that going out some indexs and its kit for being applied to diagnosing cancer of liver, such as Gao Er
Base protein GP 73, the degree of accuracy of GP73 detection kits, sensitivity, repetition in China market
Property is still treated further perfect, and existing study has independent index GP73 detection kit (patent
Application number：2014107362520), it is 69% in diagnosing cancer of liver medium sensitivity, and specificity is
75%, golgiosome glycoprotein -73GP73 is entered by fluorescent micro-ball immune chromatography technology first
Row detection, has high sensitivity and high specific concurrently, is capable of quick detection 73GP73 content.This
Outside, the detection kit has the advantages that operation is fast and convenient, result is accurate, economic and practical,
It is small by the serious blood fat of serum (or blood plasma), haemolysis interference, when serum (or blood plasma) is blood red
When albumen≤500MG/L, triglycerides≤30MG/DL, the influence variation ＜ to the degree of accuracy
10%.
It is another to announce (number of patent application：200810103943.1) using fucosylation GP73 as inspection
The corresponding antibody and kit surveyed target and researched and developed, it is 76.9% that it, which diagnoses HCC sensitiveness, special
The opposite sex is 92.8%, related development ELISA kit.
However, a kind of preferable detection reagent is present needed exist for, can be easily from serum
Early liver cancer is detected, and distinguishes liver cancer and hepatic sclerosis, hepatitis etc. with high specificity and with sensitivity
Disease；Such reagent, which should have, simultaneously easily detects, is repeatable, can be surveyed through non-invasive procedures
The features such as determining.
AFP is the biochemical marker of classical hepatocellular carcinoma, Golgi protein GP 73 and cancer
Embryonal antigen relevant cell adhesion molecule CEACAM1 be the inventors discovered that novel liver cancer mark
The Typical Representative of thing.The inventors discovered that GP73, CEACAM1 are in diagnosis of HCC
In Sensitivity and Specificity it is all more preferable than AFP, especially sensitiveness.But three kinds of indexs
A certain degree of false negative and false positive when being used alone all be present, if passing through appropriate mode
And means, while three indexs of joint-detection, complement one another, it will help further reduce liver
The false negative and false positive of cell liver cancer clinical diagnosis.
The content of the invention
The invention provides use of the group reagent in the kit for diagnosing hepatic diseases is prepared
On the way, wherein the reagent include at least one specific binding AFP protein bound fraction,
The bound fraction of at least one specific binding GP73 protein and at least one specific binding
The bound fraction of CEACAM1 protein.
According to purposes of the present invention, wherein described specific binding AFP, GP73 and
The bound fraction of CEACAM1 protein is antibody or antigen-binding fragment.Further, institute
It is monoclonal antibody or polyclonal antibody to state antibody.
On the other hand, one group of antibody of the invention is in the kit for diagnosing hepatic diseases is prepared
Purposes, the antibody includes at least one AFP antibody, at least one GP73 antibody and extremely
A kind of few CEACAM1 antibody.
Further, according to purposes of the present invention, wherein the liver diseases are selected from B-mode
Hepatitis, hepatic sclerosis and hepatocellular carcinoma.
The invention provides use of the group reagent in the kit for prognosis liver diseases is prepared
On the way, wherein the reagent include at least one specific binding AFP protein bound fraction,
The bound fraction of at least one specific binding GP73 protein and at least one specific binding
The bound fraction of CEACAM1 protein.
Further, according to purposes of the present invention, wherein the liver diseases are selected from B-mode
Hepatitis, hepatic sclerosis and hepatocellular carcinoma.
In accordance with the purpose of the invention, wherein a group reagent is used to determine in experimenter's serum
The level of AFP, GP73 and CEACAM1 protein.
Brief description of the drawings
Figure 1A, the AFP special high expression in primary hepatocyte hepatocarcinoma (HCC).
AFP expression is through LUMINEX kit measurements, with median fluorescent intensity (MFI)
Represent, as indicated on y axis.With healthy control group, hepatitis B compares with hepatic sclerosis, and AFP is in original
High level expression in hair property hepatocellular carcinoma HCC.
Figure 1B, the GP73 high expression in hepatopathy group, and the expression in every kind of hepatopathy group is not
Together.GP73 expression is through LUMINEX kit measurements, with median fluorescent intensity (MFI)
Represent, as indicated on y axis.Compared with healthy control group, GP73 high tables in all hepatopathy groups
Reach；In hepatopathy group, expression of the GP73 in liver cirrhosis group is higher than hepatitis B group；GP73 is in HCC
Expression in group is higher than hepatitis B group；
Fig. 1 C, the CEACAM1 special high expression in primary hepatocyte hepatocarcinoma (HCC).
With healthy control group, hepatitis B group compares with liver cirrhosis group, and CEACAM1 is in HCC
Expression highest.In Figure 1A -1C each point represent an individual (each group number of cases HCC as
614, CIR is 53, and HBV is 73, and healthy control group is 264), it is horizontal
Line represents mean, and error line represents standard deviation.Statistics is examined using T, significant difference point
Do not indicated with following form：*P<0.05；**,P<0.01；***,P<0.001.
