CN101735319B - Monoclonal antibody against GP73 protein, preparation method and application thereof - Google Patents
Monoclonal antibody against GP73 protein, preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a monoclonal antibody against GP73 protein, a preparation method and an application thereof. The invention also provides an immunological quantitative detection kit for detecting GP73 in a PBLs sample and a preparation method thereof. The kit in the invention can be used for monitoring the development level of chronic hepatitis and prognosis, and can be used for early diagnosis of liver cancer. By using the kit in the invention to monitor the expression level of the GP73, the prognosis level and probability to cause cirrhosis and liver cancer can be determined for patients, and the most direct support is provided for preventing, diagnosing and curing liver cancer and hepatitis.
Description
Technical field
The invention belongs to biological technical field, more particularly, the invention provides a kind of monoclonal antibody, its preparation method and application at Golgi protein GP 73.The present invention also provides immunology detection by quantitative test kit of a kind of Golgi protein GP 73 that is used for detecting the human peripheral sample and preparation method thereof.Utilize this test kit can calculate the expression contents of the Golgi protein GP 73 in the human peripheral blood quickly and accurately.
Background technology
The main nosetiology of hepatocellular carcinoma HCC (hepatocellular carcinoma) is that HBV or HCV chronic hepatopathy poison infects, and takes place arranged as last long latent period in liver cancer---promptly infect hepatovirus to the liver cancer time of origin.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 liver cirrhosis patient 3,600,000 people 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) whole world 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 global 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 blood serum designated object 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 AFP negative (sensitivity≤70%) in AFP positive (specificity≤75%) and the part liver cancer in the optimum hepatopathy of part, make to rely on AFP to carry out the HCC early warning and monitor to have certain limitation, and AFP detects for the relevant HCC sensitivity of HCV not high.Therefore wish clinically to obtain perhaps can carry out the complementary index with AFP than alpha-fetoprotein specificity and the higher new serological index of sensitivity.
In recent years, the development of protein science technology makes the new tumor markers of high flux screening become possibility, various promising new tumor markerses are found in succession, wherein Golgi apparatus protein 73 (GolgiProtein 73, GP73) most possibly become the especially blood serum designated object of early hepatocarcinoma diagnosis of better diagnosing cancer of liver.
GP73 (Golgi Protein 73) is a kind of II type gorky transmembrane protein, also is a kind of newfound albumen relevant with hepatopathy disease course.The molecular weight of GP73 near 73KD (Kladney et al., 2000, Gene 249,53-65).GP73 in the liver cell of viral infection high expression level (Kladney, et al., 2000, Gene 249,53-65), expression arranged also in the bile epithelial cell, and expression amount is seldom in normal liver cell.Opposite, liver problem sufferer's liver cell shows the strong immune response to GP73 antibody.In GP73 mRNA and the albumen HepG2 malignant tumor of liver cell after the various viruses of transfection (comprising adenovirus) high expression level is arranged also.Facilitate hepatopathy such as alcoholic liver disease suffering from viral liver disease or non-viral, among the patient of autoimmune liver disease etc., the expression amount of GP73 significantly raise (Kladney, et al., 2002, Hepatology 35 (6): 1431-40).
(National Cancer Institute, NCI) application of GP73 as the hepatocarcinoma early diagnosis index proved to American National ICR in 2005 by a seminar of Zi Zhuing.For the detection of early hepatocarcinoma, the remolding sensitivity AFP of GP73 exceeds 2-3 doubly, is considered to a following most promising liver cancer early warning New Set.To studies show that of 352 routine liver problem sufferers, GP73 is 69% to the sensitivity of HCC diagnosis, and specificity is 75%.And its sensitivity to the early hepatocarcinoma diagnosis is 62%, is significantly higher than AFP (25%).AFP is lower than among the HCC patient of 20ng/ml, has the patient GP73 above half (57%) to raise, and GP73 is to the HCC early diagnosis in prompting, and especially the diagnosis of the early stage HCC of AFP feminine gender has significant superiority.
What but this research was adopted is the Western blot method, and western blotting method is a semi-quantitative analysis, and labor capacity is too big, is unsuitable for the high throughput analysis that large sample amount checking institute needs.In addition, what this method adopted is how anti-detection method, and this method is not set up quantitative criterion.
Purpose of the present invention lacks the deficiency of GP73 detection by quantitative test kit as the hepatocarcinoma early diagnosis detection means at present clinically in order to solve just, a kind of highly active anti-GP73 monoclonal antibody and sensitive detection kit easy and simple to handle, accurate and measuring method are provided, so that observe the GP73 expression contents from protein level, can take effective comprehensive therapeutic plan as early as possible, the generation of prevention liver cirrhosis and liver cancer.
Summary of the invention
In order to solve the problems of the technologies described above,, provide a kind of highly active anti-GP73 monoclonal antibody in a first aspect of the present invention.
In another aspect of this invention, provide the preparation method of antibody of the present invention and be used to detect the proteic application of GP73.
In another aspect of this invention, provide a kind of enzyme-linked immune quantitative detection reagent box, described test kit comprises anti-GP73 monoclonal antibody of the present invention.
In another aspect of this invention, provide a kind of chemical luminescent analysis reagent kid, described test kit comprises anti-GP73 monoclonal antibody of the present invention.
In another aspect of this invention, a kind of quick diagnosis reagent kit also is provided, comprise anti-GP73 monoclonal antibody of the present invention and the GP73 protein standard substance of demarcating content in the described test kit, wherein said protein standard substance has at least two kinds, GP73 content value in wherein a kind of standard substance is the threshold value of normal people and liver problem sufferer's GP73 content, and the GP73 content value in the another kind of standard substance is the threshold value of optimum liver problem sufferer and pernicious hepatopathy (liver cancer) patient's GP73 content.
Use monoclonal antibody of the present invention and detection kit, can calculate the expression amount of GP73 in the peripheral blood exactly, and then hepatocarcinoma early diagnosis is judged.And, use monoclonal antibody of the present invention and detection kit, can reflect the level that exists of GP73 in the serum truly, there is not cross reactivity, specificity is good, is convenient to quality control, and demonstrates high sensitivity.
