CN109557312A - Hepatocarcinoma early diagnosis method and diagnostic kit - Google Patents
Hepatocarcinoma early diagnosis method and diagnostic kit Download PDFInfo
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Abstract
The method that the present invention provides a kind of to carry out hepatocarcinoma early diagnosis in subject comprising: 1) from the subject obtain biological sample；2) content of multiple biomarkers in the biological sample is detected, two or more in AFP, AFP-L3, GP73, Fuc-GP73, DCP, CEA, CA19-9, TK1, DKK1, γ-GT, ALP, AFU, ALT and Fuc-Kininogen 1 of the multiple biomarker；3) testing result based on step 2) calculates the probability that the subject suffers from liver cancer；4) probability is compared with given threshold, wherein when the probability is more than or equal to given threshold, then it is assumed that the subject suffers from liver cancer；When the probability is less than given threshold, then it is assumed that the subject does not suffer from liver cancer.The present invention also provides corresponding detection kit and detection devices.The present invention improves the sensitivity of hepatocarcinoma early diagnosis, specificity using the joint-detection of multiple markers, provides relatively reliable foundation for the clinical diagnosis of liver cancer.
Description
Technical field
The present invention relates to the united diagnosing cancer of liver methods of the method for hepatocarcinoma early diagnosis, especially polyprotein marker.This
Invention further relates to kit and equipment for hepatocarcinoma early diagnosis.
Background technique
Hepatocellular carcinoma HCC (hepatocellular carcinoma) is clinically common one of malignant tumour, master
The teiology wanted is HCV or HBV Chronic Liver virus infection.Currently, the Symptomatic chronic hepatitis B patients of China are about 2000
Ten thousand people, wherein nearly 25% to 30% chronic hepatitis B patient can develop as cirrhosis.Since numerous hepatopaths and liver cancer are high-risk
Crowd, Chinese onset of liver cancer rate are in first of the whole world.World Health Organization's report in 2015 points out that there are 78.2 ten thousand neopathies in the liver cancer whole world
Example, 74.5 ten thousand deaths.Wherein, Chinese neopathy number of cases and death number account for about 50%.
Clinical treatment discovery turns even if recurrence occurs in row radical operation still has more than 50% patient in postoperative 5 years
It moves.Once there is relapse and metastasis, the recent follow-up survival rate of patient is extremely low.Since liver cancer early symptom and sign are unobvious, to
It was found that when major part patient be middle and advanced stage, miss best occasion for the treatment, and early stage makes a definite diagnosis, early treatment can effectively prolong
The life cycle of long patient.It is very important so establishing early diagnosis and monitoring mechanism in people at highest risk.
Tumor markers (tumor markers, TM) are used as an important indicator, play in the early diagnosis of tumour
Increasingly important role.TM refers to be occurred to be synthesized, discharged by tumour cell itself, or by machine in breeding in tumour
Body reacts tumour cell and the signal tumor of generation exists and a substance of growth.These substances are not deposited in normal adult
It is significantly higher than normal person in the level either occurred in cancer patient.Tumor markers detection technique is considered early at present
Phase finds that the unique channel of asymptomatic micro- stove tumour, this detection technique can be prior to X-rays, ultrasound, CT, MRI or PET-CT etc.
Physical inspection finds tumour, can be used for the screening of people at highest risk's malignant tumour, diagnosing tumor and antidiastole, assesses the effect for the treatment of
Fruit, prediction or monitoring tumor recurrence or transfer.
At present AFP AFP for the most-often used tumor markers of hepatocarcinoma early diagnosis, due to AFP have with
The relative specificity of HCC, therefore be used widely at present, but in the benign hepatopathy in part AFP be it is positive (specificity≤
75%) and in the liver cancer of part AFP is negative (sensitivity≤70%), so that single carries out HCC early warning and monitoring tool by AFP
There is certain limitation, and AFP detection HCC sensitivity relevant for HCV is not high.
In addition to AFP, can be used for the marker of diagnosing cancer of liver, there are also AFP heteroplasmons (AFP-L3), Gorky's transmembrane protein
(GP73), Golgi apparatus protein heteroplasmon (Fuc-GP73), serum abnormal factor (DCP), carcinomebryonic antigen (CEA), cancer are anti-
Former 19-9 (CA19-9), thymidine kinase 1 (TK1), secreted protein (Dikkopf-1, DKK1), gamma glutamyl transpeptidase (γ-
GT), alkaline phosphatase (ALP), alpha-L-fucosidase (α-L-Fucosidase, AFU), glutamic-pyruvic transaminase (ALT), kininogen
1 heteroplasmon (Fuc-Kininogen 1) etc., but each single index checkout and diagnosis liver cancer sensitivity, specificity it is lower.
Summary of the invention
In order to overcome the above problem, on the one hand, the present invention provides one kind to carry out hepatocarcinoma early diagnosis in subject
Method comprising:
1) biological sample is obtained from the subject；
2) detect the content of multiple biomarkers in the biological sample, the multiple biomarker be selected from AFP,
AFP-L3, GP73, Fuc-GP73, DCP, CEA, CA19-9, TK1, DKK1, γ-GT, ALP, AFU, ALT and Fuc-
Two or more in Kininogen 1；
3) testing result based on step 2) calculates the probability that the subject suffers from liver cancer；
4) probability is compared with given threshold, wherein then recognizing when the probability is more than or equal to given threshold
Liver cancer is suffered from for the subject；When the probability is less than given threshold, then it is assumed that the subject does not suffer from liver cancer.
