CN109957006A - A kind of plain polypeptide artificial antigen of metabolism and antibody and application - Google Patents
A kind of plain polypeptide artificial antigen of metabolism and antibody and application Download PDFInfo
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- CN109957006A CN109957006A CN201711407757.2A CN201711407757A CN109957006A CN 109957006 A CN109957006 A CN 109957006A CN 201711407757 A CN201711407757 A CN 201711407757A CN 109957006 A CN109957006 A CN 109957006A
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- antibody
- polypeptide
- metabolism
- artificial antigen
- plain
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- 229920001184 polypeptide Polymers 0.000 title claims abstract description 53
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 53
- 230000004060 metabolic process Effects 0.000 title claims abstract description 28
- 239000000427 antigen Substances 0.000 title claims abstract description 26
- 108091007433 antigens Proteins 0.000 title claims abstract description 26
- 102000036639 antigens Human genes 0.000 title claims abstract description 26
- 230000004913 activation Effects 0.000 claims abstract description 11
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 10
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 10
- 150000001875 compounds Chemical class 0.000 claims abstract description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 210000002966 serum Anatomy 0.000 claims description 12
- 238000001514 detection method Methods 0.000 claims description 5
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- 238000012360 testing method Methods 0.000 abstract description 8
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- 108090000573 Osteocalcin Proteins 0.000 description 3
- 239000006180 TBST buffer Substances 0.000 description 3
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 3
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- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 102000004067 Osteocalcin Human genes 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
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- OVBICQMTCPFEBS-SATRDZAXSA-N (2s)-1-[(2s)-2-[[(2s)-2-[[(2r)-2-[[(2s)-2-[[(2s)-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-acetamido-3-naphthalen-2-ylpropanoyl]amino]-3-(4-chlorophenyl)propanoyl]amino]-3-pyridin-3-ylpropanoyl]amino]-3-hydroxypropanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-6-[bi Chemical compound CC(O)=O.CC(O)=O.C([C@@H](C(=O)N[C@H](CCCCN=C(NCC)NCC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN=C(NCC)NCC)C(=O)N1[C@@H](CCC1)C(=O)N[C@H](C)C(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](CC=1C=NC=CC=1)NC(=O)[C@@H](CC=1C=CC(Cl)=CC=1)NC(=O)[C@@H](CC=1C=C2C=CC=CC2=CC=1)NC(C)=O)C1=CC=C(O)C=C1 OVBICQMTCPFEBS-SATRDZAXSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- UGPCUUWZXRMCIJ-KKUMJFAQSA-N Cys-Tyr-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CS)N UGPCUUWZXRMCIJ-KKUMJFAQSA-N 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- LJPIRKICOISLKN-WHFBIAKZSA-N Gly-Ala-Ser Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O LJPIRKICOISLKN-WHFBIAKZSA-N 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- OGUUKPXUTHOIAV-SDDRHHMPSA-N Leu-Glu-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N OGUUKPXUTHOIAV-SDDRHHMPSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102100031475 Osteocalcin Human genes 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 229920002535 Polyethylene Glycol 1500 Polymers 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- YFNOUBWUIIJQHF-LPEHRKFASA-N Pro-Asp-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC(=O)O)C(=O)N2CCC[C@@H]2C(=O)O YFNOUBWUIIJQHF-LPEHRKFASA-N 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- SSYBNWFXCFNRFN-GUBZILKMSA-N Val-Pro-Ser Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O SSYBNWFXCFNRFN-GUBZILKMSA-N 0.000 description 1
- 229930003448 Vitamin K Natural products 0.000 description 1
- VLSOAXRVHARBEQ-UHFFFAOYSA-N [4-fluoro-2-(hydroxymethyl)phenyl]methanol Chemical compound OCC1=CC=C(F)C=C1CO VLSOAXRVHARBEQ-UHFFFAOYSA-N 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 125000003647 acryloyl group Chemical group O=C([*])C([H])=C([H])[H] 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
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- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
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- 238000004364 calculation method Methods 0.000 description 1
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- 108700032141 ganirelix Proteins 0.000 description 1