Fig. 2A -2C, compared with healthy control group, AFP, GP73 and CEACAM1 individually (scheme
2A) or joint (Fig. 2 B) detection detects the ROC curve of effect to primary hepatocyte hepatocarcinoma
Areal analysis (Fig. 2 C) under analysis and ROC curve.
Fig. 3 A-3C, compared, AFP, GP73 with other liver diseases groups (hepatitis B, hepatic sclerosis)
And individually (Fig. 3 A) or joint (Fig. 3 B) detect to primary liver cell liver CEACAM1
Areal analysis (Fig. 3 C) under the ROC curve analysis of cancer detection effect and ROC curve.
Fig. 4 A-4C, compared with healthy control group, AFP, GP73 and CEACAM1 individually (scheme
4A) or joint (Fig. 4 B) detection to early stage HCC detect effect ROC curve analysis and
Areal analysis (Fig. 4 C) under ROC curve.
Fig. 5 A-5C, compared, AFP, GP73 with other liver diseases groups (hepatitis B, hepatic sclerosis)
And individually (Fig. 5 A) or joint (Fig. 5 B) detection are detected and imitated CEACAM1 to early stage HCC
Areal analysis (Fig. 5 C) under the ROC curve analysis of fruit and ROC curve.
Fig. 6 A-6C, compared with healthy control group, GP73 and CEACAM1 are individually (Fig. 6 A)
Or joint (Fig. 6 B) detection detects the ROC curve analysis of effect for the HCC of AFP negative
With areal analysis under ROC curve (Fig. 6 C).
Embodiment
The reagent that the present invention uses is purchased from ORIGENE, ROCKVILLE, MD, USA.
Samples sources used in experiment in clinical laboratory of BJ Union Hospital, 307 hospitals digestion oncology,
Zhong Shan tumour hospital.The acquisition of serum sample passed through correlation Institutional Review Board (hospital/
Basis institute of medical courses in general institute) there is acquisition is informed to exempt.For healthy sample, be liver biochemistry detection value just
Often, no Hepatic disease, no excessive drinking history, without other entity tumors person；For HBV cases,
HBSAG (+) is required under the premise of requirements above；Cirrhosis case has liver impedance rheograph by oneself
Or clinic, laboratory, imaging diagnosis；Primary hepatocyte hepatocarcinoma, which is made a definite diagnosis, passes through histopathology
(ultrasound, computed tomography, magnetic resonance imaging, blood vessel are made for or two kinds of different images
Shadow art) display artery enhancing lesion.Sample, which is derived from, prohibits trencherman's forearm antecubital vein for 12 hours, quiet
Arteries and veins puncture promotees solidifying pipe collection sample after collecting sample, takes serum, carries out 10000R/MIN at 4 DEG C
10MIN is centrifuged, packing afterwards is frozen in -80 DEG C, in case being used during detection.
It is every in 96 hole filter membrane plates (being purchased from Pall Corporation, CATA#8049) during detection
Hole adds 100 μ L mixture of microspheres, and the mixture of microspheres contains while is coated with AFP antibody
(being purchased from R＆D SYSTEMS, CATA#LXSAH-01), GP73 antibody (are purchased from
ORIGENE, CATA#TA600480), (paired antibody is purchased from R＆D to CEACAM1 antibody
SYSTEMS DUOSET, CATA#DY2244, wherein HUMAN CEACAM-1
CAPTURE ANTIBODY PART#842283) polyethylene microballoon, and remove liquid,
Afterwards 50 μ L capture buffer solutions (being purchased from ORIGENE companies) are added per hole by row's plate design
And 50 μ L standard items, the standard items contain AFP, GP73, CEACAM1 purifying protein
(this experiment Plays product use 4 times of gradient dilutions, from Origene Technology Inc.,
Wherein, AFP CATA#TP306622；GP73 TP300086；CEACAM1's
CATA#TP710040；), or dilution after measuring samples (picked up from subject's by sample product
Serum sample, with sample diluting liquid 1:10 dilutions), shaking table is incubated 2 hours, with washing lotion (purchase
From ORIGENE companies, AR100010) clean 3 times and remove；Detection antibody is added per hole,
The detection antibody contains biotinylated AFP antibody and (comes from BIOCHECK, P/N
70647), GP73 antibody (coming from ORIGENE, TA700480 2D6, the anti-GP73 of mouse),
CEACAM1 antibody (comes from R＆D SYSTEMS DUOSET, HUMAN CEACAM-1
DETECTION ANTIBODY PART#842285), 50 μ L, it is small that shaking table is incubated 1
When, washing lotion is cleaned 3 times and removed afterwards；50 μ L detection reagents, the detection are added per hole
Reagent contains the Avidin (being purchased from ORIGENE companies) of phycoerythrin mark, and shaking table is incubated
30 minutes, afterwards washing lotion clean and 3 times and remove；Use LUMINEX BIO-PLEX200 couple
Detection hole fluorescence intensity is directly detected, and median fluorescent intensity is converted by standard curve and obtained
Corresponding protein concentration.The direct detected value of instrument is fluorescence intensity (FI), as a result with median fluorescent
Intensity (MFI) and concentration value (returning conversion by 5 points to obtain concentration value by median fluorescent intensity)
Report.
