CN104610452B - Anti-human CD83 monoclonal antibody and its preparation, identification and application - Google Patents
Anti-human CD83 monoclonal antibody and its preparation, identification and application Download PDFInfo
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Abstract
The invention discloses anti-human CD83 monoclonal antibody, specificity is directed to the amino acid sequence of people CD83 extracellular fragment the 20th to 145.The invention also discloses the hybridoma cell strains for producing the monoclonal antibody.The invention also discloses the preparation method of the monoclonal antibody and identification methods.The invention also discloses the application of the monoclonal antibody and its exploitations of detection kit, anti-human CD83 monoclonal antibody of the invention can be used for the immunological experiments such as immunoblotting, immunoprecipitation, immunohistochemistry, ELISA and flow cytometry, to the qualitatively and quantitatively detection including CD83 or sCD83 in human serum, human tissue cell, people's tissue specimen, human secretion and people's cell culture, it can also be used to express activating or inhibiting and for antagonism and the effect for blocking CD83 for the human tissue cell of CD83.
Description
Technical field
The present invention relates to monoclonal antibody technique field more particularly to anti-human CD83 monoclonal antibodies, specifically, being directed to
20th to 145 amino acids sequence of people's CD83 extracellular fragment has reactive monoclonal antibody.The invention further relates to generate the list
The hybridoma cell strain of clonal antibody.The invention further relates to the preparation method of the monoclonal antibody and identification methods.The present invention is also
It is related to application and its kit of the monoclonal antibody.
Background technique
CD83 is a kind of surface glycoprotein that molecular weight is 45kDa, contactin.There are two kinds by CD83
Molecular forms, (the soluble CD83, sCD83) of (membrane CD83, mCD83) and solubility that film combines, the two has
Different biological functions.SCD83 only includes the extracellular section of mCD83, has immune suppression function.A large amount of experiment in vitro confirm
It falls off from cell surface or the sCD83 of vitro recombination is proliferated with apparent immunosuppressive action, animal the T cell that DC is mediated
Experiment in vivo is it has also been found that recombinate sCD83 albumen to mice with experimental autoimmune encephalomyelitis (EAE) and allograft skin
Skin, which transplants acute rejection, has prevention or therapeutic effect well.The prompt of these results, recombination sCD83 are likely in organ
The effect that important anti-immunity is repelled is played in transfer operation.In addition, suffering from Malignancy and rheumatic arthritis
In person, it was found that high-caliber sCD83.However, the mechanism of sCD83 release behind is not still elucidated with.
In order to more study the function of mCD83 and sCD83 in detail, accurate quickly detection blood or tissue local are developed
The tool of the protein expression of mCD83 and sCD83 and further exploitation have important face by the biological therapy of target spot of CD83
Bed meaning.
Summary of the invention
It is an object of the present invention to provide anti-human CD83 monoclonal antibodies, specifically, for people CD83 extracellular fragment
20th to 145 amino acids sequence has reactive monoclonal antibody.
It is also another object of the present invention to provide the hybridoma cell strains for generating above-mentioned anti-human CD83 monoclonal antibody.
A further object of the present invention is to provide the preparation method and identification method of above-mentioned anti-human CD83 monoclonal antibody.
A further object of the present invention be to provide above-mentioned anti-human CD83 monoclonal antibody in immunoblotting, immunoprecipitation, exempt from
The application of CD83 is detected in the test of epidemic disease group, ELISA or flow cytometry.
For this purpose, the BALB/c female of 8-10 week old is immunized using Pichia anomala expression CD83 extracellular domain recombinant protein by the present invention
After mouse, the spleen cell of mouse is taken to be merged in vitro under polyethylene glycol effect with myeloma SP2/0 cell, by limited
Dilution method and indirect enzyme-linked immunosorbent assay (ELISA) screen positive hybridoma cell strain.Conventional preparation ascites, is used
MabSelect affinitive layer purification antibody.And titration, the survey of affinity costant are carried out to anti-human CD83 monoclonal antibody
The identification of fixed, Ig subclass measurement and specificity.The preparation of anti-human CD83 monoclonal antibody, be CD83 molecule detection and
The research of CD83 function provides the foundation, and anti-human CD83 monoclonal antibody can be widely applied in the research of molecular immunology.
