CN107746430A - A kind of preparation and its application of GP73 C-terminals antigen - Google Patents

Abstract

The present invention provides a kind of GP73 C-terminals albumen, thus the monoclonal antibody of anti-GP73 C-terminals is prepared, in order to solve clinically to lack deficiency of the GP73 immue quantitative detection reagent boxes as early diagnosis of liver cirrhosis detection means at present, another object of the present invention is to provide a kind of sensitive, steady quality easy to operate, accurate, the detection kit that can be mass-produced and its assay method, so as to the GP73 expression contents from protein level, effective comprehensive therapeutic plan can be taken as early as possible, effectively prevent the further canceration of hepatic sclerosis.

Description

A kind of preparation and its application of GP73 C-terminals antigen

Technical field

The present invention relates to bio-pharmaceutical engineer technology domain, and in particular to the preparation and its application of GP73 C-terminal antigens.

Background technology

Hepatic sclerosis is the late stage of various chronic liver diseases development, long-term or repeated action is formed by one or more causes of disease Diffusivity hepatic lesion.It is posthepatitic cirrhosis in most of China, small part is that alcoholic cirrhosis and blood fluke property liver are hard Change.There are extensive necrosis of liver cells, regeneration of remaining liver cell nodules, connective tissue proliferation and fiber in Histopathology every shape Into, cause lobuli hepatis structure destroy and pseudolobuli formed, liver gradually deforms, is hardened and develops into hepatic sclerosis.Early stage is due to liver Dirty compensation more by force can non-evident sympton, stage has multisystem using hepatic disorder and portal hypertension as main performance Involvement, it is concurrent that UGB, hepatic encephalopathy, scabies secondary infection, hypersplenia, ascites and canceration etc. often occurs in late period Disease.

Cause the cause of disease of hepatic sclerosis a lot, viral cirrhosis, alcoholic cirrhosis, metabolic hepatic sclerosis, courage can be divided into The cholestatic hepatic sclerosis of juice, vena hepatica backflow obstruction hepatic sclerosis, autoimmune cirrhosis, poisonous substance and drug induced liver cirrhosis, battalion Support not benign hepatic sclerosis and cryptogenic cirrhosis etc..

Child-Pugh grade scales are a kind of liver reserve function amounts of progress to liver cirrhosis patient clinically commonly used Change the grade scale assessed, the standard was proposed by Child in 1964 earliest, and Child was by 5 indexs of patient (including one at that time As situation, ascites, serum bilirubin, seralbumin concentration and prothrombin time) different conditions be divided into three levels, point Not remember with 1 point, 2 points and 3 points, and 5 index score are added, summation is minimum to be divided into 5 points, and highest is divided into 15 points, so as to It is divided into A, B, C three-level according to more major general's liver reserve functions of the summation, imply that the liver damage of three kinds of different orders of severity (fraction is higher, and liver reserve function is poorer).But because the general status item of patient is usually not easy to score, subsequent Pugh proposes to use The presence or absence of hepatic encephalopathy and its degree replace general status, i.e., nowadays clinical conventional Child-Pugh improvement stagings.

Hepatic sclerosis can be divided into compensatory phase and decompensated liver cirrhosis again.Decompensated cirrhosis is marked equivalent to Child-Pugh A levels in standard, Decompensated stage is equivalent to B and C levels.

A chronic process is presented in hepatic sclerosis disease.In Child-Pugh any level, or compensatory phase and Decompensated stage, Have in its Histopathology extensive necrosis of liver cells, regeneration of remaining liver cell nodules, connective tissue proliferation and fiber every Formed, cause lobuli hepatis structure to be destroyed and formed with pseudolobuli, liver gradually deforms, is hardened and develops into hepatic sclerosis.

Liver fibrosis is a process being continuously in progress in disease development, and chronic liver disease develops into the centre of hepatic sclerosis Only stage which must be passed by.

It is clinically more difficult completely to distinguish both liver fibrosis and hepatic sclerosis, but the two has the difference of essence.Liver fiber Change is not a kind of independent disease, is only a kind of hepatic disease of adjoint a variety of chronic liver diseases, liver fibrosis be by liver more Caused by unrestrained property extracellular matrix (particularly collagen) over-deposit, hepatic sclerosis is then that excess fibrosis makes liver atrophy be hardened Caused by.

Liver fibrosis is the cercinoma prophase pathologic change of hepatic sclerosis, and its early stage is reversible, and hepatic sclerosis is liver fibrosis further sends out The result of exhibition, it is that the whole latter stage of a variety of liver diseases shows, generally more difficult reverse.

The pathological characteristic of liver fibrosis is to have a large amount of proliferations of fibrous tissue and deposition, but not yet shape in portal area and lobuli hepatis It is spaced in into leaflet, hepatic sclerosis then has pseudolobuli to be formed, and central vein area and portal area are spaced, and the normal configuration of liver meets with To destruction, further development is hepatic sclerosis for liver fibrosis.

In a word, hepatic sclerosis is a kind of chronic progressive hepatopathy.Early diagnosis helps to carry out effectively in liver fibrosis stage Treatment, make liver diseases turn into it is reversible, it is poor that no person develops into hepatic sclerosis prognosis.

The aided detection method of current diagnosis hepatic sclerosis mainly includes：The serum of imageological examination, liver biopsy and laboratory Learn detection.Imageological examination reliable results, but only liver organization structure occurs to find during significant change, thus it is unfavorable In the early diagnosis of hepatic sclerosis.Liver biopsy can establish clarifying a diagnosis for hepatic sclerosis, but because its is traumatic, it is more difficult to suffer from Person receives.And the Serologic detection in laboratory is liver fibrosis detection at present, it is difficult to provide clarifying a diagnosis for hepatic sclerosis.With liver Exemplified by four inspections (III procollagen type, type Ⅳ collagen, laminin and hyaluronidase) of fiberising stage, liver fibrosis Four check because the inflammatory effect by liver is larger, therefore its specificity and sensitivity is not high.

GP73 also known as Golm1 or Golph2 is the type golgiosome of one kind II that was found first in 2000 of researcher across Memebrane protein.Molecular weight about 45KD, about 73KD, its Serial No. 51280 in GeneBank after glycosylation.Because in electrophoresis When display 7.3 × 10⁴Molecular weight, thus referred to as GP73.Coding GP73 gene is located at Chromosome 9, total length totally 3080 Individual nucleotides, the open reading frame containing 1200bp in its gene, 402 amino acid are encoded, there are 5 glycosylation sites. GP73 is a transmembrane protein being made up of intracellular, cross-film and extracellular three regions.Immunohistochemical study confirms：Various kinds of cell can GP73 is expressed, and normal liver cell expression quantity is not expressed even seldom.Under normal circumstances, GP73 is one kind on Golgi membrane AQP-CHIP, intracellular and cell surface can be secreted under disease event.There is researcher's confirmation：The clipped enzymes of GP73 are made It is extracellular with being secreted into, into peripheral blood.

Further there is researcher's discovery again：Chronic hepatitis B, chronic hepatitis C, alcoholic hepatitis and autoimmunity Property hepatitis liver organization in GP73 is horizontal is higher than 70 times of normal person.GP73 expression has rise in various hepatopathys, prompts GP73 is horizontal unrelated with viral infection.Further study show that：In all cells, all hepatopathys trigger GP73 to raise, and It is especially pronounced on connective tissue periphery and cirrhotic nodule position.