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- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- FSQQTNAZHBEJLS-UPHRSURJSA-N maleamic acid Chemical compound NC(=O)\C=C/C(O)=O FSQQTNAZHBEJLS-UPHRSURJSA-N 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- SHUZOJHMOBOZST-UHFFFAOYSA-N phylloquinone Natural products CC(C)CCCCC(C)CCC(C)CCCC(=CCC1=C(C)C(=O)c2ccccc2C1=O)C SHUZOJHMOBOZST-UHFFFAOYSA-N 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 238000013102 re-test Methods 0.000 description 1
- 108010048734 sclerotin Proteins 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 238000002444 silanisation Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- POECFFCNUXZPJT-UHFFFAOYSA-M sodium;carbonic acid;hydrogen carbonate Chemical compound [Na+].OC(O)=O.OC([O-])=O POECFFCNUXZPJT-UHFFFAOYSA-M 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 235000019168 vitamin K Nutrition 0.000 description 1
- 239000011712 vitamin K Substances 0.000 description 1
- 150000003721 vitamin K derivatives Chemical class 0.000 description 1
- 229940046010 vitamin k Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- 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
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/35—Valency
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Endocrinology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to antibody arts, in particular to a kind of plain polypeptide artificial antigen of metabolism and antibody and application.A kind of plain polypeptide artificial antigen of metabolism, is obtained by the carrier protein couplet that polypeptide is activated with aminated compounds；The amino acid sequence of the polypeptide is as shown in SEQ ID NO:1.The plain polypeptide artificial antigen of metabolism provided by the invention, polypeptide therein contains cysteine, it is obtained through test, the macro-molecular protein KLH of the polypeptide and activation is coupled to form the comlete antigen with immunogenicity, and the intracorporal immune system of mouse can be excited to generate antimetabolic element monoclonal antibody.
Description
Technical field
The present invention relates to antibody arts, in particular to a kind of plain polypeptide artificial antigen of metabolism and antibody and application.
Background technique
Metabolism element is one section of conserved sequence in Mouse Bone calcium cellulose protein molecule, and analyzing this segment polypeptide by sequence has
Antigenicity.It includes albumen (bone gamma-carboxyglutamic-acid- that osteocalcin, which is also known as bone γ-carboxylic glutamic acid,
containing proteins,BGP).The albumen just gathers after the bone mineralising peak phase.Using vitamin K antagon,
Content of this albumen in bone can be made to reduce, but have no effect on the content of its proline, nor affect on the mechanical strength of bone.Bone calcium
Element is secreted by osteoblast, and content is different with the variation at age, will be seen that by the content of serum osteocalcin
The state of osteoblast has great importance for the judgement etc. of osteoporosis.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of plain polypeptide artificial antigen of metabolism, which contains cysteine, pass through
Test obtains, and the macro-molecular protein KLH of the polypeptide and activation is coupled to form the comlete antigen with immunogenicity, can excite
The intracorporal immune system of mouse generates antimetabolic element monoclonal antibody.
The second object of the present invention is to provide antibody made from above-mentioned antigen, which can be used in detecting mouse blood
The relevant peptide molecule of metabolism element in clear.
The third object of the present invention is to provide the kit containing above-mentioned antibody, for the relevant peptide molecule of metabolism element
Detection provide convenience.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of plain polypeptide artificial antigen of metabolism, is obtained by the carrier protein couplet that polypeptide is activated with aminated compounds；It is described
The amino acid sequence of polypeptide is as shown in SEQ ID NO:1.
The plain polypeptide artificial antigen of metabolism provided by the invention, polypeptide therein contain cysteine, obtain through test, this is more
Peptide and the macro-molecular protein KLH of activation are coupled to form the comlete antigen with immunogenicity, can excite mouse is intracorporal to exempt from
Epidemic disease system generates antimetabolic element monoclonal antibody.
