CN103074302B - Anti-C-peptide monoclonal antibody and use thereof in detection of C-peptide content - Google Patents
Anti-C-peptide monoclonal antibody and use thereof in detection of C-peptide content Download PDFInfo
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- CN103074302B CN103074302B CN201210555678.7A CN201210555678A CN103074302B CN 103074302 B CN103074302 B CN 103074302B CN 201210555678 A CN201210555678 A CN 201210555678A CN 103074302 B CN103074302 B CN 103074302B
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Abstract
The invention discloses an anti-C-peptide monoclonal antibody and a use thereof in detection of C-peptide content. According to the invention, C-peptide-GST and C-peptide-Trx are used as immunogens, two hybridoma cell strains are obtained by cell fusion technique-based screening, the microbial preservation numbers of the two hybridoma cell strains are respectively CGMCC NO. 6804 and CGMCC NO.6805, two monoclonal antibodies secreted by the two hybridoma cell strains can mutually matched, the content of C-peptide in serum is detected by double-antibody sandwich ELISA method, and the detection result has high accuracy and sensitivity.
Description
Technical field
The present invention relates to monoclonal antibody, particularly relate to the anti-C-peptide monoclonal antibody of pairing and secrete the hybridoma cell strain of this anti-C-peptide monoclonal antibody, the invention further relates to them preparing the purposes in the reagent or medicine detecting or diagnose C peptide, belonging to detection or the diagnostic field of C peptide.
Background technology
C peptide (C-Peptide), also known as connection peptides, is the secretory product of beta Cell of islet, and it and pancreas islet have a common precursor---proinsulin.The proinsulin of a molecule, after enzyme is cut, is cracked into the Regular Insulin of a molecule and the C peptide of a molecule.Measure the function that C Toplink learns islet cells, the Diagnosis and Treat of diabetes is had a very big significance, C peptide does not have the function of Regular Insulin, and the Regular Insulin of islet β cell and C peptide are equimolecular relation, this that is, secrete several insulin molecule, several C peptide molecule must be secreted simultaneously.In blood, C peptide concentration can indirect reaction insulin concentration.C peptide is not by liver enzyme deactivation, and the transformation period is longer than Regular Insulin, directly drains in urine through kidney, therefore in blood, the concentration of C peptide can reflect the function of pancreas islet better.The removing of C peptide is mainly through kidney degraded and excretion, and the concentration of C peptide in urine is higher than the concentration in blood plasma.The Regular Insulin for the treatment of is not containing C peptide, and therefore, C peptide is not by the impact of exogenous insulin, and this is part more superior to Regular Insulin in research islet beta cell function.C peptide release test can replace insulin release test in some sense, can be used for the patient just accepting insulinize, and the mensuration of C peptide concentration is diagnosis and the useful indicators understanding diabetes clinical condition.
C peptide is made up of 31 amino-acid residues, and molecule is less, and the C stabilized peptide obtained by the mode of Peptide systhesis is very poor, easily degrades, no matter as immunizing antigen or screening antigen all improper.
Summary of the invention
An object of the present invention is to provide two kinds of anti-C-peptide monoclonal antibody of pairing mutually;
Two of object of the present invention is to provide the hybridoma cell strain secreting described anti-C-peptide monoclonal antibody;
Three of the object of the invention described anti-C-peptide monoclonal antibody is applied to diagnosis or detects C peptide;
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The present invention adopts C peptide-GST to do immunogen, and C peptide-Trx, as screening antigen, screens the strain of acquisition 2 strain of hybridoma, 3G1 and 5F7; Adopt C peptide-Trx to do immunogen, C peptide-GST, as screening antigen, screens the strain of acquisition 3 strain of hybridoma, 2A9,5C8 and 5B12.
The present invention finds to adopt C peptide-GST to do immunogen by further test and screen monoclonal antibody secreted by the hybridoma cell strain 5F7 that obtains and employing C peptide-Trx and do immunogen and screen monoclonal antibody secreted by the hybridoma cell strain 5B12 that obtains, therebetween can mutually match, as the monoclonal antibody clonal antibody of a pair pairing, be applied to by double crush syndrome method and detect C peptide, detected result has higher accuracy and sensitivity.
