CN101062939A - Device and reagent case for detecting hepatic carcinoma fucosido-fucosyl gorky protein GP73 - Google Patents
Device and reagent case for detecting hepatic carcinoma fucosido-fucosyl gorky protein GP73 Download PDFInfo
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Abstract
The invention discloses a prepack eccentric column of fucosido-fucosyl Golgi protein 73 (or Golgi protein 73 heterogeneous body) relative to liver cancer separation, which is characterized by the following: comprising upper separator tube and lower collecting pipe; packing affinity agent of coupling small lentils agglutinine (LCA); assembling a filter cloth on the bottom of the upper separator tube; packing buffer solution in lower collecting pipe; binding small lentils agglutinine (LCA) and sugar chain fucosido-fucosyl Golgi protein 73; centrifuging through adding into eluent; proceeding purify Fuc-GP73 part. This invention discloses a chemoluminescence testing agent case with the eccentric column and enzyme joint immunoassay agent case with the column and preparing and using method. This agent case can test the content of liver cancer fucosido-fucosyl Golgi protein 73, which can provide support for early stage diagnosis.
Description
Technical field
The present invention relates to a kind of separation liver cancer fucosylated Golgi protein GP 73 pre-mounted eccentric column of (or being called Golgi protein 73 heteroplasmons), also related to a kind of chemiluminescence detection kit and a kind of preparation method who contains enzyme-linked immune quantitative detection reagent box and these test kits of this pre-mounted eccentric column who contains this pre-mounted eccentric column, belong to the medical product technical field, be used for the early diagnosis of carrying out liver cancer.
Background technology
The main nosetiology of hepatocellular carcinoma HCC (hepatocellular carcinoma) is that HBV or HCV chronic hepatopathy poison infects, and in liver cancer long latent period-promptly infect hepatovirus to the liver cancer time of origin takes place to have as last.According to the introduction of on January 26th, 2006 " Chinese Medicine " report and " cancer " 2005 the 6th interim articles, China has hepatitis B virus carriers about 1.2 hundred million now, accounts for world hepatitis B virus carriers's 1/3; Symptomatic chronic viral hepatitis B patient 2,000 ten thousand people, China also has 3,600,000 people's liver cirrhosis patients in addition.Because numerous liver problem sufferer and liver cancer high risk population, the hepatocellular carcinoma sickness rate of China is the highest.Estimate that according to international cancer research institution of the World Health Organization (IARC) global onset of liver cancer number was 56.4 ten thousand in 2000, dead 54.9 ten thousand people; China's onset of liver cancer number 30.6 ten thousand, dead 30.0 ten thousand people account for the whole world 50%.
It is very important setting up early diagnosis and monitoring mechanism in the high risk population.Early discovery, early treatment are the keys that improves patient's survival rate, and Most patients middle and advanced stage when going to a doctor has lost best occasion for the treatment, and the high-risk factor comprises chronic hepatitis and the liver cirrhosis that is caused by hepatitis B virus and hepatitis C virus.Will effectively be treated in addition, the clinical efficiency of examination and generaI investigation depends on early diagnosis.
At present, the monitoring traditionally of HCC disease is health check-up, and ultrasonic image analysis of liver and serum mark series detect.
Wherein the serologic marker thing of normal use is alpha-fetoprotein AFP, because alpha-fetoprotein AFP has the relative specificity with HCC, therefore be used widely at present, but in the optimum hepatopathy of part in the positive and part liver cancer of AFP AFP negative, the feasible AFP of dependence is used for the HCC early warning and monitors and bring certain restriction, and the HCC that is correlated with at detection HCV is not very sensitive.Clinical hope obtains the more special and new serological index of sensitive more than alpha-fetoprotein.
Disease glycobiology result of study shows that glycosylation changes the characteristics of morbid state often.Malignant cell and Normocellular substance metabolism are very different, participate in the change that vigor all takes place metabolic a lot of enzyme, particularly the grow enzyme of relevant metabolic process of cell proliferation often obviously increases, and this metabolic process is accelerated, to adapt to the needs of the continuous division growth of tumour cell.Wherein glycosyltransferase also is the enzyme that class vigor in malignant cell has obvious change.Because the structure of sugar chain is determined by the vigor that participates in the various glycosyltransferases of sugar chain synthetic, what of a certain glycosyl in the sugar chain increase and decrease of a certain glycosyltransferase vigor can directly influence, so the variation of glycosyltransferase vigor must bring the respective change of sugar chain structure on the cell synthetic glycoprotein in the malignant tumour.
These cacoplastic sugar chains can appear on the glycoprotein of tumor cell surface, and these cells releases or some soluble sugar conjugates that come off can be brought into play immunosuppression or antagonistic action.This immunosuppression or antagonistic action comprise the processing of disturbing tumor associated antigen and present, suppress lymphocytic propagation, and eliminate cytotoxic reaction that cancer cells is killed and wounded etc.In addition, some glycoprotein or the Saliva Orthana that discharge from cancer cells, also can in and corresponding anticancrin and weaken the antitumor immune reaction in the cancer patient body.This process and then cause the change of cell adhesion, invasion and attack and transfer ability, this is to cause tumour cell to have a major reason of aggressive and transfer ability.
Numerous liver cancer that studies show that are accompanied by the core fucosylation of multiple protein.This with liver cancer cell in fucosyltransferase relevant unusually, usually the albumen with the core fucosylation is called its heteroplasmon.
At present the alpha-fetoprotein variant AFP-L3 as tumour early warning mark of success is exactly an exemplary, FDA in October, 2005 approval clinical with alpha-fetoprotein variant AFP-L3 as the liver cancer warning index.Alpha-fetoprotein variant AFP-L3 is a component of the sugar chain abnormal that takes place when cell carcinogenesis of alpha-fetoprotein, significantly improves for the generation specificity that detects liver cancer.Discover except the alpha-fetoprotein sugar chain abnormal to also have antitrypsin, Transferrins,iron complexes when canceration of hepatic cell, also to supervene sugar chain abnormal at present.Japanese scientist Akira Naitoh adopted the glycosylation of lectin affinity electrophoresis developing method detection hepatopathy unusual in 99 years, discovery is between liver cancer and hepatitis, liver cirrhosis, and alpha-fetoprotein variant, antitrypsin heteroplasmon, the heteroplasmonic content of Transferrins,iron complexes and ratio have significant difference.It is significant for the early diagnosis of liver cancer to detect alpha-fetoprotein variant, antitrypsin heteroplasmon, Transferrins,iron complexes heteroplasmon.