The data-searching carried out according to inventor, other are there is no both at home and abroad about detecting double monoclonal antibodies for GP73C ends The report of sandwich ELISA method.

The content of the invention

In order to inquire into Sensitivity and Specificity of the monoclonal antibody detection GP73 albumen in liver cirrhosis diagnosis, the present invention is done A large amount of clinical tests.As a result show that serum GP73 can be as the auxiliary diagnostic index of hepatic sclerosis, particularly in chronic hepatitis During switching to hepatic sclerosis, with more important diagnostic value.

It is an object of the present invention to provide a kind of GP73 C-terminals albumen, its amino acid sequence is SEQ ID NO:1.

Another object of the present invention is to, there is provided a kind of preparation method of GP73 C-terminals albumen, mainly comprise the following steps：

(1) PCR is expanded：Using containing BamHI, EcoRI restriction enzyme site primer PCR amplification GP73 C-terminals gene (1019~ 1336bp), digestion is connected with the PGEX-4T-2 plasmids after BamHI, EcoRI digestion after purification, is transformed into Escherichia coli BL21；

(2) expression-form is determined：Select positive colony, 25 DEG C of IPTG pyrans of use 1% at random Glucosides (IPTG) chemical induction, 1%NP-40 cracking, -80 DEG C of multigelations 3 times, centrifugation, supernatant precipitation is taken to carry out respectively SDS- polyacrylamide gel electrophoresises (SDS-PAGE) are identified, determine expression-form；

(3) culture and purifying are expanded：Choose positive colony and be enlarged culture, 1%NP-40 cracking, -80 DEG C of multigelations 3 times, using purification by chromatography GP73C ends antigen.

Wherein, the sense primer in step (1) is that GC GCT GGA TCC CAG CTG GCC TCA (5 ' -3 ', draw by downstream Thing is GC GAA TTC TGA TGTGAG ATGATT.

Another object of the present invention is to, there is provided a kind of GP73 C-terminals protein monoclonal antibody preparation method, mainly include Following steps：

(1) animal immune

Take restructuring GP73C end antigen protein 300mg/L that emulsifying agent is made with Split completely mixed in equal amounts, it is immune altogether Balb/c mouse 5；Only, subcutaneous multi-point injection, it is immune to cannot be used up full freund adjuvant the 2nd time after 2 weeks by 1st immune 100 μ g/, Dosage, approach are identical with the 1st time；2 weeks rear molding venous blood samplings determine potency, and potency is up to 1：1000~1：For fusion when 5000； Before fusion 3 days with the direct abdominal cavity spleen area booster immunization of antigen 1 time；

(2) using indirect elisa method measure potency

The purification of Recombinant GP73C ends antigen concentration for being coated with XPS is 5mg/L, and enzymic-labelled antibody is sheep anti-mouse igg (Sigma Products, working concentration 1：5000), ELIASA 450nm wavelength measure OD values；P/N value >=2.1 times are the positive, 2.1 times of ＜ is negative (N values<0.05, calculated by 0.05)；

(3) preparation of immune spleen cell

Take and Balb/c mouse are immunized, eyeball bloodletting is for detecting antibody；Spleen is taken out in sterile working, gently cleans and goes Except connective tissue, crush and grind on copper mesh is placed in, and is extruded into by mesh in solution, 1000r/min, 5min is centrifuged, abandons supernatant, It is resuspended in solution and splenocyte suspension is made, adds platform to expect that blue dye liquor makees cell count；

(4) prepared by myeloma cells liquid

Myeloma cell is placed in 37 DEG C of 5%CO₂Expand culture in incubator；The fusion same day, which collects, is in exponential phase SP2/O myeloma cell, centrifuge 1000r/min, 5min, abandon supernatant, make cell count after being resuspended in liquid；

(5) cell fusion

Splenocyte is drawn respectively and myeloma cell's suspension presses 5：1 mixing, is placed in centrifuge tube after fully mixing and centrifuges, abandon Supernatant, gently attack ttom of pipe, makes sedimentation cell loose uniformly into pasty state；50% polyethylene glycol (PEG) 0.7ml is taken to add mixing Promote fusion in cell, the strict control action time in 2~3min, adds endless full nutrient solution dilution, stop PEG immediately Effect；800r/min, 7min are centrifuged, abandons supernatant；Add 10mL HAT nutrient solutions and cell suspension is made, and add and be paved into raising In 96 orifice plates of cellular layer, per hole 0.1mL, put in 37 DEG C of 5%CO2 incubators and cultivate, liquid is changed after 4~5d 1 time, take during 8~10d Supernatant, potency is surveyed with ELISA method, carries out primary dcreening operation, positive hole is subcloned；

(6) prepared by the cloning (limiting dilution assay) of hybridoma and ascites

After the hybridoma for doing cloning is blown and beaten uniformly repeatedly with sample injector, a little cell suspension will be taken in 96 orifice plates Put in another sterile vials；This cell suspension is serially diluted exactly, until every milliliter contains 10 cells；It will dilute Cell suspension inoculation in being covered with 96 orifice plates of feeder layer, per hole 0.1mL, i.e. every Kong Hanyi cell puts 37 DEG C 5%CO₂Cultivated in incubator, 5d or so observes cell clone growing state under inverted microscope；Clone 3 times repeatedly, it is determined that Positive monoclonal cell line, finally clonal cell line is numbered, puts in liquid nitrogen and preserves.

Another object of the present invention is to, there is provided a kind of reagent for diagnosing hepatic diseases, wherein containing GP73 C-terminals Protein monoclonal antibody.

Another object of the present invention is to, there is provided for the kit of diagnosing hepatic diseases, wherein containing GP73 C-terminal eggs White monoclonal antibody.

Another object of the present invention is to, there is provided above-mentioned reagent and/or kit is used for diagnosing hepatic diseases in preparation Application in product.

Specifically, being directed to GP73 after birth outskirt different epitopes, the present invention screens 3 pairs of preferable monoclonal antibodies of pairing.

And the invention discloses said monoclonal antibody i) to pass through GP73 contents kit side in ELISA method detection serum The application in face；Ii the application in terms of) detecting GP73 content kits by magnetic bead chemoluminescence method；Iii) examined by ELISA method Survey the application in terms of fucosylation GP73 content kits in serum.

Wherein, the sandwich ELLSA quantitative detecting methods of GP73 C-terminals albumen, to be directed to GP73 C-terminal albumen difference tables with two plants The monoclonal antibody of position, by substrate color developing detection GP73 albumen, passes through calibration object respectively as coated antibody and detection antibody The standard curve of preparation determines the content of GP73 albumen.

Wherein, coated antibody coated elisa plate, antibody labeling horseradish peroxidase is detected.

It should be noted that：GP73 C-terminal peptide has preferable hydrophily and accessibility, and the amino acid of coding is positioned at thin Extracellular regions, it is unique not comprising other trans-membrane regions, and in human protein storehouse；Meanwhile serum GP73 is by not Woods protease is discharged into extracellular after digestion at the 55th amino acids, contains complete C-terminal.Therefore, by GP73 C-terminals One section of sequence as epitope, prokaryotic expression GP73 C-terminal albumen, the monoclonal antibody thus prepared is directed to GP73 C-terminals, for producing Kit can improve the recall rate of GP73 in sample.