In some embodiments, step 2) is also included in gender and/or the year of the subject when calculating the probability
Age.For example, women is 0, male 1.
In some embodiments, step 3) is using based on the biomarker testing result and/or the subject
Gender and/or the Multiple regression model at age come calculate the subject suffer from liver cancer probability.
In some embodiments, the biological sample is serum or blood plasma.
On the other hand, the present invention also provides a kind of in subject carries out the detection kit of hepatocarcinoma early diagnosis, packet
Include the reagent of the content for detecting multiple biomarkers in the biological sample from the subject, the multiple biology
Marker be selected from AFP, AFP-L3, GP73, Fuc-GP73, DCP, CEA, CA19-9, TK1, DKK1, γ-GT, ALP, AFU, ALT,
Two or more in Fuc-Kininogen 1.
In some specific embodiments, the reagent in the kit for detecting the biomarker includes as follows
Two or more:
1) the magnetic bead coupled antibody of the enzyme labelled antibody of AFP and/or AFP；
2) the magnetic bead coupled antibody of the enzyme labelled antibody of GP73 and/or GP73；
3) the magnetic bead coupled antibody of the enzyme labelled antibody of CEA and/or CEA；
4) the magnetic bead coupled antibody of the enzyme labelled antibody of CA19-9 and/or CA19-9；
5) the magnetic bead coupled antibody of the enzyme labelled antibody of DCP and/or DCP；
6) the magnetic bead coupled antibody of the enzyme labelled antibody of DKK1 and/or DKK1；
7) the magnetic bead coupled antibody of the enzyme labelled antibody of TK1 and/or TK1；
8) the magnetic bead coupled antibody of the enzyme labelled antibody of ALT and/or ALT；
9) the magnetic bead coupled antibody of the enzyme labelled antibody of AFU and/or AFU；
11) the magnetic bead coupled antibody of the enzyme labelled antibody of γ-GT and/or γ-GT；
12) the magnetic bead coupled antibody of the enzyme labelled antibody of Kininogen 1 and/or Kininogen 1；
13) enzyme mark agglutinin LcA；And
14) reaction substrate of ALP.
In some embodiments, the reagent is placed in test strip.
In some embodiments, the kit further includes the biomarker as standard items.
In some embodiments, the kit further includes kit operation instructions, the kit operation instruction
Secretary is loaded with kit application method and the property using testing result and/or the subject based on the biomarker
Not and/or the age Multiple regression model come calculate the subject suffer from liver cancer probability.
On the other hand, the present invention also provides a kind of equipment for carrying out hepatocarcinoma early diagnosis in subject, including
Detection module, data processing module and display module, wherein the detection module be used for using the detection kit detection come
From the content of multiple biomarkers in the biological sample of the subject, the multiple biomarker is selected from AFP, AFP-
L3, GP73, Fuc-GP73, DCP, CEA, CA19-9, TK1, DKK1, γ-GT, ALP, AFU, ALT and Fuc-Kininogen
Two or more in 1；The data processing module is built-in with logistical regression algorithm, for being based on the multiple life
The gender and/or age of the testing result of object marker and the subject calculate the probability that the subject suffers from liver cancer；
And the display module is for exporting calculated result.
The present invention improves the sensitivity of hepatocarcinoma early diagnosis, specificity using the joint-detection of multiple protein marker,
Relatively reliable foundation is provided for the clinical diagnosis of liver cancer.
Detailed description of the invention
Fig. 1 is 1 test card of AFP/GP73/CEA/CA19-9/DCP/DKK1/TK1/AFU/ALT/ γ-GT/Kininogen
Bar schematic diagram.
Fig. 2 is that Fuc-Kininogen 1/AFP-L3/Fuc-GP73/AFU tests strip schematic diagram.
Fig. 3 is that ALP tests strip schematic diagram.
Specific embodiment
Unless otherwise indicated, all technical and scientific terms used herein has those of ordinary skill in the art institute usually
The meaning of understanding.
" subject ", which refers to, suffers from or suspects the individual (preferably people) with certain disease, alternatively, in prediction risk
When " subject " may also comprise healthy individuals.The term usually can with it is mutual with " patient ", " test object ", " treatment object " etc.
Change use.
" early diagnosis " used when referring to liver cancer herein refer to can prior to X-ray, ultrasound, CT, etc. physical inspection means
Carry out the method that diagnosing tumour whether there is.
