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CN107857815A - Monoclonal antibody reacted with glycopeptide and application thereof - Google Patents

Monoclonal antibody reacted with glycopeptide and application thereof Download PDF

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Publication number
CN107857815A
CN107857815A CN201710202207.0A CN201710202207A CN107857815A CN 107857815 A CN107857815 A CN 107857815A CN 201710202207 A CN201710202207 A CN 201710202207A CN 107857815 A CN107857815 A CN 107857815A
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Prior art keywords
antibody
glycopeptide
afp
amino acid
fucosylation
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Inventor
井口由起子
野村雄
深川耕次
江头由里子
比嘉友纪子
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Sysmex Corp
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Sysmex Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/471—Pregnancy proteins, e.g. placenta proteins, alpha-feto-protein, pregnancy specific beta glycoprotein

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  • Life Sciences & Earth Sciences (AREA)
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Abstract

The present invention relates to monoclonal antibody reacted with glycopeptide and application thereof.The present invention provides a kind of monoclonal antibody with both the amino acid of fucose moiety and peptide moiety for epitope.The antibody of the present invention is the monoclonal antibody with glycopeptide reaction, the glycopeptide contains core fucose and from the asparagine of additional sugar chain to amino acid more than continuous 4 residues of C ends side, the antibody with the core fucose of the glycopeptide and glycopeptide from the asparagine of additional sugar chain to both amino acid more than 3 residues of C ends lateral extent for epitope.

Description

Monoclonal antibody reacted with glycopeptide and application thereof
Technical field
The present invention relates to monoclonal antibody reacted with glycopeptide and application thereof.
Background technology
It is said that when being developed from hepatitis, hepatic sclerosis to liver cancer, in Biosample, combined with LcA (LCA) Alpha-fetoprotein (AFP) (LCA associativity AFP) increase.Patent document 1 describes a kind of for detecting LCA associativities AFP's Antibody.According to records, the antibody of patent document 1 shows reactivity to associativity AFP, and AFP non binding to LCA does not show reaction Property.
Prior art literature
Patent document
Patent document 1：Japanese Unexamined Patent Application 63-307900 publications
The content of the invention
The invention problem to be solved
Described in patent document 1, fucose (Fucose) in the sugar chain for the AFP that LCA is combined be present.In Biosample The AFP fractions combined with LcA be referred to as AFP-L3 fractions.AFP-L3 fractions are by fucosylation AFP (AFP Asparagine residue on be attached with core fucose (N-acetyl-glucosamine (GlcNAc) α with the reduction end of N-type sugar chain 1-6 combine fucose) AFP) form.
Obtained in the embodiment of patent document 1 with LCA associativities AFP (AFP-LCA-R) combine but not with the non-knots of LCA The antibody that conjunction property AFP (AFP-LCA-NR) is combined (with reference to embodiment 1, table 1, Fig. 1).But the epitope of the antibody is failed to understand.It is real Apply in example and point out, LCA associativities and non binding as described above due to the presence or absence of fucose, the associativity possibility with antigen Independent of the sequence of peptide moiety, and it is to rely on fucose moiety.In this case, it is possible to not only combined with AFP, and And also combined with the protein non-specific with fucose beyond it.Therefore, it is desirable to develop the fucose portion with glycopeptide Divide the monoclonal antibody that both amino acid with peptide moiety is epitope.
For solving the scheme of problem
The present invention provides a kind of antibody, and it is the monoclonal antibody with glycopeptide reaction, and above-mentioned glycopeptide contains core fucose With, to amino acid more than continuous 4 residues in C ends side, the antibody is with above-mentioned glycopeptide from the asparagine of additional sugar chain Core fucose and glycopeptide from the asparagine of additional sugar chain to both amino acid more than 3 residues of C ends lateral extent For epitope.
The present invention provides a kind of method for producing above-mentioned antibody, and it contains following process：Will contain core fucose and from The asparagine of additional sugar chain is risen to the glycopeptide antigen of amino acid more than continuous 4 residues in C ends side exempts to animal Epidemic disease.
The present invention provides a kind of method for producing the hybridoma for producing above-mentioned antibody, and it contains following process：Core will be contained Heart fucose and from the asparagine of additional sugar chain to the glycopeptide antigen pair of amino acid more than continuous 4 residues in C ends side Animal is immunized.
The present invention provides a kind of monoclonal antibody, and it reacts with following glycopeptide,
[chemical formula 1]
Do not reacted with following glycopeptide,
[chemical formula 2]
Do not reacted with following glycopeptide,
[chemical formula 3]
The present invention provides a kind of method for producing above-mentioned antibody, contains following process：Glycopeptide containing following structure is resisted Animal is immunized original,
[chemical formula 4]
(X is arbitrary sugar chain).
The present invention provides a kind of method for producing the hybridoma for producing above-mentioned antibody, contains following process：It will contain following Animal is immunized the glycopeptide antigen of structure,
[chemical formula 5]
(X is arbitrary sugar chain).
The present invention provides a kind of fucosylation AFP assay method, and it uses above-mentioned antibody.
The present invention provides a kind of fucosylation AFP detection kits, and it, which contains fucosylation AFP seizure, uses Antibody, fucosylation AFP detection antibody, solid phase, above-mentioned seizure antibody or above-mentioned detection antibody are described anti- Body.
The present invention provides a kind of hybridoma, and its international deposit number is NITE BP-02263 or NITE BP-02264.
Invention effect
The present invention provides a kind of monoclonal with both amino acid of the fucose moiety of glycopeptide and peptide moiety for epitope and resisted Body.
Brief description of the drawings
Fig. 1 is the figure of clone 1F8-A4 and 2F11-2A9 protein imprinted result for showing to obtain in embodiment 1 (swimming lane 1：Fucosylation AFP (AFP-L3/ recombinants) (positive antigen), swimming lane 2：Non- fucosylation AFP (comes from people's blood Clear AFP (LEE biosolutions) the non-adsorbed fraction of LCA agglutinins) (Negative antigens), swimming lane 3：Fucosylation ALP (Oriental yeast/47787055)) (Negative antigens).
The survey of the OD450 under various antigen concentrations when Fig. 2 is the clone I2-1F8 for being shown with obtaining in embodiment 1 The figure of definite value.
OD450 measure under various antigen concentrations when Fig. 3 is the clone I2-2F11 for being shown with obtaining in embodiment 1 The figure of value.
Fig. 4 is the figure (swimming lane 1 of clone I2-1F8 protein imprinted result for showing to obtain in embodiment 1：Restructuring AFP-L3, swimming lane 2：Non- fucosylation AFP, swimming lane 3：Natural human AFP (ミ ュ ー タ ス ワ コ ー AFP-L3 standard items 1), swimming lane 4：Natural human AFP-L3 (ミ ュ ー タ ス ワ コ ー AFP-L3 standard items 2)).