Further, the carrier protein is spoon sky hemocyanin.
Further, the aminated compounds is maleimide.
The plain polypeptide artificial antigen of metabolism provided by the invention can be prepared by the following method:
The plain polypeptide dissolution of the metabolism as shown in SEQ ID NO:1, obtains being metabolized the polypeptide that plain peptide concentration is 6-9mg/ml
Solution；
The carrier protein of activation dissolves, and obtains the carrier protein solution that the activated carrier protein concentration is 4-6mg/ml；
The polypeptide solution is that 1:2-3 is mixed with volume ratio with the carrier protein solution, is coupled；
The product of the coupling is isolated and purified to obtain the plain polypeptide artificial antigen of the metabolism.
The preparation method provided by the invention for being metabolized plain polypeptide artificial antigen, it is simple and easy to do, and be coupled and imitate through this method
Rate is high, 90% or more.
Further, the solvent for being metabolized plain polypeptide dissolution is 20 ± 1mM sodium phosphate buffer, wherein also containing 230
± 5mM NaCl, 2 ± 0.2mM EDTA, 80 ± 2mM sucrose, pH6.5-6.7.
Further, the solvent of the carrier protein dissolution of the activation is 20 ± 1mM sodium phosphate buffer, wherein also containing
100 ± 5mM EDTA, 80 ± 2mM sucrose, pH 6.5-6.7.
Further, coupling condition is to continue 2 hours or 2-8 DEG C overnight in room temperature under stiring.
The present invention also provides antibody made from above-mentioned metabolism element polypeptide artificial antigen.
Further, the antibody is monoclonal antibody.
Further, the hypotype of the monoclonal antibody is G2a.
Further, the potency of the monoclonal antibody is 1:4000.
The present invention also provides answering in the plain polypeptide fragment of antibody metabolism present in detection serum or tissue
With.
The present invention also provides the kits for containing the antibody.
Further, the kit is ELASA kit.
ELISA kit provides a kind of specific serological and refers to for the diagnosis and identification of adult's sclerotin and related disease
Mark, to improve the accuracy of diagnosis.
Further, the kit further includes ELISA Plate, yin and yang attribute referring to product, two corresponding anti-solution, developing solution, reaction terminating
Liquid, concentrated cleaning solution, sample diluting liquid, sealing plate film and Fresco Bag, product description and product quality report any one of or
It is a variety of.
Compared with prior art, the invention has the benefit that
(1) a kind of plain polypeptide artificial antigen of metabolism provided by the invention, the polypeptide contain cysteine, obtain through test,
The polypeptide and the macro-molecular protein KLH of activation are coupled to form the comlete antigen with immunogenicity, can excite in Mice Body
Immune system generate antimetabolic element monoclonal antibody.
(2) monoclonal antibody provided by the invention can be used in detecting the relevant polypeptide point of metabolism element in mice serum
Son, specificity and sensitivity are good.
(3) present invention provides the kit for containing above-mentioned antibody, and the detection for the relevant peptide molecule of metabolism element provides
It is convenient.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 is the KLH structural schematic diagram of maleimide activation in the embodiment of the present invention 1；
Fig. 2 is the potency result linear graph that number is 21 monoclonal antibodies in the embodiment of the present invention 3；
Fig. 3 is the result figure of monoclonal antibody (21#) and the plain polypeptide identification specificity of metabolism in the embodiment of the present invention 4.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
In order to better understand the present invention, special to be stated the preparation process of monoclonal antibody, unless otherwise specified,
For conventional method.Agents useful for same material is conventional biochemical reagent unless otherwise specified in case study on implementation.
Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.Used in embodiment
Phosphate buffer is the PBS buffer solution of pH 7.4,0.01M.Carbonate buffer solution used in embodiment be pH 9.6,
The sodium carbonate buffer of 0.05M.Bovine serum albumin(BSA) abbreviation BSA, spoon sky hemocyanin are referred to as KLH.
