CN101074263B - Recombinant proteinA monoclonal antibody, its production and use - Google Patents
Recombinant proteinA monoclonal antibody, its production and use Download PDFInfo
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- CN101074263B CN101074263B CN2007101014887A CN200710101488A CN101074263B CN 101074263 B CN101074263 B CN 101074263B CN 2007101014887 A CN2007101014887 A CN 2007101014887A CN 200710101488 A CN200710101488 A CN 200710101488A CN 101074263 B CN101074263 B CN 101074263B
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Abstract
The present invention provides an anti-protein A mono-clone antibody, a DNA molecule for encoding antibody, a carrier for expressing antibody and eukaryotic host cell converted by the expression carrier. Light-chain variable zone has amino-acid sequence with SEQ ID NO: 1, and heavy-chain variable zone has amino-acid sequence with SEQ ID NO:2. The anti-protein A mono-clone antibody provided by the present invention can inspect protein A concentration in decontaminated product fast.
Description
Technical field
The present invention relates to the antibody technique field.Specifically, the present invention relates to a kind of new anti-proteinA monoclonal antibody, and its production and use.
Background technology
(Staphylococal ProteinA is a kind of from the isolating protein of aureus cell wall SPA) to SP.1940, Vevwey found to contain a kind of material in some streptococcus aureus, in double diffusion test, can form precipitation with normal human serum.Jensen (1959) has also found similar phenomenon, with its called after A antigen.
Lofkvist in 1963 etc. have separated A antigen, and prove that it is a kind of protein, and have any different with sugar; Grov (1960) is called for short SPA albumin A (ProteinA) with its called after SP.The gene of coding SPA was cloned and at expression in escherichia coli (Duggleby in nineteen eighty-three, C.J and Jones, SA:cloning and expression of the Staphylococcus aureus protein A gene in Escherichiacoli.Nucl Acids Res.11 (1983) 3065-3076; Lofdahl, S., Guss, B., et al; Gene forStaphylococcal protein A.Proc.Natl.Acid.Sci.USA 80 (1983) 697-701).To discovering of SPA 26S Proteasome Structure and Function, the SPA molecule comprises A, B, C, D, five homeodomains of E, each structural domain all have can with the autonomous bonded ability of IgG, the SPA gene has 1600bp.
Owing to have 5 structural domains to combine on the SP, have Fc section bonded ability with most of Mammals IgG with the Fc district of IgG.As a kind of affinity ligand, albumin A is fixed on above the agarose, and 5 structural domains above it just can freely combine with the Fc of IgG, and a part fixed albumin A can combine with bimolecular IgG, its C end of recombinant protein A has a Gelucystine, and is stronger in conjunction with the ability of IgG.Recombinant protein A is widely used in the separation and purification and the detection of monoclonal antibody or polyclonal antibody.When using recombinant protein A affinity chromatography technology, need to detect the concentration of recombinant protein A in the product, to determine its quantity residual in product, therefore, press for a kind of can be effectively, the product of rapid detection proteinA.
Summary of the invention
One of technical issues that need to address of the present invention provide a kind of anti-proteinA monoclonal antibody of reorganization, and this antibody is used for the concentration of vitro detection proteinA.
Second technical problem that the present invention need solve provides a kind of dna molecular of the above-mentioned anti-proteinA monoclonal antibody of encoding.
The 3rd technical problem that the present invention need solve provides a kind of expression vector.
The 4th technical problem that the present invention need solve provides a kind of eukaryotic host cell.
The 3rd technical problem that the present invention need solve provides a kind of purposes of said monoclonal antibody.
The 4th technical problem that the present invention need solve provides a kind of this MONOCLONAL ANTIBODIES SPECIFIC FOR method.
To achieve the above object of the invention, one aspect of the present invention provides a kind of anti-proteinA antibody of reorganization, this antibody contains variable region of heavy chain and variable region of light chain, it is characterized in that, variable region of light chain has the aminoacid sequence shown in the SEQ ID NO:1, and variable region of heavy chain has the aminoacid sequence shown in the SEQ ID NO:2.
The present invention provides the dna molecular of coding said monoclonal antibody on the other hand.
