CN107202885A

CN107202885A - Antibody and kit for detecting α L fucosidases in serum

Info

Publication number: CN107202885A
Application number: CN201710191320.3A
Authority: CN
Inventors: 马杰; 吴云
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-03-28
Filing date: 2017-03-28
Publication date: 2017-09-26
Anticipated expiration: 2037-03-28
Also published as: CN107202885B

Abstract

The present invention relates to serum antigen detection technique, and in particular to a kind of antibody and kit for being used to detect α L fucosidases in serum.There is provided the antibody pair for detecting liver cancer marker α L fucosidases in test serum, it is characterised in that be made up of first antibody and secondary antibody；The first antibody, as coated antibody, is CGMCC NO by deposit number：13583 hybridoma secretion；The secondary antibody, as detection antibody, is CGMCC NO by deposit number：13584 hybridoma secretion.The antibody to high specificity, can simultaneously with other marks are antibody combined uses, uninterruptedly detect AFU in serum；Sensitivity is high, detectable concentration as little as 250pg/ml AFU, AFU albumen that can be in accurate quantitative analysis serum.

Description

Antibody and kit for detecting alpha-L-fucosidase in serum

Technical field

The present invention relates to serum antigen detection technique, and in particular to a kind of to be used to detect alpha-L-fucosidase in serum Antibody and kit.

Background technology

The incidence of disease of the cancer in the whole world is in rising trend.On 2 3rd, 2014, what the World Health Organization delivered《Global cancer Report 2014》It has been shown that, the newly-increased cases of cancer height of China is ranked first in the world, and the new cases and death toll of wherein liver cancer are occupied First place in the world.Hepatocellular carcinoma (hepatocellularcarcinoma, HCC) is one of global most common malignant tumour, China It is hepatitis B big country, onset of liver cancer rate accounts for the 55% of global sum.Clinically the diagnosis of liver cancer mainly passes through Virus monitory first tire Albumen (Alpha-fetoprotein, AFP), B ultrasound finds perfused rat liver model, and then CT and MRI is checked.But AFP positive rates are about 60~70%, and not all hepatocellular carcinoma all secretes substantial amounts of AFP, about 40% early primary hepatocarcinoma and 15%~20% late period The serum afp of Patients with Primary is normal, therefore only still has the possibility failed to pinpoint a disease in diagnosis by AFP diagnosing liver cancers, causes clinic 80% patient has belonged to middle and advanced stage when making a definite diagnosis.Mid and late liver cancer is substantially without effective treatment method, to people at highest risk such as hepatitis B, hepatic sclerosis The examination and early diagnosis for carrying out hepatic carcinoma mark are the most effective measures for solving liver cancer high mortality.

Alpha-L-fucosidase (AFU) is a kind of lysosomal hydrolase, is widely present in various histocytes and body fluid, Participate in the catabolism of the various bioactivators such as glycoprotein and glycolipid.Liver is one of organ rich in lysosome, works as liver During cell carcinogenesis, AFU synthesis increases, and tumour cell membrane permeability increases, and the enzyme amount being discharged into blood increases, and degraded speed Degree slows down, thus causes serum AFU concentration to raise.Serum AFU activity performance graphs are to judging liver cancer treatment effect, estimating prognosis There are extremely important meaning, even better than AFP with forecast recurrence.But serum AFU vitality tests are in some metastatic hepatic carcinomas, lung There are some overlapping between cancer, breast cancer, ovary or uterine cancer, or even in some Non-cancerous illness such as hepatic sclerosis, chronic hepatitis Also there is slight rise with hemorrhage of digestive tract etc., should simultaneously be determined with AFP when using AFU, the diagnosis of primary carcinoma of liver can be improved Positive rate has preferable complementation up to 93.1%.

Luminex Suspension array techniques are a kind of many work(that Luminex companies of the U.S. develop in 1990s mid-term The liquid-phase chip analysis platform of energy, also referred to as liquid chip.It organically incorporates coloured microballoon, laser technology, newest height Speed digital signal processing and computer technology, the fluorescence-encoded micro-beads of application resist with the specificity for different target molecule Body, different microballoons can simultaneously be detected with independent assortment and be for up to 100 kinds of differences in one 25-50 microlitres of sample Detection project, with repeatability with stability is good, high flux, Testing index can be selected flexibly, and high sensitivity and high letter Make an uproar many advantages, such as comparing, it is adaptable to the analysis of antigen albuminoid and the quantitative analysis of major disease antigen markers in blood plasma, It is the medical science being significant an auxiliary detection means.Compared with traditional solid phase chip, solid phase chip is overcome big Kinetics is disturbed by surface tension, three-dimensional effect etc. during Molecular Detection, makes the stability and repeatability of testing result It is greatly improved.

Therefore, AFU specific antibodies are obtained, AFU contents in serum are examined using Luminex Suspension array techniques Survey, it will help improve the recall rate of early liver cancer.

The content of the invention

In order to improve the recall rate of early liver cancer, the present invention is provided to detect the antibody of alpha-L-fucosidase in serum And kit, its high specificity, sensitivity height, it can accurately detect the content of alpha-L-fucosidase in serum.

Claimed technical scheme is as follows：

For the antigen protein for the antibody for preparing anti-alpha-L-fucosidase, it is characterised in that its amino acid sequence such as SEQ Shown in ID NO.1.

Encode the gene of the antigen protein, it is characterised in that its nucleotide sequence is as shown in SEQ ID NO.2.

Monoclonal antibody for detecting liver cancer marker alpha-L-fucosidase in test serum, it is characterised in that by Deposit number is secreted for CGMCC NO.13584 hybridoma.

Secrete the hybridoma of the monoclonal antibody, it is characterised in that its deposit number is CGMCC NO.13584.

Antibody pair for detecting liver cancer marker alpha-L-fucosidase in test serum, it is characterised in that by first Antibody and secondary antibody composition；The first antibody is as coated antibody, by the hybridization that deposit number is CGMCC NO.13583 Oncocyte is secreted；The secondary antibody is as detection antibody, by hybridoma point of the deposit number for CGMCC NO.13584 Secrete.

Kit for hepatocarcinoma early diagnosis, it is characterised in that including the monoclonal antibody or the antibody pair, with And the common reagent for immune detection.

The kit, it is characterised in that including the antibody pair, the first antibody is coupled with magnetic bead, and described second Antibody biotin labeling, the common reagent for immune detection is SA-PE.

The kit, it is characterised in that standard items also including alpha-L-fucosidase and/or with the α-L- rock algaes Standard curve or linear regression equation specification that the standard items of glycosidase make.