Embodiment
As long as the antibody of present embodiment is in the measure system using the Biosample of antibody using present embodiment etc. It is middle to play specificity.For example, even if with the material that does not include in Biosample there occurs non-specific binding, if this The antibody of embodiment plays specificity in usually used environment, then also plays the action effect of the present invention.It is specific and Speech, in the material during the blood samples such as whole blood, serum, blood plasma are detected by ELISA, as long as showing in ELISA measure systems Specificity is shown, combined with the material being typically free of in blood sample, ELISA reagents also possible.
The antibody of present embodiment be with glycopeptide reaction monoclonal antibody, above-mentioned glycopeptide contain core fucose and from The asparagine of additional sugar chain is risen to amino acid more than continuous 4 residues in C ends side, and the antibody is with the core of above-mentioned glycopeptide Heart fucose and glycopeptide to both amino acid more than 3 residues of C ends lateral extent are table from the asparagine of additional sugar chain Position.
As long as with the asparagine of the glycopeptide of the antibody response of present embodiment containing core fucose and from additional sugar chain Rise to amino acid more than continuous 4 residues in C ends side.
The antibody of present embodiment is produced by the method containing following process, and the process is：Core rock algae will be contained Sugar and animal is entered to the glycopeptide antigen of amino acid more than continuous 4 residues in C ends side from the asparagine of additional sugar chain Row is immune.
The N-terminal of glycopeptide antigen can combine the boiomacromolecules such as KLH, BSA via PEG etc..In addition, these glycopeptides resist Former C-terminal can be with amidatioon etc..Known method can be utilized in the method for N-terminal combination boiomacromolecule.In addition, C is last The amidatioon at end can also utilize known method.
Glycopeptide antigen is carried out to immune process to animal can be using pair in known monoclonal antibody production method Animal carries out immune process.As monoclonal antibody production method, can enumerate for example：Mouse spleen method, mouse ilium lymph Connection (with reference to No. 4098796 publications of Japanese Patent No.) etc..
Animal be immunized is not particularly limited, suitably selected from non-human animal with reference to monoclonal antibody production method Select.
Specifically, when using mouse iliac lymph nodes method as monoclonal antibody production method, carry glycopeptide and KLH etc. Body protein is coupled, and is blended with the emulsion of Freund's adjuvant etc. with glycopeptide solution (conversion of (2.5mg/mL) carrier protein) 0.06mL/ Amount only is at least immune 1 time to the root of the tail portion of mouse.
After animal is immunized, making, screening of hybridoma etc. can be carried out according to known methods and obtain this The antibody of embodiment.
Screen hybridoma when, can suitably by the use of the glycopeptide as antigen, core is eliminated from the glycopeptide as antigen The material of fucose, from glycopeptide of the glycopeptide as antigen with identical sugar chain structure but with different peptide chains as sun Property antigen or Negative antigens.The glycopeptide that the above-mentioned amino acid residue for making the glycopeptide as antigen tails off is preferably for example by glycopeptide C ends side be set to the glycopeptides of continuous 3 residues from the asparagine of additional sugar chain.
For the screening benchmark of hybridoma, for example, during using ELISA, the OD450 values of positive antigen and Negative antigens Difference be more than 0.05, and the OD450 values of Negative antigens be less than 0.05.
The isotype of the antibody of present embodiment is not particularly limited.In addition, the antibody of present embodiment also includes containing There is F (ab ')2, Fab ', Fab, CDR fragment such as peptide.
The antibody of present embodiment can also carry out the markings such as biotinylation, ALPization.
The antibody of present embodiment described above has above-mentioned property, thus, for example with containing core in partial sequence Fucose and from the asparagine of additional sugar chain to amino acid more than continuous 4 residues in C ends side glycoprotein specificity Reaction.
The antibody of present embodiment can be used for ELISA, protein imprinted, immuning tissue's dyeing, immunosedimentation etc..
The monoclonal antibody of another embodiment and following glycopeptide (sequence number 13) specific reaction.
[chemical formula 1]
The monoclonal antibody is not reacted with following glycopeptide (sequence number 14),
[chemical formula 2]
Do not reacted with following glycopeptide (sequence number 15).
[chemical formula 3]
That is, the antibody of present embodiment and the antigen without core fucose and with amino acid " EIQ " is (above-mentioned (2)) do not react, do not reacted with the antigen (above-mentioned (3)) with core fucose and without amino acid sequence " EIQ ".Therefore Think, the epitope of the antibody of present embodiment contains both core fucose and amino acid sequence " EIQ ".
It should be noted that in this specification, the antibody " being reacted with glycopeptide " of present embodiment refers to that glycopeptide and antibody lead to Cross antigen-antibody reaction and combine.
Further, above-mentioned antibody does not preferably react with following glycopeptide (sequence number 16).
[chemical formula 4]
Further, above-mentioned antibody preferably reacts with fucosylation AFP.
Further, above-mentioned antibody can be with the denaturation fucose that is pre-processed with the denaturants such as SDS, heating etc. Base AFP is combined.When using the solution containing SDS as pretreatment, to what is be fully denatured for making fucosylation AFP SDS concentration (following, to be referred to as " SDS concentration during pretreatment ") is not particularly limited, preferably 0.03 mass/mass % (with More than down referred to as " % ").On the other hand, if including excessive denaturant during antigen-antibody reaction, antibody is also denatured, It is possible to produce antigen-antibody reaction undesirable influence, therefore the concentration of denaturant is preferably reduced by dilution etc..Make By the use of when containing SDS solution as denaturant, (hereinafter referred to as " final SDS is dense for SDS concentration during to antigen-antibody reaction Degree ") it is not particularly limited, preferably less than 0.025%.
It should be noted that in this specification, the antibody " being reacted with fucosylation AFP " of present embodiment refers to rock Algae glycosylation AFP is combined with antibody by antigen-antibody reaction.Fucosylation AFP can be in recombinant, natural materials It is any.Natural fucosylation AFP is, for example, the AFP being present in people's blood.People AFP sequence is for example with GenBank Accession No.NM_001134 are registered, and have the amino acid sequence of sequence number 25.
Further, above-mentioned antibody preferably reacts with the fucosylation AFP being denatured in the presence of SDS and DTT.Denaturation Condition be in the presence of 2% SDS, 50mM DTT normal temperature (25 DEG C) under reaction.
Further, above-mentioned antibody is not preferably anti-with the non-fucosylation AFP that is denatured in the presence of SDS and DTT Should.The condition of denaturation is the reaction under SDS, 50mM for having 2% DTT normal temperature (25 DEG C).
As the CDR of the antibody of present embodiment, for example following CDR can be enumerated.
＜ CDR-A ＞
The CDR of heavy chain contains the amino acid sequence shown in sequence number 1, the amino acid sequence shown in sequence number 2, sequence Amino acid sequence shown in column number 3.
The CDR of light chain contains the amino acid sequence shown in sequence number 4, the amino acid sequence shown in sequence number 5, sequence Amino acid sequence shown in column number 6.
＜ CDR-B ＞
The CDR of heavy chain contains the amino acid sequence shown in sequence number 7, the amino acid sequence shown in sequence number 8, sequence Amino acid sequence shown in column number 9.
The CDR of light chain contain the amino acid sequence shown in sequence number 10, the amino acid sequence shown in sequence number 11, Amino acid sequence shown in sequence number 12.