Description of drawings
Fig. 1 is GP73 gene complete sequence figure (SEQ ID NO:1), and wherein the sequence in the sequence front end frame is Nde I site, and the sequence in the terminal frame of sequence is BamH I site.
Fig. 2 is the structural representation of employed plasmid vector pET-15b-GP73.
Fig. 3 identifies electrophorogram for the Pet-15b-GP73 carrier.
Fig. 4 is the electrophorogram of expression of GP73 bacterium and GP73 protein purification, 1-6 swimming lane: select the high transformed bacteria of GP73 expression amount; The 7-10 swimming lane: No. 2 bacterial strains of selecting are expressed, the GP73 albumen of purifying, about molecular weight 40KD.
Fig. 5 is the bilogarithmic graph that the 3E12 monoclonal antibody detects standard substance, and the X-coordinate among the figure is the natural logarithm of absorbancy, and ordinate zou is the natural logarithm of concentration.
The bilogarithmic graph that Fig. 6 detects standard substance for import monoclonal antibody YA-14, the X-coordinate among the figure is the natural logarithm of absorbancy, ordinate zou is the natural logarithm of concentration.
Fig. 7 is the GP73 protein content of enzyme linked immunological kit mensuration of the present invention and the graph of a relation between hepatopathy or the liver cancer.
The ROC curve that Fig. 8 exempts from quantitative assay hepatopathy serum diagnosis liver cancer for the GP73 enzyme, X-coordinate are (1-specificity per-cent), and ordinate zou is a sensitivity per-cent, records area under curve AUC=0.88.
Fig. 9 is the GP73 protein content of chemical luminescence reagent kit mensuration of the present invention and the graph of a relation between hepatopathy or the liver cancer.
Figure 10 is the ROC curve of GP73 chemoluminescence quantitative assay hepatopathy serum diagnosis liver cancer, and X-coordinate is (a 1-specificity per-cent), and ordinate zou is a sensitivity per-cent, records area under curve AUC=0.9.
Embodiment
The invention provides a kind of enzyme-linked immune quantitative detection reagent box at Golgi protein GP 73, described test kit comprises anti-GP73 monoclonal antibody of the present invention.
On the other hand, the present invention also provides a kind of chemical luminescent analysis reagent kid at Golgi protein GP 73, and described test kit comprises anti-GP73 monoclonal antibody of the present invention.
Monoclonal antibody of the present invention is screened the hybridoma clone and is obtained animal immune, the natural GP73 antigen of employing by using solubility conformation type Golgi protein GP 73 antigen.Particularly, monoclonal antibody of the present invention is prepared by following method: with the pure product of Golgi protein GP 73 recombinant antigen with conformation type epi-position the Balb/c mouse is carried out immunity, the spleen cell and the murine myeloma cell SP2/0 that get immune mouse merge, use the special hybridoma clone of the Golgi protein GP 73 antigen selection GP73 with conformation type epi-position who from serum, extracts again, be injected to mouse peritoneal, collect ascites, monoclonal antibody purification.In some specific embodiments of the present invention, described monoclonal antibody is the secreted monoclonal antibody of hybridoma 3E12 strain that filters out, and described hybridoma cell strain is preserved in Chinese common micro-organisms DSMZ (CGMCC) on November 11st, 2008 with preserving number CGMCC No.2742.
In certain embodiments of the invention, the standard substance that also comprise Golgi protein GP 73 in the described test kit.In certain embodiments of the invention, the proteic standard substance of described GP73 are the GP73 full-length proteins, and are in the protein protection stable liquid.Described GP73 standard substance can carry out recombinant expressed the preparation by genetically engineered, molecular biology and biochemical means.A kind of preparation method comprises: use the escherichia coli expression Golgi protein GP 73, utilize the albumen label of amalgamation and expression to do affinity purification, obtain the GP73 standard substance.
In some preferred embodiment of the present invention, the mentioned reagent box can also contain enzyme plate, horseradish peroxidase (HRP) mark polyclonal antibody and auxiliary reagent.Wherein, chromogenic substrate tetramethyl benzidine (TMB) can be contained in the described auxiliary reagent,, luminous substrate luminol (luminol,3-aminophthalic acid cyclic hydrazide) can be contained in the described auxiliary reagent for chemiluminescence detection kit for enzyme-linked immunologic detecting kit.In other preferred embodiments of the present invention, described monoclonal antibody is to be coated on the enzyme plate.
In specific embodiments of the present invention, the preparation method of mentioned reagent box also is provided, described method comprises the preparation of antibody sandwich plate, the preparation of enzyme labelled antibody and the preparation of auxiliary reagent.
Monoclonal antibody bag in the test kit of the present invention be the mouse-anti GP73 monoclonal antibody that mouse immune obtained with the pure product of Golgi protein GP 73 albumen by plate to flat board wrap by and preparation.Described flat board can be selected commercially available enzyme plate or chemoluminescence plate for use; Specification can be dull and stereotyped or 12 * 8,12 * 4 removable battens in 96 holes.
The step of a kind of enzyme plate that can be used for preparing monoclonal antibody bag quilt of the present invention or chemoluminescence plate is as follows:
1) prepares monoclonal antibody: after routinely the Balb/c mouse being carried out immunity with the Golgi protein GP 73 recombinant antigen, the spleen cell and the murine myeloma cell SP2/0 that get immune mouse merge, with the special hybridoma clone of the Golgi protein GP 73 antigen selection GP73 that extracts in the serum, the injection mouse peritoneal is collected ascites and promptly obtain anti-GP73 monoclonal antibody behind reorganization proteinG prepackage chromatography column purifying.
2) bag quilt: with monoclonal anti body and function 0.05M, the carbonic acid buffer dilution back of pH9.5 adds each hole of enzyme plate or chemoluminescence plate, every hole 100 μ l, absorption is spent the night, wash plate with tween-phosphate buffered saline buffer, spend the night with tween-phosphate buffered saline buffer sealing again, dry after the drying, promptly obtain the enzyme plate or the chemoluminescence plate of monoclonal antibody bag quilt.