In order to solve the above-mentioned technical problem the present invention, provides the following terms：
The present invention provides anti-human CD83 monoclonal antibodies, are directed to the 20th to 145 amino acids of people CD83 extracellular fragment
Sequence has reactivity.
Specifically, anti-human CD83 monoclonal antibody is IgG1, κ subclass.
Specifically, it is CCTCC NO that anti-human CD83 monoclonal antibody, which is by deposit number,：The hybridoma cell strain of C2014186
The monoclonal antibody CD83-1H10 that 1H10 secretion generates.
Specifically, it is CCTCC NO that anti-human CD83 monoclonal antibody, which is by deposit number,：The hybridoma cell strain of C2014187
The monoclonal antibody CD83-11A7 that 11A7 secretion generates.
Specifically, it is CCTCC NO that anti-human CD83 monoclonal antibody, which is by deposit number,：The hybridoma cell strain of C2014188
The monoclonal antibody CD83-14E4 that 14E4 secretion generates.
The present invention also provides mouse hybridoma cell strain 1H10, deposit number is CCTCC NO：C2014186.
Invention further provides mouse hybridoma cell strain 11A7, deposit number is CCTCC NO：C2014187.
Invention further provides mouse hybridoma cell strain 14E4, deposit number is CCTCC NO：C2014188.
Invention further provides application of the above-mentioned anti-human CD83 monoclonal antibody in detection CD83, wherein the detection is
It is carried out by immunoblotting, immunoprecipitation, immunohistochemistry, ELISA or flow cytometry.
Invention further provides the kits for detecting CD83, and it is anti-that it includes above-described anti-human CD83 monoclonals
Body.
Specifically, mentioned reagent box is used to detect the sCD83 in the CD83 or blood sample on immunocyte.
Invention further provides the method for the CD83 on detection cell a kind of, this method includes：It (1) will be above-described anti-
People CD83 monoclonal antibody is contacted with the lysate of isolated cell or the cell to be detected；(2) it judges whether there is
Positive reaction.
Specifically, above-mentioned positive reaction is carried out by immunoblotting, immunoprecipitation, ELISA or Flow cytometry
Judgement.
Invention further provides the method for the sCD83 in detection fluid sample a kind of, this method includes：(1) by the above institute
The anti-human CD83 monoclonal antibody stated is contacted with blood sample to be detected；(2) positive reaction is judged whether there is.
Specifically, above-mentioned positive reaction is carried out by immunoblotting, immunoprecipitation, ELISA or Flow cytometry
Judgement.
Summary of the invention is described in detail：
One aspect of the present invention is related to the monoclonal antibody for having specificity to people CD83, and the anti-human CD83 monoclonal is anti-
Body acupuncture has reactivity to CD83 (Genbank registration number CAG33300.1) the 20th sequence into 145 amino acids；It is preferred that
Ground, the anti-human CD83 monoclonal antibody are CD83-1H10, CD83-11A7 and CD83-14E4.
Another aspect of the present invention is related to the mouse hybridoma cell strain of the anti-human CD83 monoclonal antibody of stably excreting
1H10,11A7 and 14E4, deposit number are followed successively by CCTCC NO：C2014186,CCTCC NO：C2014187 and CCTCC NO：
C2014188。
Another aspect of the present invention is related to the preparation method of the anti-human CD83 monoclonal antibody, utilizes Pichia pastoris table
After the BALB/c female mice of the recombinant C D83 protein immunization 8-10 week old reached, the spleen cell and myeloma SP2/0 of mouse are taken
Cell is merged in vitro under polyethylene glycol effect, and it is thin to screen positive hybridoma by limiting dilution assay and indirect elisa method
Born of the same parents' strain.Conventional preparation ascites, with MabSelect affinitive layer purification antibody.