Alpha-fetoprotein (Alpha-fetoprotein, AFP) is a kind of glycoprotein, belongs to albumin family, mainly by fetus
Liver cell and yolk bag synthesis.AFP concentration with higher in fetal circulation, then declines after birth, until birth after 2 to
More difficult detection in 3 months blood, therefore content is extremely low in adult serum.When liver cell or the generation of gonad embryonic tissue are pernicious
When lesion, AFP gene is reactivated, and makes the cell for having lost synthesis AFP ability originally start to synthesize again, then blood
In can be detected AFP concentration significantly increase, therefore the detection of AFP concentration have to hepatocellular carcinoma and trophocyte malignant tumour it is important
Clinical value.With biochemistry and the progress and application of Correlation Analysis Technique, it is found that AFP molecule is not single molecule,
But it is variant with the affinity of ectogenous agglutinine, that is, there is sugar chain heterogeneity.Using different agglutinin affinity electrophoresis or
They can be divided into several components (heteroplasmon) using isoelectric focusing technique by person.Wherein, AFP heteroplasmon AFP-L3 is small flat
Beans agglutinin (LcA) mating type AFP, and it is related to hepatocellular carcinoma.It is thin that the detection of AFP-L3 in approval in 2005 is used for liver by FDA
The early warning of born of the same parents' cancer.
Gorky's transmembrane protein (Golgi protein 73, GP73), also known as II type golgiosome memebrane protein (GOLPH2)
Or Golgi membrane protein I (GOLM1), it is transmembrane glycoprotein, the suitable face of golgiosome is positioned at, mainly by intracellular region, cross-film
Area and extracellular region three parts composition.The study found that GP73 has obvious expression in hepatitis, cirrhosis and liver cancer, especially with liver cancer
It is the most significant.GP73 also has heterogeneity, and including the GP73 of core fucosylation modification, i.e. Golgi apparatus protein is heterogeneous
Body Fuc-GP73 is in research in the application value in terms of diagnosis of hepatoma.
Serum abnormal factor, or be de--γ-carboxyl factor (Des-gamma-carboxy-
Prothrombin, DCP), the incomplete carboxylation of one or more glutaminic acid residues is γ-carboxyl in γ-carboxyglutamic acid structure
Glutamic acid causes it to lose normal coagulation function.DCP positive rate in the serum of Patients with Primary is high, it is considered to be one
The strong primary liver cancer markers of species specificity.
Carcinomebryonic antigen (carcinoembryonic antigen, CEA) be nineteen sixty-five by Gold and Freedman first from
A kind of tumor associated antigen extracted in colon cancer and embryonic tissue is a kind of acid sugar egg with human embryos antigenic characteristic
It is white, it is present in the cancer cell surface that endoderm cell differentiates, is the structural proteins of cell membrane.It is formed in cytoplasm,
It is secreted by cell membrane extracellularly, subsequently into ambient body fluid.In the past using CEA as early diagnosis colon cancer and the carcinoma of the rectum
Specific marker, through a large amount of clinical practice, the malignant tumour CEA value of discovery not only gastrointestinal tract can be increased, breast cancer,
Also there is raising in the serum of lung cancer and other malignant tumours.Therefore, carcinomebryonic antigen is a kind of broad-spectrum tumor marker,
CA 19-9 (cancer antigen 19-9, CA19-9), also referred to as sugar antigen CA19-9
(carbohydrate antigen 19-9) is a kind of glycoprotein tumor markers of mucin type, reason mouse monoclonal antibody
116NS19-9 is identified and is named.CA 19-9 is the high marker of a kind of pair of cancer of pancreas sensibility.
Thymidine kinase 1 (thymidine kinase 1, TK1) is one of two kinds of forms of thymidine kinase, is present in thin
It is isodynamic enzyme with the thymidine kinase 2 being present in mitochondria in cytoplasm.TK1 content in proliferative cell is high, and in the cell cycle
The S phase reach peak value, content is extremely low in resting cell.It has been found that the raised serum levels and malignant proliferative lesion of TK1
It is related.Relative to normal person, TK1 content can increase 2 to 200 times in malignant proliferation lesion patients serum.
Secreted protein (Dickkopf-related protein 1, DKK1) is Dickkopf protein family member, is
It is a kind of containing there are two the secreting glycoprotein of rich cysteine regions, it and the Wnt access receptor being located on cell membrane, low-density
Lipoprotein receptor relative protein 5 (LRP5)/LRP6 and DKK1 co-receptor Kremen1 or Kremen2 is combined, and forms endosome,
Inhibit Wnt signal path, adjusts platform development and tumor development.Its high expression in a variety of human tumors, and and Partial tumors
Prognosis it is closely related.
Gamma glutamyl transpeptidase (γ-glutamyl-transferase, γ-GT) is a kind of liver enzyme, for transporting paddy
The sweet peptide of Guang and amino acid enter cell, and the γ-glutamyl being catalyzed on glutathione is transferred to another peptide or another amino
On acid.γ-GT is distributed widely in tissue, especially rich content in kidney, liver and pancreas.In normal human serum
γ-GT is mainly from liver.When primary or metastatic hepatic carcinoma, γ-GT is significantly raised in blood, the reason is that cancer cell generated
γ-GT increases to be acted on cancerous tissue itself or surrounding inflammatory stimulus, increases the permeability of liver plasma membrane, so that in blood
γ-GT increases.
Alkaline phosphatase (alkaline phosphatase, ALP) belongs to heterodimeric protein, and molecular weight is about
56KDa.Phosphate group on substrate molecule can be removed corresponding substrate dephosphorylation by it by hydrolysis phosphate monoester,
And phosphate anion and free hydroxyl are generated, this kind of substrate includes nucleic acid, albumen, alkaloid etc..ALP is mainly used for obstructive
The inspection of jaundice, primary carcinoma of liver, secondary carcinoma of liver, Cholestatic hepatitis etc..When suffering from these diseases, liver cell is excessively manufactured
ALP enters blood through lymphatic channel and sinus hepaticus, simultaneously because biliary tract bile excretion disorder, reflux enter blood and cause serum levels of ALP in liver
It is significantly raised.