Enzyme mark polyclonal antibody in the test kit of the present invention can prepare with horseradish peroxidase (HRP) mark GP73 polyclonal antibody.GP73 resists by the routine immunization method more is adopted the immune new zealand white rabbit of the pure product of GP73 to obtain.
A kind of method that can be used for preparing enzyme mark polyclonal antibody of the present invention is as follows:
A) with NaIO
4-glycol method is carried out the oxidation of HRP, reaches final concentration 10mg/ml.
B) in alkaline carbonic acid salt buffer (0.05M, the carbonate buffer solution of pH9.5), dialysed 6 hours, realize the mark of HRP, use NaBH after reaction finishes polyclonal antibody
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 polyclonal antibody.
In enzyme linked immunological kit of the present invention, auxiliary reagent can comprise integrated enzyme reaction substrate solution, colour developing liquid, reaction terminating liquid and cleaning buffer solution.A kind of auxiliary reagent that can be used for the mentioned reagent box is as follows:
A) 3% superoxol of substrate solution A:50mmol/L phosphoric acid-citrate buffer solution (pH5.0) preparation;
B) colour developing liquid B: tetramethyl benzidine (TMB) methanol solution, concentration is 0.1mg/ml;
C) reaction terminating liquid: 2mol/L sulfuric acid;
D) cleaning buffer solution (20 times of concentrated solutions, 20 *): 0.05% polysorbas20 solution of 20mmol/L PBS (pH7.4) preparation.
E) sample diluent: 2%BSA (bSA)
The present invention also provides the method for using above-mentioned enzyme linked immunological kit to detect the Golgi protein GP 73 protein content, and described method comprises the steps:
A) antigen-antibody reaction: every hole adds 100 μ l sample diluents in the micropore of the antibody sandwich plate that test kit provides, add 50 μ l test serum samples or 50 μ l more respectively and contain different concns (0,20ng/ml, 40ng/ml, 100ng/ml, GP73 protein standard substance 250ng/ml), 37 ℃ of water bath heat preservations 30 minutes are washed plate.HRP-polyclonal antibody solution is added each hole, every hole 100 μ l, 37 ℃ of water bath heat preservations 30 minutes.Repeat to wash plate operation 4 times.
B) color reaction: every hole adds substrate solution A successively, each 50 μ l of colour developing liquid B, and 37 ℃ of water bath heat preservations 10 minutes, every hole adds 50 μ l reaction terminating liquids again and finishes reaction.
C) colorimetric:, measure OD value and record at 450nm with microplate reader with the light absorption value zeroing in blank hole.
D) result judges:
A. for microplate reader that can not automatic data processing, the data that can handle with the computer computed in software.
B. hand computation result: the absorbance value that deducts the blank well background calculates mean absorbance (y axle) and its corresponding concentration value (x axle) of each reference liquid to correct absorption value, connects into a typical curve.Calculate the concentration value of unknown patient specimen according to this typical curve.
In chemical luminescence reagent kit of the present invention, auxiliary reagent can comprise chemiluminescence reaction substrate solution, colour developing liquid and sample diluent, and a kind of auxiliary reagent of mentioned reagent box is as follows:
1) substrate solution A:EDTA (1.0 * 10
-2M), H
2O
2(7.5 * 10
-3M), HCl (1.0 * 10
-2M) and Tween20 (1%).
2) colour developing liquid B:50mM pH9.6 carbonate buffer solution contains luminol 5.0 * 10
-4Mol/L;
3) sample diluent: the 20mM pH 7.4 PBS damping fluids that contain 2%BSA
The present invention also provides the method for using above-mentioned chemical luminescence reagent kit to detect Golgi protein GP 73, and described method comprises the steps:
A) antigen-antibody reaction: in the micropore of the antibody sandwich plate that test kit provides, add 100 μ l sample diluents respectively, add 10 μ l test serum samples or 10 μ l then and contain different concns (0,20ng/ml, 40ng/ml, 100ng/ml, GP73 protein standard substance 250ng/ml), 37 ℃ of water bath heat preservations 30 minutes are washed plate.HRP-polyclonal antibody solution is added each hole, every hole 100 μ l, 37 ℃ of water bath heat preservations 30 minutes.Repeat to wash plate operation 4 times.
B) color reaction: every hole adds substrate solution A successively, each 50 μ l of colour developing liquid B, the value of reading.
C) result calculates:
A drawing standard curve: with standard substance concentration is X-coordinate, and the values of chemiluminescence 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 0.98 o'clock;
B calculates the test serum sample concentration: the Golgi protein GP 73 concentration that calculates the test serum sample according to the values of chemiluminescence of testing sample from typical curve.
In another aspect of this invention, a kind of quick diagnosis reagent kit also is provided, contain the monoclonal antibody of anti-GP73 and the GP73 protein standard substance of demarcating content in the described test kit, wherein said protein standard substance has at least two kinds, GP73 content in wherein a kind of standard substance is the threshold value of normal people and liver problem sufferer's GP73 content, and the GP73 content in the another kind of standard substance is the threshold value of optimum liver problem sufferer and pernicious hepatopathy (liver cancer) patient's GP73 content.In some particularly preferred embodiment of the present invention, the threshold value of described normal people and liver problem sufferer's GP73 content is set to 40ng/ml, and the threshold value of described optimum liver problem sufferer and pernicious hepatopathy (liver cancer) patient's GP73 content is set to 100ng/ml.
Test kit of the present invention has the following advantages:
A) test kit of the present invention as catching monoclonal antibody, has higher sensitivity and specific degree with reorganization GP73 antigen immune and the monoclonal antibody that obtains with natural GP73 protein screening.
B) the matched standard substance of test kit can calculate the proteic content of GP73 contained in the blood sample sample by accurate quantification.
C) owing to the threshold value of the GP73 content that normal people and liver problem sufferer are provided in the standard substance of test kit of the present invention and optimum liver problem sufferer and pernicious hepatopathy (liver cancer) patient's threshold value, therefore after reading the observed value of tested sample, only need directly this observed value and threshold value (for example 40ng or 100ng) to be compared, can tentatively judge tested patient's hepatopathy development, detecting operation is fast and convenient, the accuracy rate height.