The kit, it is characterised in that also the antibody including anti-alpha-fetoprotein, the antibody of anti-Gorky's glycoprotein 73, The antibody of anti-vascular endothelial growth factor and/or anti-cell epimatrix Metalloproteinase Inducer CD147 antibody, and its phase The immunologic function test reagent answered.

The kit, it is characterised in that also including alpha-fetoprotein, Gorky's glycoprotein 73, VEGF And/or extracellular matrix metalloproteinase CD147 standard items, and/or with alpha-fetoprotein, Gorky's glycoprotein 73rd, the standard that VEGF and/or extracellular matrix metalloproteinase CD147 standard items make is bent Line or linear regression equation specification.

The present invention uses ammonia of the DNA Star sequence analysis softwares to liver cancer serum mark alpha-L-fucosidase (AFU) Base acid sequence (NCBI sequence numbers：NP_000138.2) analyzed, consider the antigenicity, specificity, albumen of protein sequence Expression and the factor such as ease of purifying, have chosen 252-441aa peptide fragment as antigen, its amino acid sequence such as SEQ ID Shown in NO.1, gene coded sequence is as shown in SEQ ID NO.2.

Antigen protein and immune mouse are obtained by the method for albumen pronucleus expression, filters out and is capable of specific bond AFU's Monoclonal antibody, and the monoclonal antibody of acquisition is matched by the experiment of ELISA double antibodies sandwiches, screen and can be used in The antibody pair that serum AFU is quantitatively detected, is made up of first antibody (coated antibody) and secondary antibody (detection antibody), and it hybridizes Oncocyte preserving number is CGMCC NO.13583 and CGMCC NO.13584 respectively.The antibody (can be to high specificity simultaneously With other marks are antibody combined uses, uninterruptedly detect AFU in serum, see that embodiment 4 is recorded), sensitivity height (can Concentration as little as 250pg/ml AFU is detected, sees that embodiment 4 is recorded), AFU albumen that can be in accurate quantitative analysis serum.

The kit of the present invention, can also include removing alpha-fetoprotein, Gorky's glycoprotein 73, alpha-L-fucosidase, blood The specificity of other tumor markerses outside endothelial tube growth factor and extracellular matrix metalloproteinase CD147 Antibody, and its corresponding immunologic function test reagent.

Using the antibody pair or kit of the present invention, with reference to Luminex technologies, the immunological technique and mark of double antibodies sandwich Directrix curve realizes the quantitative detection of AFU in serum.The Cleaning Principle of this method is：To be coupled the magnetic bead of first antibody with it is dilute 4 DEG C of overnight incubations of test serum sample after releasing, form antigen-specific antibodies compound, are washed away and do not tied with lavation buffer solution The antigen of conjunction；The secondary antibody of biotin labeling is added, incubation at room temperature forms specific antibody-antigen-antibody complex, used Lavation buffer solution washes away uncombined antibody；Add SA-PE (SAPE), incubation at room temperature, formation antibody-anti- Antigen-antibody-biotin-SAPE compounds, uncombined SAPE is washed away with lavation buffer solution；Add after determining buffer solution and be placed in liquid MFI values are read in phase suspension chip system；AFU in sample is calculated in the equation that MFI values are finally substituted into standard curve Concentration, the extension rate for being multiplied by blood serum sample draws AFU actual concentrations.

Compared with common detection methods ELISA, method of the invention has quick, sensitive advantage, and can be one Other liver cancer related neoplasms marks, such as AFP, AFP-L3, GP73, VEGF, CD147 etc. are detected in individual reaction system simultaneously.Profit Luminex liquid microarrays technologies are used, according to being actually needed for diagnosis, antithesis is associated with the glimmering of different tumor markers specific antibodies Pumped FIR laser microballoon carries out independent assortment, and once experiment can complete the quantitative analysis of a variety of liver cancer related neoplasms marks simultaneously, Have the advantage that：(1) the minimum 10ul of one-time detection blood plasma consumption, 6 kinds of plasma proteins can be detected simultaneously, blood is greatly reduced Consumption is starched, time saving and energy saving, testing cost is low；(2) liquid-phase chip technology reaction system is conducive to keeping biological under liquid phase environment The activity of macromolecular so that reaction system is more stablized, testing result is relatively reliable accurate；(3) the liver cancer phase in plasma sample Closing tumor markers AFP, AFP-L3, GP73, AFU, VEGF, CD147 albumen joint-detection makes phase between Diagnostic Value of Several Serum Tumor Markers The analysis of closing property is more accurate, can improve the diagnosis of liver cancer early stage, to formulate therapeutic scheme, extension patient's life in time Life.

Biological deposits information

Preservation mechanism：China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)

Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica

Brief description of the drawings

Fig. 1 .luminex liquid-phase chip Cleaning Principle schematic diagrames；

Fig. 2 .GP73 single-factor standard items testing results；

Fig. 3 .AFU single-factor standard items testing results；

Fig. 4 .VEGF single-factor standard items testing results；

Fig. 5 .CD147 single-factor standard items testing results；

Fig. 6 .AFP single-factor standard items testing results；

Fig. 7 .CD147, VEGF double factor standard items testing results；

CD147 and VEGF double factors react in same system, without generation cross reaction between antibody, each factor Reactivity worth does not have significant change.

The factor standard product testing result of Fig. 8 .CD147, VEGF, AFP tri-；

The factor of CD147, VEGF and AFP tri- is reacted in same system, between antibody without occur cross reaction, it is each because The reactivity worth of son does not have significant change.

The factor standard product testing result of Fig. 9 .CD147, VEGF, AFP, AFU tetra-；

The factor of CD147, VEGF, AFP and AFU tetra- is reacted in same system, without generation cross reaction between antibody, The reactivity worth of each factor does not have significant change.

The factor standard product testing result of Figure 10 .CD147, VEGF, AFP, AFU, GP73 five；

The factor of CD147, VEGF, AFP, AFU and GP73 five is reacted in same system, does not have to intersect between antibody Reaction, the reactivity worth of each factor does not have significant change.

Wherein, abscissa is the concentration of standard items, and ordinate is fluorescence intensity.

Embodiment

Below by specific embodiment, the present invention is described in detail, it is to be understood that following embodiments are used as solution Release and illustrate, the scope that the invention is not limited in any way.

Biomaterial

BALB/c mouse, purchased from Jie Sijie experimental animals Co., Ltd；

Hela cells, purchased from Guangzhou Sai Ku Bioisystech Co., Ltd；

Myeloma cell, the culture of Beijing Qing Yuanshengkang biological medicines Science and Technology Ltd. and preservation；

E.coli DH5 α competent cells, E.coliBL21 competent cells are that this laboratory is preserved, can be by commercially available Obtain.