As CDR, the hybridoma for producing the antibody of the present embodiment with above-mentioned CDR-A, CDR-B is respectively designated as I2-1F8, I2-2F11, and (be also referred to as in independent administrative legal person's products assessment technique basal disc organization on May 20th, 2016 NPMD- National Technicals assess association) patent Organism Depositary (total sickle in the Mu Geng Jinshi City of 〒 292-0818 Chiba, Japan Foot 5 kinds of 2 fourth mesh ground 8 122nd room) as NITE BP-02264, NITE BP-02263 carry out international accession.
The antibody of present embodiment is obtained by the method containing following process, and the process is：Following knot will be contained Animal is immunized the glycopeptide antigen (sequence number 17) of structure.
[chemical formula 5]
X is arbitrary sugar chain, to be usually incorporated in the sugar chain of glycoprotein, glycopeptide, is not particularly limited.
As glycopeptide antigen (sequence number 18), more preferably following glycopeptide.
[chemical formula 6]
X is arbitrary sugar chain, to be usually incorporated in the sugar chain of glycoprotein, glycopeptide, is not particularly limited.
The N-terminal of glycopeptide antigen can combine the boiomacromolecules such as KLH via PEG etc..In addition, the C of these glycopeptide antigens End can be with amidatioon etc..Known method can be utilized in the method for N-terminal combination boiomacromolecule.In addition, C-terminal Amidatioon can also utilize known method.
Glycopeptide antigen is immunized to the process of animal can utilize entering to animal in known monoclonal antibody production method The immune process of row.As monoclonal antibody production method, can enumerate for example：Mouse spleen method, mouse iliac lymph nodes method (with reference to No. 4098796 publications of Japanese Patent No.) etc..
Animal be immunized is not particularly limited, suitably selected from non-human animal with reference to monoclonal antibody production method Select.
When specifically, using mouse iliac lymph nodes method as monoclonal antibody production method, carry glycopeptide and KLH etc. Body protein is coupled, and is blended with the emulsion of Freund's adjuvant etc. with glycopeptide solution (conversion of (2.5mg/mL) carrier protein) 0.06mL/ Amount only is at least immune 1 time to the root of the tail portion of mouse.
After animal is immunized, making, screening of hybridoma etc. can be carried out according to known methods and obtain this The antibody of embodiment.
When hybridoma screens, can suitably by the use of the glycopeptide as antigen, core is eliminated from the glycopeptide as antigen The material of heart fucose, make glycopeptide that the amino acid residue of the glycopeptide as antigen tails off, be denatured in the presence of SDS and DTT Fucosylation AFP, be denatured in the presence of SDS and DTT non-fucosylation AFP, with core fucose and with Glycopeptide or glycoprotein (for example, fucosylation ALP etc.) of the polypeptide of the amino acid sequence different from AFP etc. are as positive Antigen or Negative antigens.The glycopeptide that the above-mentioned amino acid residue for making the glycopeptide as antigen tails off is preferably for example by the C of glycopeptide Last side is set to the glycopeptide of continuous 3 residues from the asparagine of additional sugar chain.
For the screening benchmark of hybridoma, for example, during using ELISA, the OD450 values of positive antigen and Negative antigens Difference be more than 0.05, and the OD450 values of Negative antigens be less than 0.05.
The isotype of the antibody of present embodiment is not particularly limited.In addition, the antibody of present embodiment also includes containing There is F (ab ')2, Fab ', Fab, CDR fragment such as peptide.
The antibody of present embodiment can also carry out the markings such as biotinylation, ALPization.
The antibody of present embodiment has above-mentioned property, therefore is reacted with fucosylation AFP, with non-fucosylation AFP does not react.Therefore, the antibody of present embodiment can for example determine the fucosylation AFP in Biosample.
As Biosample, can enumerate for example：Whole blood, serum, blood plasma by subject's collection etc..The Biosample It can be centrifuged, the pretreatment such as denaturation treatment.Biosample preferably carries out denaturation treatment.The condition of denaturation treatment is The reaction of normal temperature (25 DEG C) in the presence of 2% SDS, 50mM DTT.
During using the antibody of present embodiment to determine fucosylation AFP, known immunologic assay side can be utilized Method.As Radioimmunoassay of vascular endothelial growth, can enumerate for example：Enzyme linked immunosorbent assay (ELISA method), immune complex transfer assay Method (with reference to Japanese Unexamined Patent Publication 1-254868 publications), immunoturbidimetry, immunochromatography, Latex Agglutination etc..As measure One of process, the situation that explanation passes through the fucosylation AFP concentration in Sandwich ELISA measure Biosample below.
First, formed in solid phase containing the antibody for catching the fucosylation AFP in Biosample (below Referred to as " seizure antibody "), the antibody (hereinafter also referred to " detection antibody ") for detecting fucosylation AFP and rock algae Glycosylate AFP complex., can be by the way that Biosample, seizure be used when containing fucosylation AFP in Biosample Antibody and detection are mixed with antibody, to form the complex.Then, make the solution containing complex and seizure use can be caught The solid phase contact of antibody, so as to form above-mentioned complex in solid phase.Or it can also use and be fixed with seizure in advance With the solid phase of antibody.That is, contacted by making to be fixed with seizure with the solid phase of antibody, Biosample and detection with antibody, so as to To form above-mentioned complex in solid phase.The antibody of present embodiment can be as in seizure antibody and detection antibody At least one is used.
Seizure is not particularly limited with fixed form of the antibody in solid phase.For example, can both make seizure antibody and Solid phase is directly in conjunction with can also make seizure be combined indirectly via other materials with antibody and solid phase.As directly in conjunction with can be with Enumerate such as physical absorption.Combine, can be enumerated for example as indirect：Via biotin and avidin or Streptavidin The combination of the combination of (hereinafter also referred to " Avidin class ").In this case, by being caught in advance with biotin modification with anti- Body, Avidin class is incorporated into solid phase in advance, so as to via biotin and Avidin class with reference to and make seizure with anti- Body and solid phase combine indirectly.
The material of solid phase is not particularly limited, can be from such as organic high molecular compound, inorganic compound, biology Selected in macromolecule etc..As organic high molecular compound, can enumerate：Latex, polystyrene, polypropylene etc..As inorganic Compound, it can enumerate：Magnetic (iron oxide, chromium oxide and ferrite etc.), silica, aluminum oxide, glass etc..As Boiomacromolecule, it can enumerate：Insoluble agarose, insoluble glucan, gelatin, cellulose etc..Can be by 2 in these Kind combination of the above uses.The shape of solid phase is not particularly limited, can be enumerated for example：Particle, film, microwell plate, micro-pipe, examination Pipe etc..
By using method known to the technical field, to detect the complex being formed in solid phase, it is hereby achieved that raw The measured value of fucosylation AFP in thing sample.For example, it is used as detection antibody using the antibody marked by the use of mark substance When, by detecting signal caused by the mark substance, fucosylation AFP measured value can be obtained.Or using For detection antibody mark secondary antibody when, can also equally obtain fucosylation AFP measured value.