Software analysis is defined as 302~401 amino acids, as a result shows that fragment antigen binding site exposure is more, because And there is stronger antigenicity, and it is hydrophilic solvable, the fragment contains 100 amino acid, and molecular weight is larger, is easy to store steadily in the long term Deposit.GP73 C-terminal albumen is prepared using gene engineering method, genetic method is：Coding schedule is amplified using PCR method and reaches GP73 The DNA sequence dna of C-terminal, then the sequence is inserted into the multiple cloning sites of expression vector, builds recombinant expression plasmid, then convert To expressive host bacterium, structure obtains engineered strain, and engineered strain isolates and purifies to obtain GP73C ends recombinant protein through induced expression. Prepared with the immune Balb/C mouse of the GP73C ends recombinant protein of expression synthesis and screen hybridoma cell strain, it is simultaneously pure to prepare ascites Change monoclonal antibody.

Measure kit for GP73 antigens is prepared using double antibody sandwich method, quantitative inspection is carried out to GP73 in human serum Survey.With the monoclonal antibody coated elisa plate at GP73C ends, test serum is added, the Dan Ke at another plant of GP73C end is added after reaction The enzyme conjugates of grand antibody, then acted on tmb substrate, colored intensity is related to the concentration of GP73 in sample.Using HRP-mAb2 Replacement HRP-rabbit is more anti-or rabbit resists more and the association reaction of HRP-goat anti-rabbit antibody.

The invention has the advantages that providing a kind of GP73 C-terminals albumen, the monoclonal for thus preparing anti-GP73 C-terminals resists Body, in order to solve clinically to lack deficiency of the GP73 immue quantitative detection reagent boxes as early diagnosis of liver cirrhosis detection means at present, Another object of the present invention is to the detection examination that provides a kind of sensitive, steady quality easy to operate, accurate, can be mass-produced Agent box and its assay method, so as to the GP73 expression contents from protein level, effective complex treatment side can be taken as early as possible Case, effectively prevent the further canceration of hepatic sclerosis；

In addition, GP73C ends provided by the invention kit test limit≤1.00ng/ml；The range of linearity：1.00~ 450.00ng/ml coefficient correlation >=0.9900；Criticize interior, interassay coefficient of variation CV≤15%；The rate of recovery 85~115%；With AFP (alpha-fetoprotein), GPC3 (glypican-3) and human serum albumins cross reaction value≤1.00ng/ml；2~ 8 DEG C storage-stable 9 months.

Brief description of the drawings

Fig. 1 is the standard curve drawn in embodiment with GP73 calibration objects log concentration with OD values logarithm.

Fig. 2 analyzes for hepatic sclerosis sample ROC curve.

Embodiment

The invention is further described below by specific embodiment.It should be understood that these embodiments are only used for Illustrate the present invention rather than limit the scope of the present invention.The experimental method of unreceipted actual conditions in following embodiments, generally According to normal condition, or the condition that those skilled in the art can deduce in knowledge, or according to proposed by manufacturer Condition.The reagent and instrument apparatus being related in following embodiments, it is usually commercial commercially available product, or can be public by other Open the product of approach acquisition.

The GP73 C-terminal antigen preparation procedures of embodiment 1

In order to prepare specific monoclonal antibody good, affinity is high and then be applied to clinical GP73 detection kits, First have to express GP73C ends antigen.GP73 genes are located at Chromosome 9, total length 3042bp, include unique opening code-reading frame (1200bp), there are 402 amino acid of coding.GP73 is different in structure from serum GP73 (sGP73).GP73 is by one section Formed in kytoplasm by the N-terminal of myristoylation, single transmembrane region and one section long of C-terminal function of extracellular area.Its N-terminal is dredged Water, glycosylation, containing single transmembrane region and signal peptidase cleavage site (aa28-aa29), and includes two spiral-helical structures Domain.Its C-terminal is rich in acidic amino acid, includes the acylated continuous sequence (GLGNGRRS) of 14 (alkane) and has 5 glycosylation sites. Immediately transmembrane region has the coiled coil of the interphase interaction of several participation protein.These properties are prompted, and the protein passes through thin Had an effect with other protein extracellular regions.GP73 C-terminal peptide has preferable hydrophily and accessibility, the amino of coding Acid is located at extracellular space, is unique not comprising other trans-membrane regions, and in human protein storehouse；Meanwhile serum GP73 is to be discharged into by furin after the 55th amino acids digestion extracellular, contains complete C-terminal.Therefore, will One section of sequence of GP73C ends is directed to GP73 C as epitope, prokaryotic expression GP73 C-terminal albumen, the monoclonal antibody thus prepared End, the recall rate of GP73 in sample can be improved for producing kit.Software analysis is defined as 302~401 amino acids, as a result Show that fragment antigen binding site exposure is more, thus there is stronger antigenicity, and it is hydrophilic solvable, the fragment contains 100 Amino acid, molecular weight is larger, is easy to store steadily in the long term.

In numerous protein expression systems, prokaryotic expression system has that easily operated, growth is rapid, yield is high and expense With the advantage such as cheap, therefore this experiment employs prokaryotic expression system.Select suitable carrier and by thalline to expressing external source base Because extremely important, it can be changed expressing for adjustment autogene according to outside environmental elements by thalline, make own growth and breeding In optimum state, it is coupling to be additionally, since prokaryotes own transcription and translation, and prokaryotes are to extraneous environmental change Reaction is also suitable fast.Using PGEX-4T-2 as expression vector, it carries GSTTag labels, can be used for the present embodiment Purify expressing protein.

Specific preparation method is as follows：

1. analyze epitope：

GP73 nucleotides and overall amino acid sequence is downloaded from NCBI websites, sequence is compared using Biosun biological softwares Row, analyze epitope and screen representative epitope section.It is defined as 302~401 amino acids, it is corresponding finds the section Nucleotide sequence, carry out the synthesis of next step.Software analysis result shows that fragment antigen binding site exposure is more, thus With stronger antigenicity, and it is hydrophilic solvable, the fragment contains 100 amino acid, and molecular weight is larger, is easy to store steadily in the long term.

2. obtain GP73C ends antigen gene：

According to GP73 gene orders, primer of the design for GP73 1019~1336bp sequences.Sense primer：GC GCT GGA TCC CAG CTG GCC TCA (5 ' -3 ' anti-sense primers：GC GAA TTC TGA TGTGAG ATGATT (5 ' -3 ' with CDNA plasmids are template, by PCR synthetic gene fragments, carry out PCR primer glue reclaim purifying, conventional method conversion connect into PMD19T (sequencing vector), carry out sequence verification.After PCR amplifications, 1.5% agarose electrophoresis is identified, obtains 300bp amplification piece Section, it is consistent with expected results, multiple positive colonies are obtained after conversion is connected with carrier T, (Gen-Bank is logged in for sequencing result analysis Number AP002069 is matched completely), gene order is correct.

3. clonal expression and purifying GP73C ends antigen

(1) connection conversion：Using containing BamHI, EcoRI restriction enzyme site primer amplification GP73 C-terminals gene (1019~ 1336bp), conventional method is connected with the PGEX-4T-2 plasmids after BamHI, EcoRI digestion after purification for digestion, is transformed into BL21。

(2) expression-form is determined：Select positive colony, 25 DEG C of IPTG pyrans of use 1% at random Glucosides (IPTG) chemical induction, 1%NP-40 cracking, -80 DEG C of multigelations 3 times, centrifugation, supernatant precipitation is taken to carry out respectively SDS- polyacrylamide gel electrophoresises (SDS-PAGE) are identified, determine expression-form.As a result show, the GP73C ends antigen of purifying The great expression after induction, mainly exist in the form of soluble protein.

