CN108976301A - A kind of preparation method for antibody of the specific glycoprotein of novel serum - Google Patents

A kind of preparation method for antibody of the specific glycoprotein of novel serum Download PDF

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CN108976301A
CN108976301A CN201810905323.3A CN201810905323A CN108976301A CN 108976301 A CN108976301 A CN 108976301A CN 201810905323 A CN201810905323 A CN 201810905323A CN 108976301 A CN108976301 A CN 108976301A
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antibody
glycoprotein
serum
agglutinin
weary
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高春芳
冯惠娟
衣常红
房萌
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Oriental Hepatobiliary Surgery Hospital Second Military Medical University Of Chinese Pla
Second Military Medical University SMMU
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
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    • 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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    • G01—MEASURING; TESTING
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    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis
    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

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Abstract

Sugar chain is the pith for maintaining glycoprotein structure and function, directly takes part in the pathological processes of each major disease.The invention belongs to clinical medicine diagnostics, and it is an object of that present invention to provide a kind of preparation method for antibody of the specific glycoprotein of serum, to effectively improve the specificity and accuracy that the immunology detection technology based on agglutinin capture detects Aberrant glycosylation glycoprotein.Specifically, gained antibody specificity is high the present invention provides a kind of preparation method of specific weary glycosylated antibodies of glycoprotein, level of glycosylation is low, agglutinin capture-immunology detection of specific glycosylation albumen can be effectively used for.Agglutinin capture-immunology detection technology includes: agglutinin capture Chemiluminescence Immunoassay and agglutinin enzyme-linked immunosorbent assay.Weary glycosylated antibodies specifically include that weary core fucosylation, weary sialylated, weary divide type N-Acetyl-D-glucosamine etc. equally.

Description

A kind of preparation method for antibody of the specific glycoprotein of novel serum
Technical field
The invention belongs to clinical medicine diagnostics, relate generally to a kind of Antibody preparation side of specific glycoprotein of serum Method.More particularly, it relates to a kind of preparation method for antibody for the specific glycoprotein of serum that structure is changed based on genetic engineering, and its Purposes.
Background technique
Glycosylation is modification structure most complicated and various in protein post-translational modification, is the important composition portion of glycoprotein Point, it is played an important role in maintaining glycoprotein structure and function.At least 50% albumen is glycoprotein, serum in human plasma It is glycoprotein that 14 kinds of high-abundance proteins, which just have 11 kinds of albumen, comprising: immunoglobulin, α -1 antitrypsin, transferrins, touching Globin, fibrinogen, alpha2-macroglobulin, α 1- acidoglycoprotein, transthyretin and complement-C3.Common blood Abundance protein in clear: myosin A and golgi protein 73 also belong to glycoprotein.American National research institute deliver in the recent period one Piece report claims: glycosylation directly takes part in the pathological processes of each major disease, realizes that Personalized medicine is needed to egg White glycosylation modified carry out in-depth study.Therefore, the detection of the Aberrant glycosylation component of the specific glycoprotein of serum is to a variety of The diagnosis of disease/tumour, curative effect monitoring, Index for diagnosis etc. are of great significance.
Immunoglobulin (immunoglobulin, Ig) is also known as gamma globulin, is to have to resist in human serum and body fluid The active a kind of glycoprotein of body, is the important component that body resists disease.Ig mainly includes IgM, IgA, IgD, IgG and IgE five Seed type, wherein IgG is minimum containing sugar, is 2%~3%, IgA 8%, it is 7%~15% that remaining is various.The end research report IgA Hold the reduction of galactose content closely related with the generation of IgA type nephrosis.Wooden long narrow flag sun professor in 1985 et al. it has also been found that: rheumatoid Gala sugar amount contained by the area IgG molecule Fc sugar chain is significantly lower than normal person in arthritic's serum, and is obtained by animal model It confirms.This laboratory early period is it is also found that: horizontal the increasing of core fucosylation IgG can be used as primary carcinoma of liver and large intestine The auxiliary examining finger veins mark of cancer.
α -1 antitrypsin (α -1-antitryp sin, A1AT) is also known as α -1 protease inhibitors, is in human plasma Most important protease inhibitors accounts for 90% or more of Total plasma protein enzyme rejection ability, is mainly synthesized by liver, and sugar content is about 10%-20%.A1AT is single chain glycoprotein, and having reported has 3 N- glycosylation sites (N46, N83 and N247) on A1AT peptide chain. Nineteen ninety Li Ding state et al. discovery: with progression of the disease, LCA mating type A1AT is in health examination, oxyhepatitis, cirrhosis and liver Expression gradually rises in cancer patients serum.And the auxiliary that LCA mating type A1AT percentage > 20% can be used as liver cancer is examined Severed finger mark.