GP73 (Golgi Protein 73) is an II type gorky transmembrane protein, is a newfound albumen relevant with hepatopathy disease course.Molecular weight near 73KD (Kladney et al., 2000, Gene 249,53-65).High expression level in the liver cell of viral infection (Kladney, et al., 2000, Gene 249, and 53-65), GP73 also is expressed in the bile epithelial cell.And in the normal liver cell seldom.Opposite, liver problem sufferer's liver cell demonstration is reacted the GP73 strong immunization.Also high expression level in GP73 mRNA and the HepG2 malignant tumor of liver cell of albumen after different transfection virus comprises the transfection adenovirus.Facilitate hepatopathy such as alcoholic liver disease at viral liver disease or non-viral, and the rising of GP73 significance in the autoimmune liver disease etc. (Kladney, et al., 2002, Hepatology 35 (6): 1431-40).
Correlative study is also found to raise unusually in the liver cancer in early days that Golgi protein GP 73 can be more Zao than alpha-fetoprotein AFP, and also causes GP73 to rise at chronic virus liver diease.Discover the detection for early hepatocarcinoma, the remolding sensitivity AFP of GP73 exceeds 2-3 doubly.But GP73 also raises in the benign virus hepatopathy unusually, so the GP73 index comes predicting liver cancer that the specific degree problem takes place also to exist separately.
Richard R in 2006 etc. are at Molecular ﹠amp; Cellular Proteomics delivers document and finds that GP73 and other associated protein of liver cancer cell are similar, and behind canceration of hepatic cell, the core fucosylation also takes place GP73, and the core fucosylation of GP73 and the generation of liver cancer are more close.Experimental results show that GP73 is an index, sensitivity and specific degree are respectively 65%, 90%.And the employing mass spectrometric analysis method is an index with Fuc-GP73, and sensitivity and specific degree are respectively 90% and 100%.
Lectin is protein or the glycoprotein that a class extensively is present in the non-immunity source of natural one big class, it can with sugared specificity ground, the reversible combination in non-covalent ground, and the effect of aggegation hemocyte is arranged, so be called lectin.
Sumner and Howell in 1936 at first from sword bean (jackbean, Canavalia ensiformis) purification of seed companion ConA-ConA.Glycogen and starch in the ConA energy aggegation solution, the hemagglutinative function of ConA can be suppressed by sucrose, thereby infers the result that hemagglutinative function is and cell surface sugar acts on of ConA.
At present existing nearly thousand kind of plant are recorded has activity of lectin.In the plant, not only there is lectin in the seed, also finds to have lectin in root, stem, leaf, skin, the fruit juice.Except that plant, all there is lectin in other biology as various fungies, some virus, invertebrates, vertebrates and to the various tissues of human body and organ.
Lectin can with sugared specificity combine.By the type in conjunction with sugar, lectin can be divided into six classes: D-seminose or D-glucose at present; The N-acetylglucosamine; The N-acetylamino galactosamine; The D-semi-lactosi; The L-Fucose; Wheat germ agglutinin (WGA) can single-minded bound sialic acid.
Identify the most clearly phytohemagglutinin family be pulse family, comprise canavaline lectin ConA, LcA LCA etc.
Root of Szemao crotalaria: the formal name used at school Lens culinaris, the lectin LCA that extracts from root of Szemao crotalaria (Lens culinaris lectin) can specific combination core fucosido, and tangerine fruit powder spore lectin AAL is too to the combination of core fucosido.
The detection method that is used for Fuc-GP73 (or being called Golgi protein 73 heteroplasmons) at present mainly is lectin affinity chromatography and mass spectrometric analysis method, but this method all can't be applied in the conventional sense.Simultaneously traditional lectin affinity chromatography purifying glycosylation protein exists in the medium residue many, and the dilution during wash-out causes problems such as concentration of specimens is low.
Summary of the invention
In order to overcome the deficiencies in the prior art, the invention provides the pre-mounted eccentric column of a kind of separation liver cancer Golgi protein Fuc-GP73 (or being called Golgi protein 73 heteroplasmons) and contain the diagnostic kit of this prepacked column.Can detect the contents level of Fuc-GP73 in the serum truly by this test kit, diagnose out early primary hepatocarcinoma exactly, have high sensitivity, method is fast and convenient.
The pre-mounted eccentric column of a kind of separation liver cancer Fuc-GP73 (or being called Golgi protein 73 heteroplasmons), this pre-mounted eccentric column is made up of top separator tube and bottom collection tube, described top centrifuge tube is equipped with the affinity medium of coupling lectin, in the collection tube of described bottom buffered soln is housed, one filter cloth is arranged at described separator tube bottom, makes the filter cloth that the agarose particle can't pass.
Described lectin comprises LcA LCA and tangerine fruit powder spore lectin AAL.
Described LcA LCA and tangerine born of the same parents powder lectin AAL can be bought by commercialization and obtain, and for example buy from Sigma company.
It is the medium of carrier is carrier that described affinity medium can adopt sepharose; as sepharose sephrose 4B, sepharose sephrose 6B or sepharose sephrose FF; sepharose sephrose CL-4B, sepharose sephrose CL-6B, described buffered soln are the active protection liquid of lectin.
Described sepharose is sephrose 4B, and damping fluid is the active protection liquid of lectin, contains the CaCl of 1mmol/L
2, 1mmol/L MnCl
2With the Tris-HCL of 50mmol/L, PH 7.5.
Described filter cloth is to stop that coupling has the affinity medium of lectin to pass and the filter cloth that passes of barrier liquid and protein not.
The described sepharose that the coupling lens culinaris agglutinin is housed is obtained by following steps:
Adopt Pharmacia company through CNBr activatory Sepharose 4B coupling LCA.
(1) 1.5g Sepharose 4B is soaked in 1mmol/L HCl to expanding, moves into sand stamen funnel, with the about 30min of 300mL 1mmol/L HCl washing;
(2) take by weighing 2mgLCA, be dissolved in 7.5mL coupling buffer (pH 8.3 for 0.1mmol/L NaHCO3,0.5mol/L NaCl), merge with Sepharose 4B through washing, with the 10mL test tube of band plug turn upside down mixing (room temperature, 2h);
(3) with 10mL coupling buffer flush away link coupled LCA not, measure the LCAI content in the washings, count to such an extent that the coupling rate is 98%;
(4) with 0.2mol/L glycine sealing residue activating gene;
(5) use 10mL 0.1mol/L acetate buffer solution (pH 4, contain 0.5mol/L NaCl) and 0.1mol/L Tris damping fluid (pH 8, contain 0.5mol/L NaCl) washing 3 times successively, again to contain 0.1%BSA, 1mmol/L CaCl
2With 0.1mmol/L MnCl
2PBS (PBS-BSA) washing 1 time, 4 ℃ are temporary standby.
The invention provides with the microcentrifugation column kit and use, be used for the elutriant of the Fuc-GP73 of elution of bound above LCA-Sepharose, its making method and composed as follows:
20mmTris-Hcl, NaCL 150mm, the ph7.4 damping fluid wherein contains 500mm Alpha-Methyl-D-mannoside, for guaranteeing the long-time not long bacterium that preserves, adds sanitas Proclin 300 to 0.1%.
Elution step: when wash-out, above-mentioned solution is added wherein, incubation is 30 minutes in incubator, and centrifugal collection reaches the purpose of wash-out.