Embodiment 1
A, maleimide activation KLH and polypeptide are coupled
1. opening the KLH addition 1ml 20mM sodium phosphate buffer (as shown in Figure 1) of a pipe maleimide activation
(230mM NaCl, 2mM EDTA, 80mM sucrose, pH 6.6), which is that the KLH of maleimide activation is reconstructed,
Obtain the solution of 5mg/ml, matching while using；
2. preparing 20mM sodium phosphate buffer (100mM EDTA, 80mM sucrose, pH 6.6), as coupling buffer；
3. 4mg polypeptide is dissolved in 0.5ml coupling buffer, polypeptide solution is obtained, 50 μ l polypeptide solutions is left and taken and is used for really
Determine coupling efficiency (total cys), saves to 2-8 DEG C.
4. immediately mixing the KLH solution that 0.5ml polypeptide solution and 1ml maleic amide activate in reaction tube, stir, room
Warm (15-25 DEG C) continues 2 hours or 2-8 DEG C overnight (8-15 hours), and the polypeptide after obtaining coupling KLH is named as later
NH2-KLH；
5. retaining 100 μ l coupling reaction liquid for determining coupling efficiency (free cys).
B, the separation of KLH conjugate
1. 0.01% Sodium azide, long-term preservation is added in 1L PBS；
2. Sephadex G-25M gel column is fixed on suitable beaker；
3. removing the lid of gel column, the bottom end of gel column is cut off, allows excessive liquid to flow out, forbids liquid flow in column
It is dry；
4. 30ml PBS balanced gel column is used, if after upper and lower lid please not cover using gel column immediately, saved extremely
2-8℃；
5. the liquid after reaction is added to gel column, lower end trickle is collected；
6. being washed with total volume 10ml PBS, eluate is collected, 0.5ml-1.0ml is collected each time, is measured at 280nm
Absorbance value；
Contain protein eluate 7. collecting, -20 DEG C freeze.
C cysteine standard curve
1.DTNB analytical solution (0.1mM): add the Tris buffer of 99 volume 0.25M by 1 volume 10mMDTNB titer
It is formulated, matching while using；
2. one water object of 32mg L-cysteine hydrochloride is dissolved in 1ml distilled water, gradient dilution range 0.4-
0.04mg/ml；
3. prediluted 50 μ l of cysteine standard liquid is added in testing tube, 50 μ l distilled waters are added in blank tube；
4. 0.1ml distilled water is added in all pipes, 0.1ml DTNB examination is added in 0.75ml pH8.0DTNB buffer immediately
Agent solution makes total volume 1ml, mixes；
5. measuring absorbance value at 412nm, standard curve is made, in triplicate.
The measurement of D coupling efficiency
1. 50 μ l polypeptide solutions (total cys) are added in 0.1ml distilled water；
2. by following 50 μ l solution be added testing tube in: DTNB buffer (blank), diluted polypeptide sample (total cys,
KLH combination), polypeptide-K LH (free cys, KLH combination), diluted polypeptide sample (total cys, BSA combination), polypeptide-BSA (trip
From cys, BSA combination)；
3. 0.1ml distilled water is added into all test tubes, 0.1ml is added in the DTNB buffer of 0.75ml pH8.0 immediately
DTNB reagent solution, final volume 1ml are mixed；
4. absorbance value is measured at 412nm, if absorbance value is greater than 1.4, dilute sample retest；
5. calculation formula: coupling efficiency (%)=(total cys- dissociate cys)/total cys ╳ 100%
Obtained coupling efficiency is 90% or more.
Embodiment 2
Polypeptide is metabolized the preparation of plain monoclonal antibody
BALB/c female mice is purchased from Guangdong Province's Experimental Animal Center.