Transferrins (transferrin, Tf) is also known as transferrin, is main iron protein in blood plasma, main negative The iron that duty delivery is absorbed by digest tube and the iron discharged by red blood cell degradation.Having reported has 2 N- glycosylation sites on Tf peptide chain (N413 and N611) can form the Tf hypotype of single sialic acid to eight sialic acids.The Tf of different subtype and personal Alcohol Consumption Status phase It closes.Sialic acid is reduced in Tf sugar chain in wine-head's serum and cerebrospinal fluid, forms asialo or bifunctional sialyltransferase Tf hypotype, referred to as sugared Deletional transferrins (CDT).It prohibits against alcoholic drinks after a period of time, CDT can disappear in wine-head's serum and cerebrospinal fluid.Therefore, CDT can be with Diagnosis and wine-head as Alcohol Consumption Status are relieved the effect of alcohol the monitoring for the treatment of.Recent study discovery: primary carcinoma of liver and chronic liver disease Multiple antennas sugar chain Tf increases in patients serum, occurs containing core fucose and containing type N-acetylglucosamine sugar chain Tf is divided equally, And multiple antennas sugar chain Tf content is higher than liver cirrhosis patient in serum in patients with primary hepatic, DSA mating type Tf is expected to become primary The diagnosis index of property liver cancer.
Haptoglobin (haptoglobin, HP) is also known as hoptoglobin, is one of 2 globulin fraction of Serum A acidity Glycoprotein is mainly made up of with two light chains (2 chain of 1 chain of α and α) disulfide bond two heavy chains (β chain).HP molecule is reported There are 4 N- glycosylation sites (N184, N207, N211 and N241) in β chain, is rich in sialic acid and fucose.Abnormal core rock algae Saccharification haptoglobin (Fuc-HP) content increases the potential mark that can be used as diagnosing tumor.Ang in 2006 etc. is reported for the first time With height is sialylated and the HP of height fucosylation expression is related to the occurrence and development of primary hepatoma, and joint The sensibility and specificity for detecting HP and AFP diagnosing primary liver cancer is respectively 79% and 95%.The discovery such as Shu in 2011 The increase of Lewis X-type fucosylation HP- β subunit is likely to become HCC early diagnosis marker.
Fibrinogen (Fibrinogen, Fib) is a kind of glycoprotein with coagulation function, is synthesized by liver, is fine The precursor of fibrillarin.Having reported has 2 N- glycosylation sites (N453 and N686) on Fib peptide chain, sugar-chain end is rich in saliva Acid.The sialic acid combined on fibrinogen is converted into fibrin monomer and fibrin monomer polymerization shape in fibrinogen At playing an important role during stable reticulate body, the height of bound sialic acid content affects blood coagulation function on fibrinogen Energy.The discovery of Japanese scholars in 1985 hepatopathy especially liver cirrhosis patient shows as fibrin monomer polymerization obstacle more, when blood coagulation Between extend, these features and fibrinogen bound sialic acid content increase substantial connection.Therefore, fibrinogen combination saliva The measurement of acid content can be used as an important indicator of dysfibrinogenemia diagnosis and classification.
The macromolecular glycoprotein of rich content in alpha2-macroglobulin (α 2-macroglobulin, α 2-MG or AMG) blood plasma, Sugar content about 8%.AMG is mainly a kind of protease of wide spectrum in blood plasma by synthesizing in liver cell and monokaryon-mononuclear phagocyte system Inhibitor takes part in the pathological processes of a variety of diseases.AMG is the homotetramer structure being made of four same subunits, Reported N- glycosylation site has 8 (N55, N70, N247, N396, N410, N869, N991 and N1424) on its peptide chain.It grinds Study carefully discovery: AMG glycosylation changes related to kinds cancer.The rock algae in the site N396 and N1424 of AMG in Pancreas cancer patients serum Level of glycosylation is substantially less than healthy control group.And AMG has the glycosylation of higher degree and higher in Serum In Patients With Colorectal Carcinoma The presence of multiple antennas complexity N- sugar chain.