Another aspect of the present invention is related to the identification method of the anti-human CD83 monoclonal antibody, including：The survey of potency
The fixed, measurement of affinity costant, the measurement of Subclass of antibody and specific identification.
Another aspect of the present invention relates to anti-human CD83 monoclonal antibodies to apply in immune-blotting method CD83 test.With
Anti-human CD83 monoclonal antibody of the invention detects CD83 for western blot test, the results show that anti-human CD83 monoclonal is anti-
Physical efficiency expresses CD83 albumen for detecting recombinant C D83 albumen and cellular endogenous in western blot test.
Another aspect of the present invention relates to application of the anti-human CD83 monoclonal antibody in ELISA detection CD83 test.With
The CD83 of anti-human CD83 monoclonal antibody detection various concentration of the invention, the results show that anti-human CD83 monoclonal antibody can be used
CD83 is detected in ELISA, and can be used for detecting the middle sCD83 of tumor patient blood sample.
Another aspect of the present invention relates to anti-human CD83 monoclonal antibody answering in Flow cytometry CD83 test
With.It can be combined with CD83 positive cell with anti-human CD83 monoclonal antibody of the invention, and CD83 negative cells combination of getting along well.
Show that anti-human CD83 monoclonal antibody can be used for Flow cytometry cell surface CD83.
In the present invention, term " specificity of monoclonal antibody " refer to monoclonal antibody identification antigen on defined epitope or
Person antigenic determinant and property in combination.
Term " reactivity of monoclonal antibody " refers under the appropriate reaction conditions, monoclonal antibody and antigen binding
Ability.
Term " cell strain ", which refers to, to be obtained by screening perhaps limited dilution method from primary culture or cell line
Unicellular cultures.
The present invention provides anti-human CD83 monoclonal antibody and its preparation, identification, applications.The monoclonal antibody has potency
The features such as high, specific high, application is also relatively broad.The table that clone number is directed to for the monoclonal antibody of 1H10,11A7 and 14E4
Position is the space conformation of CD83, can be advantageously used in flow cytometry and the detection of ELISA.Especially clone number list for being 1H10
Clonal antibody can identify sCD83, but cannot identify mCD83, therefore can be used for distinguishing two kinds of different types when flow cytometer detection
CD83.
Different from existing monoclonal antibody, immunogene used in anti-human CD83 monoclonal antibody prepared by the present invention is complete
The CD83 recombinant protein of red Yeast expression is directed to the CD83 extracellular fragment i.e. CD83 the 20th comprising complete function region extremely
145th amino acids sequence, by ELISA screening to the hybridoma cell strain of CD83 recombinant protein high response.
Anti-human CD83 monoclonal antibody of the invention is different from the region CD83 or site that existing antibody is directed to, effect
Valence, specificity and application change therewith.
The resulting anti-human CD83 monoclonal antibody of the present invention has many advantages, such as that potency is high, specificity is high and applied widely,
Anti-human CD83 monoclonal antibody provided by the invention can be widely used in immunoblotting, immunoprecipitation, ELISA, flow cytometry etc.
Means of different detects CD83 albumen, provides the foundation to study the function of CD83.
Detailed description of the invention
Fig. 1 is typical chromatography peak type figure during anti-human CD83 proposed by the present invention is monoclonal antibody-purified.
Fig. 2 is anti-human CD83 monoclonal antibody Purity result proposed by the present invention.
Fig. 3-A and Fig. 3-B is anti-human CD83 monoclonal antibody specificity qualification result proposed by the present invention.
Fig. 4 is the sCD83 in anti-human CD83 monoclonal antibody detection fermentation liquid proposed by the present invention.
Fig. 5-A and Fig. 5-B is that anti-human CD83 monoclonal antibody proposed by the present invention detects IM9 cell membrane surface CD83.
Fig. 6-A and Fig. 6-B is that anti-human CD83 monoclonal antibody proposed by the present invention and polyclonal antibody assemble ELISA reagent
Box optimization experiment.
Fig. 7 is that two proposed by the present invention anti-human CD83 monoclonal antibodies assemble the optimization of ELISA kit antibody combination.