The invention provides a kind of this pre-mounted eccentric column of employing and carry out purifying Fuc-GP73 step and related reagent composition thereof.
This combination comprises prepackage microcentrifugation post (coupling of interior dress agarose and its protection liquid of LcA LCA); scavenging solution (20mmTris-Hcl; the PH7.4 damping fluid; 0.1%Proclin 300), elutriant (20mmTris-Hcl, NaCL 150mm; the ph7.4 damping fluid; wherein contain 500mm Alpha-Methyl-D-mannoside, 0.1%, Proclin 300).
Serum to be detected is centrifugal fully 1., no haemolysis; Take out prepackage microcentrifugation post, discard liquid in lower floor's collection tube.
2. sample dilution: draw 250ul serum in test tube, and add the 350ul cleaning solution dilution, shake up.
3. application of sample: sucking-off 450ul dilution back sample adds in the centrifuge tube of top.37 ℃ of incubators leave standstill, and the centrifuge tube lid is not please covered in this operation, and serum dilution will flow in 15 minutes in lower floor's collection tube;
4. will collect liquid in pipe discards;
5. add scavenging solution 600ul in the centrifuge tube of top, wait for that scavenging solution all flows into (about 3 minutes) in lower floor's collection tube, cover the centrifuge tube lid, 2000 changeed under (or 3000 change) room temperature centrifugal 1 minute;
6. discard liquid in lower floor's collection tube;
7. add scavenging solution 600ul in the centrifuge tube of top, wait for that scavenging solution all flows into (about 3 minutes) in lower floor's collection tube, cover the centrifuge tube lid, 2000 changeed under (or 3000 change) room temperature centrifugal 1 minute;
8. discard liquid in lower floor's collection tube;
9. add elutriant 450ul, when waiting eluting liquid number to ooze present lower floor collection tube, cover the centrifuge tube lid, place in 37 ℃ of thermostat containers incubation 30 minutes;
10. taking-up centrifuge tube, 2000 (or 3000 change) changeed under room temperature centrifugal 1 minute;
Be equipped with detection 11. collect the liquid (sample 1) that flows in lower floor's collection tube.
The invention provides a kind of chemical luminescence reagent kit that contains above-mentioned pre-mounted eccentric column and preparation method thereof.
This chemical luminescence reagent kit has comprised that the pre-mounted eccentric column, bag of separation Fuc-gp73 (Golgi protein 73 heteroplasmons) are by the anti-GP73 polyclonal antibody of rabbit of the chemoluminescence enzyme plate of GP73 monoclonal antibody, positive control, negative control thing, enzyme labelling; Substrate solution A, colour developing liquid B, Fuc-gp73 cleaning buffer solution, Fuc-gp73 elutriant, GP73 standard substance.
The making method of this test kit is as follows:
(1) bag quilt: the GP73 monoclonal antibody is (commercially available, Santa Cruze Biotechnology company) adds each hole of enzyme plate with 0.05M citrate buffer solution dilution back, every hole 100 μ l, absorption is spent the night, wash plate with the tween phosphate buffered saline buffer, spend the night with the tween phosphate buffered saline buffer sealing that contains bovine serum albumin again, dry after the drying, promptly obtain the monoclonal antibody coated elisa plate.Homemade plate of the optional usefulness of chemoluminescence enzyme plate or import plate; Specification can be dull and stereotyped or 12 * 8,12 * 4 removable battens in 96 holes;
(2) positive, negative control thing:
Positive control: collect GP73 masculine liver cancer patient ascites, filtration sterilization, packing;
Negative control thing: collect the negative normal human serum of GP73, filtration sterilization, packing;
(3) enzyme labelling resists (commercially available, Santa Cruze Biotechnology company) more: the enzyme mark polyclonal antibody in this test kit prepares with horseradish peroxidase (HRP) traget antibody, and step is as follows:
A) with NaIO
4-glycol method is carried out the oxidation of HRP, reaches final concentration 10mg/m;
B) and HRP in the alkaline carbonic acid salt buffer, dialysed 6 hours, realize the mark of HRP antagonist, reaction is used NaBH after finishing
4The solution termination reaction is again to the PBS dialysed overnight;
C) use saturated ammonium sulphate, the HRP enzyme that obtains purifying is marked anti-GP73 antibody;
(4) compound method of auxiliary reagent is as follows:
A) substrate solution A: by commercially available acquisition
B) colour developing liquid B: by commercially available acquisition
C) Fuc-GP73 cleaning buffer solution 20mmTris-Hcl, PH7.4 contains 0.1%Proclin 300 sanitass
D) Fuc-GP73 elutriant 20mmTris-Hcl, PH7.4,150mmNaCl, 500mmol/L Alpha-Methyl-D-mannoside contains 0.1%Proclin 300 sanitass
D) concentrated cleaning solution (20 times of concentrated solutions, 20 *): 0.05% polysorbas20 solution of PBS (pH7.4) preparation;
(5) pre-mounted eccentric column, bag is how anti-by the anti-GP73 of the chemoluminescence enzyme plate of GP73 monoclonal antibody, positive control, negative control thing, enzyme labelling, substrate solution A, colour developing liquid B, Fuc-GP73 cleaning buffer solution, Fuc-GP73 elutriant, GP73 standard substance are packed jointly, obtain test kit.
The present invention also provides a kind of enzyme-linked immune quantitative detection reagent box and making thereof that contains above-mentioned pre-mounted eccentric column.
This test kit has comprised the pre-mounted eccentric column of separation fucosylation GP73 (or Golgi protein GP 73 heteroplasmon); Bag is by the enzyme plate of Golgi protein GP 73 monoclonal antibody; Positive control, negative control thing; How anti-the anti-Golgi protein GP 73 of enzyme labelling is; Substrate solution A, colour developing liquid B, reaction terminating liquid, Fuc-GP73 cleaning buffer solution, Fuc-GP73 elutriant, GP73 standard substance, concentrated cleaning solution.