Hybridoma cell strain 5F7 submits to the mechanism of patent accreditation to carry out preservation by the present invention; Its microbial preservation is numbered: CGMCC NO.6804; The preservation time is: on November 9th, 2012; Its Classification And Nomenclature is: C-peptide monoclonal antibody hybridoma cell strain; Depositary institution: China Microbiological preservation management committee's common micro-organisms center; Preservation address: No. 3, No. 1, North Star West Road, Chaoyang District, BeiJing, China institute, Institute of Microorganism, Academia Sinica.
Hybridoma cell strain 5B12 submits to the mechanism of patent accreditation to carry out preservation by the present invention; Its microbial preservation is numbered: CGMCC NO.6805; The preservation time is: on November 9th, 2012; Its Classification And Nomenclature is: C-peptide monoclonal antibody hybridoma cell strain; Depositary institution: China Microbiological preservation management committee's common micro-organisms center; Preservation address: No. 3, No. 1, North Star West Road, Chaoyang District, BeiJing, China institute, Institute of Microorganism, Academia Sinica.
The present invention adopts the C peptide fusion protein with two kinds of different labels of escherichia coli expression, solves the problem that C peptide molecular weight is little, unstable.Adopt two kinds of fusion roteins respectively as antigen, immune mouse, adopts two kinds of antigen cross to screen, has both avoided the monoclonal antibody obtaining anti-label, turn increase the chance obtaining anti-different epi-position monoclonal antibody, thus add the chance of the pairing monoclonal antibody that can be used for sandwich assay detection C peptide.
For a kind of peptide C peptide of poor stability, adopt two kinds of different labels, express two kinds of fusion roteins with target protein, detect through existing immunological response, can react with existing specific antibody; Adopt wherein a kind of fusion rotein as immunogen, with another fusion rotein as screening antigen, to obtain monoclonal antibody, and carry out pairing experiment.Because the molecular chaperones of expressed fusion protein is different, the conformation of albumen may difference to some extent, thus may obtain the monoclonal antibody for different epi-position, and the chance of successful matching is increased.
The present invention is respectively used to immunity and detection by the fusion rotein of expression two kinds of different labels, the probability obtaining matching monoclonal antibody is increased, and then obtains the ability of anti-different epi-position, thus enhance the susceptibility and accuracy that detect C peptide.Compared with prior art, two kinds of fusion roteins with label obtaining as immunogen, because the protein conformation of different tag fusion protein is different, are which increased the chance that anti-different epitope antibodies produces by the present invention in the present invention; Using the fusion rotein with label different from immunogen as screening antigen, avoid the selection result instability that C peptide instability causes.Meanwhile, use GST and Trx as label, solubility and the stability of albumen can be increased, and be convenient to purifying.
Accompanying drawing explanation
Fig. 1 is the schema of preparation pairing antibody.
Fig. 2 carries out as pairing antibody the result that double crush syndrome detects C peptide using 5F7/5B12.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
Embodiment 1 prepares C peptide-GST and C peptide-Trx fusion rotein
1) experiment material
Expression vector: pGEX-4T-1 is purchased from GE healthcare, and pET32 is purchased from invitrogen; Expressive host bacterium: e. coli bl21 (DE3), is purchased from Beijing Tian Gen Bioisystech Co., Ltd; Yeast powder and peptone: be Oxide Products;
2) experimental technique
By C peptide gene sequence, (nucleotides sequence is classified as shown in SEQ ID No.1, coded aminoacid sequence is shown in SEQ ID No.2) 5 ' end band has BamHI site, 3 ' end band has XhoI site, be inserted on pMD-18-T carrier, by on pGEX-4T-1 and the pET32a carrier that is inserted into respectively after goal gene BamHI and XhoI double digestion with same procedure process, be built into GST-C peptide expression vector pGEX-C peptide and Trx-C peptide expression vector pET32a-C peptide, plasmid is proceeded to e. coli bl21 (DE3) competent cell, by selecting positive colony by the method for bacterium colony PCR, the positive colony obtained is cultivated in LB substratum, with isopropylthiogalactoside (IPTG) abduction delivering.Bacterium liquid after abduction delivering carries out cytoclasis.For cytoclasis, conventional method of cell disruption can be adopted, such as under the condition of 8000-12000 rev/min, centrifugal 5-10 minute, supernatant after centrifugal 0.22 μm of membrane filtration, after filtration after membrane filtration, carry out affinity purification with GST post or nickel post, GSTrap FF or the HisTrap FF prepacked column of preferred GE company carry out purifying, the method provided by purification column filler product specification sheets carries out purifying, obtains C peptide-GST and C peptide-Trx fusion rotein.