(3) culture and purifying are expanded：Choose positive colony and be enlarged culture, 1%NP-40 cracking, -80 DEG C of multigelations 3 times, using purification by chromatography GP73C ends antigen.

4. detect purified product：

Using the GP73 antigens of Abnova companies as control, enter simultaneously with the GP73C ends antigen of the present embodiment purifying gained Row gum concentration be 12% conventional electrophoretic, transferring film.5% skimmed milk power room temperature closes 1h, with Golgi protein polyclonal antibody A01, monoclonal antibody M06, M04 are primary antibody, and confining liquid dilution is after 4 DEG C of overnight incubations.(contain 0.05%Tween 20 with washing lotion Tris salt buffers, TBST) room temperature washes film 3 times.The sheep anti-mouse igg of HRPO mark is secondary antibody, is incubated at room temperature 1h, TBST room temperatures wash film 3 times, are as a result presented through electrochemistry super quick luminous (ECL), carry out western blot analysis technology (Western Blot) detect.As a result show that GP73C ends antigen is preferable for 3 kinds of antibody responses, and the reactivity of two kinds of antigen is without obvious Difference.

5. detect antigen immune activity：

Detected using EUSA (enzyme-linked immunosorbent assay, ELISA).It is high That base protein polyclone antibody A01, monoclonal antibody M06 and M04 are as primary antibody, the sheep anti mouse of horseradish peroxidase-labeled IgG is secondary antibody, the immunocompetence of detection purifying gained antigen.The GP73C ends antigen carbonate buffer solution for purifying gained is dilute Release to 2 μ g/ml and be coated with ELISA microwell plates, per the μ l of hole 100,4 DEG C overnight after abandon liquid, with cleaning solution (containing 0.05%Tween-20's Phosphate buffer, PBS) liquid is abandoned after board-washing 3 times, pat dry；Confining liquid (the phosphate-buffered containing bovine serum albumin(BSA) is added per hole Liquid, BSA-PBS100 μ l, 4 DEG C overnight, are abandoned liquid；The primary antibody that 50 μ l concentration are 100ng/ml, 37 DEG C of warm bath after mixing are added per hole 1h, cleaning solution board-washing 5 times, is patted dry；Per hole be separately added into ten thousand times of 50 μ l1,20,000 times dilution horseradish peroxidase-labeled sheep Anti- mouse IgG, through 37 DEG C of warm bath 30min, cleaning solution board-washing 5 times, pat dry；The μ l of one pack system developer 100 are added, 37 DEG C of warm bath, are kept away Light colour developing 10min；Add 50 μ l terminate liquids per hole, 450nm light absorption values are read with ELIASA.Primary antibody uses Normal Mouse Serum conduct Negative control, cut-off values are 2.1 times of negative control light absorption value, and testing result is determined as the positive more than cut-off value persons.

When the monoclonal antibody mouse IgG of horseradish peroxidase-labeled dilutes for 20,000 times, the GP73C ends of the present embodiment purifying resist It is former weaker to A01 reactivity with purchased antigen, it is preferable for the reactivity of other several antibody.Work as horseradish peroxidase When the monoclonal antibody mouse IgG of enzyme mark dilutes for 10,000 times, the GP73C ends antigen of this research purifying has with purchased antigen to 3 kinds of antibody There is good reactivity, show that expressed GP73C end proteins have stronger immunocompetence.

It is prepared by the monoclonal antibody at the GP73C ends of embodiment 2

1. animal immune

Take restructuring GP73C end antigen protein 300mg/L that emulsifying agent is made with Split completely mixed in equal amounts, it is immune altogether Balb/c mouse 5.Only, subcutaneous multi-point injection, it is immune to cannot be used up full freund adjuvant the 2nd time after 2 weeks by 1st immune 100 μ g/, Dosage, approach are identical with the 1st time.2 weeks rear molding venous blood samplings determine potency, and potency is up to 1：1000~1：For fusion when 5000. Before fusion 3 days with the direct abdominal cavity spleen area booster immunization of antigen 1 time.

2. using conventional indirect elisa method measure potency

The purification of Recombinant GP73C ends antigen concentration for being coated with XPS is 5mg/L, and enzymic-labelled antibody is sheep anti-mouse igg (Sigma Products, working concentration 1：5000), ELIASA 450nm wavelength measure OD values.P/N value >=2.1 times are the positive, 2.1 times of ＜ is negative (N values<0.05, calculated by 0.05).

3. the preparation of immune spleen cell

Take and Balb/c mouse are immunized, eyeball bloodletting is for detecting antibody.Spleen is taken out in sterile working, gently cleans and goes Except connective tissue, crush and grind on copper mesh is placed in, and is extruded into by mesh in solution, 1000r/min, 5min is centrifuged, abandons supernatant, It is resuspended in solution and splenocyte suspension is made, adds platform to expect that blue dye liquor makees cell count.

4. prepared by myeloma cells liquid

Myeloma cell is placed in 37 DEG C of 5%CO₂Expand culture in incubator.The fusion same day, which collects, is in exponential phase SP2/O myeloma cell, centrifuge 1000r/min, 5min, abandon supernatant, make cell count after being resuspended in liquid.

5. cell fusion

Splenocyte is drawn respectively and myeloma cell's suspension presses 5：1 mixing, is placed in centrifuge tube after fully mixing and centrifuges, abandon Supernatant, gently attack ttom of pipe, makes sedimentation cell loose uniformly into pasty state.50% polyethylene glycol (PEG) 0.7ml is taken to add mixing Promote fusion in cell, the strict control action time in 2~3min, adds endless full nutrient solution dilution, stop PEG immediately Effect.800r/min, 7min are centrifuged, abandons supernatant.Add 10mL HAT nutrient solutions and cell suspension is made, and add and be paved into raising In 96 orifice plates of cellular layer, per hole 0.1mL, 37 DEG C of 5%CO are put₂Cultivated in incubator, liquid is changed after 4~5d 1 time, taken during 8~10d Supernatant, potency is surveyed with ELISA method, carries out primary dcreening operation, positive hole is subcloned.

6. prepared by the cloning (limiting dilution assay) of hybridoma and ascites

After the hybridoma for doing cloning is blown and beaten uniformly repeatedly with sample injector, a little cell suspension will be taken in 96 orifice plates Put in another sterile vials.This cell suspension is serially diluted exactly, until every milliliter contains 10 cells.It will dilute Cell suspension inoculation in being covered with 96 orifice plates of feeder layer, per hole 0.1mL, i.e. every Kong Hanyi cell puts 37 DEG C 5%CO₂Cultivated in incubator, 5d or so observes cell clone growing state under inverted microscope.Clone 3 times repeatedly, it is determined that Positive monoclonal cell line, last clone strain numbering, puts in liquid nitrogen and preserves.Monoclonal antibody cell line potency result such as table 1.

16 plants of GP73McAb titration results of table

Monoclonal antibody is numbered	Ascites antibody potency
		GP73C ends -1	1:120000
GP73 C-terminals -2	1:3200000
		GP7 C-terminals 3-3	1:1280000
GP73 C-terminals -4	1:640000
		GP73 C-terminals -5	1:1600000
GP73 C-terminals -6	1:800000

ELISA results show, the identification of the monoclonal antibodies of GP73 C-terminals to antigen has high susceptibility, highest potency Reach 1:3200000.The 6 plants of GP73McAb obtained only produce reaction with GP73 antigen proteins, with other unrelated proteins without friendship Fork reaction, the GP73 albumen with can identify native state in the immunofluorescence experiment of HepG2 cells very well, prompts what is prepared GP73McAb has good specificity and immunocompetence.