The making method of this test kit is as follows:
(1) bag quilt: the GP73 monoclonal antibody is (commercially available, Santa Cruze Biotechnology company) adds each hole of enzyme plate with 0.05M carbonic acid buffer dilution back (weaker concn is 20ug/ml), every hole 100 μ l, absorption is spent the night, wash plate with the tween phosphate buffered saline buffer, spend the night with the tween phosphate buffered saline buffer sealing that contains bovine serum albumin again, dry after the drying, promptly obtain the monoclonal antibody coated elisa plate.Enzyme plate is the polystyrene enzyme plate, can select homemade plate or import plate for use; Specification can be dull and stereotyped or 12 * 8,12 * 4 removable battens in 96 holes;
(2) positive, negative control thing:
Positive control: collect GP73 masculine liver cancer patient ascites, filtration sterilization, packing;
Negative control thing: collect the negative normal human serum of GP73, filtration sterilization, packing;
(3) enzyme labelling GP73 resists (commercially available, Santa Cruze Biotechnology company) more: the enzyme labelled antibody in this test kit prepares with horseradish peroxidase (HRP) traget antibody, and step is as follows:
A) with NaIO
4-glycol method is carried out the oxidation of HRP, reaches final concentration 10mg/ml;
B) antibody and HRP dialysed 6 hours in the alkaline carbonic acid salt buffer, realized the mark of HRP to monoclonal antibody, used NaBH after reaction finishes
4The solution termination reaction is again to the PBS dialysed overnight;
C) use saturated ammonium sulphate, the HRP enzyme that obtains purifying is marked anti-GP73 antibody;
(4) compound method of auxiliary reagent is as follows:
A) substrate solution A: 3% superoxol of phosphoric acid-citrate buffer solution (pH5.0) preparation;
B) colour developing liquid B: tetramethyl benzidine (TMB) methanol solution, concentration is 0.1mg/ml;
C) GP73 cleaning buffer solution 20mmTris-Hcl, PH7.4 contains 0.1%Proclin 300 sanitass
D) GP73 elutriant 20mmTris-Hcl, PH7.4,150mmNaCl, 500mmol/L Alpha-Methyl-D-mannoside contains 0.1%Proclin 300 sanitass
E) concentrated cleaning solution (20 times of concentrated solutions, 20 *): 0.05% polysorbas20 solution of PBS (pH7.4) preparation;
F) reaction terminating liquid: 2mol/L sulfuric acid;
(5) pre-mounted eccentric column, bag is how anti-by the anti-GP73 of the enzyme plate of GP73 monoclonal antibody, positive control, negative control thing, enzyme labelling, substrate solution A, colour developing liquid B, Fuc-GP73 cleaning buffer solution, Fuc-GP73 elutriant, reaction terminating liquid, GP73 standard substance are packed jointly, obtain test kit.
The present invention has overcome in the past loaded down with trivial details, the consuming time length of detection method operation steps, has needed shortcomings such as support equipment is many, the operator only needs simple operationss such as centrifugal and incubation, in 2 hours, just can finish detection, direct quantitative calculates the content of Fuc-GP73 contained in the blood sample sample (Golgi protein 73 heteroplasmons), surpassing 5ng/ml with Fuc-GP73 content is the positive judgement of liver cancer, and method is easy, detects quick, the result is accurate, for prevention, diagnosis, the treatment of liver cancer provides support.
Description of drawings
Accompanying drawing is the cross-sectional view of pre-mounted eccentric column of the present invention.
Embodiment
Further specify the present invention with embodiment below.It should be understood that embodiments of the invention are to be used to illustrate the present invention rather than limitation of the present invention.Essence according to the present invention all belongs to the scope of protection of present invention to the concrete improvement that the present invention carries out.
Referring to accompanying drawing, the pre-mounted eccentric column of a kind of separation liver cancer Fuc-GP73 provided by the invention (Golgi protein 73 heteroplasmons), form by superposed separator tube 1 and the collection tube 3 that is positioned at the bottom, usually can adopt the mode that separator tube is plugged on the collection tube to realize both connections, described top separator tube is equipped with the affinity medium 4 of coupling lectin (for the purpose of drawing is clear, what mark among the figure is the interior space of pipe that is used to adorn the affinity medium, do not draw the medium material object), buffered soln is housed (for the purpose of drawing is clear in the collection tube of described bottom, what mark among the figure is the interior space of pipe that is used to adorn buffered soln, do not draw the solution material object), described separator tube bottom mainly is a filter cloth, this filter cloth adopts and can stop that coupling has the affinity medium of lectin to pass and the filter cloth that passes of barrier liquid and protein not, so that when centrifugation, liquid of separating and protein can see through this filter cloth and enter following collection tube.Can use existing this filter cloth, and, filter cloth be fixed by a clamping plastic packing being set in the separator tube bottom.
Embodiment 1: detect the making of the enzyme-linked immune quantitative detection reagent box of liver cancer Fuc-GP73 (Golgi protein 73 heteroplasmons):
This test kit (96 person-portion) is formed and is comprised:
Each 1 bottle of GP73 feminine gender, positive control;
GP73 monoclonal antibody bag is by 1 of plate (96 hole);
The GP73 of horseradish peroxidase (HRP) mark resists 1 bottle, the 6ml/ bottle more;
1 bottle of GP73 standard substance;
Each 1 bottle of substrate solution A, colour developing liquid B, the 5ml/ bottle;
1 bottle of reaction terminating liquid, the 5ml/ bottle;
Fuc-GP73 cleaning buffer solution 25ml/ bottle
Fuc-GP73 elutriant 15ml/ bottle
Concentrated cleaning solution (20 times of concentrated solutions, 20 *) the 20ml/ bottle
Concrete operations are as follows:
1. prepare GP73-feminine gender, positive control:
1) collect serum: secure good health from hospital or blood station normal human serum, hepatocarcinoma patient ascites, standby in-70 ℃ of preservations;
2) packing:
The GP73 positive control: liver cancer positive patients ascites, under aseptic condition, divide in the 1.5mleppendorf pipe of packing into every pipe 0.5ml.Be stored in 4 ℃;
The negative control thing: normal human serum is the GP73 feminine gender through calibrating.Get more than many parts serum and merge in batch, through 60 ℃ handled in 1 hour after, filtration sterilization.Under aseptic condition, divide in the 1.5ml eppendorf pipe of packing into every pipe 0.5ml.Be stored in 4 ℃.
2. make GP73 monoclonal antibody bag by plate:
A) bag quilt:
Enzyme plate adopts import or home-made 12 * 8 removable battens.With GP73 monoclonal anti body and function 0.05mol/L carbonate buffer solution dilution is to add each hole of enzyme plate behind the 20 μ g/ml, every hole 100 μ l, and absorption is spent the night, wash plate with cleaning buffer solution, spend the night with this confining liquid 2%BSA sealing again, dry after the drying, promptly obtain the monoclonal antibody coated elisa plate.By 96 holes/piece packaging of aluminium foil bag, vacuum sealing;
B) how anti-the anti-GP73 of rabbit of preparation horseradish peroxidase (HRP) mark is:
The anti-GP73 of rabbit resists more can be buied in commercialization.
3. the mark of antibody:
1) oxidation of enzyme (whole process lucifuge):
A) take by weighing HRP 5mg, add ddH
2O 250 μ l dissolving;
B) take by weighing NaIO
45mg adds ddH
2O 250 μ l dissolve, and are mixed with the concentration of 20mg/mL;
C) in HRP solution, dropwise add NaIO
4Solution, the limit edged stirs;
D) solution that mixes is placed 4 ℃, left standstill 30 minutes;
E) get 5ml ethylene glycol and be dissolved in 25 μ l ddH
2Among the O, dropwise add in the above-mentioned mixing solutions, the limit edged stirs;
F) room temperature left standstill 30 minutes;
G) the oxydasis process is finished, and the HRP final concentration is 10mg/ml.