Further specify the present invention with embodiment below.It should be understood that following embodiment is used to illustrate the present invention rather than limitation of the present invention.
Embodiment 1: MONOCLONAL ANTIBODIES SPECIFIC FOR of the present invention and sign
1) GP73 MONOCLONAL ANTIBODIES SPECIFIC FOR:
A) structure of Golgi protein GP 73 recombinant antigen (immunogen):
By gene order AF236056 synthetic GP73 gene complete sequence, according to pET-15b carrier multiple clone site, two ends are introduced Nde I and BamH I site respectively, and the sequence total length that obtains is shown in Fig. 1 (SEQ ID NO:1).The sequence of gained is inserted among the prokaryotic expression carrier pET-15b, and pET-15b-GP73 plasmid structure collection of illustrative plates is shown in Fig. 2.Recombinant plasmid identifies with Nde I and BamH I double digestion whether insert fragment by 1.2% agarose gel electrophoresis inspection correct, and qualification result is shown in Fig. 3, and wherein the 1st swimming lane is M.Takara DL2, and 000Marker, the 2nd swimming lane are the double digestion product.Screen correct recombinant plasmid transformed e. coli bl21, be layered on the Amp+LB plate culture medium, 37 ℃ of incubated overnight, picking list bacterium colony is put in the 5ml Amp+LB substratum and was cultivated 2-4 hour, treats that bacterium liquid shows slightly muddiness and OD
600Value reaches at 0.6 o'clock and adds the extremely concentration 0.5mmol/L of sec.-propyl-β-D-sulfo-semi-lactosi (IPTG), 37 ℃ of inducing culture 4 hours, get the cracking of bacterium liquid and do polyacrylamide gel electrophoresis (12%SDS-PAGE), have obvious expresser then to carry out great expression (Fig. 4) at the 40KD place.
Get the bacterium liquid that 5 μ l have identified, add in the 5ml Amp+LB substratum, 37 ℃ of overnight incubation, transfer to again in the 200ml Amp+LB substratum, cultivated 2 hours for 37 ℃, add IPTG (0.5mmol/L) and induce, collect thalline after 4 hours, the condition of abduction delivering is 37 ℃, 0.5mmol/L IPTG, 4h.Centrifugal collection thalline, ultrasonic (SONICS﹠amp; MATERIALS company, Uilbra-Cell VCX500 type Ultrasonic Cell Disruptor) fragmentation, ultrasonic supernatant is behind 30% saturated ammonium sulphate, with 1 * PBS (20mmol/L, pH7.4) dissolving, to 1 * PB (pH7.45) dialysed overnight, centrifugal back supernatant adopts the GE Histrap of company nickel ion affinity chromatograph purification column to carry out purifying, and the purity of protein behind the purifying reaches electrophoresis pure (more than 95%).
Recombinant antigen has only solubility just might have the conformation type epi-position, clone full-length gene GP73 albumen, in intestinal bacteria with affinity purification label his-tag amalgamation and expression, through nickel post affinity chromatography, the GP73 antigen of the solubility of the purifying of acquisition.This antigen is used for immune mouse to obtain monoclonal antibody.
B) natural GP73 antigen prepd: collect the hepatopathy human serum, store for future use.The gene recombination GP73 protein immunization New Zealand white rabbit of purifying obtains GP73 antigen polyclonal antibody.Utilize the GP73 antigen polyclonal antibody that obtains to prepare affinity column, the hepatopathy human serum is crossed affinity column, wash-out obtains the GP73 natural structure albumen behind the purifying, and this antigen is used to screen monoclonal antibody to be used.
C) immunity and cytogamy: mix with Freund's complete adjuvant as the recombinant immune of a) step of equivalent preparation is former, obtain oil emulsion.With this emulsion with 0.2 milliliter dosage subcutaneous be applied to BALB/C mice (product of CLEAJapan, 6 the week ages male) the site, back.The enhancing immunity after 7 days or 14 days of immunity for the first time, in cytogamy preceding 3 days then, intraperitoneal was used 0.2 milliliter of above-mentioned immunogen.Last enhancing immunity was downcut spleen cell from each mouse after 3 days, and (the SP2/0-Ag14 strain RikenGeneBank) mixes with 10:1, merges with 50% Macrogol 4000 with the myeloma cell.Go up selectivity at HAT substratum (product of Gibco) and cultivate hybridoma.
D) hybridoma screening: be preferred high reactivity cell strain, adopt HAT to select to cultivate, screen with the ELISA method, be specially the natural GP73 albumen that bag is extracted on wrapping by plate, the murine antibody of absorption GP73, the anti-mouse two of rabbit that adds the HRP mark resists the positive clone of positive person.Be specially: after fusion the 12nd day, measure antibody activity in each culture supernatant with the antigen coated ELISA method of natural GP73.The culture supernatant of respectively getting the fused cell of 200 microlitres is added to the product of 96 hole elisa plate Coaster) the hole in, wherein every hole is fixed with the antigen of 2 mcg/ml, being reflected at 37 ℃ carried out 1 hour, then with PBS (washing lotion) washing that contains 0.05%Tween 20, anti-mouse IgG (the Cappel product that in each hole, adds 200 microlitre peroxidase labellings, 1:20,000).Be reflected at 37 ℃ carry out 1 hour after, with above-mentioned washing lotion wash plate.In each hole, add 200 microlitre substrate solutions (0.1M tetranitro-diphenyl amine TMB and 0.012% aqueous hydrogen peroxide solution) then, reaction was at room temperature carried out 15 minutes.Then, in each hole, add 50 microlitre 3.5mol sulfuric acid and stop enzyme reaction, measure the absorbancy of wavelength 492 nanometers, selection can with the aitiogenic antibody of natural GP73 antigen, and the results absorbancy is not less than 0.15 hybridoma clone.With the restriction dilution method each clone is carried out two time clonings.Hybridoma behind the clone is transplanted in the BALB/C mice, found that 32 hybridoma clones produce the various monoclonal antibodies that ascites reclaims that can be used as.That wherein absorbancy is the highest is the clone of numbering 3E12, and this positive colony continues to use its numbering 3E12 through 3 time cloning acquisition cell strain of monoclonal antibody.This hybridoma cell strain is preserved in Chinese common micro-organisms DSMZ (CGMCC) on November 11st, 2008 with preserving number CGMCC No.2742.