7. subgroup identification

96 orifice plate bars are coated with GP73 C-terminals antigen (2.5mg/L), 4 DEG C overnight.After 2%BSA-PBS-Tween20 closings, Not homophyletic cell culture supernatant (0.1ml/ holes) is added, room temperature 2h, adds anti-mouse Ig after washing and its rabbit of subclass is immunized Serum, 37 DEG C of incubation 1h, enzyme mark goat-anti rabbit Ig antibody is added after washing, 37 DEG C of 1h, is washed, colour developing, judged result.

Subgroup identification result：

GP73McAb supernatants anti-to 6 plants carry out IgG subgroup identifications respectively, and 6 plants of monoclonal antibodies belong to IgG1, K chains (Kapa chains), Stably excreting antibody is remained to after being stored in liquid nitrogen repeatedly recovery passage, is not reduced using ELISA measure potency.

8.GP73C the monoclonal antibody Characterization of antigenic epitopes at end

The monoclonal antibody coated elisa plate (2mg/L) at GP73C ends, GP73 C-terminals expression antigen is added, is examined with double antibody sandwich method Survey GP73.3 pairs of preferable monoclonal antibodies of pairing are screened, for GP73 after birth outskirt different epitopes, are respectively：GP73C ends-1- GP73C ends-2, GP73C end-1-GP73C ends-5, GP73C end-3-GP73C ends-6.With GP73C ends-1-GP73C ends-2 It is paired into most preferably, is also matched in the production of follow-up kit using this 2 kinds of monoclonal antibodies, is used as coated antibody to be coated with by the use of GP73C ends -1 ELISA Plate, detection antibody labeling horseradish peroxidase is used as by the use of GP73C ends -2.

The preparation of the monoclonal antibody (enzyme labelled antibody) at the GP73C ends of the HRP of embodiment 3 marks

The monoclonal antibody of HRP marks is prepared using improvement Over-voltage protection, is comprised the following steps that：

1. taking 5mg HRP to be dissolved in the double distilled waters of 0.5ml, the 0.06Mol/L NaIO newly prepared are added₄(10ml is double for the aqueous solution Distilled water+128mg NaIO₄) 0.5ml, mix, put 4 DEG C of 30min；

2. 0.16Mol/L glycol waters (10mlH is added after taking out₂O+0.09ml ethylene glycol) 0.5ml, room temperature placement 30min；

3. adding the aqueous solution 1ml of the monoclonal antibody at the end containing 5mg purifying GP73C, mix, and load bag filter, it is right The carbonate buffer solutions of 0.05Mol/L pH 9.5 slowly stir dialysis 6h (or staying overnight), are allowed to combine；

4. add NaBH₄Solution (5mg/ml) 0.2ml, mix, put 4 DEG C of 2h；

5. being slowly added to isometric saturated ammonium sulfate solution in above solution, mix, 4 DEG C of 30min, centrifugation, go Clearly, precipitate and dissolved with a little 0.02Mol/L pH7.4PBS liquid, load bag filter, stayed overnight with same liquid in 4 DEG C of dialysis desalinations；

6. next day takes out centrifugation, to remove insoluble matter, enzyme-antibody (HRP-IgG) conjugate is produced, with 0.02Mol/L PH 7.4PBS liquid adds to 5ml；

7. after titration is qualified, adding the high-quality glycerine of equivalent, bottle, Cord blood are dispensed.

The main production material of the kit of embodiment 4 and production technology

1.GP73C hold the production technology of antibody coating plate

1.1 neck material

Coating buffer, antibody stoste, ELISA Plate and the shrouding film of respective amount are got according to production ordering.

1.2 coating

GP73C end -1 purified on ELISA Plate by 2 μ g/mL concentration coating, is incubated in 2~8 DEG C and is no less than 16 hours.

1.3 closing

The coating buffer in ELISA Plate is got rid of, is washed 1 time with cleaning solution (phosphate buffer, PH 7.2), is got rid of cleaning solution, add Enter confining liquid, be incubated and be no less than 16 hours in 2~8 DEG C.

1.4 dry

The confining liquid in ELISA Plate is got rid of, vacuum sealing after drying, in middle 2~8 DEG C of preservations of product temporary room.

2. the production technology of kit

2.1 neck material

Required reagent and consumptive material are got according to production ordering and agent prescription, is shown in Table 2.

The kit production technology material list of table 2

The present embodiment uses one pack system TMB nitrite ions (substrate solution in domestic goods kit for 2 kinds, using AB liquid Method that is separately formulated, being remixed before use, complex operation, difference between batch are larger), reduce operating procedure.

Horseradish peroxidase (HRP) and its label (enzyme labelled antibody, antigen, polypeptide etc.) are in low concentration in the present embodiment Under it is unstable, easily inactivate.Therefore, the dilution of HRP and its label and the preparation of stable type working solution carry to scientific research personnel Higher requirement is gone out.

Horseradish enzyme marker dilution manufactured in the present embodiment contains several HRP stabilizers, to HRP and its label and resists Body activity all has very strong protective effect, makes the HRP of dilution, the reagent such as enzyme marker and antibody is extremely low concentration (ng is horizontal) Under, room temperature use is still very stable, so as to meet that the scientific research personnel for being engaged in the experiments such as ELISA prepares enzyme and its label stable type The requirement of working solution.

Though ELISA detection method operating procedure is simple in the present embodiment, influence factor is more, and closing is wherein most important One of influence factor.The improper susceptibility and specificity that can directly affect ELISA detection method is closed, produces false positive knot Fruit；Good confining liquid can also play stably agent and protective agent effect, improve the storage life of ELISA Plate；Bad confining liquid can also Disturb the reaction of various reagents.

Confining liquid manufactured in the present embodiment is coated with again using the irrelevant protein solution of high concentration, can be allowed uncorrelated Protein fill these and be not coated with gap, so as to reduce adsorbing again for interfering material in ELISA subsequent steps.

2.2 weigh

This batch of product dosage, reagent needed for precise/absorption are calculated according to production ordering.

Points for attention：Concentration enzyme conjugates belongs to easily pollution reagent, and pays attention to being kept in dark place.

Sulfuric acid solution has corrosivity, liquid should be avoided to spill on skin and clothing.

2.3 prepare

Reagent alleged by dissolving in order, with deionized water constant volume, determine pH value.Confining liquid, enzyme storage liquid, concentration enzyme combine Thing dilution and calibration object dilution need to be filtered with 0.22 μm of filter.It is labelled on container, indicate title, lot number, Capacity and the term of validity etc., in middle 2~8 DEG C of preservations of product temporary room.

2.4 packing semi-finished product

2.4.1 neck material

The semi-finished product and raw material of respective amount are got according to production ordering, semi-finished product need to place (18~28 DEG C) balances of room temperature Component table is dispensed to specifications after 1 hour.

Note：All semi-finished product have passed through the inspection of semifinished product.

3. the requirement of main raw material(s)

3.1 antigens, antibody

3.1.1 source

Recombinate GP73C end proteins (hereinafter referred to as GP73C ends antigen), GP73C ends monoclonal antibody is Tianjin gold rainbow biotechnology Development corporation, Ltd..