A animal immune
The NH2-KLH immunogene prepared in embodiment 1 is used for the immune of BALB/c female mice, initial immunity presses 60 μ g
Albumen/mouse amount, 4 SPF Balb/c female mices of subcutaneous inoculation, number 1-4 is primary every 2 weeks booster immunizations,
It is specific as follows:
Subcutaneous first time booster immunization, the amount of being immunized are 30 μ g albumen/only；
Subcutaneous second of booster immunization, be immunized amount be 30 μ g albumen/only；
Subcutaneous third time booster immunization, the amount of being immunized are 30 μ g albumen/only；
Subcutaneous 4th booster immunization, be immunized amount be 30 μ g albumen/only.
Each mouse orbit takes blood, surveys serum titer.
Immunizing potency detection:
Step: using " NH2-BSA ", and 2 μ g/ml, 4 DEG C of coatings are overnight；2% milk, 37 DEG C of closing 2h；Serum is opened from 200 times
Begin 2 times of gradient dilutions, and blank control (blank) is PBS, and negative control (negative) is 200 times of negative serum dilutions.Potency
For the corresponding dilution of the minimum OD reading greater than maximum OD/2.
The results are shown in Table 1 for potency.
1 potency result of table
From determination data as can be seen that the Mouse titers of number 4 are excellent, therefore, chooses impact 4# mouse and do cell fusion reality
It tests.
B cell fusion experiment
Mouse boosting cell and SP2/0 cell are taken, is merged using PEG method.Cell semisolid culturemedium has been merged (to contain
HAT screening and culturing) is carried out.
1. experiment equipment
The surgical instrument of sterilizing: three scissors, three tweezers, a cell sieve, the inner core of syringe, one it is flat
Ware.Wet box, 2 500ml beakers, 2 50ml centrifuge tubes, 3 15ml centrifuge tubes.
2. experiment reagent
IMDM culture medium；IMDM complete medium (contains 15% serum)；2.2% methylcellulose, producer SIGMA, article No.:
M0262-100G；Newborn bovine serum: 10ml；PEG1500, producer Roche, article No.: 78364；HAT: producer Sigma, article No.:
H0262-10VL。
3. fusion experiment step
A) it is blown and beaten from culture bottle wall by sp2/0 cell in good condition is soft, is drawn into 50ml centrifuge tube.
B) 50 μ g of NH2-KLH immunogene is used, #4 mouse is impacted in abdominal cavity, and the neck that breaks is put to death, and is put into 75% alcohol and is impregnated
5min。
C) IMDM that a small amount of serum-free is poured into plate, cell sieve and plunger are put into plate.Use scissors
The spleen that mouse is removed with tweezers, is put into cell sieve.Lightly spleen is sufficiently pulverized with the inner core of syringe, it is thin by what is ground
Born of the same parents are drawn into the centrifuge tube of dress sp2/0, are centrifuged 1500r/min, 5min.
D) thymus gland that mouse is removed with scissors and tweezers, pulverizes.The thymocyte ground is put into 15ml centrifuge tube, then
The HAT of 1ml is added, is placed on spare in incubator.
E) cell that will be centrifuged outwells supernatant, and cell is carefully gently blown to even, centrifugation with the IMDM of serum-free,
1500r/min, 5min.
F) cell conditioned medium being centrifuged is outwelled as far as possible.The abundant suspension cell in centrifuge tube bottom is patted, centrifuge tube is put into 37
In DEG C warm water, it is slowly added to the PEG of 1ml in 1 minute, after adding, 1min is stood in warm water.Then it is slowly added in 2min
The IMDM of the serum-free of 2ml is then slowly added to the IMDM of 8ml serum-free in 2min.It is centrifuged 1000r/min, 5min.
G) supernatant is outwelled, the serum of 10ml is added, careful blow cell is even, pours into the ready thymocyte in front.
The sterilized semisolid culturemedium of 25ml is added, is mixed well.Then it uniformly pours into 30 Tissue Culture Dish.Cell is trained
Feeding ware is put into wet box, is then placed in incubator and is cultivated.