Experiment reagent

Restriction enzyme EcoR I and Xho I, buy from Takara companies, article No. D1040A, D1094A；

Carrier pET32a, buys from Novagen companies, article No. VYN0176；

T4 DNA ligases, buy from Takara companies, article No. D2011A；

Plasmid extraction kit, buys from takara companies, article No. 9760；

HyClone modified form RPMI-1640 culture mediums, purchased from Hyclone companies, article No. AB10113944；

Blood serum of newborn calf without mycoplasma (super), purchased from Zhejiang Tian Hang bio tech ltd, article No. 22012-8612；

Dual anti-(100X), purchased from Beijing Lei Gen Bioisystech Co., Ltd, article No. CA0075；

Glu (100X), purchased from Amresco companies, article No. Amresco 0374；

PEG (50%w/v polyethylene glycol 1,500), purchased from Hampton companies, article No. HR2-525；

HAT culture medium additives (50X), purchased from gibco companies, article No. 21060-017；

Sheep anti-mouse igg, article No. A0286 biological purchased from the green skies；

Sodium acetate, purchased from Beijing chemical reagents corporation, article No. 10018892；

Octanoic acid, purchased from Beijing Jin Long chemical reagent Co., Ltd；

Streptavin-HRP (Streptavidin-horseradish peroxidase), purchased from the green skies, article No. A0303；

Sulfo-NHS (N- hydroxy thiosuccinimides), purchased from SIGMA companies, article No. 56485；

EDC (1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides), purchased from SIGMA companies, article No. 22980；

MES (2- (N- morpholines) ethyl sulfonic acid), purchased from SIGMA companies, article No. M2933；

PBS-TBN (closing/storage buffer solution), PBS, containing 0.1%BSA (BSA is biological purchased from Kang Yuan), 0.02%TWEEN 20 (TWEEN 20 is purchased from Amresco 0777), 0.05%Azide (Azide is purchased from SIGMA S8032)；

Assay buffer (measure buffer solution), PBS, containing 1%BSA (BSA is biological purchased from Kang Yuan) pH7.4；

Wash buffer (lavation buffer solution), PBS, containing 0.02%TWEEN 20, (TWEEN 20 is purchased from Amresco 0777), pH7.4；

SAPE (SA-PE), purchased from ebioscience companies, article No. 12-4317；

Biotin, purchased from sigma companies, article No. H1759；

Alpha-fetoprotein standard items, purchased from Nat'l Pharmaceutical ＆ Biological Products Control Institute, production code member 150542-1.

Instrument and consumptive material

Magnetic bead, purchased from LUMINEX companies, article No. MC10030-01, MC10034-01, MC10036-01, MC10038-01, MC10044-01；

Magnetic separator, purchased from Biorad companies；

Luminex200, purchased from BioRad companies, model LUMINEX-200.

In following embodiments, not specified biological chemical reagent is this area conventional reagent, can be according to conventional side Method is prepared and obtained or commercially available, and specification is the pure level in laboratory；Not specified experiment equipment is that this area is conventional Experiment equipment, it is commercially available.

It is prepared by the monoclonal antibody of the liver cancer related neoplasms mark of embodiment 1.

(1) prepared by antigen

1. it is prepared by the antigen of Gorky's glycoprotein 73 (GP73)

Recombinant antigen is designed and preparation method is documented in：Pangli monarch, the gene cloning and prokaryotic expression of Golgi apparatus protein, Medical forum's magazine, 2015 (9):1-2.The antigen GP73-F3R3 of high-purity is prepared according to the method described in article, its Culture presevation is expressed in Beijing Qing Yuanshengkang biological medicines Science and Technology Ltd..

2. it is prepared by the antigen of alpha-L-fucosidase (AFU)

2.1 ANTIGEN DESIGNThe

466 amino acid of alpha-L-fucosidase total length, α-L-fucose is analyzed using DNA Star sequence analysis softwares Amino acid sequence (the NCBI sequence numbers of glycosides enzyme：NP_000138.2), secondary structure, hydrophilic and hydrophobic, antigenicity of albumen etc. are obtained Information.Consider protein sequence antigenicity, specificity, Protein expression and purification it is ease, choose 252-441aa Peptide fragment is as antigen A FU-F2R2, and its amino acid sequence is as shown in SEQ ID NO.1, gene coded sequence such as SEQ ID NO. 2 It is shown.

2.2 antigen presentation

2.2.1 the acquisition of purpose fragment

(1) design of primers：EcoR I restriction enzyme sites are introduced at 5 ' ends of forward primer, 5 ' ends of reverse primer introduce Xho I restriction enzyme sites, obtained primer sequence is as follows：

AFU-F2：5’-CGGAATTCGACAGCCCTGTCAAGGATGAG-3’；

AFU-R2：5’-CCGCTCGAGGAAGAGACCTTTATCTGGATC-3’。

(2) acquisition of template DNA

Genomic DNA is extracted from Hela cells：Method and steps referring to《Molecular Cloning:A Laboratory guide》.

Reverse transcription obtains cDNA：Method and steps referring to《Molecular Cloning:A Laboratory guide》.

(3) purpose fragment is expanded according to following PCR system and program：

PCR system：20ul

PCR programs：

2.2.2 vector construction

(1) endonuclease reaction：Using restriction enzyme EcoR I and Xho I, respectively to purpose fragment and expression vector PET32a carries out double digestion.

Digestion system (100ul)：

EcoRI：5ul

Xho I：5ul

10 × buffer solution：10ul

Plasmid (8ng/ul)：55ul

H₂O：25ul

Digestion condition：37 C overnights, obtain linearisation PET-32a carriers and PCR digestion products.

(2) coupled reaction：

Linked system (12ul)：

Linearize PET-32a carriers (EcoR I/Xho I, 8ng/ul)：0.5ul

T4 DNA ligases：1ul

10 × buffer solution：1.2ul

PCR digestion products (1ng/ul)：9.3ul

Condition of contact：Room temperature connects 10h, obtains connection product.

2.2.3 protein expression and purifying

(1) Transformed E .coli DH5 α competent cells

E.coli DH5 α competent cells are placed after thawing on ice, add connection product, 30min is stood on ice.42 DEG C of heat 90s is handled, 2min is stood on ice.Add the μ L of LB culture mediums 600,37 DEG C, 150rpm shaking table cultures 45min.Take 200 μ L bacterium solutions It is coated on amicillin resistance (Amp⁺) on LB flat boards, 37 DEG C of incubated overnights.