In present embodiment, preferred pair fucosylation AFP is pre-processed like that according to above-mentioned.Make as pretreatment During with solution containing SDS, SDS concentration is not particularly limited during to pretreatment, and preferably more than 0.03%.In antigen-antibody During reaction, as described above, it is preferred to reduce the concentration of denaturant by dilution etc..The solution for containing SDS is being used as denaturation During agent, final SDS concentration is not particularly limited, preferably less than 0.025%.In present embodiment, preferably carry out this kind of The measured value for handling and making above-mentioned antibody that fucosylation AFP is reacted, obtained with above-mentioned fucosylation AFP.
In present embodiment, between complex formation process and complex detection process, it can be removed and not formed B/F (Bound/Free) separation of the unreacted free composition of complex.Unreacted free composition refers to not form compound The composition of body.It can enumerate for example：Fucosylation AFP in the antibody that is not combined with fucosylation AFP, Biosample Material (impurity) in addition etc..To B/F separation means be not particularly limited, if solid phase is particle, can by from Heart separation only recovery catches the solid phase for having complex, so as to carry out B/F separation.If solid phase is the containers such as microwell plate, micro-pipe, B/F separation then can be carried out by removing the liquid containing unreacted free composition.In addition, it is magnetic particle in solid phase In the case of, it can will contain unreacted dissociate into nozzle in the state of magnetic particle is fettered by magnetic with magnet The liquid suction divided removes and carries out B/F separation.After unreacted free composition is removed, the suitable water such as PBS can be used Property medium washing catch and have the solid phase of complex.
In this specification, " detection signal " includes：Qualitatively the presence or absence of detection signal, signal intensity is carried out it is quantitative and The semi-quantitatively intensity of detection signal.Semi-quantitatively detection refer to as " not producing signal ", " weak ", " in ", press " strong " etc. The mode of signal intensity is represented according to the stage.In present embodiment, the preferred intensity of detection signal quantitatively or semi-quantitatively.
As long as mark substance produces detectable signal and is not particularly limited.For example, it may be its own produces signal Material (hereinafter also referred to " signal generation material ") or be catalyzed other materials reaction and make its produce signal thing Matter.Material is produced as signal, can be enumerated for example：Fluorescent material, radio isotope etc..As catalysis other materials React and it is produced the material of detectable signal, can enumerate such as enzyme.As enzyme, can enumerate：Alkaline phosphatase, Peroxidase, beta galactosidase, luciferase etc..As fluorescent material, can enumerate：Fluorescein isothiocynate (FITC), fluorescin such as fluorchrome, GFP such as rhodamine, Alexa Fluor (registration mark) etc..As the same position of radioactivity Element, it can enumerate：125I、14C、32P etc..In these, as mark substance, preferred enzyme, particularly preferred alkaline phosphatase and peroxide Compound enzyme.
The method of detection signal is in itself known in the technical field.In present embodiment, according to from above-mentioned mark Remember that the species of the signal of material suitably selects assay method.For example, when mark substance is enzyme, light splitting can be used Device known to photometer etc. come determine because of the reaction of the enzyme-to-substrate and caused by the signal such as light, color, so as to carry out.
The substrate of enzyme can suitably select according to the species of the enzyme from known substrate.For example, using alkaline phosphatase When enzyme is as enzyme, as substrate, it can enumerate：CDP-Star (registration mark) (the chloro- 3- of 4- (methoxyl group spiral shell [1,2- dioxas Cyclobutane -3,2'- (5'- chlorine) three rings [3.3.1.13,7] decane] -4- bases) disodium phenylphosphate), CSPD (registration mark) (3- (4- methoxyl groups spiral shell [1,2- dioxetanes -3,2- (5'- chlorine) three rings [3.3.1.13,7] decane] -4- bases) phenyl phosphorus Acid disodium) etc. the chloro- 3- indolyl phosphates (BCIP) of the bromo- 4- of chemical luminous substrate, 5-, the chloro- indolyl phosphates two of the bromo- 6- of 5- The chromogenic substrates such as sodium, p-nitrophenyl phosphoric acid.In addition, during using peroxidase as enzyme, as substrate, can enumerate：Shandong The chemical luminous substrate such as minot and its derivative, 2,2'- connection nitrogen bases double (3- ethyl benzo thiazole phenanthroline -6- ichthyodins) (ABTS), The chromogenic substrate such as 1,2- phenylenediamines (OPD), 3,3', 5,5'- tetramethyl benzidines (TMB).
When mark substance is radio isotope, device known to scintillation counter etc. can be used to determine conduct The radioactive ray of signal.In addition, when mark substance is fluorescent material, can be surveyed using device known to fluorescence microplate reader etc. It is set for the fluorescence for signal.It should be noted that excitation wavelength and wavelength of fluorescence can be according to the kinds of used fluorescent material Class suitably determines.
The testing result of signal can use as fucosylation AFP measured value.For example, in quantitative detection letter Number intensity when, can be using the measured value of signal intensity in itself or by the value that the measured value obtains as fucosylation AFP's Measured value uses.As the value of the measured value acquisition by signal intensity, can enumerate for example：By fucosylation AFP measure Value subtracts value etc. obtained by the measured value of negative control sample or the value of background.Negative control sample can be selected suitably, can be with Enumerate such as the Biosample obtained as Healthy People.
In present embodiment, fucosylation can be obtained to multiple standard specimens known to fucosylation AFP concentration AFP measured value, the standard for making the relation for the measured value for representing fucosylation AFP concentration and fucosylation AFP are bent Line.The fucosylation AFP obtained by Biosample measured value is substituted into the standard curve, can be obtained in Biosample Fucosylation AFP concentration value.
, can also be by using the seizure antibody for being fixed on magnetic particle and with mark substance mark in present embodiment The Sandwich ELISA of the detection antibody of note, to determine the fucosylation AFP concentration in Biosample.In this case, Measure can use the commercially available full-automatic immunoassay apparatus such as HISCL serial (SYSMEX Co. Ltd. systems) to carry out.
In addition, the antibody of present embodiment can be used for fucosylation AFP detection kits.Present embodiment Fucosylation AFP detection kits contain seizure antibody, detection antibody, solid phase.As seizure antibody or detection With antibody, the antibody of present embodiment can be used., can be in seizure with antibody and detection antibody in sandwich immunoassays The antibody of present embodiment is used in any one.
For the fucosylation AFP detection kits of present embodiment, further, in detection antibody When mark substance is enzyme, the fucosylation AFP detections kit of present embodiment can further contain the bottom for enzyme Thing.The form of mark substance and substrate is not particularly limited, can be solid (such as powder, crystallization, freeze-drying product Deng), or liquid (such as solution, suspension, emulsion etc.).
In the fucosylation AFP detection kits of present embodiment, in order to be carried out to above-mentioned fucosylation AFP Pretreatment, it can further contain the pretreating reagent containing more than 0.03% SDS.Mentioned reagent contains more than 0.03% with above-mentioned SDS solution is same.