The hybridoma chromosome examination: adding final concentration in cell culture medium is the colchicine of 0.3 μ g/ml, continues to cultivate 2h in 37 ℃, and collecting cell adds 0.075mol/L KCl, 37 ° of C insulation 30min.Fix 3 times film-making, Giemsa dyeing with methyl alcohol and Glacial acetic acid mixed solution (both ratios are 3: 1).The chromosome number that records the 3E12 clonal cell line is 95, and big portion is kinetochore, an end karyomit(e), and kinetochore, middle part and submetacentric chromosome are arranged.
Antibody purification method: adopt protein A Sepharose 4B affinitive layer purification.Record that protein content is 7-11g/L behind the purifying.
The monoclonal antibody titration: adopt indirect elisa method to measure ascites McAb and tire, concrete steps are as follows: (1) uses the antigen coated enzyme plate of GP73 of 10mg/L, every hole 100 μ l, and 4 ℃ are spent the night; (2) (contain the PBS that mass concentration is the Tween-20 of 0.5g/L, 20mmol/L pH7.4) washes plate 3 times with washings; (3) with concentration be the BSA-PBS sealing 2h of 2mg/ml under 37 ℃ of conditions; (4) wash plate 3 times with above-mentioned washings; (5) sample to be checked (ascites) that adds different extension rates leaves standstill 1.5h, simultaneously with normal nutrient solution as negative control; (6) wash plate 3 times with above-mentioned washings; (6) in every hole, add the anti-mouse IgG of HRP-antibody, leave standstill 1h, measure with the TMB Color Appearance System then.Recording tiring of 3E12 hybridoma ascites monoclonal antibody is 6 * 10
5
Antibody subtype is identified: the IgG subclass that records 3E12 with the two expansion methods of ELISA is the IgG1 type.
Characteristic test: (SANTA CRUZ BIOTECHNOLOGY, INC. monoclonal antibody cell strain YA-14) carries out parallel comparison with external monoclonal antibody.Concrete grammar is for wrapping respectively simultaneously by on polystyrene board, concentration is 200 μ g/ml, adopt the many anti-spikes of carrying out of enzyme mark GP73 of preparation, adopt the GP73 recombinant protein of 20 liver cancer, 20 hepatitis, 20 normal people's samples and different concns (0,20ng/ml, 40ng/ml, 100ng/ml and 250ng/ml) to detect, relatively import monoclonal antibody and 3E12 are to the effect (table 1) of hepatopathy differential diagnosis, the linearity test effect (table 2 of quantitative criterion product, Fig. 5, Fig. 6).This result shows with optical density(OD) light absorption value OD, and the OD value shown in the table is (the OD value that the actual OD value-GP73 protein concentration that records is 0 control sample).
Table 1: two kinds of monoclonal antibodies are to the detection of hepatopathy disease
| Detected result (OD 450Value) | Liver cancer | Hepatitis | The normal people |
| 3E12 | 2.02±0.75 | 0.43±0.37 | 0.13±0.07 |
| Monoclonal antibody YA-14 | 0.74±0.66 | 0.51±0.35 | 0.23±0.17 |
The result shows: 3E12 has significant difference, P＜0.001 to liver cancer, hepatitis, normal people's detection OD value; And YA-14 does not have significant difference, P=NS to the detection OD value of 3 kinds of serum.
Table 2: the activity contrast of two kinds of monoclonal antibodies
| Antigen concentration | The 3E12 monoclonal antibody | Import monoclonal antibody YA-14 |
| Numbering | Concentration (ng/ml) | Ln (concentration) | Absorbancy OD 450 | Ln (absorbancy) | Absorbancy OD 450 | Ln (absorbancy) |
| 1 | 20 | 2.99 | 0.331 | -1.105636904 | 0.051 | -2.975929646 |
| 2 | 40 | 3.68 | 0.573 | -0.556869562 | 0.233 | -1.456716825 |
| 3 | 100 | 4.60 | 1.156 | 0.14496577 | 0.864 | -0.14618251 |
| 4 | 250 | 5.52 | 2.034 | 0.710004298 | 0.912 | -0.092115289 |
The result shows: the R of YA-14
2Value is significantly less than the R of 3E12
2Value illustrates that the linear relationship of YA-14 is relatively poor, can't reach the requirement of detection by quantitative reagent.
Embodiment 2: the preparation of Golgi protein GP 73 enzyme-linked immune quantitative detection reagent box
Present embodiment has prepared a kind of Golgi protein GP 73 enzyme-linked immunologic detecting kit of the present invention (96 person-portion), and its composition comprises:
5 bottles of Golgi protein GP 73 standard substance;
GP73 monoclonal antibody bag is by 1 of plate (96 hole);
1 bottle of the GP73 polyclonal antibody of horseradish peroxidase (HRP) mark, the 10ml/ bottle;
Each 1 bottle of substrate solution A, colour developing liquid B, each 5ml/ bottle;
1 bottle of reaction terminating liquid, the 5ml/ bottle;
1 bottle of cleaning buffer solution (20 * concentrate), the 50ml/ bottle.
Concrete operations are as follows:
1. prepare the Golgi protein GP 73 standard substance:
1) employed GP73 albumen is embodiment 1 the 1st) the total length people GP73 albumen of band His label of synthetic in the part.
2) packing: by 0,20ng/ml, 40ng/ml, 100ng/ml, the different concns of 250ng/ml are diluted in 2% the BSA solution, add sanitas procline 300 (available from SUPELCO companies) to ultimate density 0.1% with GP73 albumen.
Filtration sterilization, and under aseptic condition, divide in the 1.5ml tubule of packing into every pipe 0.5ml.Be stored in 4 ℃.
2. make GP73 monoclonal antibody bag by plate:
1) employed anti-GP73 monoclonal antibody is the secreted monoclonal antibody of hybridoma 3E12 strain, and antibody is through protein A agarose medium purification.