(2) positive clone identification

Picking single bacterium colony, is inoculated in LB/Amp⁺Fluid nutrient medium in, 200rpm, 37 DEG C cultivate 8 hours, 8000rpm from Heart 5min collects thalline.Using plasmid extraction kit, to specifications in operating procedure extract plasmid.According to step 2.2.1 PCR system and program in enter performing PCR checking.PCR is identified that correct recombinant plasmid send raw work bioengineering limited Company is sequenced, and the correct recombinant plasmid of comparison result is used as expression vector.

(3) Transformed E .coli BL21 cells

E.coli BL21 competent cells are placed after thawing on ice, add expression vector, 30min is stood on ice.42 DEG C of heat 90s is handled, 2min is stood on ice.Add the μ L of LB culture mediums 600,37 DEG C, 150rpm shaking table cultures 45min.Take 200 μ L bacterium solutions It is coated on Amp⁺On resistance LB flat boards, 37 DEG C of incubated overnights.

(4) protein expression

Picking single bacterium colony, is inoculated in 5ml Amp⁺In resistance LB fluid nutrient mediums, 37 DEG C of overnight incubations take incubated overnight Thing 140ul adds the fresh Amp of 3ml⁺In resistance LB fluid nutrient mediums, 37 DEG C of cultures reach OD₆₀₀For 0.5 or so, addition 1/ 1000IPTG (0.8M), 37 DEG C, 180rmp cultures 4h.The SDS-PAGE for running 10% judges induction situation.

(5) protein purification

Using ammonium sulfate precipitation method purifying protein：Sample is centrifuged, precipitation is removed, retains supernatant and measures volume；One The addition ammonium sulfate of side stirring on one side slowly.Then, solution is placed on magnetic stirring apparatus and stirs 6h, or 4 DEG C stirred At night, protein is set fully to precipitate.By protein solution centrifugation, abandon supernatant and retain precipitation.Add 10-20ml PBS- nitrine Change sodium solution solubilising protein.After albumen precipitation dissolving, the 24-48h that dialyses is put into bag filter, dialyzate is changed every 5h and removes Remove ammonium sulfate.Dialyzate is collected, centrifugation determines the content of protein in supernatant.

3. it is prepared by the antigen of VEGF (VEGF)

3.1 ANTIGEN DESIGNThe

Amino acid sequence (the NCBI sequences of human vascular endothelial growth factor are analyzed using DNA Star sequence analysis softwares Number：NP_001020539.2), according to information such as the secondary structure of albumen, hydrophilic and hydrophobic, antigenicities, and each variant difference Effect, selects the 207-371aa peptide fragment of 165 amino acid as antigen VEGF-FR, its amino acid sequence such as SEQ ID Shown in NO.5, gene coded sequence is as shown in SEQ ID NO.6.

3.2 antigen presentation

3.2.1 the acquisition of purpose fragment

VEGF-F：5’-CGGAATTCGCACCCATGGCAGAAGGAGGAG-3’；

VEGF-R：5’-CCGCTCGAGCCGCCTCGGCTTGTCACATCTG-3’。

(2) cDNA obtained using 2.2.1 expands purpose fragment as template according to following PCR system and program：

PCR system：20ul

PCR programs：

3.2.2 vector construction

The same 2.2.2 of construction method.

3.2.3 protein expression and purifying

Host Strains and the same 2.2.3 of expression and purification method.

4. it is prepared by the antigen of extracellular matrix metalloproteinase (CD147)

4.1 ANTIGEN DESIGNThe

People CD147 amino acid sequence (protein ID are analyzed using DNA Star sequence analysis softwares： BAC76828.1), ease, the selection 25- of the antigenicity, specificity, Protein expression and purification of protein sequence is considered 177aa peptide fragment is as Antigens CD14 7-F3R3, and its amino acid sequence is as shown in SEQ ID NO.9, gene coded sequence such as SEQ Shown in ID NO.10.

4.2 antigen presentation

4.2.1 the acquisition of purpose fragment

CD147-F3:5’-CGGAATTCACAGTCTTCACTACCGTAGAAG-3’；

CD147-R3:5’-CGCTCGAGCTCCATGTTCAGGTTCTCAATG-3’。

PCR system：20ul

PCR programs：

4.2.2 vector construction

The same 2.2.2 of construction method.

4.2.3 protein expression and purifying

Host Strains and the same 2.2.3 of expression and purification method.

(2) Antibody preparation

1. immune mouse

Prepare 8-12 weeks BALB/c mouse two (every kind of two mouse of antigen immune) after birth, pressed with the antigen of high-purity Two mouse are immunized according to following method：

First immunisation, takes the emulsification of 100ug antigen+100ul Freund's complete adjuvants to mix injection mouse vola, about 80ul/ is only；Second It is secondary immune, take the emulsification of 100ul antigen+100ul Freund's incomplete adjuvants to mix injection mouse vola, about 80ul/ is only；Third and fourth, five times It is immune, it is immune with second.

2. cell fusion

All operations are completed in super-clean bench below.

(1) myeloma cell is taken out from incubator, blows open and is poured into after attached cell in centrifuge tube, 5min, 2000 is centrifuged Turn, supernatant is abandoned after centrifugation, RPMI-1640 culture mediums (dual anti-containing the 2/1) mixing for adding 40ml ice-water baths is stand-by.

(2) prepare three culture dishes, the RPMI-1640 culture mediums (dual anti-containing 2/1) of ice-water bath are poured into respectively, take mouse big Inboard leg lymphocyte, is put into first culture dish by two, starts to cut off the adipose tissue and connective torn on lymphocyte Tissue, is moved into second culture dish, is cleaned lymph node cells, then is moved into the 3rd culture dish, is started to tear up lymph node and is allowed Cell is discharged, and blown and beaten back and forth with dropper it is several under, stronger discharges cell, then starts filtration cell (with carrying The suction pipe of cotton is filled into centrifuge tube) centrifuged together to 40ml, and labeled as lymph node cells, and myeloma cell, 10min, respectively abandons supernatant 30ml, starts counting up by 2000 turns/min after centrifugation, by lymph node cells and myeloma cell in proportion (GP73 is 1:3, AFU be 2:3, VEGF be 2:3, CD147 be 1:1) mix, add the RPMI-1640 culture mediums of ice-water bath extremely 50ml, centrifuges 5min, 2000 turns/min, abandons supernatant and add the RPMI-1640 culture mediums of ice-water bath to 50ml, centrifuge 5min, 2000 turns/min, supernatant is abandoned, the RPMI-1640 culture mediums of heat is added to 50ml, centrifuges 5min, 2000 turns/min.