The fucosylation AFP detections kit of present embodiment can be further suitably containing the pre- of Biosample Treatment fluid, the cleaning solution of solid phase, enzyme reaction stopping agent, standard items etc..
For the fucosylation AFP detection kits of present embodiment, seizure antibody, detection can be used The form of the binding reagents box such as antibody, solid phase is suitably contained in container, or can also independently pack.Present embodiment In fucosylation AFP detection kits, seizure antibody can be directly combined to solid phase, can also make seizure antibody Combined indirectly via other materials with solid phase.When making seizure be combined indirectly with antibody and solid phase, in the reagent of present embodiment In box, seizure can be contained in different vessels with antibody and solid phase.Make seizure antibody and solid phase via for example raw When thing element and Avidin class combine indirectly, the seizure of biotin modification can be contained in 1 container with antibody, will be combined with The solid phase of Avidin class is contained in other container.It should be noted that Biosample, catching with antibody, detection with anti- The details of body, solid phase etc. is identical with content described in the explanation of said determination method.
Fucosylation AFP is known with Relations with Liver Cancer.Therefore, the fucosylation AFP detections of present embodiment are used Kit can be used for the diagnosis of liver cancer.
Embodiment
Describe the present invention in detail by the following examples, but the present invention is not by any restriction of these embodiments.
Embodiment 1
The acquirement of antibody：(1) acquisition of antibodyome
Obtain producing the hybridoma of antibodyome by mouse iliac lymph nodes method (Japanese Patent No. 4098796).It is specific and Speech, the glycopeptide A (sequence number 19) of the structure described in table 1 below is synthesized, itself and KLH are coupled, mixed with Freund's complete adjuvant Merging reaches 2.5mg/mL (carrier protein conversion), and thus obtained emulsion is only carried out with 0.06mL/ to the root of the tail portion of mouse 1 injection, so as to carry out.In addition, after immune 17 days, it is only that 2.5mg/mL (carrier protein conversion) KLH is even with 0.06mL/ Join glycopeptide solution A and 1 injection is carried out to the root of the tail portion of mouse, so as to carry out supplementary immunization.Further, in supplementary immunization 4 days Afterwards, from the isolated lymphocyte of iliac lymph nodes, cell fusion is carried out with myeloma, obtains hybridoma.
[table 1]
Circle represents mannose, square expression N-acetyl-glucosamine, triangular representation fucose.
The N-terminal of glycopeptide is KLH-PEG4, and C-terminal has carried out amidatioon.
(2) primary screening
By using the positive antigen (glycopeptide A) described in table 2 or Negative antigens (non-fucosylation glycopeptide A：Sequence Numbering 20) antigen solid phase ELISA, from the hybridoma obtained by above-mentioned (1) selection with positive antigen display reaction but with Negative antigens react few hole.Antigen solid phase ELISA is carried out by the following method.Show the result in table 3.
[table 2]
Circle represents mannose, square expression N-acetyl-glucosamine, triangular representation fucose.
The N-terminal of glycopeptide is BSA-PEG4, and C-terminal has carried out amidatioon.
＜ methods ＞
(1) in 96 orifice plates (nunc Maxisoap/446612), it is (dilute that each screening antigen 1 μ g/ml are added with 50 μ l/ holes Release liquid, 10mM phosphate buffers, pH7), in 37 DEG C of immobilizations 1 hour.
(2) each hole is washed in μ l/ hole × 5 time of PBST 300.
(3) the μ l/ holes of 1%BSA-PBS 100,4 DEG C, overnight and close each hole.
(4) each hole is washed in μ l/ hole × 5 time of PBST 300.
(5) antibody culture supernatant (primary antibody) is diluted 10 times with 1%BSA-PBS, be added with 50 μ l/ holes, RT reactions 1 hour.
(6) each hole is washed in μ l/ hole × 5 time of PBST 300.
(7) by anti-mouse IgG-HRP (JIR/715-035-151) and anti-mouse IgG Lchain-HRP (JIR/115- 20,000 times 035-174) is diluted with 1%BSA-PBS, is added with 50 μ l/ holes, RT reacts 0.5 hour.
(8) each hole is washed in μ l/ hole × 5 time of PBST 300.
(9) developed the color with 100 μ l/ holes addition HRP chromogenic substrates.
(10) stop solution is added with 100 μ l/ holes, stops colour developing.
(11) OD450 is determined.
(12) by visual observation, select that the difference of signal to positive antigen and the signal to Negative antigens is big, resists with feminine gender 7 few holes of former reaction, for postsearch screening.
[table 3]
(3) postsearch screening
By using the protein imprinted of following antigen, the selection identification core rock algae from the hole obtained by above-mentioned (2) The hole of sugar and both amino acid, is cloned.It is protein imprinted to be carried out as follows.
＜ materials ＞
Screen antigen
Positive antigen：Fucosylation AFP (AFP-L3/ recombinants)
Negative antigens 1：Non- fucosylation AFP (the LCA aggegations of the AFP (LEE biosolutions) from human serum The non-adsorbed fraction of element)
Negative antigens 2：Fucosylation ALP (Oriental yeast/47787055)
Primary antibody
Screening antibodies：The positive culture supernatant of primary screening
Negative control：Hybridoma medium
Secondary antibody
Anti-mouse-IgG (Fc) Ab-HRP (BET/A90-131P)
Anti-mouse-IgM Ab-HRP (SBA/1020-05)
Sealer：PVDF Blocking Reagent for Can Get Signal (TOYOBO/NYPBR01)
Cleaning solution：TBST
Dilution：1%BSA-TBST
Pvdf membrane (iBlot (registration mark) 2Transfer Stacks, PVDF, mini/IB24002)
Blotter (iBlot2)
Luminous detection reagent ECL prime Western Blotting Detection Reagent (GE healthcare/RPN2232)
＜ methods ＞
(1) NuPAGE LDS Sample Buffer (4X) (Thermo/ of 1/4 amount is added in each screening antigen NP0008), the NuPAGE Sample Reducing Agent (10X) (Thermo/NP0009) of 1/10 amount and mix.
(2) electrophoresis (SDS-PAGE) is carried out with following amount of antigen to molecular weight marker and each screening antigen of (1).
Swimming lane 0：Molecular weight marker (MagicMark (trade mark) XP Western Protein Standard/LC5602)
Swimming lane 1：Positive antigen：Fucosylation AFP (AFP-L3) 50ng
Swimming lane 2：Negative antigens 1：Non- fucosylation AFP 50ng
Swimming lane 3：Negative antigens 2：Fucosylation ALP 50ng
(3) trace is in pvdf membrane.
(4) 1 hour is impregnated in PVDF Blocking Reagent for Can Get Signal at room temperature and is entered Row closing.
(5) washed 3 times with TBST.
(6) primary antibody is diluted with 1%BSA-TBST according to following dilution ratios, reacted overnight at 4 DEG C.
Screening antibodies：The 10 times of dilutions of positive culture supernatant are screened for the first time
Negative control：10 times of dilutions of Hybridoma medium
(7) washed 3 times with TBST.
(8) secondary antibody (2 kinds of mixing) is diluted with 1%BSA-TBST according to following dilution ratios, reacted at room temperature 1 hour.