2) preparation of antibody and mark (lucifuge):
A) adjust antibody concentration (protein concentration cross low then concentrate) to about the 5mg/ml, with 50mmol/L CB (the 1mol/L NaHCO about pH9.5 with PEG20000
3With 1mol/L Na
2CO
3By 10: 1 mixed, use preceding with 20 times of distilled water dilutings) to dialyse and remove glycerine or impurity (as Tris), 4 ℃ of following dialysed overnight are wherein changed liquid 3 times;
B) antibody is mixed by 1: 4 with HRP, dialysis was changed liquid once in preceding two hours more than 6 hours in 50mmol/L pH9.5 CB;
C) with the 1mg NaBH of fresh configuration
4The solution termination reaction.Shake up, 4 ℃ left standstill 2 hours, and shake once per half an hour, NaBH
4The amount that solution adds is suitable;
D) the 10 mM PBS (Na of pre-configured 0.01mol/L of usefulness pH7.2
2HPO
4And NaH
2PO
4Storing solution, the two becomes the PBS damping fluid pH value mixing as required) dialysed overnight.Changing liquid once gets final product.
3) purifying HRP enzyme is marked anti-GP73 antibody
A) finish in the GP73 antibody-solutions of mark and dropwise add saturated ammonium sulphate solution, stir while adding, be reduced to 1/3 until saturated ammonium sulphate concentration;
B) 4 ℃ left standstill 1 hour;
C) 8000rpm is centrifugal 10 minutes, and supernatant is moved in the new pipe, and precipitation suspends again with equal-volume PBS;
D) repeat above operation, saturated ammonium sulphate concentration is brought up to 40%, collect respectively and go up cleer and peaceful precipitation;
E) repeat above operation, saturated ammonium sulphate concentration is brought up to 50%, collect respectively and go up cleer and peaceful precipitation;
F) repeat above operation, saturated ammonium sulphate concentration is brought up to 60%, collect respectively and go up cleer and peaceful precipitation;
G) collect isolating each component, SDS-PAGE identifies purity;
H) how anti-to the PBS dialysed overnight HRP-of Ti Chuning is;
I) how anti-the centrifugal concentrated and purified HRP-of ultrafiltration pipe is, obtains mole ratio and mark anti-monoclonal antibody near 1: 8 enzyme.
4) packing: mark anti-GP73 with the damping fluid dilution that contains 10% foetal calf serum by the enzyme of step 3) acquisition and resist to suitable working concentration more, press the packing of 6ml/ bottle, be stored in 4 ℃.
4. the preparation of auxiliary reagent:
1) substrate solution A: 3% superoxol of phosphoric acid-citrate buffer solution (PH5.0) preparation, press the packing of 5ml/ bottle;
2) colour developing liquid B:TMB (0.1mg/ml) methanol solution is pressed the packing of 5ml/ bottle;
3) reaction terminating liquid: 2mol/L H
2SO
4, press the packing of 3ml/ bottle;
4) Fuc-GP73 cleaning buffer solution 20mmTris-Hcl, PH7.4 contains 0.1%Proclin 300 sanitass
5) Fuc-GP73 elutriant 20mmTris-Hcl, PH7.4,150mmNaCl, 500mmol/L Alpha-Methyl-D-mannoside contains 0.1%Proclin 300 sanitass
6) concentrated cleaning solution (20 times of concentrated solutions, 20 *): 0.05% polysorbas20 solution of PBS (pH7.4) preparation;
Embodiment 2: detect the making of the chemiluminescence detection kit of liver cancer fucosido GP73 (Golgi protein 73 heteroplasmons)
This test kit (48 person-portion) is formed and is comprised:
Each 1 bottle of GP73 feminine gender, positive control;
GP73 monoclonal antibody bag is by 1 of plate (96 hole);
The GP73 of horseradish peroxidase (HRP) mark resists 1 bottle, the 6ml/ bottle more;
1 bottle of GP73 standard substance
Each 1 bottle of substrate solution A, colour developing liquid B, the 5ml/ bottle;
Fuc-GP73 cleaning buffer solution 25ml/ bottle
Fuc-GP73 elutriant 15ml/ bottle
Concentrated cleaning solution (20 times of concentrated solutions, 20 *) the 20ml/ bottle
Concrete operations are as follows:
1. prepare GP73-feminine gender, positive control:
1) collect serum: obtain normal human serum, hepatocarcinoma patient ascites from hospital or blood station, standby in-70 ℃ of preservations;
2) packing:
The GP73 positive control: liver cancer positive patients ascites, under aseptic condition, divide in the 1.5mleppendorf pipe of packing into every pipe 0.5ml.Be stored in 4 ℃;
The GP73 negative control sera: normal human serum is the GP73 feminine gender through calibrating.Get more than many parts serum and merge in batch, through 60 ℃ handled in 1 hour after, filtration sterilization.Under aseptic condition, divide in the 1.5mleppendorf pipe of packing into every pipe 0.5ml.Be stored in 4 ℃.
2. make GP73 monoclonal antibody bag by plate:
A) bag quilt:
The chemoluminescence enzyme plate adopts import or home-made 12 * 8 removable battens.With step 1) AFP monoclonal anti body and function 0.05mol/L citrate buffer solution dilution is to add each hole of enzyme plate behind the 20 μ g/ml, every hole 100 μ l, and absorption is spent the night, wash plate with cleaning buffer solution, spend the night with this confining liquid damping fluid sealing again, dry after the drying, promptly obtain the monoclonal antibody coated elisa plate.By 96 holes/piece packaging of aluminium foil bag, vacuum sealing;
B) how anti-the anti-GP73 of preparation horseradish peroxidase (HRP) mark is:
The anti-GP73 antibody of rabbit can be buied in commercialization.
3. the mark of antibody:
1) oxidation of enzyme (whole process lucifuge):
A) take by weighing HRP 5mg, add ddH
2O 250 μ l dissolving;
B) take by weighing NaIO
45mg adds ddH
2O 250 μ l dissolve, and are mixed with the concentration of 20mg/mL;
C) in HRP solution, dropwise add NaIO
4Solution, the limit edged stirs;
D) solution that mixes is placed 4 ℃, left standstill 30 minutes;
E) get 5ml ethylene glycol and be dissolved in 25 μ l ddH
2Among the O, dropwise add in the above-mentioned mixing solutions, the limit edged stirs;
F) room temperature left standstill 30 minutes;
G) the oxydasis process is finished, and the HRP final concentration is 10mg/ml.
