CN108285893A - The monoclonal antibody of hybridoma cell strain and anti-human CTRP3b and its application - Google Patents
The monoclonal antibody of hybridoma cell strain and anti-human CTRP3b and its application Download PDFInfo
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- CN108285893A CN108285893A CN201711460128.6A CN201711460128A CN108285893A CN 108285893 A CN108285893 A CN 108285893A CN 201711460128 A CN201711460128 A CN 201711460128A CN 108285893 A CN108285893 A CN 108285893A
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- Prior art keywords
- ctrp3b
- antibody
- cell strain
- hybridoma cell
- human
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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
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- 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
Abstract
The present invention relates to the monoclonal antibodies of hybridoma cell strain and anti-human CTRP3b and its application.The deposit number of involved hybridoma cell strain is respectively CCTCC NO:C201797 and CCTCC NO:C201798.Involved antibody is the antibody of the hybridoma cell strain secretion of the present invention.The antibody of the involved invention that bases on practicality is used to detect the application of people CTRP3b and the antibody of the present invention is used to prepare the application of people's CTRP3b detection kits.
Description
Technical field
The present invention relates to hybridoma cell strain, antibody and its applications, and in particular to the hybridoma for detecting people CTRP3b
Cell strain, antibody and its application.
Background technology
C1q and tumor necrosis factor related protein 3 (CTRP3) are mainly by fatty thin
The Adipocyte Factor of born of the same parents or fibroblasts to secrete, in the organs such as kidney, small intestine, lungs and fibroblast, histocyte
In also have it is expressed.CTRP3 is made of secreting signal peptide, collagenous domain and C-terminal globular domain, and maturation protein is free of its N
End signal peptide (22 amino acid residues).C-terminal globular domain is similar with C1q structures, can be with other albumen or receptor phase
Interaction, therefore be considered as its functional domain.CTRP3 is with the following functions：(1) in glycolipid metabolism adjusting, inflammatory reaction
Inhibition, cardiovascular protection and reduction Apoptosis and necrosis etc. have effect；(2) glucose -lipid metabolism disorder, inflammation can be mitigated
Disease reacts and the metabolism related diseases such as immunologic inadequacy；CTRP3 can reduce glycogen output, promote Adipocyte Differentiation and fat
Fat acid stores；Meanwhile the level of CTRP3 shows promise as the marker molecule of diabetes and metabolic syndrome；(3) CTRP3 can
The release for inhibiting the proinflammatory factors such as IL-6 and the TNF-α that the signal paths such as NF- κ B, TGF-β participate in, inhibits inflammatory reaction；(4)
There is induction osteosarcoma cell to generate, promote chondrocyte proliferation, inducing adipocyte differentiation, promote vascular wall smooth by CTRP3
The physiological actions such as muscle cell multiplication and myocardial preservation can mitigate the myocardial remodelling and fibrosis of rat after myocardial infarction.Except this
Except, CTRP3 is expected to play a significant role in the heart failure etc. of seeking peace for the treatment of Metabolic syndrome.
In human body, after the transcription of CTRP3 encoding genes, two kinds of CTRP3 are formed by translation by alternative splicing：
CTRP3a and CTRP3b albumen.CTRP3b albumen 70 amino acid residues more than the collagenous domain ratio CTRP3a, remaining is homogeneous
Together.Aforementioned CTRP3 progress is using CTRP3a as research object.Our result of study is shown, in certain pathology feelings
Under condition, CTRP3b levels have significant change, prompt may have important researching value.
Currently, it is less for the research of people's CTRP3b albumen, it there is no the detection kit of specific detection CTRP3b.
Invention content
In view of the drawbacks of the prior art or insufficient, the present invention provides a pair for generating anti-human CTRP3b monoclonal antibodies
Hybridoma cell strain：
XA297-7, the hybridoma cell strain are preserved in China typical culture collection center, deposit number：CCTCC NO：
C201797, address：Wuchang, wuhan Luo Jia Shan Wuhan University；
XA97-11, the hybridoma cell strain are preserved in China typical culture collection center, deposit number：CCTCC NO：
C201798, address：Wuchang, wuhan Luo Jia Shan Wuhan University.
The antibody sequence that the hybridoma cell strain of the present invention generates：XA297-7 weight chain variabl area sequences such as SEQ.ID.NO.1
It is shown；Light-chain variable sequence is as shown in SEQ.ID.NO.2.