C chooses clone
10 plate × 93 cell monoclonals are chosen, are incubated at 96 porocyte culture plates (in advance with thymocyte bed board, 100 μ l/
Hole).
The screening of D monoclonal cell
(1) experiment reagent
Coating buffer: sodium carbonate-bicarbonate buffer, pH 9.6；
Confining liquid: 2% milk in PBS washing lotion；
Developing solution: 1%A liquid+10%B liquid (A liquid: 1%TMB in DMSO；B liquid: 0.1%H2O2In citrate buffer solution)；
Terminate liquid: 2M sulfuric acid；
Secondary antibody: goat anti-mouse IgG/HRP
(2) experimental procedure
1) " NH2-BSA " is diluted with coating buffer, final concentration of 2 μ g/ml, 100 holes μ l/, 4 DEG C, overnight；It is washed afterwards with washing lotion
3 times.
2) 2% milk confining liquid is closed, 200 holes μ l/, 37 DEG C of incubators, 2h；It is washed 3 times with washing lotion afterwards.
3) addition primary antibody (cells and supernatant), negative control (SP2/0 culture supernatant), blank control (PBS), the positive are right
It is 100 holes μ l/, 37 DEG C of incubators, 1h according to (200 times of positive serum PBS dilutions)；It is washed 3 times with washing lotion afterwards.
4) secondary antibody that PBS (pH7.4) dilutes 20000 times, 100 holes μ l/, 37 DEG C of incubators, 1h is added；It is washed after taking-up with washing lotion
It washs 3 times.
5) it develops the color, 100 hole μ l/ of developing solution, developing time is 5min or so.
6) every hole is added 50 μ l terminate liquids and terminates.
7) dual wavelength (450,630) surveys light absorption value, and record saves data, as shown in table 2.In table 2, different culture plate marks
Number NO1-10.
The OD numerical value of the different culture plate measurements of table 2
With " NH2-BSA " wrapper sheet, ELISA method is used to the clone selected, does and screens for the first time, in addition to positive control
Group, OD value share 36 plants of positive hybridoma cell strains greater than 0.15.
E monoclonal cell programmed screening
By 36 plants of positive cell strains, with " NH2-BSA ", wrapper sheet using ELISA method does programmed screening again, obtains
To 17 plants of positive hybridoma cell strains.
F monoclonal cell subgroup identification
Steps are as follows:
1. experiment reagent
Coated antibody: Southern Biotech；Confining liquid: 2%BSA+3% sucrose in PBS；
Developing solution :+10 μ l 30%H of 0.2ml A liquid2O2In 10ml B liquid (A liquid: 15mg/ml ABTS in H2O；B liquid:
Citrate buffer solution, pH4.0)；Various subclass secondary antibody.
2. experimental procedure
A) with 100mM PBS (pH7.4) dilution coated antibody to 0.5 μ g/ml, every hole adds 0.1ml, 4 DEG C, stays overnight.
B) PBS-T is washed 2 times, and 200 μ l confining liquids, 37 DEG C of incubation 2h are added in every hole.
C) PBS-T is washed 3 times, and 100 μ l hybridoma supematants, 37 DEG C of incubation 1h are added in every hole.
D) PBS-T is washed 3 times, with the antibody of the diluted HRP label of confining liquid 1:1000 (κ, λ) or 1:2000 (others)
The every hole 0.1ml, is separately added into hole appropriate, 37 DEG C of incubation 1h.
E) PBS-T is washed 3 times；Every hole adds 50 μ l substrate solutions, and 10-20min is interior to survey light absorption value in dual wavelength (450,630),
Record saves data.
The 17 plants of positive cell strains screened carry out subgroup identification, finally obtain the positive hybridoma cell of 14 plants of IgG
Strain.It is specific as shown in Table 3 and Table 4.