Alpha-L-fucosidase (α-L-fucosidase, AFU) is a kind of lysosomal acid hydrolase, and molecular weight is about
270~390KD is primarily involved in the catabolism of the macromolecular substances such as various glycolipids, glycoprotein, the mucopolysaccharide containing fucosido.
It is widely present in the various histocyte lysosomes of human body and body fluid in.Usual liver cancer patient alpha-L-fucosidase obviously rises
Height, it is considered as a kind of tumor marker of primary carcinoma of liver at present.
Glutamic-pyruvic transaminase (alanine transaminase, ALT) is present in various cells, is especially most with liver cell.
When normal, as long as in liver cell in a small amount of ALT release people's blood, the enzymatic activity in serum can be significantly raised.Various viral
When the acute stage of hepatitis, drug poisoning necrosis of liver cells, ALT is largely released into blood, thus it be Diagnosis Virus Hepatitis,
The important indicator of toxic hepatitis.The concentration of ALT is 1000 to 3000 times higher than serum in liver cell.As long as there is 1% liver cell
Necrosis can make enzymatic activity in blood significantly raised, therefore ALT is the Sensitive mark of acute hepatocellular damage.
Prokineticin 1 (Kininogen 1, KNG1), also referred to as α -2 thiol protease inhibitor (alpha-2-thiol
Proteinase inhibitor) or high molecular weight kininogen (HMWK), mainly produced by liver cell and a small amount of vascular endothelial cell
It is raw, it is one of the coded product of KNG1 gene.KNG1 gene generates two different albumen by alternative splicing: high molecular weight swashs
Peptide former (HMWK) and low molecular weight kininogen (LMWK).Assembling of the HMWK for blood clotting and kallikrein kinin system
Be important, with the active bradykinin of various biological (bradykinin) be high molecular weight kininogen enzymolysis product it
One, and low molecular weight kininogen and it is not involved in blood clotting.1 heteroplasmon of Kininogen be (core fucosylation modification
Kininogen 1) Fuc-Kininogen 1, it is related with hepatocellular carcinoma, there is application well in terms of diagnosis of hepatoma
Potentiality.
In specific embodiment below, the detection method of above-mentioned 14 kinds of markers is provided.These methods include horseradish
The method of peroxidase HRP labelled antibody, the method for magnetic bead coupled antibody, the production method for testing strip and on instrument
Detection method.
Marking fluid, flushing liquor involved in this paper, biological sample dilutions liquid and preservation liquid are preferably phosphate buffer, carbon
One of phthalate buffer, Tris-HCl buffer and morpholino b acid buffer are a variety of.
10mM carbonate buffer solution (the 10mM CB of the preferred pH 9.5 of HRP marking fluid involved in Examples below
Buffer, pH 9.5)；0.1M morpholino b acid buffer (the 0.1M MES of the related preferred pH 6.0 of marked by magnetic bead liquid
Buffer, pH 6.0)；The related preferred pH 7.4 of HRP flushing liquor 10mM phosphate buffer (10mM PB buffer,
PH 7.4,0.15M NaCl, 0.1%Tween 20)；The 25mM Tris-HCl of the related preferred pH7.2 of magnetic bead flushing liquor is slow
Fliud flushing (25mM Tris-HCl buffer, pH 7.2,0.15M NaCl, 0.1%Tween 20)；Related immune response punching
10mM phosphate buffer (10mM PB buffer, pH 7.4, the 0.15M NaCl, 0.1%Tween of the preferred pH 7.4 of washing lotion
20)；The related preferred pH 7.4 of biological sample dilutions liquid 10mM phosphate buffer (10mM PB buffer, pH 7.4,
0.15M NaCl, 1%BSA)；Related HRP labelled antibody saves the 10mM phosphate buffer (10mM of the preferred pH 7.4 of liquid
PB buffer, pH 7.4,0.15M NaCl, 1%BSA, 0.05%PC300)；It is excellent that related magnetic bead coupled antibody saves liquid
Select pH 7.2 25mM Tris-HCl buffer (25mM Tris-HCl buffer, pH 7.2,0.15M NaCl, 0.05%
Tween 20,0.05%PC300).
Used subject's biological sample is serum or blood plasma.
1 AFP of embodiment GP73 CEA CA19-9 DCP DKK1 TK1 AFU ALT TK1 γ-GT Kininogen
1 detection
Principle:
The detection of AFP is carried out in test strip using enzymatic sandwich immunoassay chemiluminescence.AFP in biological sample
After antigen and the AFP monoclonal antibody of magnetic bead coupling incubate in reacting hole, it is compound that Ag-Ab is formed by immune response
Object.After Magneto separate and flushing, the compound being incorporated on magnetic bead is adsorbed in magnetic field, and unbonded material is then rinsed.It will
The antigen-antibody complex is drawn onto the reacting hole of the AFP monoclonal antibody containing horseradish peroxidase (HRP) label, temperature
Antibody-antigen-antibody sandwich complex is formed after educating, unbonded material is rinsed after Magneto separate.The sandwich complex and HRP
Catalytic luminescence substrate reactions generate chemiluminescence signal, and signal strength is directly proportional to the concentration of AFP in sample.