2) bag quilt:
12 * 8 removable battens that enzyme plate adopts Costar company to produce.With the described monoclonal anti body and function of step 1) 0.05mol/L, the carbonate buffer solution dilution of pH9.5 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, with cleaning buffer solution (PBST solution, comprise 20mmol/LPBS, 0.5%Tween-20 pH7.4) washes plate, seal (the 2%BSA that spends the night with this confining liquid damping fluid again, be diluted in the PBST solution), dry after the drying, promptly obtain the monoclonal antibody coated elisa plate.By 96 holes/piece packaging of aluminium foil bag, vacuum sealing.
3. the anti-GP73 polyclonal antibody for preparing horseradish peroxidase (HRP) mark
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 polyclonal antibody and mark (lucifuge)
A) adjust antibody concentration (cross lowly then concentrate as protein concentration) to about the 5mg/ml, with 50mmol/L carbonate buffer solution (the 1mol/L NaHCO about pH9.5 with PEG20000
3With 1mol/L Na
2CO
3Press the mixed of 10:1, 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) polyclonal antibody is mixed by 1:4 with HRP, dialysis two hours was changed liquid once more than 6 hours in 50mmol/L pH9.5 carbonate buffer solution.
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 antigen polyclonal antibody
A) finish in the polyclonal antibody 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) the HRP-polyclonal antibody of Ti Chuning is to the PBS dialysed overnight.
I) the centrifugal concentrated and purified HRP-polyclonal antibody of ultrafiltration pipe obtains mole ratio and marks anti-GP73 antigen polyclonal antibody near the enzyme of 1:8.
4) packing: mark anti-GP73 antigen polyclonal antibody to suitable working concentration with the enzyme that damping fluid (20mmol/L PBS) dilution that contains 10% foetal calf serum is obtained by step 3), press the packing of 6ml/ bottle, be stored in 4 ℃.
4. prepare substrate solution A: (50mmol/L, PH5.0) Pei Zhi 3% superoxol is pressed the packing of 5ml/ bottle with phosphoric acid-citrate buffer solution.
5. preparation colour developing liquid B:TMB (0.1mg/ml) methanol solution is pressed the packing of 5ml/ bottle.
6. preparation reaction terminating liquid: 2mol/L H
2SO
4, press the packing of 5ml/ bottle.
7. 1% polysorbas20 solution of preparation cleaning buffer solution (20 times of concentrated solutions): PBS (pH7.4) preparation is pressed the packing of 50ml/ bottle.
The using method of the quantitative enzyme-linked immunologic detecting kit of embodiment 3:GP73
1. cleaning buffer solution preparation: 20 times of dilutions of concentrated cleaning buffer solution adding distil water that test kit is provided.
2. preparation standard substance: the standard substance in the test kit comprise the GP73 antigen standard substance of 5 different concns, and each standard substance concentration is respectively 0,20ng/ml, 40ng/ml, 100ng/ml, 250ng/ml.
3. antigen-antibody reaction: in the micropore of the antibody sandwich plate that test kit provides, add 100 μ l sample diluents respectively, add standard substance or each 10 μ l of test serum sample of different concns then, 37 ℃ of water bath heat preservations 30 minutes.Repeat to wash plate 4 times with cleaning buffer solution then, each 3 minutes.
4. integrated enzyme reaction: GP73 polyclonal antibody HRP solution is added each hole, every hole 100 μ l, 37 ℃ of water bath heat preservations 30 minutes.Repeat to wash plate operation 4 times, each 3 minutes.
5. color reaction: every hole adds each 50 μ l of substrate solution A, TMB colour developing liquid B successively, 37 ℃ of water bath heat preservations 10 minutes, and every hole adds 50 μ l reaction terminating liquids again and finishes reaction.
6. colorimetric:, measure OD mean value at 450nm with microplate reader with the light absorption value zeroing in blank hole; Write down each hole light absorption value; Calculate the mean value of diplopore standard substance OD value.
7. the result calculates
1) drafting of typical curve: with standard substance concentration is that the average OD value that X-coordinate, standard substance are measured is an ordinate zou, draws the typical curve of this mensuration; Base of calculation curvilinear regression coefficients R 2 is as R2〉this experiment is effectively 0.98 the time.
2) calculate testing sample concentration: when typical curve and quality control product all are determined when effective, calculate GP73 antigen concentration the test serum sample from typical curve according to the OD value of sample to be tested.
Embodiment 4: the quality examination of the quantitative enzyme-linked immunologic detecting kit of GP73 antigen of the present invention
1) accuracy: the detected result of 15 parts of GP73 antigen negative quality controlled serums (comprising the specificity control serum) reference material, non-false positive occurs.The reference material detected result of 10 parts of GP73 antigen positive quality controlled serums does not have false negative and occurs.
2) precision: randomly draw 20 box different batches test kits, use with a GP73 antigen positive quality controlled serum by specification operation steps and carry out replication.Calculate each measurement result, obtain average, SD and variation coefficient CV.CV " 15% between the Precision test result demonstration is criticized.
3) detection sensitivity: according to the recombinant expressed antigen gradient dilution of GP73 antigen measurement result, the detection sensitivity of this test kit is 5ng/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.Measure and calculation result by the method described in the embodiment 3.Calculate jamming rate by the interference test calculation formula then.The mushing error of sample # 1, #2, #3 is all less than 1.5%.
5) rate of recovery: using by concentration is 20ng/ml, and 40ng/ml, 100ng/ml, the standard substance of 250ng/ml detect the absorbancy that obtains and return the value that typical curve obtains and carry out the rate of recovery relatively, and the rate of recovery is between 89-132%.
The preparation of embodiment 5:GP73 antigen chemiluminescence quantification kit
Present embodiment has prepared a kind of Golgi protein GP 73 chemiluminescence detection kit of the present invention (96 person-portion), and its composition comprises:
5 bottles of Golgi protein GP 73 standard substance;
GP73 monoclonal antibody bag is by 1 of plate (96 hole);
1 bottle of the GP73 polyclonal antibody of horseradish peroxidase (HRP) mark, the 10ml/ bottle;
1 bottle of substrate solution: EDTA (1.0 * 10
-2M), H
2O
2(7.5 * 10
-3M), HCl (1.0 * 10
-2M) and Tween20 (1%)
1 bottle of colour developing liquid: 1uminol 5.0 * 10
-4Mol/L;
1 bottle of sample diluent: the 20mM pH 7.4 PBS damping fluids that contain 2%BSA
Concrete operations are as follows:
1. prepare the Golgi protein GP 73 standard substance:
1) employed GP73 albumen is embodiment 1 the 1st) the total length people GP73 albumen of band His label of synthetic in the part.