In a preferable example, this dna molecular contains the nucleotide sequence of the described monoclonal antibody variable region of light chain of the coding shown in the SEQID NO:3, and the nucleotide sequence of the described monoclonal antibody variable region of heavy chain of the coding shown in the SEQIDNO:4.
Third aspect present invention provides a kind of expression vector, the expression regulation sequence that this expression vector contains above-mentioned dna sequence dna and links to each other with this series of operations.
Fourth aspect present invention provides a kind of host cell, it is characterized in that, it is transformed by above-mentioned expression vector.In a preferable example, this host cell is a Chinese hamster ovary celI.
Fifth aspect present invention provide a kind of anti-proteinA monoclonal antibody detect proteinA concentration method, it is characterized in that described method be the ELISA method of routine.
Sixth aspect present invention provides a kind of method for preparing said monoclonal antibody, it is characterized in that, this method comprises:
A) provide an expression vector, the expression regulation sequence that this expression vector contains above-mentioned dna sequence dna and links to each other with this series of operations;
B) with the described expression vector transformed host cell of step a);
C) host cell of gained culturing step b under the condition that is fit to described monoclonal antibody expression); With
D) separation and purification obtains described monoclonal antibody.
The invention has the advantages that monoclonal antibody of the present invention can be used for the residual quantity of proteinA in the rapid detection purified product.Antibody of the present invention detects proteinA high specificity, highly sensitive with the ELISA method, significantly is better than commercially available antibody.
Description of drawings
Fig. 1: carrier pMG18, and indicated element and restriction enzyme site wherein, wherein, hCMV pro is the main early promoter of human cytomegalic inclusion disease virus; Ck is a people κ constant region of light chain gene; IgG1 constant region behaviour γ 1 weight chain constant area gene; PA is the poly-adenosine signal; DHFR is a dihydrofolate reductase gene; AmpR is an ampicillin resistance gene.
Fig. 2: the pM carrier that the present invention is constructed, wherein, Ck still is people κ chain (light chain) constant region gene, IgG1 constant is mouse γ 1 weight chain constant area gene.
Fig. 3: pM (H+L) carrier that the present invention is constructed, contain anti-proteinA heavy chain of antibody total length of reorganization and light chain full-length gene.
Fig. 4: anti-proteinA monoclonal antibody of the present invention and commercially available antibody are respectively applied for the sensitivity comparative result (■ is a monoclonal antibody of the present invention, and zero is commercially available antibody) that detects proteinA concentration.
Embodiment
The present invention relates to a kind of anti-proteinA antibody of reorganization, this antibody comprises variable region of heavy chain and variable region of light chain, it is characterized in that variable region of light chain has the aminoacid sequence shown in the SEQ ID NO:1, variable region of heavy chain has the aminoacid sequence shown in the SEQ ID NO:2.
Monoclonal antibody can make with the whole bag of tricks well known to those skilled in the art.For example, monoclonal antibody can use hybridoma method (by Kohler etc., Nature, 256:495 (1975) at first proposes) to make, or with recombinant DNA method (U.S. Patent No. 4,816,567) make the also available for example Clackson of monoclonal antibody etc., Nature, 3 52:624-628 (1991) and Marks etc., J.Mol.Biol., the described technology of 222:581-597 (1991) is separated acquisition from phage antibody library.
The present invention also provides the dna molecular of the anti-proteinA monoclonal antibody of code book invention reorganization.In a preferable example, this dna molecular contains the nucleotide sequence of the described monoclonal antibody variable region of light chain of coding shown in the SEQ ID NO:3, and the nucleotide sequence of the described monoclonal antibody variable region of heavy chain of coding shown in the SEQ ID NO:4.
Behind the nucleotide sequence that obtains code book invention anti-proteinA monoclonal antibody variable region of heavy chain of reorganization and variable region of light chain, can prepare monoclonal antibody of the present invention by the following method usually.
At first, provide nucleotide sequence that contains code book invention monoclonal antibody and the expression vector of the expression regulation sequence that links to each other with this series of operations.