Operation:
1.HRP marks AFP antibody
A) 10mg HRP is weighed, is dissolved in 2mL distilled water, HRP enzyme solutions are prepared；
B) the above-mentioned HRP enzyme solutions of 0.5mL are taken, the 0.088M NaIO that 50 μ L now match is added4In solution, it is protected from light mixing；
C) room temperature is protected from light 15min；
D) solution after reaction is added in the 30kD super filter tube of 500 μ L, is centrifuged 3min in 7000rpm, adds 200 μ L
HRP marking fluid, ultrafiltration 5 times；
E) HRP solution is collected, it is 1mL that HRP marking fluid to total volume, which is added,；
F) it takes 50 μ L AFP antibody (1mg/mL) to be added in the 50kD super filter tube of 500 μ L, 450 μ L HRP label is added
Liquid is centrifuged 3min in 7000rpm, adds 200 μ L HRP marking fluids into inner tube every time, be repeated 3 times；
G) back-off super filter tube inner tube is centrifuged 1min in 3000rpm, throws away liquid into new casing；It is added into inner tube
200 μ L HRP marking fluids, are centrifuged again；The HRP solution for taking 20 μ L to activate is added in the casing for collecting antibody-solutions, room temperature
React 2h；
H) liquid moves in inner tube after reacting in casing, is centrifuged 3min in 7000rpm, adds 200 μ L HRP punching every time
Washing lotion, ultrafiltration 5 times；
I) back-off super filter tube inner tube is centrifuged 1min in 3000rpm, throws away liquid into new casing；It is added into inner tube
200 μ L HRP flushing liquors, are centrifuged again；
J) HRP labelled antibody is added into casing and saves liquid, total volume is adjusted to 500 μ L, is sub-packed in 0.6mL EP pipe,
Every 50 μ L of pipe.It is placed in 4 DEG C of refrigerators and saves.
2. magnetic bead is coupled AFP antibody
A) it takes 10 μ L (100mg/mL) magnetic beads to be added in 2mL EP pipe, 1mL marking fluid is added, vortex oscillation mixes, will
EP pipe is placed on magnetic frame, after Magneto separate, is moved and is abandoned supernatant, repeats this step 2 time；
B) 0.5mL marked by magnetic bead liquid is added, vortex oscillation mixes, and 5 μ L EDC (10mg/mL, now with existing is added into EP pipe
With), 13 μ L NHS solution (10mg/mL, ready-to-use), vortex oscillation mixes, and is incubated at room temperature 15min；
C) EP pipe is placed on magnetic frame, after Magneto separate, moves and abandon supernatant, 0.5mL marked by magnetic bead liquid, vortex oscillation is added
It mixes；
D) 20 μ L antibody (1mg/mL) are added into EP pipe, vortex oscillation mixes, and is incubated at room temperature 3h；
E) EP pipe is placed on magnetic frame, after Magneto separate, moves and abandon supernatant, 1mL magnetic bead flushing liquor is added, repeats the step
4 times；
F) 1mL magnetic bead coupled antibody is added and saves liquid, vortex oscillation is mixed, stored in 4 DEG C of refrigerators.
The production of 3.AFP test strip (see Fig. 1)
A) No. 1 hole for testing strip is well；
B) No. 2 holes for testing strip are sample dilution holes；
C) 10 times of dilutions of the above-mentioned magnetic bead coupled antibody of 50 μ L are taken, are lyophilized in No. 3 holes of test strip；
D) 100 times of dilutions of the 100 above-mentioned enzyme labelled antibodies of μ L are taken, are lyophilized in No. 4 holes of test strip；
E) 600 μ L immune response flushing liquor is added in No. 5 holes of test strip；
F) 100 μ L HRP catalytic luminescence substrate solutions are added in No. 6 holes；
G) No. 7 holes for testing strip are waste liquid hole；
H) No. 8 holes for testing strip are instrument connection.
The detection of 4.AFP blood sample
A) 10 μ L blood samples are added in No. 1 hole of test strip；
B) from taking 190 μ L of biological sample dilutions liquid to be added in No. 1 hole in No. 2 holes, sample is diluted 20 times；
C) be added in No. 3 holes from drawing 100 μ L in the sample after dilution, oscillation mix after in 37 DEG C of incubations 15min, general
Reaction solution stands 1min in magnetic field, and waste liquid is discharged after Magneto separate；
D) from drawn in No. 5 holes 100 μ L immune response flushing liquor be added in No. 3 holes, concussion mix after it is quiet in magnetic field
1min is set, waste liquid is discharged after Magneto separate, repeats the step 2 time；
E) from drawn in No. 5 holes 100 μ L immune response flushing liquor mixed with previous step reactant after be added in No. 4 holes,
In 37 DEG C of incubation 15min after oscillation mixing, reaction solution is stood into 1min in magnetic field, waste liquid is discharged after Magneto separate；
F) it is added in No. 4 holes from drawing 100 μ L immune response flushing liquor in No. 5 holes, stands 1min in magnetic field, magnetic point
Waste liquid is discharged from after；Repeat the step 3 time.