Embodiment 2 prepares anti-C peptide pairing monoclonal antibody
1) experiment material
SP 2/0 is purchased from the biochemical institute in Shanghai cell centre; Balb/c mouse is purchased from Chinese Academy of Medical Sciences's Experimental Animal Center; Foetal calf serum: Hangzhou folium ilicis chinensis Products; DMEM, HAT and HT are Invitrogen Products; 50%PEG1450 solution is Sigma Products; Other medicine is analytical pure grade.
2) experimental technique
2.1 mouse immune
C peptide-GST and the C peptide-Trx fusion rotein of choosing 6-8 6-8 female Balb/c mouse escherichia coli expression in age in week carry out immunity respectively.3 immunity are carried out before cytogamy.Head exempts from: be expelled to mouse carotid dorsal sc, every injected in mice 0.2ml after getting the antigen of 50 μ g/ mouse and the emulsification of equal-volume Freund's complete adjuvant; Two exempt from: head is expelled to mouse carotid dorsal sc, every injected in mice 0.2ml after getting the antigen of 50 μ g/ mouse and the emulsification of equal-volume Freund's incomplete adjuvant after exempting from 3 weeks; Three exempt from: two exempt from 3 weeks after, be expelled to mouse carotid dorsal sc, every injected in mice 0.2ml after getting the antigen of 50 μ g/ mouse and the emulsification of equal-volume Freund's incomplete adjuvant.Three exempt from latter one week, and mouse docking blood sampling, adopts indirect ELISA method to measure antiserum titre.
2.2 serum antibody titers detect
Adopt indirect ELISA to tire to immune serum to measure, adopt the serum of the front mouse of immunity as negative control.The albumen of two kinds of different tag expression detects mutually, namely C peptide-GST does immunogenic mouse, tire and adopt C peptide-Trx coated elisa plate to detect, the mouse of C peptide-Trx immunity detects with C peptide-GST, affects the detection of tiring to target protein to avoid the non-specific antibody of anti-label protein.
With 0.05M pH9.6 carbonate buffer solution by antigen diluent to 0.1-10 μ g/ml, every hole 100 μ l is added in enzyme plate, and 4 DEG C of refrigerators are placed and spent the night, and make Antigen adsorption in enzyme plate, with tween 20 phosphate buffered saline buffer (PBST) detersive enzyme target 3 times.The mice serum collected is made 1/3 serial dilution with PBST from 1/1000 and sets up 1 hole diluent contrast, do 8 holes altogether, antiserum(antisera) dilute is added to by every hole 100 μ l and wraps by good enzyme plate, and 37 DEG C of incubations 60 minutes, wash plate 3 times with PBST.Sheep anti mouse-HRP is diluted to PBST the working concentration that specification sheets provides, every hole 100 μ l is added to enzyme plate, and 37 DEG C of incubations 30 minutes, wash plate 3 times with PBST.Every hole adds 100 μ l Urea Peroxides-tetramethyl benzidine substrate (H
2o
2-TMB) develop the color as substrate, after adding stop buffer termination reaction, microplate reader reads the light absorption value under 450nm.We by OD value for more than negative control twice time antibody most highly diluted multiple be decided to be tiring of this antibody.Serum titer reaches 10
4more than can choose higher mouse of tiring and carry out cytogamy experiment.
2.3 Monoclonal Antibody Cell fusion experiments
According to serum antibody titer measurement result, preferred antibody higher mouse of tiring carries out cytogamy experiment.The last time after immune 3-4 week, carry out impact immunity, 50 μ g antigen abdominal injections, the marrow that broken by mouse after 3 days is put to death, collect spleen, make single cell suspension, with myeloma cell after PEG1450 merges, screening and C peptide have the hybridoma of specific binding, thus the cell of monoclonal antibody is produced in preparation.
Fusion method: splenocyte is mixed with the ratio of SP2/0 oncocyte in 5-10:1, centrifugal remove supernatant after, in cell, add 1ml PEG1450, within 1 minute, add, act on after 1 minute, stop merging with serum free medium and promote the effect of reagent.Centrifugal to abandon the perfect medium containing HAT of cell suitable volumes after supernatant resuspended, adds 96 porocyte culture plates, 5%CO2 gas, 37 DEG C, saturated humidity cultivates.