The subtype data of 3 17 plants of positive cell strains of table measurement
The positive hybridoma cell strain of 4 14 plants of IgG of table
Embodiment 3
Antibody titer measurement-ELISA method
1. antigen coat: polypeptide antigen is diluted to 2 μ g/ml of concentration, is added 96 orifice plates, every 100 μ l of hole, in 4 DEG C of wet box
Coating is overnight；
2. washing: washing coated ELISA Plate, 200 holes μ l/, washing 3 with the PBS buffer solution containing 0.05% Tween-20
It is secondary, 5min/ times；
3. closing: being closed with 1%BSA solution to 96 orifice plates, 250 holes μ l/, 37 DEG C of wet box inner sealing 2h；
4. washing: ibid washing methods；
5. primary antibody be incubated for: by rabbit anteserum polyclonal antibody according to 1:1000,1:2000,1:4000,1:8000,1:16000,
1:32000,1:64000 are diluted with PBS buffer solution, while blank control and negative serum control is arranged, 100 holes μ l/, and 37
1h is incubated in DEG C wet box；
6. washing: ibid washing methods；
7. secondary antibody is incubated for: the HRP goat anti-mouse igg antibody marked being diluted with PBS buffer solution according to 1:5000,100 μ l/
Hole, 37 DEG C of wet box are interior to be incubated for 1h；
8. washing: ibid washing methods；
9. adding substrate: 96 orifice plates are added according to 100 holes μ l/ in TMB, are protected from light 20min in 37 DEG C of wet box；
10. terminating reaction: using 2M H2SO496 orifice plates are added according to 50 holes μ l/ and terminate reaction, with microplate reader in 450nm wave
Absorbance value is measured when long, the OD positive/OD feminine gender > 2.1 is serum titer criterion.
11. it is as shown in Figure 2 to screen the potency result that obtained number is 21 monoclonal antibodies.
Obtaining the antibody titer is 1:4000.
Embodiment 4
Monoclonal antibody identifies specificity-Western blot method
1. the preparation of protein sample:
Will after the pipe high pressure sterilization of silanization be added 0.1%BSA and 0.9%NaCl mixed solution after soaked overnight,
Solution was sucked out to and was made pipe natural air drying in second day, polypeptide is added in air-dried pipe and freezes.Pipette tips are equally used 0.1%
The mixed solution soaked overnight of BSA and 0.9%NaCl uses ddH in second day2O rinses (3 times) well high pressure sterilization and dries standby afterwards
With.
2.SDS-PAGE
It selects stand-by after suitable glass plate wiped clean.First with prepared separation gel is added after 1.5% agar edge sealing,
Space needed for reserving concentration glue, is added on 1ml deionized water rubber cover, is allowed to and air exclusion, in favor of the cohesion of separation gel.
After the completion of separation gel polymerization, coating water is poured out, rinses the top of glue for several times to remove unpolymerized acryloyl with deionized water
Amine sucks remaining liquid at the top of glue with filter paper.Glue is concentrated in preparation, and careful injection separation gel upper layer rapidly is inserted into completely at once
Comb cannot generate bubble, place to solidifying.
After glue polymerization completely to be concentrated, electrophoretic buffer is added, carefully extracts comb.The total protein of cell sample that will be prepared
Product loading in order.1 × sample-loading buffer is added in the sample well not being loaded.With constant pressure from cathode to positive electrophoresis, bromophenol blue
Voltage is 80V in concentration glue, and voltage increases to 120V after bromophenol blue enters separation gel, continues electrophoresis and divides until bromophenol blue reaches
From glue bottom, power supply is closed, gel is unloaded.