G) it is added in No. 4 holes from drawing 100 μ L HRP catalytic luminescence substrates in No. 6 holes, concussion injects No. 8 holes after mixing
In, luminous value (RLU) is detected after 25 DEG C of incubation 2min.
The detection of GP73, CEA, CA19-9, DCP, DKK1, TK1, AFU, ALT, TK1, γ-GT and Kininogen 1 are same
Antibody is only changed into corresponding monoclonal antibody by AFP respectively.
2 Fuc-Kininogen 1 of embodiment AFP-L3 Fuc-GP73 detection
Principle:
The detection of Fuc-Kininogen 1 is carried out in test strip using enzymatic sandwich immunoassay chemiluminescence.It is raw
After 1 monoclonal antibody of Kininogen of 1 antigen of total Kininogen and magnetic bead coupling in object sample incubates in reacting hole,
Antigen-antibody complex is formed by immune response.After Magneto separate and after rinsing, the compound being incorporated on magnetic bead is adsorbed on
In magnetic field, unbonded material is then rinsed.The antigen-antibody complex is drawn onto the agglutinin (LcA) containing HRP label
In reacting hole, fucosylated 1 antigen of Kininongen forms the fucosylated antigen-LcA of antibody-in conjunction with LcA after incubation
Sandwich complex, unbonded material is rinsed after Magneto separate.The sandwich complex and HRP catalytic luminescence substrate reactions generationization
Learn luminous signal.Signal strength is directly proportional to 1 concentration of Kininogen fucosylated in sample.
Operation:
1.HRP marks agglutinin (LcA)
A) 10mg HRP is weighed, is dissolved in 2mL distilled water, HRP enzyme solutions are prepared；
B) 0.5mL HRP enzyme solutions are taken, the 0.088M NaIO that 50 μ L now match is added4Solution is protected from light mixing；
C) room temperature is protected from light 15min；
D) solution after reaction is added in the 10kD super filter tube of 500 μ L, is centrifuged 3min in 7000rpm, adds 200 μ L
HRP marking fluid, ultrafiltration 5 times；
E) HRP solution is collected, it is 1mL that HRP marking fluid to total volume, which is added,；
F) it takes 50 μ L LcA (1mg/mL) to be added in the 50kD super filter tube of 500 μ L, 450 μ L HRP marking fluids is added；?
7000rpm is centrifuged 3min, adds 200 μ L HRP marking fluids into inner tube every time, is repeated 3 times；
G) back-off super filter tube inner tube is centrifuged 1min in 3000rpm, throws away liquid into new casing；It is added into inner tube
200 μ L HRP marking fluids, are centrifuged again；The HRP solution for taking 20 μ L to activate is added in the casing for collecting LcA solution, and room temperature is anti-
Answer 2h；
H) liquid moves in inner tube after reacting in casing, is centrifuged 3min in 7000rpm, adds 200 μ L HRP punching every time
Washing lotion, ultrafiltration 5 times；
I) back-off super filter tube inner tube is centrifuged 1min in 3000rpm, throws away liquid into new casing；It is added into inner tube
200 μ L HRP flushing liquors, are centrifuged again；
J) HRP labelled antibody is added into casing and saves liquid, total volume is adjusted to 500 μ L, is sub-packed in 0.6mL EP pipe,
Every 50 μ L of pipe, is placed in 4 DEG C of refrigerators and saves.
2. magnetic bead is coupled 1 antibody of Kininogen
Magnetic bead is coupled Kininogen antibody and is coupled AFP antibody with magnetic bead, only changes antibody into corresponding Kininogen
1 antibody.
The production of the test strip of 3.Fuc-Kininogen 1 (see Fig. 2)
The production that Fuc-Kininogen 1 tests strip tests strip with AFP, and magnetic bead is only coupled AFP antibody and HRP
Label AFP antibody changes magnetic bead coupling 1 antibody of Kininogen and HRP label L cA into respectively.
The detection of 1 blood sample of 4.Fuc-Kininogen
1 blood sample testing process of Fuc-Kininogen is detected with AFP blood sample, only changes test strip into phase
The Fuc-Kininogen 1 answered tests strip.
The detection of AFP-L3 and Fuc-GP73 with Fuc-Kininogen 1 detect, only by antibody change into corresponding AFP and
GP73 monoclonal antibody.
The detection of 3 ALP of embodiment
Principle:
The detection of ALP uses direct chemoluminescence method, biological sample is mixed with ALP catalytic luminescence substrate, by direct
Enzymic catalytic reaction generates chemiluminescence signal.
Operation:
The production of 1.ALP test strip (see Fig. 3)
A) No. 1 hole for testing strip is well；
B) No. 2 holes for testing strip are sample dilution holes；
C) by 100 μ L ALP catalytic luminescence substrate kept dries in No. 3 holes.
The detection of 2.ALP blood sample
A) 10 μ L blood samples are added in No. 1 hole of test strip.
B) from taking 190 μ L of blood sample dilution to be added in No. 1 hole in No. 2 holes, sample is diluted 20 times.
C) it is added in No. 3 holes from drawing 100 μ L in the sample after dilution, oscillation is examined after 25 DEG C of incubation 2min after mixing
It surveys luminous value (RLU).