2) packing: by 0,20ng/ml, 40ng/ml, 100ng/ml, 250ng/ml different concns are diluted in 2% the BSA solution with GP73 albumen.In solution, add sanitas procline 300 (available from SUPELCO companies) to ultimate density 0.1%.
Filtration sterilization, and under aseptic condition, divide in the 1.5ml tubule of packing into every pipe 0.5ml.Be stored in 4 ℃.
2. make GP73 monoclonal antibody bag by plate:
1) employed anti-GP73 monoclonal antibody is the secreted monoclonal antibody of hybridoma cell strain 3E12, and antibody is through protein A agarose medium purification.
2) bag quilt:
12 * 8 removable battens that enzyme plate adopts Costar company to produce.With the described monoclonal anti body and function of step 1) 0.05mol/L, the carbonate buffer solution dilution of pH9.5 is to add each hole of chemoluminescence plate behind the 20 μ g/ml, every hole 100 μ l, and absorption is spent the night, with cleaning buffer solution (PBST solution, comprise 20mmol/LPBS, 0.5%Tween-20 Ph7.4) washes plate, use this confining liquid damping fluid (2%BSA again, the preparation of PBST solution) sealing is spent the night, and dries after the drying, promptly obtains the monoclonal antibody bag by the chemoluminescence plate.By 96 holes/piece packaging of aluminium foil bag, vacuum sealing.
3. the anti-GP73 polyclonal antibody for preparing horseradish peroxidase (HRP) mark
Anti-GP73 Polyclonal Antibody Preparation, purifying and the branch process of assembling of horseradish peroxidase (HRP) mark is as described in embodiment 2 the 3rd part.
4. preparation substrate solution: EDTA (1.0 * 10
-2M), H
2O
2(7.5 * 10
-3M), HCl (1.0 * 10
-2M) and Tween20 (1%); Press the packing of 5ml/ bottle.
5. preparation colour developing liquid B:20mM pH7.4PBS damping fluid contains luminol 5.0 * 10
-4Mol/L; Press the packing of 5ml/ bottle.
6. 1% polysorbas20 solution of preparation cleaning buffer solution (20 * concentrated solution): PBS (pH7.4) preparation is pressed the packing of 15ml/ bottle.
The using method of embodiment 6:GP73 chemiluminescence immune detection reagent kit
1. antigen-antibody reaction: in the micropore of the antibody sandwich plate that test kit provides, add 100 μ l sample diluents respectively, add 10 μ l test serum samples or 10 μ l then and contain different concns (0,20ng/ml, 40ng/ml, 100ng/ml, GP73 protein standard substance 250ng/ml), 37 ℃ of water bath heat preservations 30 minutes are washed plate.HRP-polyclonal antibody solution is added each hole, every hole 100 μ l, 37 ℃ of water bath heat preservations 30 minutes.Repeat to wash plate operation 4 times.
2. color reaction: every hole adds substrate solution A successively, each 50 μ l of colour developing liquid B, the value of reading.
3. the result calculates:
1) drawing standard curve: with standard substance concentration is X-coordinate, and the values of chemiluminescence 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 0.98 o'clock;
2) calculate the test serum sample concentration: the Golgi protein GP 73 concentration that calculates the test serum sample according to the values of chemiluminescence of testing sample from typical curve.
Embodiment 7: the quality examination of GP73 chemoluminescence method detection by quantitative test kit of the present invention
1) rate of recovery: be divided into three parts of pooled serum samples, every part of 1ml after getting three parts of testing samples mixing.Add 0,20 respectively, the pure product of GP73 recombinant antigen of 100ng, make recovery test serum specimen # 1, #2, #3.The by specification operation steps is measured and calculation result.Then by rate of recovery calculation formula calculate recovery rate.The rate of recovery of sample # 2, #3 is respectively 96.4% and 98.7%, and average recovery rate is 97.5%, and promptly the proportional jitter of test kit is 2.5%, and accuracy is 97.5%.
2) precision: randomly draw 20 box different batches test kits, use with a sample to be tested body by specification operation steps and carry out replication.Calculate each measurement result, obtain average, SD and variation coefficient CV.CV " 15% between the Precision test result demonstration is criticized
3) linearity range: the pure product solution that is diluted to a series of different concns with the pure product of GP73 antigen: 1000ng/ml, 500ng/ml, 250ng/ml, 100ng/ml, 40ng/ml, 20ng/ml, 5ng/ml, 2ng/ml and 0ng/ml.Operation steps is measured to specifications.With concentration is that X-coordinate, absorbancy are the ordinate zou curve plotting.The highest detection higher limit is 1500ng/ml in the linearity range, and the lowest detection lower value is 0.5ng/ml.The linearity range of test kit is 0.5-1000ng/ml.
4) detection sensitivity: according to above-mentioned linearity range measurement result, the detection sensitivity of this test kit is 0.5ng/ml.
5) 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.Measure and calculation result by the method described in the embodiment 6.Calculate jamming rate by the interference test calculation formula then.The mushing error of sample # 1, #2, #3 is all less than 1.5%.
Embodiment 8: the quantitative enzyme-linked immunologic detecting kit of GP73 of the present invention is to the detection of clinical serum sample
For judging the coincidence rate of GP73 antigen enzyme-linked immunologic detecting kit of the present invention and liver cancer detected result, get the clinical serum specimen of ditan hospital: 120 parts of known liver cancer positive samples, 120 parts of healthy serum of health check-up, 80 parts of chronic hepatitiss.Detected result is seen Fig. 7 and Fig. 8.