α 1- acidoglycoprotein (α 1-acid glycoprotein, α 1-AG or AGP) is also known as mucoprotein, is in human serum Sugar content highest, acid strongest glycoprotein, sugar content are up to 40%, are mainly synthesized by liver, and certain tumor tissues can also close At.Having reported has 5 N- glycosylation sites (N15, N38, N58, N75 and N85) on AGP peptide chain, rich in sialic acid, fucose and Multiple antennas sugar chain branches structure.The level of glycosylation of AGP is related to inflammation generation, and especially chronic liver inflammatory reaction is related. In rheumatoid arthritis and chronic hepatitis patient serum, AGP concentration increases, while core fucose and triantennary in AGP Sugar chain structure significantly increases.In chronic respiratory disease patients serum, AGP concentration is without significantly changing, multiple antennas sugar chain in AGP Structure increases.And in patients of ulcerative colitis serum, AGP concentration and its level of glycosylation nothing are substantially change.
Transthyretin (Transthyretin, TTR) is also known as prealbumin, is mainly synthesized by liver, denier by Choroid plexus and retina synthesis.TTR major function is transhipment thyroxine and retinol, and cause starch is more likely formed after gene mutation Sample substance, with the myocardium amyloidosis such as heredity thyroxine transport protein related amyloidosis and the senile amyloidosis of whole body It is related.Having reported has 1 potential N- glycosylation site (N118) on TTR peptide chain, but TTR often shows as aglycosylated modification, Because the increase of sugar chain can accelerate the degradation of TTR.
Complement-C3 (Complement C3, C3) is one group of glycoprotein with enzymatic activity, is that content is highest in serum Complement component is mainly synthesized by macrophage and liver.C3 is cracked into two segments of C3a and C3b under the action of C3 convertase, And it plays the role of a nucleus in complement Classical pathway and alternative activation pathway.Having reported has 3 N- glycosylation positions on C3 peptide chain Point (N85, N939 and N1617).Research at present about C3 sugar chain is less.
Myosin A (Fetuin A) be also known as 2-HS glycoprotein (2-Heremans-Schmid glycoprotein, It AHSG), is one of the member of cystatin superfamily, it is related with actrapid monotard's resistance, liver fat generation. Fetuin A is mainly synthesized by liver, sugar content about 35%.Having reported has 2 N- glycosylation sites on Fetuin A peptide chain (N156 and N176) is rich in fucose structure.Fucosylation Fetuin A has facilitation to growth and metastasis of tumours, Tumor colonies are mainly promoted to grow and reduce apoptosis in conjunction with tumor cell surface receptor by calcium-mediated.This laboratory It has also been found that: liver cancer, serum of cirrhosis patients fucosylation Fetuin A level are apparently higher than HBV Asymptomatic Carriers and normal Healthy control group, and good diagnostic value is shown to liver cirrhosis patient.
Golgi protein 73 (Golgi protein-73, GP73) is a kind of resident golgiosome II type transmembrane glycopeptide It is white, there is expression in the epithelial cell of human multiple tissue, but hardly express in normal liver cell.GP73 peptide is reported There are 3 N- glycosylation sites (N109, N144 and N398) on chain, and is rich in fucose structure.Research is found: primary carcinoma of liver is suffered from GP73 expression increases in person liver cell and serum, and its sensibility and specificity are higher than in the early diagnosis of primary carcinoma of liver AFP.It is also found that: core fucose GP73 (Fc-GP73) content also increases in liver cancer patient blood serum, Fc-GP73 facilitates for research The mutation and migration of liver cancer cells.But the diagnostic value in relation to GP73 still has certain dispute, it is now recognized that GP73 itself There is preferable value to the diagnosis of cirrhosis, and the GP73 of fucosylation modification is considered as the marker of diagnosing cancer of liver.
Currently, the Aberrant glycosylation level detection method of glycoprotein mainly has: agglutinin trace (Lectin-Blot, LB), Glycoprotein electrophoresis, mass spectrum (MS), agglutinin capture Chemiluminescence Immunoassay and agglutinin enzyme-linked immunosorbent assay (Lectin-ELISA) etc..Agglutinin capture Chemiluminescence Immunoassay and agglutinin enzyme-linked immunosorbent assay belong to be based on The immunology detection technology of agglutinin capture, the technology are to be made the specificity of antigen and antibody based on immunological response The very high experimental technique of a kind of sensibility being combined together with the specific effect of, sugar chain and agglutinin has sensitive, special Different, quick, efficient, stable and the features such as be easily operated automatically, the Aberrant glycosylation for being widely used in a variety of glycoprotein is horizontal Detection.