4 logistical regression model foundation of embodiment
306 parts of serum samples are collected, wherein 165 parts of liver cancer samples, 141 parts of cirrhosis samples, the training number as model
According to collection.Above-mentioned 14 kinds of markers in sample are detected according to the method for embodiment 1 to 3, and combine age (age) and the property of subject
Not (gender), totally 16 indexs establish logistical regression model as independent variable, and gender is carried out with a sub- variable
Coding, 0 represents women, and 1 represents male.Logistic re-gression analysis is carried out using R language, all samples carry out bootstrap
Random sampling is run 1000 times, and the regression coefficient of each variable and the intercept of model are estimated using maximum-likelihood method, is suffered from
The mathematical model (model 1) of the relationship of liver cancer probability and each independent variable.
The detection of 5 clinical blood sample of embodiment
208 parts of serum samples are had collected altogether, wherein 115 parts of liver cancer samples, 93 parts of cirrhosis samples.It detects in serum sample
Above-mentioned 14 kinds of markers and statistical sample age and gender.Using the Multiple regression model (model 1) in embodiment 4 come
P value is calculated, and by P value compared with threshold value 0.5, serum sample is divided into liver cancer positive group, the serum if P<0.5 if P>=0.5
Sample is divided into liver cirrhosis group, draws the AUROC of ROC curve and computation model, and all calculating processes are realized using R language.
As a result as shown in following table (table 1), it is 0.845 that single index AFP, which distinguishes the liver cancer positive and the AUROC of liver cirrhosis group serum sample,
And the AUROC of model 1 is then noticeably greater than independent AFP, is increased to 0.957.Simultaneously as can be known from Table 1, false positive rate be 5%,
10%, in 15% level, the true positive rate of independent AFP is respectively 60%, 68%, 71%, and the true positive rate of model 1 is respectively
82%, 86%, 88%.Illustrate the early diagnosis of logistical regression model (model 1) for liver cancer, reference value is significantly excellent
In single index AFP.
Table 1 compares independent AFP and combines the performance of multi objective Multiple regression model
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；The ordinary skill people of this field
Member is it is understood that be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of
Technical characteristic is equivalently replaced；And these are modified or replaceed, it does not separate the essence of the corresponding technical solution, and the present invention is each
The range of embodiment technical solution should all cover in the range of description of the invention.
Claims (10)
1. a kind of method for carrying out hepatocarcinoma early diagnosis in subject, comprising:
1) biological sample is obtained from the subject；
2) content of multiple biomarkers in the biological sample is detected, the multiple biomarker is selected from AFP, AFP-
L3, GP73, Fuc-GP73, DCP, CEA, CA19-9, TK1, DKK1, γ-GT, ALP, AFU, ALT and Fuc-Kininogen
Two or more in 1；
3) testing result based on step 2) calculates the probability that the subject suffers from liver cancer；
4) probability is compared with given threshold, wherein when the probability is more than or equal to given threshold, then it is assumed that institute
Subject is stated with liver cancer；When the probability is less than given threshold, then it is assumed that the subject does not suffer from liver cancer.
2. the method as described in claim 1, wherein step 2) is also included in the gender of the subject when calculating the probability
And/or the age.
3. method according to claim 1 or 2, wherein step 3) using based on the biomarker testing result and/or
The gender of the subject and/or the Multiple regression model at age calculate the probability.
4. it is method according to claim 1 or 2, wherein the biological sample is serum or blood plasma.
5. a kind of detection kit for carrying out hepatocarcinoma early diagnosis in subject, including in the life from the subject
Detect the reagent of the content of multiple biomarkers in object sample, the multiple biomarker be selected from AFP, AFP-L3, GP73,
Two in Fuc-GP73, DCP, CEA, CA19-9, TK1, DKK1, γ-GT, ALP, AFU, ALT, Fuc-Kininogen 1 or
It is more than two.
6. detection kit as claimed in claim 5, wherein including following for detecting the reagent of the biomarker
Two or more:
1) the magnetic bead coupled antibody of the enzyme labelled antibody of AFP and/or AFP；
2) the magnetic bead coupled antibody of the enzyme labelled antibody of GP73 and/or GP73；
3) the magnetic bead coupled antibody of the enzyme labelled antibody of CEA and/or CEA；
4) the magnetic bead coupled antibody of the enzyme labelled antibody of CA19-9 and/or CA19-9；
5) the magnetic bead coupled antibody of the enzyme labelled antibody of DCP and/or DCP；
6) the magnetic bead coupled antibody of the enzyme labelled antibody of DKK1 and/or DKK1；
7) the magnetic bead coupled antibody of the enzyme labelled antibody of TK1 and/or TK1；
8) the magnetic bead coupled antibody of the enzyme labelled antibody of ALT and/or ALT；
9) the magnetic bead coupled antibody of the enzyme labelled antibody of AFU and/or AFU；
11) the magnetic bead coupled antibody of the enzyme labelled antibody of γ-GT and/or γ-GT；
12) the magnetic bead coupled antibody of the enzyme labelled antibody of Kininogen 1 and/or Kininogen 1；
13) enzyme mark agglutinin LcA；And
14) reaction substrate of ALP.
7. detection kit as claimed in claim 6, wherein the reagent is placed in test strip.
8. detection kit as claimed in claim 5, wherein further including the multiple biomarker as standard items.