The dna sequence dna of code book invention monoclonal antibody can make with conventional means well known to those skilled in the art.For example, can be according to sequence synthetic disclosed by the invention or with PCR method amplification obtain the encoding nucleotide sequence of this monoclonal antibody variable region of heavy chain and variable region of light chain.Then, these nucleotide sequences are inserted in the suitable expression vector by selecting proper restriction site with the whole bag of tricks well known in the art, make them respectively before expression vector entrained CH encoding sequence and constant region of light chain encoding sequence, and make them in same reading frame.
Used expression vector is a various commercially available expression vector well known by persons skilled in the art among the present invention, for example available from the expression vector of Qiagen and Promega company, and the expression vector pMG 18 that can buy (" carrying out the exploitation of the instrument of environmental monitoring according to the INCP-9 plasmid sequence " (DEVELOPMENT OFTOOLS FORENVIRONMENTAL MONITORING BASED ON INCP-9PLASMIDS SEQUENCES), A.Created, R.Krasowiak, M.Titok, C.M.ThomasSchool of Biological Sciences, University of Birmingham, Edgbaston, Birmingham B152TT, UK and Faculty of Biology, Dept of Microbiology, Belarus State UniversityScoring Av.4, Minsk 220080 Belarus).
Subsequently, the expression vector with above-mentioned acquisition transforms proper host cell." host cell " generally comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.In the present invention, preferable host cell is a Chinese hamster ovary celI.Method with the expression vector transformed host cell has a variety of, used Transformation Program to depend on host to be transformed.Power one method that heterologous polynucleotide is imported in the mammalian cell is known in the art, it comprises transfection, calcium phosphate precipitation, the Polybrene (1 of dextran mediation, 5 one dimethyl, one 1,5 one phenodiazines, 11 methylene radical gather Methobromide) mediation transfection, protoplastis fusion, electroporation, liposome-mediated transfection and with the dna direct microinjection in karyon.In the present invention, preferred methods is electroporation or liposome mediated-method etc.For example can adopt the liposome method test kit of Invitrogen company to come transfection CHO cell.
Then, under the condition that is fit to monoclonal antibody expression of the present invention, cultivate the host cell that transforms gained.Use conventional immunoglobulin purification step then, as albumin A-Sepharose, conventional separation and purification means purifying well known to those skilled in the art such as light basic phosphatic rock chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, sieve chromatography or affinity chromatography obtain the anti-proteinA monoclonal antibody of reorganization of the present invention.
The gained monoclonal antibody can be identified with conventional means.The binding specificity of monoclonal antibody can be measured with immunoprecipitation or external combination test (as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)).The binding affinity of monoclonal antibody is available Munson etc. for example, Anal.Biochem., and the Scatchard of 107:220 (1980) analyzes and measures.
Below in conjunction with embodiment the present invention is described in further detail.Yet should be appreciated that and enumerate these embodiment, and be not to be used for limiting the present invention just for an illustration.
Although the invention describes following concrete example, having a bit is significantly to those skilled in the art, promptly can do various variations and change to the present invention under the premise without departing from the spirit and scope of the present invention.Therefore, claims have covered all these changes within the scope of the present invention.
Do not indicate among the embodiment that being of source is commercially available.
Embodiment 1 screens the antibody gene variable region of proteinA from antibody library
1) structure of mouse source antibody library
ProteinA (available from GE company) and freund's adjuvant immunity Balb/C mouse, after the immunity 4 times, 1: 500 dilution back of mice serum shows strong positive reaction with FL, get immunity back mouse spleen, according to people J.Mol.Biol. such as Marks, 222,581-597.Hoogenboom and Winter, J.Mol.Biol., 227, people J Immunol Methods.2001 Nov 1 such as 381-388.Haidaris CG; 257 (1-2): 185-202, Griffiths, people EMBO J. such as A.D., 13,3245-3260 (1994) .Nissim, people EMBO J. such as A., 13, the method for describing among the 692-698 (1994) makes up mouse source antibody library.
2) screening
The antibody library bacterial strain of recovery is added 14 milliliters of fresh LB substratum for 1 milliliter, in 50 milliliters of triangular flasks, cultivated 16 hours for 37 ℃.