Currently, the antibody for the detection of glycoprotein Aberrant glycosylation level is mostly IgG type antibody.IgG molecule is mainly by two Heavy chain and two light chains form two and antigen reactive Fab segment by being covalently united with non covalent forms With a Fc segment.(Asn297-X-Ser/Thr, X can be except proline the consensus sequence in the area CH2 in the Fc segment of IgG Arbitrary amino acid residue) the complicated double antenna type containing fucose and sialic acid can be formed by amido bond and sugar chain covalent bond Nuclear structure.There is also N- glycosylations for Fab segment in 30% IgG.Therefore, the level of glycosylation of antibody itself can influence to be based on Detection of the immunology detection technology of agglutinin capture to glycoprotein Aberrant glycosylation level, if the glycoprotein glycosylation of detection Type is similar to the type of glycosylation on antibody, then the detection technique can cause the inaccuracy of testing result, especially false positive.
Summary of the invention
It is an object of that present invention to provide a kind of preparation method for antibody of the specific glycoprotein of serum, are based on agglutination to effectively improve The immunology detection technology of element capture especially reduces the specificity and accuracy of the Aberrant glycosylation component detection of glycoprotein False positive or detection background.Specifically, the present invention provides a kind of preparation method of weary glycosylated specific sugar protein antibodies, Preparation process is simple, yield is big, antibody specificity is high, level of glycosylation is low.
In order to achieve the above object, technical method of the invention specifically includes that
(1) by a kind of comprising providing the albumen containing glycosylated specific sugar protein antibodies by method disclosed herein And gene order；
(2) light, again containing glycosylated specific sugar protein antibodies comprising providing by method disclosed herein by a kind of Chain expression plasmid；
(3) by a kind of comprising by method disclosed herein, providing the light, again of weary glycosylated specific sugar protein antibodies Chain expression plasmid；
(4) by a kind of comprising providing weary glycosylated specific sugar protein antibodies albumen by method disclosed herein.
Detailed description of the invention
Fig. 1 is overview flow chart of the present invention.
Fig. 2 is method disclosed by the invention haptoglobin antibody DNA analysis result obtained.
Fig. 3 is the coomassie brilliant blue staining result of method disclosed by the invention haptoglobin antibody albumen obtained.
Fig. 4 is the western blot (Western in method disclosed by the invention haptoglobin antibody albumen obtained Blot, WB) result.
Fig. 5 is the agglutinin trace (Lectin in method disclosed by the invention haptoglobin antibody albumen obtained Blot, LB) result.
Specific embodiment
The present invention will be described in detail with reference to the accompanying drawings and embodiments.
Fig. 1 is overview flow chart of the present invention:
1. immune mouse:
Mouse is immunized using humanized's haptoglobin sterling.First immunisation is that BALB/C mice is only injected intraperitoneally in 100 μ g/, Freund's complete adjuvant (Sigma, F5881).It is immunized and is only injected intraperitoneally for 50 μ g/ for the second time, Freund's incomplete adjuvant (Sigma, F5506).Third and fourth time immune with second.Booster immunization is that (Sigma, MS6722) is only injected intraperitoneally in 50 μ g/.Docking acquisition After mice serum carries out antibody titer analysis, takes out mouse boosting cell and carry out specificity antibody screening.
2. specificity antibody screening: following two method can be used.
(1) authentic monoclonal antibody method: after taking out mouse boosting cell, mouse boosting cell and myeloma cell are melted It closes.By the methods of HAT culture screening, affinity screening, specificity screening and subgroup identification, high-affinity and special is filtered out The hybrid tumor cell monoclonal containing glycosylated haptoglobin antibody of property.It is obtained after extracting hybridoma total serum IgE and reverse transcription CDNA is obtained, expands and light chain and heavy chain DNA sequences is simultaneously obtained by unique Sequence Detection.
(2) mouse immune source phage displaying antibody library method: after taking out mouse boosting cell, extracting mouse boosting cell gene, To construct phage displaying antibody library in conjunction with bacteriophage.By it is affine screening, ELISA positive monoclonal select, ELISA method and Fortbio interaction of molecules filters out the bacteriophage Dan Ke of the haptoglobin antibody containing glycosylation of high-affinity and specificity It is grand.CDNA is obtained after extracting bacteriophage total serum IgE and reverse transcription, expand and light chain and heavy chain are obtained by unique Sequence Detection DNA sequence dna.