9. detection kit as claimed in claim 5, wherein further including kit operation instructions, which is recorded:
It is returned using based on the multiple biomarker testing result and/or subject's gender and/or the logistic at age
Return model to calculate the probability that the subject suffers from liver cancer；And the probability is compared with given threshold, wherein when
When the probability is more than or equal to given threshold, then it is assumed that the subject suffers from liver cancer；When the probability is less than given threshold,
Then think that the subject does not suffer from liver cancer.
10. a kind of equipment for carrying out hepatocarcinoma early diagnosis in subject, including detection module, data processing module and aobvious
Show module, wherein the detection module is used to come from institute using detection kit detection described in any one of claim 5 to 9
State the content of multiple biomarkers in the biological sample of subject, the multiple biomarker be selected from AFP, AFP-L3,
In GP73, Fuc-GP73, DCP, CEA, CA19-9, TK1, DKK1, γ-GT, ALP, AFU, ALT and Fuc-Kininogen 1
Two or more；The data processing module is built-in with logistical regression algorithm, for being based on the multiple biology
The gender and/or age of the testing result of marker and the subject calculate the probability that the subject suffers from liver cancer；With
And the display module is for exporting calculated result.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110320370A (en) * | 2019-07-31 | 2019-10-11 | 万东山 | The synchronous immunity detection reagent of hepatic carcinoma marker AFP, alpha-fetoprotein variant, GP73 antigen, method and application |
| CN111781357A (en) * | 2020-07-10 | 2020-10-16 | 万东山 | Kit for simultaneously detecting four tumor markers of lung cancer and application thereof |
| CN112646039A (en) * | 2021-01-06 | 2021-04-13 | 华瑞同康生物技术（深圳）有限公司 | TK1 antibody, kit and application thereof |
| CN114167060A (en) * | 2021-11-25 | 2022-03-11 | 辽宁师范大学 | Liver cancer biomarker and kit for detecting liver cancer |
| CN114420284A (en) * | 2021-12-20 | 2022-04-29 | 海南松林生物科技有限责任公司 | Liver cancer screening method and device |
| CN114550921A (en) * | 2020-11-26 | 2022-05-27 | 华瑞同康生物技术（深圳）有限公司 | Risk of illness prediction modeling method based on LI-RADS classification |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102081100A (en) * | 2010-07-20 | 2011-06-01 | 李伯安 | Liver cancer multi-marker micro-array kit as well as preparation method and application thereof |
| EP2977760A1 (en) * | 2013-03-20 | 2016-01-27 | Seoul National University R&DB Foundation | Biomarker for diagnosing liver cancer |
| CN106596523A (en) * | 2015-10-20 | 2017-04-26 | 南京南泰生物科技有限公司 | Discovery and applications of sCD40L liver cancer early screening and diagnosis marker |
-
2018
- 2018-12-14 CN CN201811534109.8A patent/CN109557312A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102081100A (en) * | 2010-07-20 | 2011-06-01 | 李伯安 | Liver cancer multi-marker micro-array kit as well as preparation method and application thereof |
| EP2977760A1 (en) * | 2013-03-20 | 2016-01-27 | Seoul National University R&DB Foundation | Biomarker for diagnosing liver cancer |
| CN106596523A (en) * | 2015-10-20 | 2017-04-26 | 南京南泰生物科技有限公司 | Discovery and applications of sCD40L liver cancer early screening and diagnosis marker |
Non-Patent Citations (4)
| Title |
|---|
| 原丽莉等: "《新编消化内科住院医师问答》", 31 January 2017, 华中科技大学出版社 * |
| 宗迎迎等: "Logistic 回归和 ROC 曲线分析血清 DKK1、GP73和 AFP 在原发性肝癌诊断中的价值", 《检验医学》 * |
| 彭彦辉: "《肝癌多学科总和诊疗学》", 31 December 2017, 河北科学技术出版社 * |
| 秦继宝等: "血清AFP、TK1、DKK1联合检测对原发性肝癌的诊断价值", 《临床检验杂志》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110320370A (en) * | 2019-07-31 | 2019-10-11 | 万东山 | The synchronous immunity detection reagent of hepatic carcinoma marker AFP, alpha-fetoprotein variant, GP73 antigen, method and application |
| CN111781357A (en) * | 2020-07-10 | 2020-10-16 | 万东山 | Kit for simultaneously detecting four tumor markers of lung cancer and application thereof |
| CN111781357B (en) * | 2020-07-10 | 2024-01-19 | 万东山 | Kit for simultaneously detecting four tumor markers of lung cancer and application of kit |
| CN114550921A (en) * | 2020-11-26 | 2022-05-27 | 华瑞同康生物技术（深圳）有限公司 | Risk of illness prediction modeling method based on LI-RADS classification |
| CN112646039A (en) * | 2021-01-06 | 2021-04-13 | 华瑞同康生物技术（深圳）有限公司 | TK1 antibody, kit and application thereof |
| CN114167060A (en) * | 2021-11-25 | 2022-03-11 | 辽宁师范大学 | Liver cancer biomarker and kit for detecting liver cancer |
| CN114420284A (en) * | 2021-12-20 | 2022-04-29 | 海南松林生物科技有限责任公司 | Liver cancer screening method and device |
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