12000rpm high speed centrifugation 10 minutes is transferred to supernatant liquor in 50 milliliters of aseptic centrifuge tubes, preserves standby.Guarantee that its titre should be 2 * 10
11More than.At first use the human IgG1 as antigen, the ordinary method bag is by 25 ml cells culturing bottles.Add in the cell bottle behind the bag quilt and be no less than 3 * 10
10Phage, 37 ℃ of incubations 1 hour.Collect the supernatant liquor in the culturing bottle, standby.ProteinA is as antigen, with the ordinary method bag by 25 ml cells culturing bottles.Bag by after the cell bottle in add to go up the supernatant liquor that the step collects, 37 ℃ of incubations 1 hour.Outwell the liquid in the bottle, wash culturing bottle 10 times with 10 milliliters of PBS that added 1%Tween-20.The TG1 cell that adds 1 milliliter of logarithmic phase in culturing bottle, 37 ℃ of incubation concussions were cultivated 16 hours.
Repeat the described step 4 of epimere time.
Cell dilution to 10 with above-mentioned acquisition
5Cultivate being added with on 1.5% agar plate of 0.1 penbritin after the cells/ml, obtain mono-clonal.
Get being cloned on the 96 hole depth orifice plates on the above-mentioned flat board and cultivate, the clone in every hole does 960 clones (10 96 orifice plates) altogether.
Above-mentioned deep-well plates after centrifugal 20 minutes, is transferred to new aseptic deep-well plates with supernatant at 5000RPM on the 96 orifice plate whizzers, be preserved in after sealing 4 the degree standby.
Get 10 of 96 orifice plates, add in every hole the conventional bag of proteinA (10 mcg/ml) 10 microlitres by after, add supernatant 10 microlitres of above-mentioned preservation respectively, 37 ℃ of incubations after 1 hour with the PBS washing that contains 1%Tween-20 20 times.The goat-anti M13 monoclonal antibody (available from Pharmacia company) that adds 1 microlitre HRP mark, 37 ℃ of incubations use the PBS of 1%Tween-20 to wash 10 times after 30 minutes.
Add and contain PBS 200 microlitres of 0.025%DAB developer and the H of 1 microlitre 1%
2O
2The 37 ℃ of incubation colour developing was read 595 nanometers after 20 minutes on plate reading machine photoabsorption.
Determine the hole that color reaction is strong according to the photoabsorption reading, the corresponding clone in these holes is the stronger antibody variable region clone of avidity.This test-results filters out 325 positive colonies altogether, determines wherein 5 the strongest clones of avidity according to its reading, is used for next step research.
3) the antibody variable region encoding sequence is to the clone of expression vector
Above-mentioned 5 clones' bacterial strain is increased in 100 milliliters of LB substratum, then according to manufacturer's specification sheets with the plasmid DNA extracting and purifying test kit of Promega company (
Plus SV Minipreps DNAPurification System) plasmid DNA purification.
With XbaI with on 1.5% agarose gel electrophoresis, separate endonuclease bamhi after NheI Restriction Enzyme enzyme is cut above-mentioned plasmid DNA, get band about 357bp and carry out glue and reclaim, the gained fragment is variable region of heavy chain.Behind the pcr amplification, carry out sequencing.With XbaI with on 1.5% agarose gel electrophoresis, separate endonuclease bamhi after BsiWI Restriction Enzyme enzyme is cut above-mentioned plasmid DNA, get band about 336bp and carry out glue and reclaim, the gained fragment is variable region of light chain.Behind the pcr amplification, carry out sequencing.After determining correct sequence, chemical synthesis is synthesized the light chain full length sequence, 5 ' end design XbaI enzyme cutting site, and 3 ' end design BamHI restriction enzyme site, constant region is a mouse κ constant region of light chain sequence.
The PCR method is from mouse boosting cell amplification IgG1 CH sequence, and the CH1 initial amino acid sported Ala-Ser, contained the NheI restriction enzyme site simultaneously, 3 ' end design BamHI restriction enzyme site, after amplification back sequence verification is errorless, NheI and BamHI double digestion IgG1 constant region fragment and pMG18 are connected into pMG18 with the IgG1 constant region, the novel vector called after pM of structure.