The screening of 2.4 monoclonal antibodies
2.4.1 indirect elisa method screens positive hole
Merge plate or clone plate after cell cultures 7-10 days, adopt indirect ELISA to measure monoclonal antibody culture supernatant, identical with bioactivity, different label protein cross detection.With 0.05M pH9.6 carbonate buffer solution by antigen diluent to 0.1-10 μ g/ml, every hole 100 μ l is added in enzyme plate, and 4 DEG C of refrigerators are placed and spent the night, and make Antigen adsorption in enzyme plate, with tween 20 phosphate buffered saline buffer (PBST) detersive enzyme target 3 times.From each culture hole, take out 100 μ l supernatants be added to bag by good enzyme plate, 37 DEG C of incubations 60 minutes, wash plate 3 times with PBST.Sheep anti mouse-HRP is diluted to PBST the working concentration that specification sheets provides, every hole 100 μ l is added to enzyme plate, and 37 DEG C of incubations 30 minutes, wash plate 3 times with PBST.Every hole adds 100 μ l Urea Peroxides-tetramethyl benzidine substrate (H
2o
2-TMB) develop the color as substrate, after sour termination reaction, microplate reader reads the light absorption value under 450nm.
2.4.2 monoclonal antibody cloning and build strain
The positive hole screened adopts limiting dilution assay to carry out cloning, cultivates after 7-10 days, selects monoclonal hole, and indirect elisa method detects culture supernatant, again carries out cloning, until detect aperture total positives, now can set up hybridoma cell strain to positive hole.Enlarged culturing is carried out in the hole that growth selection state is good, and frozen.
2.5 antibody preparations, purifying and mark
2.5.1 ascites preparation and purification
Choose individual female Balb/c mouse bigger than normal, abdominal injection sterilising liq paraffin, every injected in mice 0.5ml.7-21 days pneumoretroperitoneum injection 0.5-1 × 10
6individual monoclonal antibody hybridoma, gathers ascites after waiting mouse web portion to swell.With the monoclonal antibody in the Protein G prepacked column affinity purification ascites of GE company, concrete grammar is see purification column product description.
2.5.2 monoclonal antibody biotin marks
Preparation 10mM Sulfo-NHS-LC-Biotin, with 1mg/ml antibody according to 20:1(mol ratio) mix, 900 rotating speeds shake 90 minutes, add 10%BSA, be stored in 4 DEG C for subsequent use.
2.6 monoclonal antibodies prepare result
Adopt C peptide-GST to do immunogen, C peptide-Trx, as screening antigen, merges the strain of acquisition 2 strain of hybridoma, 3G1 and 5F7; Adopt C peptide-Trx to do immunogen, C peptide-GST, as screening antigen, merges the strain of acquisition 3 strain of hybridoma, 2A9,5C8 and 5B12.
Test example 1 monoclonal antibody sandwich ELISA detects the effect test of C peptide
1. test materials
Monoclonal antibody secreted by hybridoma cell strain 5F7; Monoclonal antibody secreted by hybridoma cell strain 5B12;
2. test method
With the monoclonal antibody secreted by hybridoma cell strain 5F7 and the pairing of the monoclonal antibody secreted by hybridoma cell strain 5B12, set up double crush syndrome method and detect C peptide, concrete test method is as follows:
With 0.05M pH9.6 carbonate buffer solution, monoclonal antibody is diluted to 1-10 μ g/ml(preferably 4 μ g/ml), every hole 100 μ l is added in enzyme plate, 4 DEG C of refrigerators are placed and are spent the night, and make Antigen adsorption in enzyme plate, with tween 20 phosphate buffered saline buffer (PBST) detersive enzyme target 3 times.C peptide calibration object (outsourcing) is diluted with PBST and sets up 1 hole diluent contrast, establish 6 concentration altogether, be respectively 25ng/ml, 10.25 ng/ml, 3.75 ng/ml, 1.25 ng/ml, 0.5 ng/ml and 0 ng/ml, the calibration object diluted is added to bag by good enzyme plate by every hole 100 μ l, 37 DEG C of incubations 60 minutes, wash plate 3 times with PBST.Biotin marked monoclonal antibody dilution working concentration with PBST, every hole 100 μ l is added to enzyme plate, and 37 DEG C of incubations 30 minutes, PBST washes plate 3 times.Dilute HRP labelled streptavidin to working concentration with PBST, every hole adds 100 μ l, and 37 DEG C of incubations 30 minutes, wash plate 3 times.Every hole adds 100 μ l Urea Peroxides-tetramethyl benzidine substrate (H
2o
2-TMB) develop the color as substrate, after sour termination reaction, measure the light absorption value under 450nm, with Excel Software on Drawing graphic representation.