The main raw material(s) purposes of table 3 and source

Title	Purposes	Source
			GP73C ends antigen	Prepare calibration object	Escherichia coli prokaryotic expression
GP73C ends -1	Coated elisa plate	Balb/c mouse
			GP73C ends -2	Mark horseradish peroxidase	Balb/c mouse

3.1.2 outward appearance

Visually observe, raw material answer clear, no precipitation.

3.1.3 purity and molecular weight

Purity is determined using SDS-PAGE, applied sample amount is 10 μ g.GP73C ends antigen purity is more than 90%, and molecular weight exists 12KD or so；Antibody purity is all higher than 90%, and a colour band is shown respectively in 25KD and 55KD or so.

3.1.4 protein concentration

Using protein concentration in the antigen of BCA methods measure GP73C ends.GP73C ends -1 after purification and horseradish peroxidase (HRP) the GP73C ends -2 of mark determine OD with ultraviolet specrophotometer₂₈₀, by (OD₂₈₀× 0.625)=_ mg/mL calculates egg White concentration.

GP73C ends antigen and the after purification protein concentration of GP73C ends monoclonal antibody should be greater than 0.5mg/mL, HRP marks GP73C ends monoclonal antibody gram molecule ratio is between 1~2.

It is 3.1.5 linear

3.1.5.1 GP73C ends antigen linear determination

2ug/mL coated elisa plates are pressed with the GP73C ends -1 of purifying, calibration is prepared with the GP73C ends antigen of expression and purification Product, detected after kit is made.

For the range of linearity between 1.00~450.00ng/mL, coefficient correlation is not less than 0.9900.

3.1.6 the monoclonal antibody titer of ascites at GP73C ends

Respectively with the monoclonal cell strain 1 at GP73C ends, No. 2 injection mouse peritoneals, obtained ascites is diluted, uses table Up to the antigen coat ELISA Plate at the GP73C ends of purifying, detected using ELISA indirect methods.2.1 times of negative control OD value are judgement Positive threshold value, the greatest dilution of positive reaction is monoclonal antibody titer of ascites.

The monoclonal antibody titer of ascites at GP73C ends is not less than 1:10⁵。

The kit correlation finished product of embodiment 5 and semi-finished product performance indications and inspection procedure

First, end properties index and inspection procedure

1. performance indications

1.1 outward appearance

Kit outward appearance should be complete, no breakage；ELIAS strip should smooth, non-trimming, surface smoothness be good, bottom non-ripple And scratch；Liquid component should be as clear as crystal, no floccule, ne-leakage；Label is clearly easy to identify.

1.2 test limit

Test limit≤1.00ng/mL.

It is 1.3 linear

In the range of 1.00ng/mL~450.00ng/mL, coefficient correlation >=0.9900.

It is repeated in 1.4 batches

Coefficient of variation CV≤15%.

It is repeated between 1.5 batches

Coefficient of variation CV≤15%.

1.6 the degree of accuracy

The rate of recovery is in the range of 85%~115%.

1.7 stability

Took effect phase kit, detection outward appearance, test limit, linear, repeatability and the degree of accuracy, it as a result should meet 1.1~ 1.4th, 1.6 requirement.

1.8 traceability

This product calibration object can trace to the source to restructuring GP73C end proteins (hereinafter referred to as GP73C ends antigen).

2. the method for inspection

2.1 outward appearance

Test method：Visually inspect, its result should meet 1.1 requirement.

Result of the test is shown in Table 4.

Table 4

2.2 test limit

Test method：Detected by the use of zero-dose calibration object as sample, replication 20 times, draw 20 measurement results (X values), its average value (M) and standard deviation (SD) are calculated, draws the X values corresponding to M+2SD, brings X values into calibration curve equation In, corresponding concentration value, as test limit are obtained, its result should meet 1.2 requirement.

Result of the test is shown in Table 5.

Table 5

It is 2.3 linear

Test method：With exceeding or diluted by a certain percentage at least close to the sample of range of linearity upper concentration (activity) 5 diluted concentration (xi) points, wherein low value concentration should be close to range of linearity lower limits.Tested respectively with test kit, it is each dilute Release concentration determination 3 times, obtain the average (yi) of measurement result respectively.It is equal with measurement result with diluted concentration (xi) for independent variable Value (yi) is that dependent variable obtains equation of linear regression.The coefficient correlation (r) of linear regression is calculated by formula (1).

Its result should meet 2.3 requirement.

Result of the test is shown in Table 6.

Table 6

Lot number	First	Second batch	3rd batch
				Linearly	0.9942	0.9919	0.9948

It is repeated in 2.4 batches

Test method：With kit test high (100.00ng/mL~300.00ng/mL), in (30.00ng/mL~ 100.00ng/mL), low (1.00ng/mL~30.00ng/mL) three level concentration samples, retest 20 times (n=20), are obtained Go out 20 measurement results (X values), calculate the average value (M) and standard deviation (SD) of each 20 measured values of concentration samples respectively, count The coefficient of variation (CV) is calculated, its result should meet 2.4 requirement.

Result of the test is shown in Table 7.

Table 7

Lot number	Low concentration CV (%)	Middle concentration C V (%)	High concentration CV (%)
				First	5.33	3.53	4.55
Second batch	5.94	8.53	5.02
				3rd batch	2.56	3.34	1.69

It is repeated between 2.5 batches

Test method：Take the kit of three different lot numbers, test respectively with a sample (concentration be 30.00ng/mL~ 100.00ng/mL), each lot number retest 10 times, 30 measurement results (X values) are drawn, calculates being averaged for 30 measured values It is worth (M) and standard deviation (SD), calculates the coefficient of variation (CV), its result should meets 2.5 requirement.

Result of the test is shown in Table 8.

Table 8

Lot number	First, second batch, the 3rd batch
		Middle concentration C V (%)	8.33

2.6 the degree of accuracy

Test method：Certain low concentration serum sample (standard liquid body is added in high standard solution or sterling Product should be not more than 1 with sample volume ratio:9 or its volume ratio will not produce the change of matrix, sample is always dense after adding standard liquid Degree must be in the kit measurement range of linearity), each concentration replication 3 times, the rate of recovery, its result are calculated by formula (4) 2.6 requirement should be met.

In formula：R-the rate of recovery；

V-addition titer volume；

The volume of V0-low concentration sample；

The measure concentration that C-low concentration sample is added after titer；

The measure concentration of C0-low concentration sample；

The concentration of Cs-titer.

Result of the test is shown in Table 9.

Table 9

Lot number	First	Second batch	3rd batch
				The rate of recovery (%)	109.04	105.71	108.46

2.7 stability

Effect phase kit was taken, detects 2.1~2.4,2.6, its result should meet 1.7 requirement.

2nd, semi-finished product performance indications and inspection procedure

3.1 GP73C ends antibody coating plate

Appearance test：GP73C ends antibody coating plate bar should smooth, non-trimming, surface smoothness be good, bottom non-ripple and draws Wound.

The kit for having completed to examine is taken, with semi-finished product GP73C ends antibody coating plate to be checked, is tested jointly in the kit 6 bottles of calibration objects, every bottle of retest 3 times respectively of two kinds of GP73C ends antibody coating plates, take the averages of each point OD values, calibration object The OD value relative deviations of each concentration point are in the range of ± 15%.Vacuum packaging, 2~8 DEG C of preservations.

3.2 reagent outward appearances

Appearance test：Liquid component should be as clear as crystal, no floccule, ne-leakage.