Cut expression vector pM with XbaI and NheI Restriction Enzyme enzyme.The restriction enzyme mapping of this expression vector as shown in Figure 2.Above-mentioned variable region of heavy chain is inserted in the XbaI/NheI site of this expression vector then.Equally, utilize XbaI and BamHI Restriction Enzyme the light chain of antibody full-length cDNA to be inserted in the XbaI/BamHI site of pM carrier, thereby make up the expression vector pM (H+L) that is contained anti-proteinA heavy chain of antibody total length of reorganization and light chain full-length gene respectively, as shown in Figure 3.
4) screening of the transfection of Chinese hamster ovary celI and recombinant clone
The expression vector that has antibody gene of above-mentioned structure is inoculated in 100 milliliters of LB substratum in the transformed into escherichia coli DH5 α bacterial strain respectively and increases, with ultrapure plasmid DNA purification kit (Ultrapure Plasmid DNA Purification Kit) the extracting and purifying plasmid DNA of Qiagen company.The liposome method test kit of employing Invitrogen company (lipofectamine 2000 transfection reagent, 11668-027), with the plasmid DNA cotransfection Chinese hamster ovary celI of above-mentioned purifying, carry out with reference to the specification sheets of producer by operation.
The Chinese hamster ovary celI that transforms carries out the selection in continuous 9 weeks on HAT selection substratum, cultivate in the enterprising limit by row dilution of 96 orifice plates at last, carries out continuously 3 times, carry out mono-clonalization.
The monoclonal cell of choosing ties up on RPMI 1640 substratum and cultivates, and supernatant is carried out the test of Western trace, judges expression intensity according to staining reaction, picks out 12 and expresses strong clone as the candidate cell strain.
5) Purification of Monoclonal Antibodies
Go out said monoclonal antibody with the direct separation and purification from cells and supernatant of ProteinA affinity column, prove that through the SDA-PAGE electrophoresis products therefrom purity is greater than 90%.The product of affinity chromatography passes through sieve chromatography once more, has obtained the sample of purity＞98%.These samples are used for following further analysis and research.
The expression intensity of embodiment 2 antibody genes in Chinese hamster ovary celI
12 high expression level candidate clones that above-mentioned screening is obtained are incubated in the tissue culture ware of 10cm, the expression amount of measuring antibody with the ELISA method as described below.Sheep anti-mouse igg (Fc) is wrapped quilt in elisa plate, 4 ℃ are spent the night, sealed 2 hours in 37 ℃ through 2%BSA, add culture supernatant to be measured and standard substance (mouse IgG1), 370C was hatched 2 hours, added HRP-sheep anti-mouse igg (κ) and carried out association reaction, hatched 1 hour for 37 ℃, add TMB in 37 ℃ of effects 10 minutes, use H at last
2SO
4Termination reaction is surveyed A
450Value.The expression amount that has shown 12 candidate clones that above-mentioned screening obtains in the following table 1.
The expression amount of table 1 candidate clone in Chinese hamster ovary celI
| Cell strain number | 1A5 | ?3B6 | ?3C3 | ?4E9 | ?5H4 | ?4K1 | ?7D2 | ?8E5 | ?9A1 | ?8H3 | ?9D4 | ?6B2 | ?5E3 |
| Expression amount (mcg/ml) | 179.4 | ?152.3 | ?112.9 | ?286.4 | ?178.9 | ?143.8 | ?301.5 | ?201.4 | ?273.1 | ?132.9 | ?243.2 | ?132.9 | ?169.3 |
As can be seen from Table 1,4E9,7D2 and 9A1 have very high expression level.
The dna sequencing of embodiment 3 anti-proteinA monoclonal antibody genes
According to pedigree, the anti-proteinA antibody gene of the 7D2 cell strain of above-mentioned acquisition is carried out dna sequencing.The result is as follows: SEQ ID NO:3 has shown that the aminoacid sequence of its chain variable region gene sequence (5 ' to 3 ') its supposition of 336bp is presented among the SEQ ID NO:1; SEQ ID NO:3 has shown 7D2 heavy chain variable region gene sequence ((5 ' to 3 ') 357 bp), and the aminoacid sequence of its supposition is presented among the SEQ ID NO:2.