3. test-results
Test-results is shown in Fig. 2.Double crush syndrome experiment shows, with the monoclonal antibody secreted by hybridoma cell strain 5F7 and the pairing of the monoclonal anti physical efficiency secreted by hybridoma cell strain 5B12, double crush syndrome method good linearity when detecting the C peptide calibration object antigen that 25ng/ml, 10.25 ng/ml, 3.75 ng/ml, 1.25 ng/ml, 0.5 ng/ml and 0 ng/ml constant gradient dilute that pairing is set up, has good application potential.
Claims (6)
1. the purposes of a C-peptide monoclonal antibody hybridoma cell strain in preparation diagnosis or detection C-peptide content reagent or medicine, it is characterized in that, C-peptide monoclonal antibody hybridoma cell strain is the combination that microbial preservation is numbered that the hybridoma cell strain of CGMCC NO.6804 and microbial preservation are numbered the hybridoma cell strain of CGMCC NO.6805.
2. the purposes of a C-peptide monoclonal antibody hybridoma cell strain in preparation diagnosis or detection diabetes medicament, it is characterized in that, C-peptide monoclonal antibody hybridoma cell strain is the combination that microbial preservation is numbered that the hybridoma cell strain of CGMCC NO.6804 and microbial preservation are numbered the hybridoma cell strain of CGMCC NO.6805.
3. the purposes of a monoclonal antibody in preparation diagnosis or detection C-peptide content reagent or medicine, it is characterized in that, described monoclonal antibody is selected from microbial preservation and is numbered the combination of monoclonal antibody that the monoclonal antibody of the hybridoma cell strain secretion of CGMCC NO.6804 and microbial preservation are numbered the hybridoma cell strain secretion of CGMCC NO.6805.
4. the purposes of a monoclonal antibody in preparation diagnosis or detection diabetes medicament, it is characterized in that, described monoclonal antibody is selected from microbial preservation and is numbered the combination of monoclonal antibody that the monoclonal antibody of the hybridoma cell strain secretion of CGMCC NO.6804 and microbial preservation are numbered the hybridoma cell strain secretion of CGMCC NO.6805.
5. for diagnosing or detect a double antibody for C-peptide content or diabetes, it is characterized in that: the monoclonal antibody that the hybridoma cell strain that described double antibody is numbered CGMCC NO.6804 by microbial preservation is secreted and the monoclonal antibody that the hybridoma cell strain that microbial preservation is numbered CGMCC NO.6805 is secreted are composed of.
6. a test kit for diagnosis or detection C-peptide content or diabetes, is characterized in that: the monoclonal antibody that the hybridoma cell strain that the monoclonal antibody of secreting containing the hybridoma cell strain being numbered CGMCC NO.6804 by microbial preservation and microbial preservation are numbered CGMCC NO.6805 is secreted.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103852584B (en) * | 2014-03-28 | 2016-04-13 | 重庆中元生物技术有限公司 | A kind of latex enhancing immune of quantitative detection C peptide is than turbid kit |
| CN107176997A (en) * | 2017-06-08 | 2017-09-19 | 广东工业大学 | A kind of C peptides immunizing antigen and anti-C peptides Yolk antibody and its preparation and application |
| CN109705221B (en) * | 2018-12-27 | 2021-03-09 | 美康生物科技股份有限公司 | C peptide immunogen, monoclonal antibody pair thereof and application of antibody pair in C peptide magnetic particle chemiluminescence immunoassay reagent |
| CN111690064A (en) * | 2020-06-24 | 2020-09-22 | 合肥天麦生物科技发展有限公司 | Detection method of proinsulin precursor protein and preparation method of PPI monoclonal antibody thereof |
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