3. the acquisition of the plasmid construction and albumen of the haptoglobin antibody containing glycosylation:
Gene order obtained is cloned into expression vector plasmid, the haptoglobin antibody containing glycosylation is obtained and expresses matter Grain.By plasmid transfection into antibody expressing cells.And collect enrichment and purifying that culture supernatant carries out antibody.Using dodecyl Sodium sulphate polyacrylamide gel (SDS-PAGE) electrophoresis and the dyeing of coomassie brilliant blue staining liquid carry out antibody purity detection.Specifically Are as follows: after BCA method kit (Thermo, 23225) detection antibody concentration, will be owned using phosphate buffered saline solution (PBS) Every kind of sample of 20 μ L and is fitted into the sample well of gel by sample preparation at the concentration of 0.1mg/mL.Runing time and electricity Pressure is set as 80V 90mim and 120V 60min.It is dyed, is judged with coomassie brilliant blue staining liquid (Beyotime, P0017) Antibody purity.Using the specificity and level of glycosylation of WB and LB detection antibody, specific steps are as described in Fig. 4 and Fig. 5.
4. the acquisition of the plasmid construction and albumen of weary glycosylation haptoglobin antibody:
By the expression plasmid of the weary glycosylation haptoglobin antibody Fab section of prokaryotic expression system building expression, pass through gene Change structure and eukaryotic expression system constructs the expression plasmid of weary haptoglobin antibody Fc sections of glycosylation.By above-mentioned plasmid transfect respectively to In E.coli bacterial strain and antibody expressing cells, and collect enrichment and purifying that culture supernatant carries out antibody.Using SDS-PAGE electricity Swimming and the dyeing of coomassie brilliant blue staining liquid carry out antibody purity detection.Using the specificity and glycosylation water of WB and LB detection antibody Flat, specific steps are as described in Fig. 4 and Fig. 5.
Fig. 2 is method disclosed by the invention haptoglobin antibody DNA analysis result obtained:
1% Ago-Gel is prepared, all samples are configured to the concentration of 0.1mg/mL using PBS, and by 10 μ L's Every kind of sample is fitted into the sample well of gel.Runing time and voltage are set as 140V 20mim.Swimming lane 1 is DNA Marker, Swimming lane 2 is the light chain plasmids DNA of glycosylation haptoglobin antibody obtained, and swimming lane 3 is glycosylation haptoglobin obtained The heavy chain plasmid DNA of antibody.Swimming lane 4 is the light chain plasmids DNA of weary glycosylation haptoglobin antibody obtained, and swimming lane 5 is institute The heavy chain plasmid DNA of the weary glycosylated haptoglobin antibody obtained.
Fig. 3 is the coomassie brilliant blue staining result of method disclosed by the invention haptoglobin antibody albumen obtained:
10% PAGE gel is prepared, all samples are configured to the concentration of 0.1mg/mL using PBS, and by 20 μ Every kind of sample of L is fitted into the sample well of gel.Runing time and voltage are set as 80V 90mim and 120V 60min.It will coagulate It is put into after glue cutting after dyeing 30min in Coomassie brilliant blue dye liquor, clear water is washed to no background color.Swimming lane 1 is albumen Haptoglobin antibody (Abcam, ab131236) is commercialized in Marker, swimming lane 2, and swimming lane 3 is is obtained glycosylated haptoglobin Antibody protein (reduced form), swimming lane 3 are weary glycosylation haptoglobin antibody albumen (reduced form) obtained.
Fig. 4 is the WB result in method disclosed by the invention haptoglobin antibody albumen obtained:
10% PAGE gel is prepared, humanized's haptoglobin sterling is configured to by the dense of 0.1mg/mL using PBS It spends, 20 μ L is added in each sample aperture.Runing time and voltage are set as 80V 90mim and 120V 60min.Gel is cut After carry out transferring film, runing time and voltage are set as 90V 90mim.Film is closed overnight with 5% BSA solution, it will using PBS Each sample is diluted to 4 μ g/mL 1/mL, is added into film after being incubated for 4h, 0.1% TW-20/TBS solution washing 4 times, every time 10min.After being incubated for 1h using the anti-murine antibody solution of the fluorescent marker of 4 μ g/mL of 1mL, 0.1% TW-20/TBS solution Washing 4 times, each 10min.M is albumen Marker, and 1 is commercialized haptoglobin antibody as a result, 2 is are obtained glycosylated touching pearl Protein antibodies are as a result, 3 to be obtained weary glycosylated haptoglobin antibody result.