Embodiment 4 monoclonal antibody avidity researchs
Cell culture fluid to each clone carries out purifying by the following method.Centrifugal cell and the cell debris removed of 10000rpm, filter membrane ultrafiltration and concentration to 1/10 volume of 100kD molecular weight cut-off, the ultrafiltration damping fluid is 100mMTris-HCl, pH7.5.Cross the SPA-sepharose affinity column, sample solution is 100mM Tris-HCl, and pH7.5, elutriant are the 20mM citric acid, pH3.0,100mM NaCl.Molecular sieve (Sephadex G200) chromatography.Elutriant is 100mM Tris-HCl, and pH7.5 gets pure product.
Avidity measure to adopt the Scatchard analytical method (people such as Munson, 1980, Anal.BioChem. 107:220) carries out.The result shows, 4E9, and the avidity of 7D2 and three kinds of monoclonal antibodies of 9A1 reaches 5.6 * 10 respectively
-9, 5.78 * 10
-10With 7.52 * 10
-8
The single gram detection of embodiment 5 anti-proteinA proteinA reaches the comparison with the commercial reagent box
1. reagent and material
1.1 anti-proteinA monoclonal antibody: the engineering cell of structure (7D2) is through the serum-free fermentation, and the proteinA affinitive layer purification obtains.
1.2 commercially available proteinA detection kit (Cygnus Technologies, Inc.Catalog#F050).
1.3 the anti-proteinA polyclonal serum of rabbit, proteinA and freund's adjuvant immunity new zealand white rabbit are got serum and are detected, and after the dilution in 1: 2000, are strong positive reaction with proteinA, and it is standby to get serum.
1.4 how anti-goat anti-rabbit igg (H+L) is, HRP mark, extent of dilution 1: 1000-4000, Southern Biotech.
1.5HRP chromogenic substrate: TMB, Shanghai Xiamen Kehua divides A liquid and B liquid, face with before, both equal-volumes mix.
1.6 stop buffer: 0.5 mol/L sulfuric acid.
1.7pH=7.2 PBS: take by weighing KH
2PO
40.21g, NaCl 9.0g, Na
2HPO
4.12H
2O 0.97g adds the injection water and is dissolved to 1000ml.
1.8 bag is cushioned the sodium carbonate buffer of liquid: 20mmol/lpH=9.6.
1.9 sealing damping fluid: take by weighing the 3g bovine serum albumin, the PBS that adds pH7.2 is dissolved to 100ml.
1.10 lavation buffer solution: get 1ml Tween20, add PBS to 1 liter.
1.11 microplate reader.
1.12 low absorption 96 hole enzyme plate: Nunc.
1.13 other: multiple tracks sample injector, suction nozzle etc.
2. method: sandwich ELISA method
Anti-proteinA monoclonal antibody is diluted in to wrap and is cushioned in the liquid 2.1 recombinate, and concentration 1.0 μ g/ml wrap by 96 hole enzyme plates 0.1ml/well, 37 ℃ of 2h.
2.2 washing: lavation buffer solution washing 2 times all pats dry on thieving paper at every turn.
2.3 sealing: the sealing damping fluid is filled it up with 37 ℃ of 2h behind the hole.
2.4 washing: lavation buffer solution washing 3 times all pats dry on thieving paper at every turn.
2.5 add antigen: adding is diluted to 100,50 with the sealing damping fluid, the ProteinA standard substance of 25,12.5,6.25 (pg/mL), and 0.1ml/well establishes 2 multiple holes, adds the detected sample that suitably dilutes in the same plate, and 0.1ml/well establishes 2 multiple holes.
2.6?37℃2h。
2.7 washing: lavation buffer solution washing 5 times all pats dry on thieving paper at every turn.
2.8 how anti-add the anti-proteinA of rabbit.
2.9?37℃2h。
2.10 washing: lavation buffer solution washing 2 times all pats dry on thieving paper at every turn.
2.11 it is anti-to add the goat anti-rabbit igg two of HRP mark: add and use PBS1: two of 2000 dilutions resist 37 ℃ of 1h.
2.12 washing: lavation buffer solution washing 5 times all pats dry on thieving paper at every turn.