(3) it is initially added into PEG：Take out and mix and the lymph node cells after centrifugation and myeloma cell, abandon supernatant, blot net Remaining RPMI-1640 culture mediums in centrifuge tube, take out about 0.8ml PEG and drop by drop add in centrifuge tube, and along with light Micro- friction, rocks, after addition is finished, and is put into 37 degree of water baths and is incubated one minute, PEG is easier bonding.

(4) cell after water-bath is taken out, starts the slow RPMI-1640 culture mediums for adding heat, one after another drop of addition and companion With rocking, up to 35ml, then 50ml is added to, centrifuge 10min, 1000 turns/min.

(5) 200ml nutrient solution (culture medium additive containing HAT (50X), dual anti-(100X), Glu (100X) are prepared And 20% blood serum of newborn calf without mycoplasma), feeder cells containing 10ml.

(6) supernatant is removed after cell centrifugation, adds 200ml nutrient solutions, mix, bed board 185ul/ holes, 10 blocks of plates, mark 1 is arrived No. 10, it is put into incubator.6th day viewing fusion plate after fusion, and cell hole and monoclonal hole are marked, to be looked into after detecting Look for.

3. the screening and detection of hybridoma

(1) it is coated with fast testing plate：With antigen coat, 2ug/ml, 50ul/ holes, totally two ten blocks of plates.Use 1xPBS dilutions Added to after mixing antigen in detection plate, 37 degree incubations 2 hours are washed five times, add confining liquid after patting dry, 180ul/ holes, and 37 degree incubate Educate 1 hour, pat dry, be put into 4 degree it is stand-by.

(2) ten pieces of fast testing plates are taken out and 1 to No. 10 fusion plates are taken out, one piece of detection plate correspondence, one block of fusion plate is carried out ELISA detect, from fusion plate in take cells and supernatant add detection plate in, 50ul/ holes, leave and take last four hole respectively as Two negative holes and positive hole, 37 degree are incubated 1 hour, take out detection plate, abandon supernatant, board-washing five times is patted dry, and add 1:10000 times The sheep anti mouse secondary antibody (dilution PBST-BSA) of dilution, 50ul/ holes, 37 degree 1 hour, board-washing 5 times is patted dry, add TMB colour developing Ten minutes, terminate liquid is added, reading marks positive cell hole.

Take out ten pieces of new fast testing plates within (2) second days, ELISA detections are carried out again to 1 to No. 10 fusion plates, read Number, marks positive cell hole.The cell hole for detecting all be positive cell hole and readings more than 1 twice is selected, GP73 is 17 plants, AFU is 12 plants, and VEGF is 10 plants, and CD147 is 10 plants, prepares clone.

(3) detection of monoclonal antibody：With 400ml nutrient solutions (culture medium additive containing HAT (50X), dual anti-(100X), Glu (100X), 20% blood serum of newborn calf without mycoplasma), feeder cells containing 20ml.Micro- Microscopic observation is thin per hole Born of the same parents' quantity, the cell quantity of taking-up about 80, average bed board, 190ul/ holes are put into incubator, are cultivated seven days, viewing clone's plate, Monoclonal hole is recorded, and carries out ELISA detections, according to OD₄₅₀>0.8 screening criteria, selects monoclonal antibody strain.

(4) select after monoclonal antibody strain, add new nutrient solution (containing feeder cells), be put into incubator and cultivate. After four days, clone's plate viewing cell quantity and nutrient solution color are opened, cell concentration, which is covered with, accounts for hole 2/3, watch cell hole In cell state.

GP73 singlings are：2F10A4,4D3D5,7B7B3,1C10B5,1G9C8,3D10B5,4G8D7,8F10D1, 10B9F11,10C11B1,4G8E12 and 7E11D11.

AFU singlings are：1E1D1,3B6C3,3F12D8,1A4H4,3F12E4,10E11G10,10F10G10,5F2C3, 7B9H5,4G1H4,5E5A5,5E5G5,5F2D7.

VEGF singlings are：1D2E5,3B1D8,4C3D12,4D3E5,5C7F10,5E1C3,7A11D4,7A11E10, 8E10D8。

CD147 singlings are：1A2D8,3D6C6,4A4C3,5A4E9,5C9G1,6A1D4,6B7F12.

(5) it is coated with the cell in one piece of fast testing plate, 24 holes of detection.

4. prepare ascites

(1) prepare 20 mouse given birth to, inject paraffin oil, about 1ml/ is only.

(2) by incoming six hole of the cell covered with 24 holes, covered with the vial in incoming bottle again after being covered with six holes Afterwards, cell is injected in mouse abdomen.

(3) mouse web portion was watched after 15 days, ascites can be extracted by big tripe situation occur, handles ascites, centrifugation 10min, 2000 turns/min, leaves and takes supernatant, is stored in minus 20 degrees standby.

5. ascites antibody purification

Using sorvall 50ml centrifuge tubes as reaction vessel, using sad method, Purified monoclonal resists from mouse ascites Body, experimental procedure is as follows：

(1) 10ml ascites is centrifuged into 30min in room temperature 20000rpm；

(2) 10ml supernatants are taken to add the sodium-acetate buffers of 20ml 0.06M PH 4.0, then with 1M NaOH regulations PH To PH 4.8；

(3) 750ul octanoic acids are added dropwise, room temperature quickly stirs 30min；Then room temperature centrifugal mixture 20000rpm, 30min；Supernatant carefully is poured out, and siphons away with pipette tips the supernatant that sediment adheres to above；

(4) with 1L 0.1M PH 6.5NaPO4,4mM EDTA dialysis twice, change a not good liquor within 3 hours, obtain antibody-solutions, Determine protein concentration.

Embodiment 2. is used for the antibody of double-antibody method detection to pairing

(1) ELISA double antibodies sandwiches pairing experiment

In accordance with the following steps, the monoclonal antibody for purifying each obtained albumen to embodiment 1 respectively carries out pairing in fact Test：

1st, it is coated with：Coated antibody (one of monoclonal antibody that the purifying of embodiment 1 is obtained) is diluted to 5ug/ml with PBS (PH=7.4). Per hole 100ul, 37 DEG C of coating 1h.

2nd, board-washing：PBST is washed 5 times, is patted dry.

3rd, close：Confining liquid (contains 3%BSA and 4% sucrose PBS), per hole 200ul, 37 DEG C of closing 1h.

4th, dry：Board-washing, does not pat dry raffinate, air-dries 30min.