2) preparation of antibody and mark (lucifuge):
A) adjust antibody concentration (protein concentration cross low then concentrate) to about the 5mg/ml, with 50mmol/L CB (the 1mol/L NaHCO about pH9.5 with PEG20000
3With 1mol/L Na
2CO
3By 10: 1 mixed, use preceding with 20 times of distilled water dilutings) to dialyse and remove glycerine or impurity (as Tris), 4 ℃ of following dialysed overnight are wherein changed liquid 3 times;
B) antibody is mixed by 1: 4 with HRP, dialysis was changed liquid once in preceding two hours more than 6 hours in 50mmol/L pH9.5 CB;
C) with the 1mg NaBH of fresh configuration
4The solution termination reaction.Shake up, 4 ℃ left standstill 2 hours, and shake once per half an hour, NaBH
4The amount that solution adds is suitable;
D) the 10mM PBS (Na of pre-configured 0.01mol/L of usefulness pH7.2
2HPO
4And NaH
2PO
4Storing solution, the two becomes the PBS damping fluid pH value mixing as required) dialysed overnight.Changing liquid once gets final product.
3) purifying HRP enzyme is marked anti-GP73 antibody:
A) finish in the monoclonal anti liquid solution of mark and dropwise add saturated ammonium sulphate solution, stir while adding, be reduced to 1/3 until saturated ammonium sulphate concentration;
B) 4 ℃ left standstill 1 hour;
C) 8000rpm is centrifugal 10 minutes, and supernatant is moved in the new pipe, and precipitation suspends again with equal-volume PBS;
D) repeat above operation, saturated ammonium sulphate concentration is brought up to 40%, collect respectively and go up cleer and peaceful precipitation;
E) repeat above operation, saturated ammonium sulphate concentration is brought up to 50%, collect respectively and go up cleer and peaceful precipitation;
F) repeat above operation, saturated ammonium sulphate concentration is brought up to 60%, collect respectively and go up cleer and peaceful precipitation;
G) collect isolating each component, SDS-PAGE identifies purity;
H) how anti-to the PBS dialysed overnight HRP-of Ti Chuning is;
I) how anti-the centrifugal concentrated and purified HRP-of ultrafiltration pipe is, and it is anti-how anti-near 1: 8 enzyme mark to obtain mole ratio.
4) packing: mark anti-GP73 with the damping fluid dilution that contains 10% foetal calf serum by the enzyme of step 3 acquisition and resist to suitable working concentration more, press the packing of 6ml/ bottle, be stored in 4 ℃.
4. the preparation of auxiliary reagent:
1) substrate solution A:1.0ml EDTA (1.0 * 10
-2M, 1.0ml H
2O
2(7.5 * 10
-3M), 0.4ml HCl (1.0 * 10
-2M) and 0.2ml Tween20 (1%)
2) colour developing liquid B:luminol 5.0 * 10
-4Mol/L;
3) Fuc-GP73 cleaning buffer solution 20mmTris-Hcl, PH7.4 contains 0.1%Proclin 300 sanitass
4) Fuc-GP73 elutriant 20mmTris-Hcl, PH7.4,150mmNaCl, 500mmol/L Alpha-Methyl-D-mannoside contains 0.1%Proclin 300 sanitass
5) concentrated cleaning solution (20 times of concentrated solutions, 20 *): 0.05% polysorbas20 solution of PBS (pH7.4) preparation;
Embodiment 3: the preparation of pre-mounted eccentric column and use
1, pre-mounted eccentric column is made up of centrifugal post and filled media, and centrifugal post is made up of the centrifuge tube on top and the collection tube of bottom, and the two is nested together, and forms centrifugal post, and auxiliary reagent comprises Fuc-GP73 scavenging solution and Fuc-GP73 elutriant.
2, the preparation of centrifugal column material: adopt the LCA of Pharmacia company, coupling according to the following steps through CNBr activatory Sepharose4B and Sigma company:
(1) 1.5g Sepharose 4B is soaked in 1mmol/L HCl to expanding, moves into sand stamen funnel, with the about 30min of 300mL 1mmol/L HCl washing;
(2) take by weighing 50mg LCA, be dissolved in 7.5mL coupling buffer (0.1mmol/L NaHCO3, pH8.3,0.5mol/L NaCl), merge with Sepharose 4B through washing, with the 10mL test tube of band plug turn upside down mixing (room temperature, 2h);
(3) with 10mL coupling buffer flush away link coupled LCA not.LCA1 content in the washings is counted to such an extent that the coupling rate is 98% after measured;
(4) with 0.2mol/L glycine sealing residue activating gene;
(5) use 10mL 0.1mol/L acetate buffer solution (pH 4, contain 0.5mol/L NaCl) and 0.1mol/L Tris damping fluid (pH 8, contain 0.5mol/L NaCl) washing 3 times successively, again to contain 0.1%BSA, 1mmol/L CaCl
2With 0.1mmol/L MnCl
2PBS (PBS-BSA) washing 1 time, 4 ℃ are temporary standby.
3, in the centrifuge tube of top, add one deck filter cloth, and add a plastic packing and fix, the aperture of this filter cloth is less than agarose, so agarose can not pass through, but protein mass and liquid can flow through.Get 300ul the sephrose 4B of link coupled lectin add in the centrifuge tube on centrifugal post top.
4, add the 1ml-2ml lectin and preserve damping fluid, damping fluid is full of the position that has medium, and major part is present in the following collection tube, and the damping fluid in the collection tube of pre-mounted eccentric column of the present invention bottom contains the CaCl of 1mmol/L
2, 1mmol/L MnCl
2With 50mmol/L Tris-HCL, PH 7.5
The using method of this pre-mounted eccentric column is as follows:
Serum to be detected is centrifugal fully 1., no haemolysis; Take out prepackage microcentrifugation post, discard liquid in lower floor's collection tube.
2. sample dilution: draw 250ul serum in test tube, and add the 350ul cleaning solution dilution, shake up.
3. application of sample: sucking-off 450ul dilution back sample adds in the centrifuge tube of top.37 ℃ of incubators leave standstill, and the centrifuge tube lid is not please covered in this operation, and serum dilution will flow in 15 minutes in lower floor's collection tube;
4. will collect liquid in pipe discards;
5. add scavenging solution 600ul in the centrifuge tube of top, wait for that scavenging solution all flows into (about 3 minutes) in lower floor's collection tube, cover the centrifuge tube lid, 2000 changeed under (or 3000 change) room temperature centrifugal 1 minute;
6. discard liquid in lower floor's collection tube;
7. add scavenging solution 600ul in the centrifuge tube of top, wait for that scavenging solution all flows into (about 3 minutes) in lower floor's collection tube, cover the centrifuge tube lid, 2000 changeed under (or 3000 change) room temperature centrifugal 1 minute;
8. discard liquid in lower floor's collection tube;
9. add elutriant 450ul, when waiting eluting liquid number to ooze present lower floor collection tube, cover the centrifuge tube lid, place in 37 ℃ of thermostat containers incubation 30 minutes;
10. taking-up centrifuge tube, 2000 (or 3000 change) changeed under room temperature centrifugal 1 minute;
Be equipped with detection 11. collect the liquid (sample 1) that flows in lower floor's collection tube.
The quality determining method of test kit of the present invention is:
1) albumen carrying capacity: the content of coupling LCA deducts not coupling protein acquisition content by protein content before calculating coupling.Requirement is not less than 7mg/ml, can obtain result preferably at 7mg/ml-10mg/ml.