The XA297-11 weight chain variabl area sequences of the present invention are as shown in SEQ.ID.NO.3；Light-chain variable sequence is such as
Shown in SEQ.ID.NO.4.
The anti-human CTRP3b monoclonal antibodies that hybridoma cell strain secretion of the present invention generates.
Antibody of the present invention is used to detect the application of people CTRP3b.
Further, antibody of the present invention is used to prepare the application of people's CTRP3b detection kits.
The present invention also provides people's CTRP3b detection kits.The people's CTRP3b detection kits provided include above-mentioned anti-
Body.
The present invention kit further include：Sample diluting liquid, substrate dilution, substrate, cleaning solution, standard items and nonionic
Activating agent.
The present invention easy to operate, of low cost, high sensitivity, high specificity quantitative detection body fluid in CTRP3b concentration
ELISA kit and its preparation method and application.
Description of the drawings
Fig. 1 is the electrophoretogram of recombined human CTRP3b albumen, 1, Marker；2,500mM imidazole elutions；3, recombined human
CTRP3b albumen.
Fig. 2 this kit representative standard curves.
Specific implementation mode
The present invention is further elaborated by the following examples.Embodiment is that the present invention is described in detail, but
The present invention is not by these any restriction.
The horseradish peroxidase (HRP) used in following embodiment is limited purchased from Shanghai life work biotechnology service
Company；Gene order optimization, synthesis clone are completed by the prosperous bio tech ltd of Beijing AudioCodes；Antibody sequencing is by Nanjing gold
It completes the bio tech ltd Si Rui；Ni-NTA purification columns are purchased from GE companies；Endotoxin removal column purchased from the silent winged generation of match you (in
State) Co., Ltd；The pools DMEM high culture medium, fetal calf serum are Hycolon Products；The purchase of overall length CTRP3 albumen is certainly
AVISCERA BIOSCIENCE companies.
Embodiment 1：The preparation of hybridoma cell strain and antibody of the present invention
According to people CTRP3b sequences (GenBank ACCESSION NM_181435.5) open reading frame sequence, and according to
E. coli codon Preference carries out codon optimization, carries out full genome synthesis (sequence is as shown in SEQ.ID.NO.5), will close
At gene be connected between the EcoRI and HindIII of pET30a (+), using IPTG (final concentration 1mM) carry out 16 DEG C,
200rpm stays overnight induced expression, and thalline is harvested by centrifugation in 4000g.After ultrasonication, 12000g centrifuging and taking supernatants.Supernatant passes through nickel column
Affinity purification is changed to PBS buffer solution, using Millipore Utral- after imidazole concentration gradient elution by dialysis
15 concentrations, after removing endotoxin, as recombined human CTRP3b albumen.(referring to attached drawing 1)
6-8 week old Balb/C female mices are chosen, CTRP3b albumen is used as antigen to obtain high-affinity antibody
Low dose of (about 1 μ g), long time-histories, repeatedly immune method, after selection is 10 days immune, serum is detected using indirect ELISA method
Potency (envelope antigen is the albumen of digestion and purifying), potency reach 1: 5 ten thousand or more preparation fusion.
It learns from else's experience the mouse spleen of booster immunization, grinding is broken, after filtration washing, cell suspension is made and counts；Marrow
Tumor SP2/0 cell growths are collected by centrifugation and wash to logarithmic phase, mix in the ratio of 1: 10 or 1: 5 with splenocyte, adopt at room temperature
Rush fusion is carried out with PEG, 96 orifice plates containing feeder cells are added in the cell suspension after fusion, set 37 DEG C, 5%CO2, relatively
It is cultivated in saturated humidity incubator.After fusion 24 hours, 5 × HAT selections culture solution, 1 × HAT/HT selection trainings are used successively
Nutrient solution and 1 × HT culture solutions carry out fusion myeloma cell's screening.Using indirect ELISA method screening and cloning (CTRP3 is negative,
CTRP3b is positive), and carry out continuous cloning.It finally screens to obtain the hybridization for capableing of the anti-CTRP3b monoclonal antibodies of stably excreting
Tumor cell strain is respectively designated as XA297-7 and XA297-11, is preserved in China typical culture collection center, address：Wuhan
City Wuchang Luo Jia Shan Wuhan University, deposit number are respectively：CCTCC NO:C201797 and CCTCC NO:C201798.