5th, antigen is added：Antigen (obtained antigen is purified in embodiment 1) is diluted with the PBS (PH=7.4) containing 1%BSA To 2ug/ml, ELISA Plate is added, from the 1st hole (2ug/ml) doubling dilution to the 11st hole (0.2ng/ml), per hole 100ul, the 12nd Kong Jiahan 1%BSA PBS (PH=7.4) is used as negative control, 37 DEG C of incubation 40min.

6th, board-washing：PBST is washed 5 times, is patted dry.

7th, detection antibody is added：Monoclonal antibody (the embodiment 1 of biotin labeling is diluted with the PBS (PH=7.4) containing 1%BSA One of monoclonal antibody that purifying is obtained), ELISA Plate is then added, per hole 100ul, 37 DEG C of incubation 40min.

8th, board-washing：PBST is washed 5 times, is patted dry.

9th, Streptavin-HRP is added：Streptavin-HRP is diluted with the PBS (PH=7.4) containing 1%BSA, per hole 100ul, 37 DEG C of effect 30min.

10th, board-washing：PBST is washed 5 times, is patted dry.

11st, develop the color：Tetramethyl benzidine (TMB) 100ul colour developings are added per hole.

12nd, terminating reaction：1M H are added per hole₂SO₄100ul, ELIASA readings.

As a result it is as shown in the table：

Table 1.GP73 double antibodies sandwiches match experimental result

Table 2.AFU double antibodies sandwiches match experimental result

Table 3.VEGF double antibodies sandwiches match experimental result

Table 4.CD147 double antibodies sandwiches match experimental result

Using alpha-fetoprotein standard items as antigen, detect that this laboratory has according to above-mentioned ELISA double antibodies sandwiches experimental procedure Anti- AFP antibody to (coated antibody 3B11H5 and detection antibody 2G5E8), as a result show, the antigen inspection of AntiAFP antibody pair The survey limit is 1.6ng/ml.

(2) pairing antibody is verified to the Detection results of serum

Experimental procedure is matched according to above-mentioned ELISA double antibodies sandwiches, using human serum as antigen, the preferable antibody pair of checking pairing Detection results, filter out the best antibody of Detection results and send China General Microbiological culture presevation administrative center to be protected Hide, it is as a result as follows：

GP73：Coated antibody 8F10D1, detection antibody 10B9F11；

AFU：Coated antibody 3F12D8 (CGMCC NO：13583), detection antibody 10F10G10 (CGMCC NO： 13584)；

VEGF：Coated antibody 7A11D4 (CGMCC NO：13585), detection antibody 8E10D8 (CGMCC NO： 13586)；

CD147：Coated antibody 4A4C3 (CGMCC NO：13587), detection antibody 6B7F12 (CGMCC NO：13588)；

AFP：Coated antibody 3B11H5 (CGMCC NO：13581), detection antibody 2G5E8 (CGMCC NO：13582).Implement The coupling of the antibody of example 3. and mark

1st, the coupling of magnetic bead and coated antibody

(1) magnetic bead is cleaned：Suspend be not coupled magnetic bead (MC10030-01, MC10034-01, MC10036-01, MC10038-01, MC10044-01), transferase 45 .0 × 10⁶Centrifuge tube is put on magnetic separator by individual magnetic bead into centrifuge tube 30-60s is separated, supernatant is suctioned out, 100ul dH are added₂O is vortexed and suspended again magnetic bead, and 30-60s is separated on magnetic separator, is inhaled Go out supernatant.

(2) activated magnetic beads：Add 80ul 0.1M NaH₂PO₄(Ph6.2), vortex suspension magnetic bead；Add 10ul 50mg/ Ml sulfo-NHS (N- hydroxy thiosuccinimides) and 10ul 50mg/ml EDC (1- (3- dimethylamino-propyls) -3- second Base carbodiimide hydrochloride), it is vortexed and mixes, is incubated at room temperature 20 minutes to activate microballoon.Then 250ul 50mM Ph5.0MES are used (2- (N- morpholines) ethyl sulfonic acid) solution is washed twice, to remove unassembled sulfo-NHS and EDC.

(3) antibody-coupled magnetic beads：Take 100ul 50mM MES (Ph5.0) to add in the magnetic bead after activation and magnetic bead is resuspended, take 10-20ug embodiments 2 screen obtained optimal coated antibody and add in corresponding magnetic bead (a kind of a kind of magnetic bead of albumen correspondence), 500ul is settled to MES, lucifuge is reacted 2 hours at room temperature.Centrifuge tube is put on magnetic separator and separates 30-60s, is suctioned out Supernatant.

(4) magnetic bead of coupled antibody is preserved：Add 500ul PBS-TBN and be used as Block buffer, at room temperature whirling vibration It is incubated 30 minutes.Centrifuge tube is put on magnetic separator and separates 30-60s, supernatant is removed, cleaning is used as with 1ml PBS-TBN Buffer solution is washed twice, suctions out supernatant, adds 4 DEG C of preservations of 250-1000ul PBS-TBN.

(5) confirmation of antibody coupling efficiency：The magnetic bead (microballoon group) after antibody coupling is diluted with assay buffer, makes it Final concentration of 50 magnetic beads/ul, add 96 orifice plates, 50ul/ holes.The sheep anti-mouse igg of biotin labeling is diluted to 4ug/ml, 2ug/ Ml, 1ug/ml, 0.5ug/ml, 0.25ug/ml, 0.125ug/ml, 0.0625ug/ml, 50ul is added per hole, and blank control adds Enter 50ul assay buffer, room temperature concussion is incubated 30min, and wash buffer are washed 3 times, add SAPE 50ul/ holes, room temperature Oscillation incubation 30 minutes, wash buffer are washed 2 times, and the machine (luminex200) on 80ul assay buffer that is eventually adding is examined Survey, it is as a result as shown in the table.

The tumor markers antibody coupling of table 6 confirms result

2nd, the biotin labeling of antibody is detected

2mg biotins (sigma) are dissolved in 200ul solvents DMSO, obtained optimum detection will be screened in embodiment 2 Antibody is dissolved to 1mg/ml 450ul with PH=9.6 carbonic acid buffer.The biology dissolved is added into detection antibody-solutions Add 500ul pure glycerins after plain 50ul (organic solvent accounts for system 10%, and biotin is excessive), room temperature reaction 4h, be stored in -20 DEG C of ice It is standby in case.

The standard curve making of embodiment 4.