2) accuracy: the detected result of 10 parts of normal negative quality controlled serums (comprising the specificity control serum) reference material, non-false positive occurs.The reference material detected result of 5 parts of liver cancer GP73 positive quality control serum does not have false negative and occurs.
3) precision: randomly draw 20 box different batches test kits, use with a liver cancer positive quality control serum by specification operation steps and carry out replication.Calculate each measurement result, obtain average, SD and variation coefficient CV.CV was less than 15% between the Precision test result demonstration was criticized.
3) detection sensitivity: according to GP73 standard substance dilution metering result, the detection sensitivity of this test kit is 1ng/ml.
4) specificity: be divided into four parts of pooled serum samples, every part of 1ml after getting four parts of testing samples mixing.Make interference test serum specimen # 1, #2, #3 after adding tissue-type plasminogen activator (tPA), plasminogen (Plasmin) or the fibronectin (FN) of 50ng dosage respectively, the #4 pooled serum sample that does not add any chaff interference is as basic sample.The by specification operation steps is measured and calculation result.Calculate jamming rate by the interference test calculation formula then.The mushing error of sample # 1, #2, #3 is all less than 5%.
Embodiment 4: the method and the step of carrying out the Fuc-GP73 content detection with the chemiluminescence detection kit of detection liver cancer Fuc-GP73 provided by the invention (Golgi protein 73 heteroplasmons) are:
One, test kit is formed
1, mouse-anti people GP73 monoclonal antibody bag is by plate
2, many antienzymes of the anti-people GP73 of rabbit marker
3, chemical luminous substrate A and B
4, concentrated cleaning solution
5, prepackage LCA-Sepharose 4B centrifuge tube
6, the LCA scavenging solution
7, the LCA elutriant
8, GP73 quantitative criterion product
9, specification sheets
10, the cover plate film
Two, sample disposal and Fuc-GP73 purifying
Serum to be detected is centrifugal fully 1., no haemolysis; Take out prepackage microcentrifugation post, discard liquid in lower floor's collection tube.
2. sample dilution: draw 250ul serum in test tube, and add the 350ul cleaning solution dilution, shake up.
3. application of sample: sucking-off 450ul dilution back sample adds in the centrifuge tube of top.37 ℃ of incubators leave standstill, and the centrifuge tube lid is not please covered in this operation, and serum dilution will flow in 15 minutes in lower floor's collection tube;
4. will collect liquid in pipe discards;
5. add scavenging solution 600ul in the centrifuge tube of top, wait for that scavenging solution all flows into (about 3 minutes) in lower floor's collection tube, cover the centrifuge tube lid, 2000 changeed under (or 3000 change) room temperature centrifugal 1 minute;
6. discard liquid in lower floor's collection tube;
7. add scavenging solution 600ul in the centrifuge tube of top, wait for that scavenging solution all flows into (about 3 minutes) in lower floor's collection tube, cover the centrifuge tube lid, 2000 changeed under (or 3000 change) room temperature centrifugal 1 minute;
8. discard liquid in lower floor's collection tube;
9. add elutriant 450ul, when waiting eluting liquid number to ooze present lower floor collection tube, cover the centrifuge tube lid, place in 37 ℃ of thermostat containers incubation 30 minutes;
10. taking-up centrifuge tube, 2000 (or 3000 change) changeed under room temperature centrifugal 1 minute;
Be equipped with detection 11. collect the liquid (sample 1) that flows in lower floor's collection tube.
Three, detection by quantitative Fuc-GP73
1. design bag by the position of plate and quantity, the bag of usefulness is not airtight by plate, puts in the 2-8 ℃ of refrigerator.
2. add 50 μ l handled sample 1 to corresponding bag by in the plate micropore.
3.37 ℃ incubation 30 minutes.
4. wash 5 times, button is done bag by liquid residual on the plate.
5. the enzyme conjugates that in each hole, adds 100 μ l successively.
6.37 ℃ incubation 30 minutes
7. wash 5 times, button is done bag by liquid residual on the plate
8. add 50 μ l substrate solutions to every hole, please in advance A, B liquid equal-volume are mixed; Or add 25 μ l substrate A to every Kong Zhongxian and add 25 μ l substrate B again, clap even 5 seconds gently.
9. after adding substrate, adopt Chemiluminescence Apparatus to detect in 10 minutes.
Four, the result judges
The production standard curve: with standard substance concentration is X-coordinate, and the RLU value that standard substance are measured is an ordinate zou, makes typical curve; Base of calculation curvilinear regression coefficients R
2, work as R
2This was measured effectively in＞0.95 o'clock;
By this reagent standard curve calculation Fuc-GP73 content, Fuc-GP73 content is the positive judgement of liver cancer greater than 5ng/ml with the value of reading that detects gained.
Embodiment 5: adopt the detected result of test kit provided by the invention to the hepatocarcinoma patient positive:
For judging the coincidence rate of chemiluminescence detection kit of the present invention and enzyme linked immunological kit and clinical liver cancer detected result, get two kinds of test kits of the present invention and carried out the comparison and detection experiment.The sample that adopts Beijing You An hospital to provide compares detection: 50 parts of known primary hepatocarcinoma positive samples, and 30 parts of liver cirrhosis serum, 80 parts of hepatitis, these hepatopathy samples are clinical pathology and make a definite diagnosis.Adopt the healthy serum of 200 parts of health check-ups of Hebei red cross blood center.Detect (2 kinds of methods detect when inconsistent and calculate with the many persons of positive quantity) with chemiluminescence detection kit and enzyme-linked immune quantitative detection reagent box respectively, the results are shown in Table 1.
Table 1. recall rate in different specimens compares
| The serum source | The example number | Positive rate GP73 (greater than 5ng) | Positive rate Fuc-gp73 (greater than 5ng) |
| 200 parts of healthy people of 30 parts of liver cirrhosis of 80 parts of hepatitis of 50 parts of primary hepatocarcinoma positive samples | ?50 ?80 ?30 ?200 | 100％ 75％ 66％ 0 | 100％ 10％ 6％ 0 |
This experimental result shows, up to 100%, the specificity in liver cirrhosis is up to 94% for the positive coincidence rate of 50 parts of primary hepatocarcinoma in the present invention, and the specific degree in hepatitis is up to 90%.
Claims (12)
1, a kind of pre-mounted eccentric column that separates fucosylated Golgi protein GP 73, it is characterized in that it is made up of top separator tube and bottom collection tube, the affinity medium of coupling lectin is equipped with on described top from pipe, one filter cloth is arranged at the separator tube bottom on top, in the collection tube of described bottom buffered soln is housed.Be the albumen of the affine absorption of purifying, and supporting scavenging solution and elutriant.
2, the pre-mounted eccentric column of separation fucosylated Golgi protein GP 73 as claimed in claim 1 is characterized in that described lectin is LcA LCA or tangerine fruit powder spore lectin AAL.