20,000 times of dilutions of anti-mouse-IgG (Fc) Ab-HRP
5,000 times of dilutions of anti-mouse-IgM Ab-HRP
(9) washed 3 times with TBST.
(10) detected by chemiluminescence
(11) 5 holes that band is only detected in positive antigen are determined as the positive, cloned.
(4) clone
The cell in 5 holes selected by above-mentioned (3) is sowed by limiting dilution assay.According to the primary screening main points of (2) Its culture supernatant is screened again, obtains 2 clones.ELISA data are shown in table 4.
[table 4]
In the same manner as postsearch screening with (3), to above-mentioned 2 clone culture supernatants implement it is protein imprinted, confirm with AFP-L3 specific reactions.Protein imprinted result is shown in Fig. 1.In Fig. 1, the antigen of each swimming lane is as follows.
Swimming lane 1：Fucosylation AFP (AFP-L3/ recombinants) (positive antigen)
Swimming lane 2：(the LCA agglutinins of the AFP (LEE biosolutions) from human serum are non-by non-fucosylation AFP Adsorb fraction) (Negative antigens)
Swimming lane 3：Fucosylation ALP (Oriental yeast/47787055) (Negative antigens)
(5) confirmation of epitope
In order to which the epitope of the antibody produced to these clones confirms, according to the step same with above-mentioned (2), come Confirm whether it identifies that compared with the glycopeptide A used as immunogene sequence amino acid residue is few, the glycopeptide B shown in table 5 (sequence number 21) and non-fucosylation glycopeptide B (sequence number 22).It the results are shown in table 6.Here, with regard to positive control (PC) for, for glycopeptide B, using the AAL agglutinins of identification fucose, for non-fucosylation glycopeptide B, identification is used GlcNAc WGA agglutinins.For negative control (NC), buffer solution is used.
[table 5]
Circle represents mannose, square expression N-acetyl-glucosamine, triangular representation fucose.
The N-terminal of glycopeptide is BSA-PEG4, and C-terminal has carried out amidatioon.
[table 6]
Clone name Glycopeptide B Non- fucosylation glycopeptide B
1F8-A4 0.048 0.043
2F11-2A9 0.046 0.049
PC 0.335 3.0
NC 0.044 0.044
The glycopeptide A used in all antibody and antigen reacts, not reacted with glycopeptide B.From the result, can obtain The epitope sequences of the antibody obtained are at least containing a certain region in the sequence shown in table 7.
[table 7]
(6) confirmation of CDR sequence
For 5 clones obtained by above-mentioned (4), the CDR parsings of antibody are carried out.As a result, CDR sequence is following 2 kinds of patterns sequence.The CDR sequence (sequence number 1~6) of 1F8-A4 antibody is shown in table 8.In addition, by 2F11-2A9 The CDR sequence (sequence number 7~12) of antibody be shown in table 9.
[table 8]
Heavy chain Light chain
CDR1 GFNIKDYY KSLLYRDGKTY
CDR2 IDPEDGES LMS
CDR3 TTFFN QQLVEYPFT
[table 9]
Heavy chain Light chain
CDR1 GFNINDYF KSLLYKDGKTY
CDR2 IDPEDGET LMS
CDR3 TGGYFV QQLVEYPFT
1F8-A4 in above-mentioned resulting clone is named as 12-1F8 and carries out international accession (NITE BP- 02264).In addition, 2F11-2A9 is named as into I2-2F11, and carry out international accession (NITE BP-02263).
Embodiment 2
Influence of the structure and SDS of sandwich ELISA to reactivity：
Using the I2-1F8 or I2-2F11 obtained by embodiment 1, examined in such a way by sandwich ELISA Survey.In addition, influences of the research SDS to reactivity.
<Material>
Antibody sensitized plate (the μ L/ holes of I2-1F8, I2-2F11/2.5 μ g/mL 100)
Recombinant AFP-L3 antigens
AntiAFP antibody：Anti-alpha-fetoprotein polyclonal antibody (WLS/#PAA153Hu01)
Labelled antibody：Goat anti-rabbit immunoglobulin-HRP
Buffer A：150mM NaCl+1%BSA/10mM phosphate buffers (pH7)
Buffer B：150mM NaCl+0.05%Tween20/10mM phosphate buffers (pH7)
＜ methods ＞
(1) (antigen pretreatment) in 20 μ g/mL AFP-L3 antigenic solutions add equivalent 2%, 1%, 0.5%, 0.25%th, 0.13%, 0.06% SDS solution, more than 3 minutes are stood after mixing.(SDS concentration during pretreatment：0.03-1%)
(2) it is diluted in the way of antigen concentration reaches 1 μ g/mL, 0.5 μ g/mL, 0.25 μ g/mL with buffer A. (final SDS concentration：0.00075%-0.1%)
(3) in antibody sensitized plate, antigenic solution is added with 100 μ L/ holes, is reacted 60 minutes at room temperature.
(4) after washing each hole with buffer B, the anti-AFP antibody of 400 times of dilutions is added with 100 μ L/ holes, at room temperature instead Answer 60 minutes.
(5) after washing each hole with buffer B, the labelled antibody of 4000 times of dilutions is added with 100 μ L/ holes, at room temperature instead Answer 40 minutes.
(6) after washing each hole with buffer B, HRP chromogenic substrates is added with 100 μ L/ holes, are developed the color 20 minutes.
(7) stop solution is added with 100 μ l/ holes, stops colour developing, determine OD450.
＜ results ＞
The survey of the OD450 under various antigen concentrations during I2-1F8, final SDS concentration, pretreatment SDS concentration will be used Definite value is shown in table 10.In addition, by using under various antigen concentrations during I2-2F11, final SDS concentration, pretreatment SDS concentration OD450 measured values be shown in table 11.
[table 10]
[table 11]
In the case of I2-1F8, when final SDS concentration is less than 0.013%, signal is dependent on antigen concentration ground Rise, in the case of I2-2F11, when final SDS concentration is less than 0.025%, signal rises dependent on antigen concentration.
Embodiment 3
The specificity confirmation of sandwich ELISA：
For the sandwich ELISA constructed by embodiment 2, specificity is confirmed in such a way.
＜ materials ＞
Antibody sensitized plate (the μ L/ holes of I2-1F8, I2-2F11/2.5 μ g/mL 100)
Positive antigen：Recombinant AFP-L3 antigens
Negative antigens 1：(LCA of the AFP (LEE biosolutions) from human serum coagulates non-fucosylation AFP The non-adsorbed fraction of collection element)
Negative antigens 2：Fucosylation a-protein LP (Oriental yeast) beyond AFP
Biotinylation AntiAFP antibody：Anti- AFP, Human (mouse) (ABV/ H00000174-M01)
Detection reagent：HRP-Conjugated Streptavidin(Thermo/N100)
Buffer A：150mM NaCl+1%BSA/10mM phosphate buffers (pH7)
Buffer B：150mM NaCl+0.05%Tween20/10mM phosphate buffers (pH7)
＜ methods ＞
(1) in 20 μ g/mL each antigenic solution, add the SDS solution (denaturation) of equivalent 0.06% or be added without (non denatured), more than 3 minutes are stood after mixing.(SDS concentration during pretreatment：0.03%)
(2) according to antigen concentration reach 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 63ng/mL, 31ng/mL mode is diluted with buffer A.