As a result
AFP, GP73 and CEACAM1 are hard in primary hepatocyte hepatocarcinoma group (HCC), liver
Median fluorescent luminous intensity in change group (CIR), hepatitis B group (HBV) and healthy control group
MFI values.
The MFI values of table 1, AFP, GP73 and CEACAM1 in different groups
Table 2, individually and AFP, GP73 and CEACAM1 label is used in combination in HCC VS
HCC ROC analysis results are diagnosed in normal healthy controls.
Table 3, individually and AFP, GP73 and CEACAM1 label is used in combination in HCC VS
HCC ROC analysis results are diagnosed in CIR+HBV.
Table 4, individually and AFP, GP73 and CEACAM1 label is used in combination in early stage HCC
The ROC analysis results of early stage HCC are diagnosed in VS normal healthy controls.
Table 5, individually and AFP, GP73 and CEACAM1 label is used in combination in early stage HCC
The ROC analysis results of early stage HCC are diagnosed in VS CIR+HBV.
Table 6, individually and GP73 and CEACAM1 labels are used in combination in AFP negative
HCC ROC analysis results are diagnosed in HCC VS normal healthy controls.
(liver cancer is sent out for table 7, AFP, GP73, CEACAM1 combination HCC analysis of clinical
Open up index of correlation) result
Table 8, AFP, GP73, CEACAM1 combination HCC analysis of clinical (hepatic injuries
Index) result
Claims (10)
1. purposes of the group reagent in the kit for diagnosing hepatic diseases is prepared, wherein institute
State bound fraction, at least one spy that reagent includes at least one specific binding AFP protein
The opposite sex combines the bound fraction of GP73 protein and at least one specific binding CEACAM1
The bound fraction of protein.
2. purposes according to claim 1, wherein described specific binding AFP, GP73
Bound fraction with CEACAM1 protein is antibody or antigen-binding fragment.
3. purposes according to claim 2, wherein the antibody is monoclonal antibody or more
Clonal antibody.
4. purposes of the one group of antibody in the kit for diagnosing hepatic diseases is prepared, described anti-
Body includes at least one AFP antibody, at least one GP73 antibody and at least one CEACAM1
Antibody.
5. according to the purposes described in any one of Claims 1-4, wherein the liver diseases select
From hepatitis B, hepatic sclerosis and hepatocellular carcinoma.
6. purposes of the group reagent in the kit for prognosis liver diseases is prepared, wherein institute
State bound fraction, at least one spy that reagent includes at least one specific binding AFP protein
The opposite sex combines the bound fraction of GP73 protein and at least one specific binding CEACAM1
The bound fraction of protein.
7. purposes according to claim 6, wherein described specific binding AFP, GP73
Bound fraction with CEACAM1 protein is antibody or antigen-binding fragment.
8. purposes according to claim 7, wherein the antibody is monoclonal antibody or more
Clonal antibody.
9. according to the purposes described in any one of claim 6 to 8, wherein the liver diseases select
From hepatitis B, hepatic sclerosis and hepatocellular carcinoma.
10. according to the purposes described in claims 1 to 3 or 6 to 8 any one, wherein described one
Group reagent is used for the water for determining AFP, GP73 and CEACAM1 protein in experimenter's serum
It is flat.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110954701A (en) * | 2019-12-18 | 2020-04-03 | 重庆医科大学 | Diagnostic kit for hepatic fibrosis or cirrhosis |
| CN111487411A (en) * | 2019-01-29 | 2020-08-04 | 广州瑞博奥生物科技有限公司 | Novel application of CEACAM1 polypeptide |
| CN112567051A (en) * | 2018-06-14 | 2021-03-26 | 给科生物研究室株式会社 | Liver cancer specific biomarkers |
| CN112782297A (en) * | 2020-12-24 | 2021-05-11 | 郭继生 | Liver cirrhosis related biomarker and screening method and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112567051A (en) * | 2018-06-14 | 2021-03-26 | 给科生物研究室株式会社 | Liver cancer specific biomarkers |
| CN111487411A (en) * | 2019-01-29 | 2020-08-04 | 广州瑞博奥生物科技有限公司 | Novel application of CEACAM1 polypeptide |
| CN110954701A (en) * | 2019-12-18 | 2020-04-03 | 重庆医科大学 | Diagnostic kit for hepatic fibrosis or cirrhosis |
| CN112782297A (en) * | 2020-12-24 | 2021-05-11 | 郭继生 | Liver cirrhosis related biomarker and screening method and application thereof |
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Application publication date: 20171114 |