2.13 colour developing: add chromogenic substrate 100 μ L/ holes, lucifuge colour developing 5～20min (developing time is decided on the colour developing situation) adds stop buffer 50 μ L/ holes.
2.14 microplate reader reading: measuring the 450nm photoabsorption, is reference wavelength with 630nm.
2.15 do linear regression equation with standard substance concentration (logarithm) and A450/630nm value.
This method detects proteinA highly sensitive (6.25pg/ml), comparison with commercially available similar detection kit: Immunoenzymetric Assay for the Measurement of Protein A (CygnusTechnologies, Inc.Catalog#F050), by specification operation.
This test kit utilizes ELISA method (double antibody sandwich method) to detect Protein A equally, its sensitivity is 250pg/ml, the ELISA method that we utilize monoclonal antibody of the present invention to set up detects Protein A, sensitivity can reach 6.25pg/ml, significantly be better than (40 times) commercial like product, as shown in Figure 4.
Above result shows, antibody of the present invention compare with commercially available antibody have high specificity, advantage that avidity is high, highly sensitive as detecting antibody in commercially available antibody, therefore can substitute the commercial anti body and be used to prepare the reagent that detects ProteinA concentration.
In addition, commercially available detection kit is normally utilized ELISA double antibody sandwich method principle, in advance with antibody sandwich in test kit, be used to detect the corresponding antigen material, this method is easier, quick.Therefore, antibody of the present invention also can substitute the coated antibody in the existing detection kit, is used to prepare the test kit that detects Protein A concentration.
Sequence table
＜110〉Shanghai CP Guojian Pharmaceutical Co.,Ltd
＜120〉anti-proteinA monoclonal antibody of a kind of reorganization and its production and use
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tcctgcaagg?gttctggcta?cacattcatt?gattatcctt?tgcaatgggt?gaagcagagt 120
catgcaaaga?gtctagagtg?gattggcatt?attaaagcta?actatagtga?tactagctac 180
aaccagaagt?tcaagggcaa?ggccacaatg?actgtagaca?aatcctccag?cacagcctat 240
atggaacttg?ccagactgac?atctgaggat?tctgccatct?attactgtgc?cctctatagc 300
gacgagggca?atcctatgaa?cgactgcgag?cagggaacct?cagtcaccgt?ctcctca 357
Claims (10)
- One kind the reorganization anti-proteinA monoclonal antibody, it comprises variable region of heavy chain and variable region of light chain, it is characterized in that, the aminoacid sequence of variable region of light chain is shown in SEQ ID NO:1, the aminoacid sequence of variable region of heavy chain is shown in SEQ ID NO:2.
- 2. a dna molecular is characterized in that, the described monoclonal antibody of its coding claim 1.
- 3. dna molecular according to claim 2, it is characterized in that, this dna molecular contains the nucleotide sequence of the described monoclonal antibody variable region of light chain of coding shown in the SEQ ID NO:3, and the nucleotide sequence of the described monoclonal antibody variable region of heavy chain of coding shown in the SEQ ID NO:4.
- 4. an expression vector is characterized in that, the expression regulation sequence that it contains the described dna sequence dna of claim 2 and links to each other with this series of operations.
- 5. an eukaryotic host cell is characterized in that, it is transformed by the described expression vector of claim 4.
- 6. the described eukaryotic host cell of claim 5 is characterized in that, it is a Chinese hamster ovary celI.
- 7. a method of utilizing the described antibody test proteinA of claim 1 concentration is characterized in that, described method is the ELISA method.
- 8. one kind prepares the described monoclonal antibody method of claim 1, it is characterized in that this method comprises:A) provide an expression vector, the expression regulation sequence that this expression vector contains the described dna sequence dna of claim 2 and links to each other with this series of operations;B) transform eukaryotic host cell with the described expression vector of step a);C) eukaryotic host cell of gained culturing step b under the condition that is fit to described monoclonal antibody expression); WithD) separation and purification obtains described monoclonal antibody.
- 9. the application of the described monoclonal antibody of claim 1 in the reagent of preparation detection proteinA concentration.
- 10. the application of the described monoclonal antibody of claim 1 in the test kit of preparation detection proteinA concentration.
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