(3) in antibody sensitized plate, antigenic solution is added with 100 μ L/ holes, is reacted 60 minutes at room temperature.
(4) after washing each hole with buffer B, the biotinylation AntiAFP antibody of 480 times of dilutions is added with 100 μ L/ holes, React 60 minutes at room temperature.
(5) after washing each hole with buffer B, the detection reagent of 10000 times of dilutions is added with 100 μ L/ holes, at room temperature Reaction 60 minutes.
(6) after washing each hole with buffer B, HRP chromogenic substrates is added with 100 μ L/ holes, are developed the color 10 minutes.
(7) stop solution is added with 100 μ l/ holes, stops colour developing, determine OD450.
＜ results ＞
Measured value using the OD450 under various antigen concentrations during I2-1F8 is shown in table 12 and Fig. 2.In addition, it will make Table 13 and Fig. 3 are shown in the OD450 measured values under various antigen concentrations during I2-2F11.
[table 12]
[table 13]
Though I2-1F8 and I2-2F11 in denatured state or under non denatured state, with non-fucosylation AFP and ALP does not show reaction.On the other hand, for I2-1F8 and I2-2F11, relative to AFP-L3, it is observed that antigen is dense The signal for spending dependence rises.I2-1F8 and I2-2F11 is particularly significantly to be carried under denatured state to AFP-L3 reactivity It is high.These results show：I2-1F8 and I2-2F11 identifies AFP-L3 fucose and peptide moiety simultaneously, so as to by sandwich ELISA and AFP-L3 specific reactions.Show in addition：I2-1F8 and I2-2F11 carries out pre- by using 0.03%SDS to antigen Handle and consumingly react.
Embodiment 4
With natural human AFP-L3 reactivity：
Confirm whether I2-1F8 reacts with natural human AFP-L3 in such a way by Western blotting.
＜ materials ＞
Primary antibody：4 DEG C of O/N of I2-1F8 (10 times of dilution hybridoma culture supernatants)
Secondary antibody：Anti-mouse-IgG (Fc) Ab-HRP (BET/#A90-131P) (20,000 times of dilutions) RT1 hours
＜ methods ＞
In addition to primary antibody and secondary antibody are changed into above-mentioned antibody, carried out in the same manner as embodiment 1 (3) protein imprinted.
＜ results ＞
Protein imprinted result is shown in Fig. 4.I2-1F8 and natural human AFP-L3 displays that reaction.In Fig. 4, each swimming The antigen in road is as follows.
Swimming lane 1：Recombinant AFP-L3
Swimming lane 2：Non- fucosylation AFP
Swimming lane 3：Natural human AFP (ミ ュ ー タ ス ワ コ ー AFP-L3 standard items 1)
Swimming lane 4：Natural human AFP-L3 (ミ ュ ー タ ス ワ コ ー AFP-L3 standard items 2)
Embodiment 5
Influences of the SDS to reactivity：
Use the ELISA constructed by embodiment 2, influences of the research SDS to reactivity.
＜ materials ＞
In addition to using I2-1F8 as antibody, material same as Example 2 is used.
＜ methods ＞
(1) (antigen pretreatment) in 20 μ g/mL AFP-L3 antigenic solutions add equivalent 0.031%, 0.016%, 0.008%th, 0.004% SDS solution, more than 3 minutes are stood after mixing.
(2) carried out in the way of antigen concentration reaches 0.4 μ g/mL, 0.2 μ g/mL, 0.1 μ g/mL with buffer A dilute Release.(final SDS concentration：0.00032%-0.00001%)
(3) later operation is carried out similarly to Example 2.
＜ results ＞
The survey of the OD450 under various antigen concentrations during I2-1F8, final SDS concentration, pretreatment SDS concentration will be used Definite value is shown in table 14.
[table 14]
When to pre-process SDS concentration be less than 0.016%, can also detectable concentration dependence signal.
Sequence table
<110>Sysmex group (Sysmex Corporation)
<120>Monoclonal antibody reacted with glycopeptide and application thereof
<130> IIC170517
<150> JP 2016-108461
<151> 2016-05-31
<160> 25
<170> PatentIn version 3.5
<210> 1
<211> 8
<212> PRT
<213>Artificial sequence
<220>
<223> CDR-A
<400> 1
Gly Phe Asn Ile Lys Asp Tyr Tyr
1 5
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<212> PRT
<213>Artificial sequence
<220>
<223> CDR-A
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Ile Asp Pro Glu Asp Gly Glu Ser
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<212> PRT
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<220>
<223> CDR-A
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Thr Thr Phe Phe Asn
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<220>
<223> CDR-A
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Lys Ser Leu Leu Tyr Arg Asp Gly Lys Thr Tyr
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Leu Met Ser
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Gln Gln Leu Val Glu Tyr Pro Phe Thr
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Ile Asp Pro Glu Asp Gly Glu Thr
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Lys Ser Leu Leu Tyr Lys Asp Gly Lys Thr Tyr
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Leu Met Ser
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Gln Gln Leu Val Glu Tyr Pro Phe Thr
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<212> PRT
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<223>By formula（1）The glycopeptide of expression
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<221> CARBOHYD
<222> (4)..(4)
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Thr Lys Val Asn Phe Thr Glu Ile Gln
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<223>By formula（2）The glycopeptide of expression
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Thr Lys Val Asn Phe Thr Glu Ile Gln
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<223>By formula（3）The glycopeptide of expression
<220>
<221> CARBOHYD
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Thr Lys Val Asn Phe Thr
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<223>By formula（4）The glycopeptide of expression
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Thr Lys Val Asn Phe Thr
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<223>By formula（5）And formula（7）The glycopeptide of expression
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Asn Phe Thr Glu Ile Gln
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<223>By formula（6）And formula（8）The glycopeptide of expression
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Thr Lys Val Asn Phe Thr Glu Ile Gln
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<223>Glycopeptide A
<220>
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<222> (5)..(5)
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Gly Thr Lys Val Asn Phe Thr Glu Ile Gln
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<212> PRT
<213>Artificial sequence
<220>
<223>Non- fucosylation glycopeptide A
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<221> CARBOHYD
<222> (5)..(5)
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Gly Thr Lys Val Asn Phe Thr Glu Ile Gln
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Gly Thr Lys Val Asn Phe Thr
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<223>Epitope 1
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Asn Phe Thr Glu Ile Gln