The preparation of anti-human CTRP3b monoclonal antibodies:8-10 week old BABL/C female mices are taken, only according to 200 μ L/, abdominal cavity
Inject paraffin oil, after 1-2 weeks, hybridoma cell strain (CCTCC NO:C201797 or CCTCC NO:C201798), with 106
It is a/only amount, intraperitoneal injection, after 7-10 days, acquire mouse ascites, with indirect ELISA method detection monoclonal antibody potency.Ascites 4
DEG C, 12000rpm centrifuge 15min, mixed with the acetate buffer solutions (0.06M, pH4.8) of 2 times of volumes, caprylic acid, room be added dropwise
Temperature stirring 30min, 4 DEG C stand it and fully precipitate.It takes supernatant to filter after centrifugation, the phosphate buffer of 1/10 volume is added
(0.1M, pH7.4) is used in combination 2M NaOH to adjust pH to 7.4.Antibody is precipitated using ammonium sulfate precipitation, precipitation is taken after centrifugation, is used
It is dissolved containing 0.2mM EDTA phosphate buffers, and dialysed overnight.It is purified using ion-exchange chromatography, buffering used
Liquid：Tris-HCl buffer solutions that equilibration buffer is 20mM pH7.5, the 20mM pH7.5 that elution buffer is the NaCl containing 1.0M
Tris-HCl buffer solutions.The antibody protein that NaCl concentration gradient elution combines detects A280, collects the antibody protein of elution.
Antibody content is measured with BCA methods commonly used in the art, antibody titer is detected with indirect elisa method.
The sequencing of antibody is completed by Nanjing Genscript Biotechnology Co., Ltd., and step is：Hybridoma uses
RPMI1640 adds 20% serum free culture system for 24 hours, centrifugation, discard supernatant, using TriZol be resuspended cell, 106A cell/mL.Extraction
After DNA, heavy chain and light chain gene are expanded using RT-PCR, DNA sequencing is carried out after being cloned into carrier T.Antibody is named as：XA297-
7 heavy chain variable regions (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4)：
EVMLVESGGGFVKPGGSQKLSCVASGFTLSNYAMSWVRQTPEKRLEWVATISTGGSYIYYADSVKGRFTISRDDAKN
TLFLQMSSLRSEDTAMYYCASLYIVTVLVQDYWGHGTTLTVSS；XA297-7 light chain variable regions (FR1-CDR1-FR2-
CDR2-FR3-CDR3-FR4)：DAVMTQTPLSLPVSLGAQASISCRSSQSLVHSNGYTYLHWYLQKPGQSPKLLIYKVSN
RFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIK；XA297-11 heavy chain variable regions
(FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4)：EVQLQQSGPELVKPGASVKISCKTSGYTFNEYVIHWVKQSHGRS
LEWIGGINPNNVSTNYNQKFKGKATLTVDKSSSTAYMELRRLTSDASAVYYCVGYGYYVYWGQGTLVTVS；XA297-
11 light chain variable regions (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4)：DVLMTQTPLSLPVSLGDQASISCKSSQSIVHS
DGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCLQGSHVPWTFGGGTKL
EIK。
Embodiment 2：People's CTRP3b ELISA kits
Kit forms：The coated ELISA Plates of anti-human CTRP3b monoclonal antibodies XA297-7, standard items be (purifying
CTRP3b albumen, freeze-dried powder), detect antibody：Antibody XA297-11, sample diluting liquid, TMB chromogenic enzyme substrate liquid, cleaning solution and
Reaction terminating liquid.TMB chromogenic enzyme substrate liquid：TMB hydrochlorides, final concentration is added with 10min before in 0.03% sodium perborate
0.01%；Reaction terminating liquid：1mol/L sulfuric acid solutions；Sample diluting liquid：PBS containing 0.1%BSA and 0.1%Tween-20 is slow
Fliud flushing；Cleaning solution:PBS buffer solution containing 0.1%Tween-20.
(1) ELISA Plate is coated with：
(1.1) hybridoma cell strain XA297-7 (number CCTCC NO:C201797 monoclonal antibody XA297-7 produced by)
Make coating antibody, being diluted to 2 μ g/mL with 50mM, pH9.6 carbonate buffer solution adds to 96 orifice plates, 100 holes μ L/, 4 DEG C of coatings
Overnight；
(1.2) it washs：With phosphate buffer board-washings 3 time of the 300 μ L containing 0.1% tween, drying；
(1.3) kept dry：It is stored in the hermetic bag for adding drier, 4 DEG C save backup.
(2) enzyme-labeled antibody is detected
0.2mL 0.1M sodium periodates are added in horseradish peroxidase (HRP) 5mg/1mL, are protected from light stirring 20min at room temperature.