3.3 reagent tests, packing and preservation

3.3.1 calibration object

The kit for having completed to examine is taken, with calibration object to be checked, tests two kinds of calibration objects respectively, each 6 bottles, every bottle of repetition is surveyed Examination 3 times, takes the average of each point OD values, the OD value relative deviations of each concentration point of calibration object are in the range of ± 15%.

The calibration object dispensed loading amount of each concentration of each kit is 0.5mL, and 2~8 DEG C preserve.

3.3.3 enzyme conjugates

The kit for having completed to examine is taken, with treating check reagent, 6 bottles of calibration objects testing jointly in the kit, two kinds of examinations Agent every bottle of retest 3 times respectively, take the average of each point OD values, the OD values relative deviation of each concentration point of calibration object is in ± 15% model In enclosing.

The dispensed loading amount of each kit is 12mL, and 2~8 DEG C preserve.

3.3.4 concentrated cleaning solution (20 ×)

Cleaning solution in each kit is the phosphate buffer of 20 times of concentrations, and dispensed loading amount 50mL, 2~8 DEG C preserve.

3.3.5 one-component TMB nitrite ions

The kit for having completed to examine is taken, with treating check reagent, 6 bottles of calibration objects testing jointly in the kit, two kinds of examinations Agent every bottle of retest 3 times respectively, take the average of each point OD values, the OD values relative deviation of each concentration point of calibration object is in ± 15% model In enclosing.

The dispensed loading amount of each kit is 12mL, and 2~8 DEG C are kept in dark place.

3.3.6 terminate liquid

Dispensed loading amount in each kit is 7mL, and (18~28 DEG C) of room temperature preserves.

The clinical trial results of embodiment 6

Tianjin Jin Hong biotechnologies development corporation, Ltd. establishes GP73 levels in measure serum by substantial amounts of experiment Double antibody sandwich method.This method uses monoclonal antibody to optimize traditional handicraft for raw material, thus has higher sensitivity (1.00ng/ml) and specificity.Detection method is easy to be quick, takes less than 1.5 hours.Standard curve linear relationship is good, phase Relation number r >=0.9900, such as Fig. 1.

It is bent that ROC is drawn with the GP73 concentration of 858 hepatic sclerosis serum samples and 1111 people taking physical examination serum samples Line.Under ROC curve area be 0.959, when 8.52ng/ml be GP73 diagnose hepatic sclerosis cut-off values when, susceptibility and specifically Property be it is optimal, respectively 81.4% and 95.0%, as shown in Figure 2.GP73 concentration is more than 4.53ng/ml, moderate hepatic injury, prompts It there may be hepatopathy more than notable liver fibrosis；GP73 concentration>During 8.52ng/ml, prompting may existing for hepatic sclerosis.Simultaneously It was found that：Serum GP73 slightly raises in chronic persistant hepatitis, and rise is notable during hepatic sclerosis, its horizontal height and liver pathological changes journey Degree is proportionate.Therefore, this kit is on the basis of clinical assistant diagnosis hepatic sclerosis, also can aid prompting hepatic sclerosis early stage Liver fibrosis.

Serum GP73 concentration has conspicuousness statistics between this studies have shown that liver cirrhosis patient difference Child-Pugh classifications Learn difference.With hepatic insufficiency, serum GP73 levels also accordingly raise.Child-Pugh is the patient (32.58ng/mL of C levels ± 16.52ng/mL) be significantly higher than B levels (19.37ng/mL ± 10.23ng/mL) and A levels patient (12.28ng/mL ± 6.09ng/mL)。

GP73 positive rates are all remarkably higher than hepatitis B related liver disease (P with concentration in hepatitis disease<0.001), show to invade in HCV GP73 up-regulated expressions in the cell of dye, GP73 are the key substances in HCV secretion process, are consistent with document report.

The concentration for detecting GP73 in 6 kinds of non-liver malignancy samples respectively is 1.0ng/ml, substantially less than hepatic sclerosis mark This (P<0.05), prompt GP73 that specificity is good, can be as the blood serum designated object only for hepatic sclerosis.

It has detected 158 hepatic sclerosis and 160 people taking physical examination plasma specimens.As a result show, cirrhotic population's blood plasma The GP73 contents of sample are (9.10ng/mL ± 8.71ng/mL), and the GP73 contents of people taking physical examination plasma specimen are (1.16ng/mL ± 0.25ng/mL), the detection of plasma sample can be used for simultaneously by showing the kit of the present embodiment.

In a word, GP73 quantitative detecting reagents method is simple, quick in the present embodiment serum, and cost is low, clinical practice result Show that GP73 can diagnose a kind of mark of hepatic sclerosis as adjuvant clinical, to the progress of hepatitis, liver fibrosis and hepatic sclerosis There is monitoring effect, there is good clinical value.

The magnetic bead chemoluminescence method of embodiment 7 detects the preparation and use of GP73 kits

The magnetic immuno detection technique established using magnetic Nano microsphere as solid phase carrier is that recent domestic is important Immunoassay technology.The magnetic microsphere that surface is chemically modified passes through covalent bond coupled antibody molecule；Its huge ratio surface The more antibody of product energy coupling, and fully expose the antigen binding site of antibody and reduce steric effect；Also, nano-magnetic is micro- Ball has superparamagnetism, when without externally-applied magnetic field it is dispersed in the solution, make antigen-antibody reaction more quick, efficiently.

The magnetic bead that the present embodiment is modified with a kind of GP73C ends monoclonal antibody coupling Streptavidin of biotin labeling, Test serum is added, is reacted with the enzyme conjugates of another plant of GP73C ends antibody, then is acted on chemical luminous substrate luminol, root According to luminous value judged result.

Concrete operations are as follows：

1. sample-adding

50 μ l magnetic beads, 20 μ l calibration objects or serum sample and 50 μ l enzyme conjugates are added in respective aperture respectively, fully Vibration mixes 30 seconds.

2. incubate

Incubated 15 minutes with the rearmounted 37 DEG C of constant water bath box of shrouding film shrouding.

3. washing

Carefully take shrouding film off, washed 6 times with board-washing machine washing, should add at least 300 μ l cleaning solutions per hole every time.

4. add substrate

50 μ l chemical luminous substrate A liquid are added per hole, add 50 μ l chemical luminous substrate B liquid, adding should avoid during substrate Bubble is produced, vibration mixed 30 seconds, with rearmounted 37 DEG C of lucifuge warm bath of shrouding film shrouding 5 minutes.

5. measure

Chemiluminescence detector and host computer are opened, sets mode determination and parameter.The 5th point after substrate is added Clock determines the luminous value RLU in each hole, is 1 second per hole minute.

6. calibration curve is fitted and concentration of specimens calculates

The RLU determined according to each calibration object, using double-log fit equation, GP73 concentration-RLU regression equation is established, Using the logarithm of each calibration object concentration as ordinate (Y-axis), each calibration object values of chemiluminescence (values of chemiluminescence that S0 need to be subtracted) Logarithm is mapped for abscissa (X-axis).Bring each sample RLU values into calibration curve, sample GP73 concentration values are calculated.

Experimental results of the fucosylation GP73 of embodiment 8 in liver cirrhosis diagnosis

Glycosylation (glycosylation) is one of most common mode, about 50% in protein post-translational modification Protein all have it is glycosylation modified.Research shows, in the generation, evolution in many diseases, the glycosylation of glycoprotein Degree is all changed.Along with the development of hepatic disease, generation core fucosylation that GP73 can be different degrees of.According to LcA can have LcA with this characteristic of core fucosylation glycoprotein specific bond using coupling Ago-Gel is affine, and absorption centrifuge tube elution obtains Fuc-GP73 (fucosylation GP73), is quantified with reference to ELISA method Detect Fuc-GP73 contents in patients serum.