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<211> 609
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<213>Homo sapiens
<300>
<308> NM_001134
<309> 2016-09-10
<313> (1)..(609)
<400> 25
Met Lys Trp Val Glu Ser Ile Phe Leu Ile Phe Leu Leu Asn Phe Thr
1 5 10 15
Glu Ser Arg Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu
20 25 30
Asp Ser Tyr Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr
35 40 45
Ile Phe Phe Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser
50 55 60
Lys Met Val Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp
65 70 75 80
Glu Gln Ser Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu
85 90 95
Glu Leu Cys His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser Asp
100 105 110
Cys Cys Ser Gln Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His
115 120 125
Lys Lys Pro Thr Pro Ala Ser Ile Pro Leu Phe Gln Val Pro Glu Pro
130 135 140
Val Thr Ser Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn
145 150 155 160
Lys Phe Ile Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro
165 170 175
Thr Ile Leu Leu Trp Ala Ala Arg Tyr Asp Lys Ile Ile Pro Ser Cys
180 185 190
Cys Lys Ala Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr
195 200 205
Val Thr Lys Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys
210 215 220
Ala Val Met Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile Thr Val
225 230 235 240
Thr Lys Leu Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Glu Ile Gln
245 250 255
Lys Leu Val Leu Asp Val Ala His Val His Glu His Cys Cys Arg Gly
260 265 270
Asp Val Leu Asp Cys Leu Gln Asp Gly Glu Lys Ile Met Ser Tyr Ile
275 280 285
Cys Ser Gln Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys
290 295 300
Leu Thr Thr Leu Glu Arg Gly Gln Cys Ile Ile His Ala Glu Asn Asp
305 310 315 320
Glu Lys Pro Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp
325 330 335
Arg Asp Phe Asn Gln Phe Ser Ser Gly Glu Lys Asn Ile Phe Leu Ala
340 345 350
Ser Phe Val His Glu Tyr Ser Arg Arg His Pro Gln Leu Ala Val Ser
355 360 365
Val Ile Leu Arg Val Ala Lys Gly Tyr Gln Glu Leu Leu Glu Lys Cys
370 375 380
Phe Gln Thr Glu Asn Pro Leu Glu Cys Gln Asp Lys Gly Glu Glu Glu
385 390 395 400
Leu Gln Lys Tyr Ile Gln Glu Ser Gln Ala Leu Ala Lys Arg Ser Cys
405 410 415
Gly Leu Phe Gln Lys Leu Gly Glu Tyr Tyr Leu Gln Asn Ala Phe Leu
420 425 430
Val Ala Tyr Thr Lys Lys Ala Pro Gln Leu Thr Ser Ser Glu Leu Met
435 440 445
Ala Ile Thr Arg Lys Met Ala Ala Thr Ala Ala Thr Cys Cys Gln Leu
450 455 460
Ser Glu Asp Lys Leu Leu Ala Cys Gly Glu Gly Ala Ala Asp Ile Ile
465 470 475 480
Ile Gly His Leu Cys Ile Arg His Glu Met Thr Pro Val Asn Pro Gly
485 490 495
Val Gly Gln Cys Cys Thr Ser Ser Tyr Ala Asn Arg Arg Pro Cys Phe
500 505 510
Ser Ser Leu Val Val Asp Glu Thr Tyr Val Pro Pro Ala Phe Ser Asp
515 520 525
Asp Lys Phe Ile Phe His Lys Asp Leu Cys Gln Ala Gln Gly Val Ala
530 535 540
Leu Gln Thr Met Lys Gln Glu Phe Leu Ile Asn Leu Val Lys Gln Lys
545 550 555 560
Pro Gln Ile Thr Glu Glu Gln Leu Glu Ala Val Ile Ala Asp Phe Ser
565 570 575
Gly Leu Leu Glu Lys Cys Cys Gln Gly Gln Glu Gln Glu Val Cys Phe
580 585 590
Ala Glu Glu Gly Gln Lys Leu Ile Ser Lys Thr Arg Ala Ala Leu Gly
595 600 605
Val

Claims (18)

1. a kind of monoclonal antibody with glycopeptide reaction,
The glycopeptide contains core fucose and from the asparagine of additional sugar chain to more than continuous 4 residues in C ends side Amino acid,
The antibody with the core fucose of the glycopeptide and glycopeptide from the asparagine of additional sugar chain to C ends lateral extent 3 Both amino acid more than individual residue is epitope.
2. a kind of monoclonal antibody, it reacts with following glycopeptide,
Do not reacted with following glycopeptide,
Do not reacted with following glycopeptide,
3. antibody according to claim 2, it does not react with following glycopeptide,
4. antibody according to claim 2, it further reacts with fucosylation AFP.
5. antibody according to claim 2, wherein, in the presence of the SDS below 0.025 mass/mass %, with containing Fucosylation AFP obtained from more than 0.03 mass/mass % SDS solution is pre-processed reacts.
6. antibody according to claim 2, wherein, in the presence of 2 mass/mass % SDS, 50mM DTT, with denaturation Fucosylation AFP reaction.
7. antibody according to claim 2, wherein, in the presence of 2 mass/mass % SDS, 50mM DTT, with denaturation Non- fucosylation AFP do not react.
8. antibody according to claim 2, wherein, the CDR of heavy chain contains amino acid sequence shown in sequence number 1, sequence The amino acid sequence shown in amino acid sequence and sequence number 3 shown in column number 2,
The CDR of light chain contains the amino acid sequence shown in sequence number 4, the amino acid sequence shown in sequence number 5 and sequence and compiled Amino acid sequence shown in numbers 6.
9. antibody according to claim 2, wherein, the CDR of heavy chain contains amino acid sequence shown in sequence number 7, sequence The amino acid sequence shown in amino acid sequence and sequence number 9 shown in column number 8,
The CDR of light chain contains the amino acid sequence shown in sequence number 10, the amino acid sequence shown in sequence number 11 and sequence Amino acid sequence shown in numbering 12.
10. a kind of method for producing the antibody described in claim 2, it contains following process：By the glycopeptide containing following structure Animal is immunized antigen,
X is arbitrary sugar chain.
11. according to the method for claim 10, wherein, the glycopeptide antigen contains following structure,
X is arbitrary sugar chain.
12. a kind of method for producing the hybridoma for producing the antibody described in claim 2, it contains following process：Will contain with Animal is immunized the glycopeptide antigen of lower structure,
X is arbitrary sugar chain.
13. according to the method for claim 12, wherein, the glycopeptide antigen contains following structure,
X is arbitrary sugar chain.
14. a kind of hybridoma, its international deposit number is NITE BP-02263 or NITE BP-02264.
15. a kind of fucosylation AFP assay method, the antibody described in its usage right requirement 2.
16. the method according to claim 11, wherein, with the solution of the SDS containing more than 0.03 mass/mass % to rock Algae glycosylation AFP is pre-processed, after the treatment, with described anti-in the presence of the SDS below 0.025 mass/mass % Body is reacted with the fucosylation AFP.
17. a kind of fucosylation AFP detection kits, it contains fucosylation AFP seizure antibody, fucosido Change AFP detection antibody, solid phase,
The seizure is with antibody or the detection with the antibody described in antibody is claim 2.
18. kit according to claim 17, wherein, further contain the SDS's containing more than 0.03 mass/mass % Pretreating reagent.
CN201710202207.0A 2016-05-31 2017-03-30 Monoclonal antibody reacted with glycopeptide and application thereof Pending CN107857815A (en)

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