By above-mentioned solution to 4 DEG C of dialysed overnights of 1mM pH4.4 sodium-acetate buffers.20 μ L 0.2M pH9.5 carbonate buffer solutions are adjusted
10mg antibody XA297-11 is added (by hybridoma cell strain XA297-11, number CCTCC NO in pH to 9.0-9.5:C201798 is produced
It is raw), room temperature, which is protected from light, is gently mixed 2h.0.1mL sodium borohydride solutions (4mg/mL), mixing is added to stand 2 hours, 4 DEG C of dialysed overnights.
4 DEG C of isometric saturated ammonium sulfate solution is added dropwise and stands 1 hour, centrifuges 30min, discards supernatant.The a small amount of 0.15M of sediment
The phosphate buffer and dialysis, supernatant of pH7.4 is enzyme labelled antibody XA297-11-HRP.
(3) sample is added：Standard items (sample to be tested) are added in coating hole, shaken at room temperature reacts 1h.
(4) integrated enzyme reaction：The monoclonal antibody XA297-11-HRP of horseradish peroxidase-labeled is added after washing 4 times
1h is reacted in 100 holes μ l/.
(5) chromogenic reaction：Substrate solution (containing 0.03% sodium perborate and 0.01% hydrochloric acid TMB) 100 is added after washing 4 times
μ l, oscillation colour developing 10 minutes.
(6) reaction is terminated：It is terminated and is reacted with 1mol/L sulfuric acid.
(7) matched curve and readings：The OD that wavelength is 405nm is read in microplate reader is worth standard curve and concentration.
The embodiment kit representative standard curve is shown in attached drawing 2.
The embodiment kit index of correlation is measured using standard items：
(1) range of linearity：The range of linearity that the kit detects CTRP3b contents is 0.375-60ng/ml.
(2) rate of recovery：Using 1ng/ml, the CTRP3b solution (solvent is sample diluting liquid) of 10ng/ml, 15ng/ml are surveyed
The rate of recovery be 95.4%-104.5%.
(3) difference is 3.5%-6.2% in criticizing.
(4) differences between batches are 6.6%-10.4%.
Embodiment 3：CTRP3b in atherosclerotic and Normal group serum is detected with kit of the present invention to contain
Amount
Using kit of the present invention, CTRP3b in 50 normal persons and 50 atherosclerotic's serum is detected
Content.The results are shown in Table 1.
The testing result (x ± s, ng/ml) of CTRP3b in 1 serum of table
| CTRP3b contents in serum | Normal group | Atherosclerotic |
| ng/mL | 263.9±87.2 | 73±68.2 |
| P<0.001 |
CTRP3b contents are significantly lower than normal person, significant difference (p ＜ 0.01) in atherosclerotic's serum.More than
Detect the content of CTRP3b in humoral specimen the result shows that can be quantified using kit of the present invention, to clinical detection have compared with
High sensibility and specificity is worth to diagnosing the disease with important references, and an effectively way is provided to prevent the disease early
Diameter.
Nucleotides sequence list electronic document
<110>The Fourth Military Medical University of P.L.A
<120>The monoclonal antibody of hybridoma cell strain and anti-human CTRP3b and its application
<141>
<160>
<210>1
<211>120
<212>Amino acid
<213>XA297-7 heavy chain variable regions
<220>
<223>
<400>1EVMLVESGGGFVKPGGSQKLSCVASGFTLSNYAMSWVRQTPEKRLEWVATISTGGSYIYYADSVKGR
FTISRDDAKNTLFLQMSSLRSEDTAMYYCASLYIVTVLVQDYWGHGTTLTVSS
<210>2
<211>112
<212>Amino acid
<213>XA297-7 light chain variable regions
<220>
<223>
<400>2
DAVMTQTPLSLPVSLGAQASISCRSSQSLVHSNGYTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSG
TDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIK
<210>3
<211>115
<212>Amino acid
<213>XA297-11 heavy chain variable regions
<220>
<223>
<400>3
EVQLQQSGPELVKPGASVKISCKTSGYTFNEYVIHWVKQSHGRSLEWIGGINPNNVSTNYNQKFKGKATLTVD
KSSSTAYMELRRLTSDASAVYYCVGYGYYVYWGQGTLVTVSA