Fig. 5 is the LB result in method disclosed by the invention haptoglobin antibody albumen obtained:
10% PAGE gel is prepared, all samples are configured to the concentration of 0.1mg/mL using PBS, and by 10 μ Every kind of sample of L is fitted into the sample well of gel.Runing time and voltage are set as 80V 90mim and 120V 60min.It will coagulate Transferring film is carried out after glue cutting, runing time and voltage are set as 90V 90mim.Film is closed overnight with 5% BSA solution, is used After lentil lectin (LCA) solution of the biotin labeling of 4 μ g/mL concentration of 5mL is incubated for 4h, 0.1% TW-20/TBS solution is washed It washs 4 times, each 10min.After being incubated for 1h using the streptomycin solution of the fluorescent marker of 4 μ g/mL concentration of 5mL, 0.1% TW-20/ TBS solution washs 4 times, each 10min.Swimming lane 1 is albumen Marker, and haptoglobin antibody is commercialized in swimming lane 2, and swimming lane 3 is institute Glycosylated haptoglobin antibody is obtained, swimming lane 4 is is obtained weary glycosylated haptoglobin antibody albumen.
The present invention provides a kind of preparation method for antibody of weary glycosylated specific glycoprotein of serum, preparation process is simple, Yield is big, antibody specificity is high, level of glycosylation is low, effectively increases the immunology detection technology based on agglutinin capture to different The often specificity of glycosylation glycoprotein detection.

Claims (6)

1. a kind of preparation method for antibody of the specific glycoprotein of novel serum, this method be changed based on genetic engineering structure serum it is specific The preparation method for antibody of glycoprotein, it is characterised in that the party specifically includes the following steps:
(1) using the specific antibody of cell-fusion techniques and the Antibody library screening specific glycoprotein of serum；
(2) pass through the gene constructed weary glycosylated antibodies that the specific glycoprotein of serum is expressed with protokaryon system；
(3) pass through weary glycosylated antibodies that are gene constructed, changing structure and the eukaryon system expression specific glycoprotein of serum.
2. the specific glycoprotein of serum described in claim 1, including 11 kinds of serum high abundance glycoprotein (immunoglobulin (IgG, IgA, IgM, IgE), α -1 antitrypsin, transferrins, haptoglobin, fibrinogen, alpha2-macroglobulin, α 1- acid sugar Albumen, transthyretin and complement-C3) with abundance protein (myosin and golgi protein 73) in 2 kinds of serum.
3. cell-fusion techniques described in claim 1 and Antibody library, including authentic monoclonal antibody method are exempted from mouse Epidemic disease source phage displaying antibody library method.
4. expression system described in claim 1, including prokaryotic expression system and eukaryotic expression system.
5. gene modification described in claim 1, the N- glycosylation site gene order mutation for being based primarily upon antibody peptide chain is carried out Change structure.
6. antibody obtained by claim 1-5, for establishing immunology detection technology (the agglutinin captureization based on agglutinin capture Learn electrochemiluminescent immunoassay technology and agglutinin enzyme-linked immunosorbent assay etc.), the Aberrant glycosylation of the specific glycoprotein of quantitative detection serum Component.Can be applied to a variety of diseases, (kinds of tumors such as primary hepatoma, colorectal cancer, cancer of pancreas, itself are exempted from cirrhosis Epidemic disease disease etc.) screening and diagnosis.
CN201810905323.3A 2018-08-09 2018-08-09 A kind of preparation method for antibody of the specific glycoprotein of novel serum Pending CN108976301A (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110045126A (en) * 2019-04-03 2019-07-23 中国医学科学院北京协和医院 A kind of biomarker and application thereof for diagnosis of autoimmune pancreatitis
CN112924671A (en) * 2021-01-28 2021-06-08 中国医学科学院北京协和医院 Biomarker for diagnosing rheumatoid arthritis combined with pulmonary interstitial fibrosis and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110045126A (en) * 2019-04-03 2019-07-23 中国医学科学院北京协和医院 A kind of biomarker and application thereof for diagnosis of autoimmune pancreatitis
CN112924671A (en) * 2021-01-28 2021-06-08 中国医学科学院北京协和医院 Biomarker for diagnosing rheumatoid arthritis combined with pulmonary interstitial fibrosis and application thereof

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