The antigen that embodiment 1 is prepared carries out 4 times of gradient dilutions with assay buffer, obtains concentration and is respectively 1000ng/ml, 250ng/ml, 64ng/ml, 16ng/ml, 4ng/ml, 1ng/ml, 0.25ng/ml antigenic solution, while with Assay buffer are used as blank control.

After the magnetic bead being coupled in embodiment 3 is diluted well with assay buffer, added in the orifice plate of lucifuge 96 per hole 50ul, 96 orifice plates are put on magnetic sheet, and 120ul wash buffer are added per hole, are stood 2 minutes, are outwelled wash buffer, plus Enter the antigen of various concentrations gradient, 4 DEG C of reaction overnights (15-18h), wash buffer are washed three times；Add the biotin diluted The detection antibody of mark, 50ul/ holes, 800 revs/min of room temperature is incubated 30 minutes；Wash buffer are washed three times；Add SAPE, 50ul/ holes, 800 revs/min of room temperature is incubated 30 minutes, and wash buffer are washed twice, add machine examination on 80ul assay buffer Survey, as a result as shown in Fig. 2-6, AFP, GP73, AFU, VEGF susceptibilitys can reach 250pg/ml, and CD147 susceptibilitys can reach 64pg/ml。

CD147/VEGF double factors, the factors of CD147/VEGF/AFP tri-, CD147/VEGF/ AFP/AFU are additionally carried out Four factors, and the factor standard product examines of CD147/VEGF/AFP/AFU/GP73 five are surveyed.Specific detection method ibid, difference It is：(1) magnetic bead for being coupled different tumor markers coated antibodies is mixed into a system；(2) antigen is different tumours The mixed liquor of mark antigen；(3) detection antibody is the different tumor-marker analyte detection antibody of the biotin labeling diluted Mixed liquor.

As a result as shown in table 7-10：

Table 7.CD147, VEGF double factor standard items testing result

The factor standard product testing result of table 8.CD147, VEGF, AFP tri-

The factor standard product testing result of table 9.CD147, VEGF, AFP, AFU tetra-

The factor standard product testing result of table 10.CD147, VEGF, AFP, AFU, GP73 five

Multiple-factor testing result shows that coated antibody provided by the present invention and detection antibody have advantages below：

(1) high specificity：Multipair antibody in same reaction system simultaneously detect multiple protein when reactivity worth and list Without significant change between each factor antibody and other factors cross reaction does not occur for reactivity worth when solely detecting every kind of albumen.

(2) accuracy is good：In same reaction system, do not occur cross reaction between each factor antibody.

(3) sensitivity is high：In same reaction system, AFP is detected, GP73, AFU, VEGF sensitivity can reach 250pg/ml, detection CD147 sensitivity can reach 64pg/ml.

SEQUENCE LISTING

<110>Horse, it is outstanding

<120>Antibody and kit for detecting alpha-L-fucosidase in serum

<130>P160545/YAY

<160> 12

<170> PatentIn version 3.5

<210> 1

<211> 190

<212> PRT

<213> Artificial Sequence

<220>

<223>The amino acid sequence of alpha-L-fucosidase antigen

<400> 1

Asp Ser Pro Val Lys Asp Glu Val Val Val Asn Asp Arg Trp Gly Gln

1 5 10 15

Asn Cys Ser Cys His His Gly Gly Tyr Tyr Asn Cys Glu Asp Lys Phe

20 25 30

Lys Pro Gln Ser Leu Pro Asp His Lys Trp Glu Met Cys Thr Ser Ile

35 40 45

Asp Lys Phe Ser Trp Gly Tyr Arg Arg Asp Met Ala Leu Ser Asp Val

50 55 60

Thr Glu Glu Ser Glu Ile Ile Ser Glu Leu Val Gln Thr Val Ser Leu

65 70 75 80

Gly Gly Asn Tyr Leu Leu Asn Ile Gly Pro Thr Lys Asp Gly Leu Ile

85 90 95

Val Pro Ile Phe Gln Glu Arg Leu Leu Ala Val Gly Lys Trp Leu Ser

100 105 110

Ile Asn Gly Glu Ala Ile Tyr Ala Ser Lys Pro Trp Arg Val Gln Trp

115 120 125

Glu Lys Asn Thr Thr Ser Val Trp Tyr Thr Ser Lys Gly Ser Ala Val

130 135 140

Tyr Ala Ile Phe Leu His Trp Pro Glu Asn Gly Val Leu Asn Leu Glu

145 150 155 160

Ser Pro Ile Thr Thr Ser Thr Thr Lys Ile Thr Met Leu Gly Ile Gln

165 170 175

Gly Asp Leu Lys Trp Ser Thr Asp Pro Asp Lys Gly Leu Phe

180 185 190

<210> 2

<211> 570

<212> DNA

<213> Artificial Sequence

<220>

<223>The gene coded sequence of alpha-L-fucosidase antigen

<400> 2

gacagccctg tcaaggatga ggtggtagta aatgaccgat ggggtcagaa ctgttcctgt 60

caccatggag gatactataa ctgtgaagat aaattcaagc cacagagctt gccagatcac 120

aagtgggaga tgtgcaccag cattgacaag ttttcctggg gctatcgtcg tgacatggca 180

ttgtctgatg ttacagaaga atctgaaatc atttcggaac tggttcagac agtaagtttg 240

ggaggcaact atcttctgaa cattggacca actaaagatg gactgattgt tcccatcttc 300

caagaaaggc ttcttgctgt tgggaaatgg ctgagcatca atggggaggc tatctatgcc 360

tccaaaccat ggcgggtgca atgggaaaag aacacaacat ctgtatggta tacctcaaag 420

ggatcggctg tttatgccat ttttctgcac tggccagaaa atggagtctt aaaccttgaa 480

tcccccataa ctacctcaac tacaaagata acaatgctgg gaattcaagg agatctgaag 540

tggtccacag atccagataa aggtctcttc 570

<210> 3

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223>The PCR forward primers AFU-F2 of alpha-L-fucosidase antigen encoding sequences

<400> 3

cggaattcga cagccctgtc aaggatgag 29

<210> 4

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223>The PCR reverse primers AFU-R2 of alpha-L-fucosidase antigen encoding sequences