Specific method is as follows：

1. coupling has the making step of LCA Ago-Gel

(1) 1.7g Sepharose CL 6B are soaked in 1mmol/L HCL to expanding, sand core funnel are moved into, with 350mL Concentration is that 1mmol/L HCL are washed 25 minutes.

(2) 5mg LCA are weighed, are dissolved in 17mL coupling buffer, are merged with scrubbed Sepharose CL 6B, are used 20mL test tubes with plug turn upside down mixings, at room temperature standing 2.5 hours.Post is filled, is washed away with 15mL coupling buffers and is not coupled LCA.

(3) LCA. being coupled with the closing of 0.2mol/L glycine

(4) it is successively 0.1mol/L acetate buffer solutions with 15mL concentration and Tris buffer solutions that concentration is 0.1mol/L wash 3 times, then washed 1 time with PBS-BSA, 4 DEG C are temporary standby.

2.Fuc-GP73 purification step

(1) test serum is centrifuged completely；The pre-mounted eccentric column of 4 DEG C of preservations is taken out, discards liquid in underlying collection pipe.

(2) diluting blood sample:200 μ L serum are drawn in test tube, and add 300 μ L cleaning solution dilutions, gently concussion mixes.

(3) it is loaded:Sample adds top centrifuge tube after drawing 400 μ L dilutions, is placed in 37 DEG C of insulating boxs, this step need not Lid centrifuge tube lid, dilution is waited all to flow into underlying collection Guan Zhongyue 25 minutes.

(4) liquid in collective low pipe is discarded.

(5) 500 μ L cleaning fluids are added into top centrifuge tube, wait cleaning fluid all to flow into collective low pipe (about 5 points Clock), centrifugation lid is covered, 3000 leaves the heart 1 minute at room temperature.

(6) liquid in underlying collection pipe is discarded.

(7) (5) (6) step is repeated once.

(8) 400 μ L are added into top centrifuge tube, when wait has eluent instillation underlying collection pipe, cover centrifugation lid, It is placed in 37 DEG C of insulating boxs and incubates 30 minutes.

(9) centrifuge tube is taken out, 3000 leaves the heart 1 minute at room temperature.

(10) collect and flow into liquid (containing GP73 heteroplasmons) in underlying collection pipe, it is standby.

(11) GP73 heteroplasmon contents in collection liquid are detected, step is the same as GP73 contents in ELISA method detection serum.

Detect 156 hepatic sclerosis, 160 chronic hepatitis and 205 people taking physical examination serum Fuc-GP73 contents.As a result Show：Liver cirrhosis group Fuc-GP73 levels are significantly higher than chronic hepatitis (P<And healthy control group 0.01).By drawing ROC curve It is 0.908 to calculate Fuc-GP73 detection hepatic sclerosis TG-AUCs, and susceptibility 76.9%, specificity is 93.1%, and the positive is sentenced Disconnected value is 2.55ng/ml.

Several embodiments of the invention are described in detail above, but the content is only the invention Preferred embodiment, it is impossible to be considered as the practical range for limiting the invention.It is all according to the invention application range institute The equivalent change of work and improvement etc., all should still be belonged within the patent covering scope of the invention.

Sequence table

<110>Tianjin Jin Hong biotechnologies development corporation, Ltd.

<120>A kind of preparation and its application of GP73 C-terminals antigen

<130> 2017

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 100

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 1

Val Gln Ala Ala Leu Ser Val Ser Gln Glu Asn Pro Glu Met Glu Gly

1 5 10 15

Pro Glu Arg Asp Gln Leu Val Ile Pro Asp Gly Gln Glu Glu Glu Gln

20 25 30

Glu Ala Ala Gly Glu Gly Arg Asn Gln Gln Lys Leu Arg Gly Glu Asp

35 40 45

Asp Tyr Asn Met Asp Glu Asn Glu Ala Glu Ser Glu Thr Asp Lys Gln

50 55 60

Ala Ala Leu Ala Gly Asn Asp Arg Asn Ile Asp Val Phe Asn Val Glu

65 70 75 80

Asp Gln Lys Arg Asp Thr Ile Asn Leu Leu Asp Gln Arg Glu Lys Arg

85 90 95

Asn His Thr Leu

100

Claims (10)

1. a kind of GP73C end proteins, its amino acid sequence is SEQ ID NO:1.

A kind of 2. method for preparing GP73C end proteins as claimed in claim 1, it is characterised in that first, using PCR method Amplify the DNA sequence dna that coding schedule reaches GP73C ends；Then, the sequence is inserted into the multiple cloning sites of expression vector, structure weight Group expression plasmid；Afterwards, expressive host bacterium is transformed into, structure obtains engineered strain, and engineered strain is through induced expression；Finally, divide GP73C ends recombinant protein is obtained from purifying.

3. the preparation method of a kind of GP73C end proteins according to claim 2, it is characterised in that main to include following step Suddenly：

(1) PCR is expanded：Using containing BamHI, EcoRI restriction enzyme site primer PCR amplification GP73C end groups because (1019~ 1336bp), digestion is connected with the PGEX-4T-2 plasmids after BamHI, EcoRI digestion after purification, is transformed into Escherichia coli BL21；

(2) expression-form is determined：Select positive colony, 25 DEG C of IPTG pyranosides of use 1% at random (IPTG) chemical induction, 1%NP-40 cracking, -80 DEG C of multigelations 3 times, centrifugation, take supernatant precipitation to carry out SDS- respectively and gather Acrylamide gel electrophoresis (SDS-PAGE) is identified, determines expression-form；

(3) culture and purifying are expanded：Choosing positive colony and be enlarged culture, 1%NP-40 is cracked, -80 DEG C of multigelations 3 times, Using purification by chromatography GP73C ends antigen.

A kind of 4. preparation method of GP73C end proteins according to claim 3, it is characterised in that the upstream in step (1) Primer is that (5 ' -3 ', anti-sense primer is GC GAA TTC TGA TGTGAG to GC GCT GGA TCC CAG CTG GCC TCA ATGATT。

5. one kind prepares monoclonal antibody methodology using GP73C end proteins as claimed in claim 1, it is characterised in that uses table The immune Balb/C mouse of GP73C ends recombinant protein up to synthesis prepare and screen hybridoma cell strain, prepare ascites and simultaneously purify list Clonal antibody.

6. application of the monoclonal antibody being prepared in claim 5 in diagnosing hepatic diseases reagent is prepared.

7. application of the monoclonal antibody being prepared in claim 5 in diagnosing hepatic diseases kit is prepared.

8. such as the kit in claim 7, it is characterised in that the detection process of the kit passes through including monoclonal antibody The process of GP73 contents/fucosylation GP73 contents in ELISA method detection serum.

9. such as the kit in claim 7, it is characterised in that the detection process of the kit passes through magnetic including monoclonal antibody Process in terms of pearl chemoluminescence method detection GP73 content kits.

10. such as the kit in claim 8, it is characterised in that the process of GP73 contents in ELISA method detection serum, for Two plants of monoclonal antibodies for GP73C end protein different epitopes are shown respectively as coated antibody and detection antibody by substrate Color detects GP73 albumen, and the standard curve prepared by calibration object determines the content of GP73 albumen.