3, the pre-mounted eccentric column of separation fucosylated Golgi protein GP 73 as claimed in claim 1, it is characterized in that described affinity media is that the employing sepharose is a carrier is carrier, comprises sepharose sepharose 4B, sepharose sepharose 6B or sepharose sepharose FF, sepharose sepharose CL-4B, sepharose sepharose CL-6B.Described buffered soln is the active protection liquid of lectin.
4, the pre-mounted eccentric column of separation fucosylated Golgi protein GP 73 as claimed in claim 1 is characterized in that described filter cloth is to stop that coupling has the affinity media of lectin to pass and the filter cloth that passes of barrier liquid and protein not.
5, the pre-mounted eccentric column of separation fucosylated Golgi protein GP 73 as claimed in claim 3 is characterized in that the described sepharose that the coupling LcA is housed obtains by following steps:
(1) the Sepharose 4B with bromize hydrogen activating soaks, washs with 1mmol/L HCl;
(2) take by weighing LCA, be dissolved in the coupling buffer, with Sepharose 4B merging mixing through washing;
(3) use not link coupled LCA of coupling buffer flush away, measure the LCA content in the washings.
(4) with glycine sealing residue activating gene, washing, 4 ℃ are temporary standby.
6, the pre-mounted eccentric column of separation fucosylated Golgi protein GP 73 as claimed in claim 1 is characterized in that the damping fluid in the collection tube of bottom contains the CaCl of 1mmol/L
2, 1mmol/L MnCl
2With the Tris-HCL of 50mmol/L, PH7.5.
7, as the supporting solution of use of the pre-mounted eccentric column of claim 1 indication comprise cleaning buffer solution (20mmol/L Tris-HcL, PH7.4) and elution buffer (20mmol/LTris-HcL, PH7.4,500mmol/L Alpha-Methyl-D-mannoside).
8, a kind of detection by quantitative test kit that detects fucosylated Golgi protein GP 73, it is characterized by the contained sample processing system of test kit according to claim 1, carry out after centrifugal, wash-out obtains Fuc-GP73 through claim 1 described device, carry out detection by quantitative then, method comprises radioimmunoassay, fluorescence immunoassay (time resolved fluorescence technology), enzyme immunoassay, immuno-gold labeling technology, chemiluminescent immunoassay, electrochemiluminescence immunoassay.
9, the detection by quantitative test kit of a kind of detection fucosylation GP73 as claimed in claim 8 is characterized by sample processing system according to claim 1, detects GP73 and Fuc-GP73 content and adopts chemiluminescence method, and detection kit comprises following assembly:
(1) pre-mounted eccentric column of separation fucosylation GP73;
(2) bag is by the chemoluminescence enzyme plate of GP73 antibody;
(3) positive control, negative control thing;
(4) the anti-GP73 antibody of enzyme labelling;
(5) chemical luminous substrate solution A, chemical luminous substrate colour developing liquid B, Fuc-GP73 cleaning buffer solution, Fuc-GP73 elutriant, GP73 standard substance.
The pre-mounted eccentric column of described separation liver cancer fucosylation GP73 is made up of top separator tube and bottom collection tube, and described top separator tube is equipped with the affinity medium of coupling lectin, in the collection tube of described bottom buffered soln is housed.
10, test kit as claimed in claim 7, its making method may further comprise the steps:
(1) the GP73 monoclonal antibody bag of high titre is spent the night to enzyme plate absorption;
(2) wash plate with the tween phosphate buffered saline buffer, spend the night with the tween phosphate buffered saline buffer sealing that contains bovine serum albumin again, dry after the drying;
(3) collect GP73 masculine liver cancer patient ascites, filtration sterilization, packing promptly obtain positive control or as positive quality control serum.Collect the negative normal human serum of GP73, filtration sterilization, packing promptly obtain the negative control thing;
(4) how anti-the anti-GP73 of rabbit of acquisition enzyme labelling is;
(5) pre-mounted eccentric column, bag is how anti-by the anti-GP73 of rabbit of the chemoluminescence enzyme plate of GP73 monoclonal antibody, positive control, negative control thing, enzyme labelling, substrate solution A, colour developing liquid B, Fuc-GP73 cleaning buffer solution, Fuc-GP73 elutriant, GP73 standard substance, concentrated cleaning solution are packed jointly, obtain test kit.
11, a kind of detection by quantitative test kit of detection liver cancer fucosylated Golgi protein GP 73 as claimed in claim 8, it is characterized by sample processing system according to claim 1, detect Fuc-GP73 content and adopt the enzyme linked immunological quantitative analysis method, detection kit comprises following assembly:
(1) pre-mounted eccentric column of separation liver cancer fucosylated Golgi protein GP 73;
(2) bag is by the enzyme plate of GP73 antibody;
(3) positive control, negative control thing;
(4) how anti-the anti-GP73 of the rabbit of enzyme labelling is;
(5) substrate solution A, colour developing liquid B, Fuc-GP73 cleaning buffer solution, Fuc-GP73 elutriant, reaction terminating liquid, GP73 standard substance, concentrated cleaning solution,
The pre-mounted eccentric column of described separation liver cancer fucosylated Golgi protein GP 73 is made up of top separator tube and bottom collection tube, and described top separator tube is equipped with the affinity medium of coupling lectin, in the collection tube of described bottom buffered soln is housed.
12, test kit as claimed in claim 11, its making method may further comprise the steps:
(1) the GP73 monoclonal antibody (commercially available) of high titre bag is spent the night to enzyme plate absorption;
(2) wash plate with the tween phosphate buffered saline buffer, spend the night with the tween phosphate buffered saline buffer sealing that contains bovine serum albumin again, dry after the drying;
(3) collect GP73 masculine liver cancer patient ascites, filtration sterilization, packing promptly obtain positive control.Collect the negative normal human serum of GP73, filtration sterilization, packing promptly obtain the negative control thing.
(4) how anti-the anti-GP73 of rabbit of acquisition enzyme labelling is;
(5) pre-mounted eccentric column, bag is how anti-by the anti-GP73 of rabbit of the enzyme plate of GP73 monoclonal antibody, positive control, negative control thing, enzyme labelling, substrate solution A, colour developing liquid B, Fuc-GP73 cleaning buffer solution, Fuc-GP73 elutriant, reaction terminating liquid, GP73 standard substance, concentrated cleaning solution are packed jointly, obtain test kit.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710107508 CN101062939A (en) | 2007-05-17 | 2007-05-17 | Device and reagent case for detecting hepatic carcinoma fucosido-fucosyl gorky protein GP73 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710107508 CN101062939A (en) | 2007-05-17 | 2007-05-17 | Device and reagent case for detecting hepatic carcinoma fucosido-fucosyl gorky protein GP73 |
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|---|---|
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|---|---|---|---|
| CN 200710107508 Pending CN101062939A (en) | 2007-05-17 | 2007-05-17 | Device and reagent case for detecting hepatic carcinoma fucosido-fucosyl gorky protein GP73 |
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