<400> 4

ccgctcgagg aagagacctt tatctggatc 30

<210> 5

<211> 165

<212> PRT

<213> Artificial Sequence

<220>

<223>The amino acid sequence of VEGF antigen

<400> 5

Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys

1 5 10 15

Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu

20 25 30

Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys

35 40 45

Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu

50 55 60

Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile

65 70 75 80

Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe

85 90 95

Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg

100 105 110

Gln Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe

115 120 125

Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser

130 135 140

Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys

145 150 155 160

Asp Lys Pro Arg Arg

165

<210> 6

<211> 495

<212> DNA

<213> Artificial Sequence

<220>

<223>The gene coded sequence of VEGF antigen

<400> 6

gcacccatgg cagaaggagg agggcagaat catcacgaag tggtgaagtt catggatgtc 60

tatcagcgca gctactgcca tccaatcgag accctggtgg acatcttcca ggagtaccct 120

gatgagatcg agtacatctt caagccatcc tgtgtgcccc tgatgcgatg cgggggctgc 180

tgcaatgacg agggcctgga gtgtgtgccc actgaggagt ccaacatcac catgcagatt 240

atgcggatca aacctcacca aggccagcac ataggagaga tgagcttcct acagcacaac 300

aaatgtgaat gcagaccaaa gaaagataga gcaagacaag aaaatccctg tgggccttgc 360

tcagagcgga gaaagcattt gtttgtacaa gatccgcaga cgtgtaaatg ttcctgcaaa 420

aacacagact cgcgttgcaa ggcgaggcag cttgagttaa acgaacgtac ttgcagatgt 480

gacaagccga ggcgg 495

<210> 7

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223>The PCR forward primers VEGF-F of VEGF antigen encoding sequences

<400> 7

cggaattcgc acccatggca gaaggaggag 30

<210> 8

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<223>The PCR reverse primers VEGF-R of VEGF antigen encoding sequences

<400> 8

ccgctcgagc cgcctcggct tgtcacatct g 31

<210> 9

<211> 153

<212> PRT

<213> Artificial Sequence

<220>

<223>The amino acid sequence of CD147 antigens

<400> 9

Thr Val Phe Thr Thr Val Glu Asp Leu Gly Ser Lys Ile Leu Leu Thr

1 5 10 15

Cys Ser Leu Asn Asp Ser Ala Thr Glu Val Thr Gly His Arg Trp Leu

20 25 30

Lys Gly Gly Val Val Leu Lys Glu Asp Ala Leu Pro Gly Gln Lys Thr

35 40 45

Glu Phe Lys Val Asp Ser Asp Asp Gln Trp Gly Glu Tyr Ser Cys Val

50 55 60

Phe Leu Pro Glu Pro Met Gly Thr Ala Asn Ile Gln Leu His Gly Pro

65 70 75 80

Pro Arg Val Lys Ala Val Lys Ser Ser Glu His Ile Asn Glu Gly Glu

85 90 95

Thr Ala Met Leu Val Cys Lys Ser Glu Ser Val Pro Pro Val Thr Asp

100 105 110

Trp Ala Trp Tyr Lys Ile Thr Asp Ser Glu Asp Lys Ala Leu Met Asn

115 120 125

Gly Ser Glu Ser Arg Phe Phe Val Ser Ser Ser Gln Gly Arg Ser Glu

130 135 140

Leu His Ile Glu Asn Leu Asn Met Glu

145 150

<210> 10

<211> 459

<212> DNA

<213> Artificial Sequence

<220>

<223>The gene coded sequence of antigen

<400> 10

acagtcttca ctaccgtaga agaccttggc tccaagatac tcctcacctg ctccttgaat 60

gacagcgcca cagaggtcac agggcaccgc tggctgaagg ggggcgtggt gctgaaggag 120

gacgcgctgc ccggccagaa aacggagttc aaggtggact ccgacgacca gtggggagag 180

tactcctgcg tcttcctccc cgagcccatg ggcacggcca acatccagct ccacgggcct 240

cccagagtga aggctgtgaa gtcgtcagaa cacatcaacg agggggagac ggccatgctg 300

gtctgcaagt cagagtccgt gccacctgtc actgactggg cctggtacaa gatcactgac 360

tctgaggaca aggccctcat gaacggctcc gagagcaggt tcttcgtgag ttcctcgcag 420

ggccggtcag agctacacat tgagaacctg aacatggag 459

<210> 11

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223>The PCR forward primers CD147-F3 of CD147 antigen encoding sequences

<400> 11

cggaattcac agtcttcact accgtagaag 30

<210> 12

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223>The PCR reverse primers CD147-R3 of CD147 antigen encoding sequences

<400> 12

cgctcgagct ccatgttcag gttctcaatg 30

Claims

1. the antigen protein of the antibody for preparing anti-alpha-L-fucosidase, it is characterised in that its amino acid sequence such as SEQ Shown in ID NO.1.

2. encode the gene of the antigen protein described in claim 1, it is characterised in that its nucleotide sequence such as SEQ ID NO.2 It is shown.

3. the monoclonal antibody for detecting liver cancer marker alpha-L-fucosidase in test serum, it is characterised in that by protecting It is CGMCC NO to hide numbering：13584 hybridoma secretion.

4. secrete the hybridoma of the monoclonal antibody described in claim 3, it is characterised in that its deposit number is CGMCC NO：13584.

5. the antibody pair for detecting liver cancer marker alpha-L-fucosidase in test serum, it is characterised in that resisted by first Body and secondary antibody composition；The first antibody, as coated antibody, is CGMCC NO by deposit number：13583 hybridoma Cell is secreted；The secondary antibody, as detection antibody, is CGMCC NO by deposit number：13584 hybridoma secretion.

6. the kit for hepatocarcinoma early diagnosis, it is characterised in that including the monoclonal antibody or right described in claim 3 It is required that the antibody pair described in 5, and the common reagent for immune detection.

7. kit according to claim 6, it is characterised in that including the antibody pair described in claim 3, described first Antibody is coupled with magnetic bead, the secondary antibody biotin labeling, and the common reagent for immune detection is that strepto- is affine Element-phycoerythrin.

8. kit according to claim 6, it is characterised in that standard items also including alpha-L-fucosidase and/or The standard curve or linear regression equation specification made with the standard items of the alpha-L-fucosidase.

9. according to any described kits of claim 6-8, it is characterised in that the also antibody including anti-alpha-fetoprotein, anti-height Antibody, the antibody of anti-vascular endothelial growth factor and/or the anti-cell epimatrix metalloprotein enzyme induction of your base glycoprotein 73 because Sub- CD147 antibody, and its corresponding immunologic function test reagent.

10. kit according to claim 9, it is characterised in that also including alpha-fetoprotein, Gorky's glycoprotein 73, blood The standard items of endothelial tube growth factor and/or extracellular matrix metalloproteinase CD147, and/or with alpha-fetoprotein, The standard items of Gorky's glycoprotein 73, VEGF and/or extracellular matrix metalloproteinase CD147 The standard curve or linear regression equation specification of making.