Annotate: the ROC curve of Fig. 8 refers to experimenter's performance curve (receive operatingcharacteristic curve), it is the overall target of reflection sensitivity and specificity continuous variable, be to disclose sensitivity and specific mutual relationship with the composition method, it is by setting out continuous variable a plurality of different threshold values, thereby calculate a series of sensitivity and specificity, be that ordinate zou, (1-specificity) are depicted as curve for X-coordinate again with sensitivity, area under curve (AUC, Area under curve) big more, diagnostic accuracy is high more.On the ROC curve, the upper left point of the most close coordinate diagram is sensitivity and all higher threshold value of specificity. when AUC greater than 0.8 the time, show that this detection index has good diagnostic value.
The area under curve AUC of the ROC curve among Fig. 8 is 0.88, show that detecting GP73 albumen can play good effect to diagnosing cancer of liver, threshold value is taken from sensitivity and the pairing numerical value of specificity optimal balance point on the ROC curve, it is that normal people and liver problem sufferer are threshold value that analysis draws with 40ng, with 100ng is the threshold value of liver cancer patient and hepatitis, this experimental result shows, the present invention for the positive rate of diagnosing cancer of liver up to 73%, positive rate 17% in chronic hepatitis, specificity reaches 96% in the normal people, satisfies the Clinical Laboratory needs.
Embodiment 6: GP73 chemoluminescence detection by quantitative reagent of the present invention is to the detection of liver cancer, chronic hepatitis, normal people's sample
With 40ng is that normal people and liver problem sufferer are threshold value, with 100ng is the threshold value of liver cancer patient and hepatitis, 90 parts of liver cancer (all being AFP negative HCC sample) that detection No.302 Hospital, P.L.A. provides, 75 parts of hepatitis, 50 parts of health examination persons' serum specimen.The results are shown in Figure 9 and Figure 10.
The result shows that the liver cancer positive rate is 80%, and the hepatitis positive rate is 21%, normal people's specificity 90%.
Statistical study, the area under curve AUC of the ROC curve among Figure 10 is 0.9.Show that detecting GP73 can play the efficient diagnosis effect in the AFP negative HCC.Sequence table
＜110〉Beijing Hotgen Biotechnology Co., Ltd.
＜120〉the proteic monoclonal antibody of a kind of anti-GP73, its preparation method and application
<130>CP1080731P
<160>1
<170>PatentIn version 3.3
<210>1
<211>1212
<212>DNA
＜213〉human (homo sapiens)
<400>1
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Claims (10)
1. the monoclonal antibody of an anti-Golgi protein GP 73, described monoclonal antibody is by the secretion of 3E12 hybridoma, and described hybridoma is preserved in Chinese common micro-organisms DSMZ (CGMCC) on November 11st, 2008 with preserving number CGMCCNo.2742.
2. enzyme-linked immune quantitative detection reagent box, described test kit comprises the monoclonal antibody of claim 1.
3. according to the test kit of claim 2, described test kit also comprises the polyclonal antibody and the auxiliary reagent of enzyme plate, horseradish peroxidase (HRP) mark.
4. according to the test kit of claim 3, comprise chromogenic substrate tetramethyl benzidine (TMB) in the wherein said auxiliary reagent.
5. chemical luminescent analysis reagent kid, described test kit comprises the monoclonal antibody of claim 1.
6. according to the test kit of claim 5, described test kit also comprises the polyclonal antibody and the auxiliary reagent of enzyme plate, horseradish peroxidase (HRP) mark.
7. according to the test kit of claim 6, comprise the luminous substrate luminol,3-aminophthalic acid cyclic hydrazide in the wherein said auxiliary reagent.
8. a quick diagnosis reagent kit comprises the monoclonal antibody of claim 1 and the GP73 protein standard substance of demarcating content in the described test kit.
9. quick diagnosis reagent kit according to Claim 8, wherein said protein standard substance has at least two kinds, GP73 content in wherein a kind of standard substance is the threshold value of normal people and liver problem sufferer's GP73 content, and the GP73 content in the another kind of standard substance is the threshold value of optimum liver problem sufferer and pernicious liver problem sufferer's GP73 content.
10. according to the quick diagnosis reagent kit of claim 9, the threshold value of wherein said normal people and liver problem sufferer's GP73 content is 40ng/ml, and the threshold value of described optimum liver problem sufferer and pernicious liver problem sufferer's GP73 content is 100ng/ml.
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|---|---|---|---|---|
| US9469686B2 (en) | 2013-03-15 | 2016-10-18 | Abbott Laboratories | Anti-GP73 monoclonal antibodies and methods of obtaining the same |
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| CN104215761B (en) * | 2014-08-27 | 2016-04-20 | 广西医科大学 | Detect the kit of anti-GP73 antibody in serum |
| CN106153934B (en) * | 2015-03-26 | 2018-06-19 | 广州瑞博奥生物科技有限公司 | A kind of kit of efficient quantitative detection golgiosome 73 |
| CN107345967A (en) * | 2016-05-05 | 2017-11-14 | 中国医学科学院基础医学研究所 | Purposes of the GP73 albumen as serum markers in cancer is diagnosed |
| CN106405104B (en) * | 2016-08-31 | 2019-01-08 | 鲁凤民 | A kind of new cirrhosis or hepatic fibrosis markers |
| CN111413502B (en) * | 2017-04-27 | 2022-10-14 | 北京大学 | New application of GP73 and liver tissue inflammation activity degree detection kit based on GP73 |
| CN107271674B (en) * | 2017-06-13 | 2019-05-07 | 首都医科大学附属北京地坛医院 | A kind of target marker GP73 and detection application method for steatohepatitis detection |
| CN113425843B (en) * | 2020-03-08 | 2022-08-02 | 北京舜景生物医药技术有限公司 | Application of GP73 inhibitor in preparation of medicine for treating diabetes |
| CN111378627B (en) * | 2020-03-11 | 2023-08-25 | 海南众森生物科技有限公司 | Monoclonal antibody of Golgi apparatus protein 73, kit and application |
| CN115754290A (en) * | 2022-09-26 | 2023-03-07 | 浙江大学 | Kit for detecting early liver cancer |
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| US9469686B2 (en) | 2013-03-15 | 2016-10-18 | Abbott Laboratories | Anti-GP73 monoclonal antibodies and methods of obtaining the same |
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