3.Western-blot
1) a clip pvdf membrane sum number filter paper identical with gel size, electricity consumption, which turns liquid, which impregnates filter paper and electrophoresis, terminates
Gel afterwards, pvdf membrane are first impregnated 10 seconds with methanol, and deionized water is rinsed, and are then soaked in electricity again and are turned in liquid；
2) plastic stent of protein electrophoresis instrument is opened, one piece of sponge is respectively placed in two sides, according to from cathode to anode successively
It is placed for filter paper-gel-pvdf membrane-filter paper order, removes bubble, plastic stent clamping is put into electric turn trough, is powered on,
Turn 1h in ice bath constant current 400mA electricity；
3) sample film that electricity takes a turn for the better is placed in room temperature in the TBST containing 5% skimmed milk power and closes 1h；
4) film is placed in room temperature jog 2-3h in the primary antibody of appropriate extension rate, or overnight in 4 DEG C of reactions；
5) film is washed twice with TBST；
6) film is placed in room temperature jog 1-2h in secondary antibody, or overnight in 4 DEG C of reactions；
7) film is washed twice with TBST；
4. development
Suck excessive moisture on film, after film is reacted 1min in ECL reaction solution, suck moisture content on film, by sample film in
Developed with X-ray tabletting in darkroom.
The i.e. above-mentioned metabolism element polypeptide result specific using Western blot method verifying monoclonal antibody (21#) identification
As shown in Figure 3.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
SEQUENCE LISTING
<110>Shenzhen Xianjin Technology Academe
<120>a kind of plain polypeptide artificial antigen of metabolism and antibody and application
<130> 2010
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 16
<212> PRT
<213>artificial sequence
<400> 1
Cys Tyr Leu Gly Ala Ser Val Pro Ser Pro Asp Pro Leu Glu Pro Thr
1 5 10 15
Claims (10)
1. a kind of plain polypeptide artificial antigen of metabolism, which is characterized in that by the carrier protein couplet of polypeptide and aminated compounds activation
It obtains；The amino acid sequence of the polypeptide is as shown in SEQ ID NO:1.
2. the plain polypeptide artificial antigen of metabolism according to claim 1, which is characterized in that the carrier protein is that spoon sky blood is blue
Albumen.
3. the plain polypeptide artificial antigen of metabolism according to claim 2, which is characterized in that the aminated compounds is Malaysia acyl
Imines.
4. antibody made from the plain polypeptide artificial antigen of the described in any item metabolism of claim 1-3.
5. antibody according to claim 4, which is characterized in that the antibody is monoclonal antibody.
6. antibody according to claim 5, which is characterized in that the hypotype of the monoclonal antibody is G2a.
7. antibody according to claim 6, which is characterized in that the potency of the monoclonal antibody is 1:4000.
8. answering in the plain polypeptide fragment of the described in any item antibody of claim 4-7 metabolism present in detection serum or tissue
With.
9. the kit containing the described in any item antibody of claim 4-7.
10. kit according to claim 9, which is characterized in that the kit is ELASA kit.
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| WO2011140086A2 (en) * | 2010-05-03 | 2011-11-10 | Bristol-Myers Squibb Company | Serum albumin binding molecules |
| CN103524629A (en) * | 2013-10-25 | 2014-01-22 | 中国科学院深圳先进技术研究院 | Complete antigen of AG22 polypeptide, preparation method and antibody thereof |
| CN103554249A (en) * | 2013-10-25 | 2014-02-05 | 中国科学院深圳先进技术研究院 | Complete antigen of AG15 polypeptide as well as preparation method and antibody thereof |
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| CN106892975A (en) * | 2017-03-16 | 2017-06-27 | 深圳先进技术研究院 | Adjust polypeptide of glycometabolism and application thereof |
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| WO2011140086A2 (en) * | 2010-05-03 | 2011-11-10 | Bristol-Myers Squibb Company | Serum albumin binding molecules |
| CN103524629A (en) * | 2013-10-25 | 2014-01-22 | 中国科学院深圳先进技术研究院 | Complete antigen of AG22 polypeptide, preparation method and antibody thereof |
| CN103554249A (en) * | 2013-10-25 | 2014-02-05 | 中国科学院深圳先进技术研究院 | Complete antigen of AG15 polypeptide as well as preparation method and antibody thereof |
| CN106353506A (en) * | 2016-08-12 | 2017-01-25 | 江苏泽成生物技术有限公司 | Kit for detecting osteocalcin content and testing method thereof |
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