<210>4
<211>112
<212>DNA
<213>XA297-11 light chain variable regions
<220>
<223>
<400>4
DVLMTQTPLSLPVSLGDQASISCKSSQSIVHSDGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSG
TDFTLKISRVEAEDLGVYYCLQGSHVPWTFGGGTKLEIK
<210>5
<211>897
<212>DNA
<213>People's CTRP3b full genome composition sequences
<220>
<223>
<400>5
ATGCAAGATGAATACATGGAGGTGAGCGGAAGAACTAATAAAGTGGTGGCAAGAATAGTGCAAAGCCACCAGC
AGACTGGCCGTAGCGGCTCCAGGAGGGAGAAAGTGAGAGAGCGGAGCCATCCTAAAACTGGGACTGTGGATAATAAC
ACTTCTACAGACCTAAAATCCCTGAGACCAGATGAGCTACCGCACCCCGAGGTAGATGACCTAGCCCAGATCACCAC
ATTCTGGGGCCAGTCTCCACAAACCGGAGGACTACCCCCAGACTGCAGTAAGTGTTGTCATGGAGACTACAGCTTTC
GAGGCTACCAAGGCCCCCCTGGGCCACCGGGCCCTCCTGGCATTCCAGGAAACCATGGAAACAATGGCAACAATGGA
GCCACTGGTCATGAAGGAGCCAAAGGTGAGAAGGGCGACAAAGGTGACCTGGGGCCTCGAGGGGAGCGGGGGCAGCA
TGGCCCCAAAGGAGAGAAGGGCTACCCGGGGATTCCACCAGAACTTCAGATTGCATTCATGGCTTCTCTGGCAACCC
ACTTCAGCAATCAGAACAGTGGGATTATCTTCAGCAGTGTTGAGACCAACATTGGAAACTTCTTTGATGTCATGACT
GGTAGATTTGGGGCCCCAGTATCAGGTGTGTATTTCTTCACCTTCAGCATGATGAAGCATGAGGATGTTGAGGAAGT
GTATGTGTACCTTATGCACAATGGCAACACAGTCTTCAGCATGTACAGCTATGAAATGAAGGGCAAATCAGATACAT
CCAGCAATCATGCTGTGCTGAAGCTAGCCAAAGGGGATGAGGTTTGGCTGCGAATGGGCAATGGCGCTCTCCATGGG
GACCACCAACGCTTCTCCACCTTTGCAGGATTCCTGCTCTTTGAAACTAAGTAA
Claims (10)
1. being used to prepare the hybridoma cell strain of anti-human CTRP3b monoclonal antibodies, the deposit number of the hybridoma cell strain is：
CCTCC NO：C201797.
2. anti-human CTRP3b monoclonal antibodies caused by hybridoma cell strain as described in claim 1.
3. antibody as claimed in claim 2, which is characterized in that the weight chain variabl area sequence of the antibody such as SEQ.ID.NO.1 institutes
Show；Light-chain variable sequence is as shown in SEQ.ID.NO.2.
4. antibody described in claim 2 is used to detect the application of people CTRP3b.
5. being used to prepare the hybridoma cell strain of anti-human CTRP3b monoclonal antibodies, the deposit number which protects
For：CCTCC NO：C201798.
6. anti-human CTRP3b monoclonal antibodies caused by hybridoma cell strain as claimed in claim 5.
7. antibody as claimed in claim 6, which is characterized in that the weight chain variabl area sequence of the antibody such as SEQ.ID.NO.3 institutes
Show；Light-chain variable sequence is as shown in SEQ.ID.NO.4.
8. antibody described in claim 6 is used to detect the application of people CTRP3b.
9. a kind of people CTRP3b detection kits, which is characterized in that the kit includes claim 2 and 6 antibody.
10. the kit described in claim 9, which is characterized in that the kit further includes：Sample diluting liquid, substrate dilution
Liquid, substrate, cleaning solution, standard items and tween.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711460128.6A CN108285893B (en) | 2017-12-28 | 2017-12-28 | Hybridoma cell strain, monoclonal antibody resisting human CTRP3b and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711460128.6A CN108285893B (en) | 2017-12-28 | 2017-12-28 | Hybridoma cell strain, monoclonal antibody resisting human CTRP3b and application thereof |
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| Publication Number | Publication Date |
|---|---|
| CN108285893A true CN108285893A (en) | 2018-07-17 |
| CN108285893B CN108285893B (en) | 2021-02-05 |
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ID=62832382
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| CN201711460128.6A Active CN108285893B (en) | 2017-12-28 | 2017-12-28 | Hybridoma cell strain, monoclonal antibody resisting human CTRP3b and application thereof |
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| Country | Link |
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2017
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