Fig. 8-A and Fig. 8-B is highly sensitive ELISA kit antibody concentration optimization.
Fig. 9-A and Fig. 9-B is the optimization of muting sensitivity ELISA kit antibody concentration.
Figure 10-A and Figure 10-B is that CD83ELISA kit detects sCD83 content in Serum of Cancer Patients.
Specific embodiment
In the following, technical solution of the present invention is described in detail by specific embodiment.
The preparation of embodiment 1sCD83 recombinant protein
Preparation in the embodiment of the present invention about sCD83 recombinant protein, specifically refers to Chinese invention patent《It is soluble
The expression and preparation method of CD83》, application No. is 201310187915.3, publication date is on August 14th, 2013, Publication No.
103243119A；Or refer to research paper：Yugang Guo et al., The expression and
characterization of functionally active soluble CD83by Pichia pastoris using
High-density fermentation, PLOS ONE 9 (2):E89264,2014.
The preparation of the anti-human CD83 monoclonal antibody of embodiment 2
(China of Beijing Aibo Bio-Technology Co., Ltd. is entrusted in the specific implementation of anti-human CD83 monoclonal antibody hybridoma
Beijing, postcode 100085) it completes.The description of implementation process generality can be found in Chinese invention patent《Anti-human NKp30 monoclonal antibody
Preparation, identification and application》, application No. is 201010531489.7, publication date is on March 16th, 2011, Publication No.
101985476A。
It is summarized as follows：
1, prepare before cell fusion
The sCD83 recombinant protein of purifying is mixed with isometric complete Freund's adjuvant, the BALB/c that 8-10 week old is immunized is female
Mouse.It strengthens the immune system once every 2 weeks later, after being immunized 5 times altogether, the serum titer of ELISA detection mouse reaches 1：105When,
Impact is carried out to mouse to be immunized once, will take off white execution after the blood sampling of above-mentioned impact immunized mice eye socket after 3 days, 75% alcohol disappears
Poison takes spleen, removes connective tissue, collects splenocyte suspension, it is spare to be prepared into spleen mononuclear cell suspension.It is previous in cell fusion
It, prepares Turnover of Mouse Peritoneal Macrophages, as feeder cells, is put into cell incubator, and 37 DEG C, cultivate under the conditions of 5%C02.It is multiple
It revives SP2/0 myeloma cell (ATCC number CRL-1581), guarantees that cell is in logarithmic growth phase when fusion.In cell fusion
Before, SP2/0 cell is washed 2 times with the incomplete RPMI-1640 culture medium of 30-40ml.Cell cannots be used up full RPMI-1640 culture medium
It is resuspended spare.
2, the preparation of cell fusion and hybridoma
By above-mentioned splenocyte suspension and SP2/0 cell, 50%PEG4000 (pH=8.0-8.2) induced fusion is used.Fusion
Cell detects antibody in HAT culture solution culture 1 week, Aspirate supernatant, indirect elisa method.Positive hybridoma cell strain is screened, is adopted
Subcloning is carried out with limited dilution method.Hybridoma cell strain 1H10,11A7 and 14E4 repeatedly are obtained after screening, outside non-individual body
Culture 2 months are above or after liquid nitrogen cryopreservation 6 months, which remains to stablize and largely secrete the antibody of anti-human CD83,
The monoclonal antibody is named as CD83-1H10, CD83-11A7 and CD83-14E4.
Hybridoma cell strain 1H10,11A7 and 14E4 were preserved in Chinese Typical Representative culture guarantor on November 25th, 2014
Hiding center (CCTCC, Wuhan, China, Wuhan University, postcode：430072), deposit number is respectively CCTCC NO：C2014186,
CCTCC NO：C2014187 and CCTCC NO：C2014188.
3, a large amount of preparations of anti-human CD83 monoclonal antibody
Preparing anti-human CD83 monoclonal antibody can be used two methods, increment cultivation and mice celiac inoculation.Increment
Cultivation is that low-serum-concentration culture medium carries out mass propgation in vitro by the cell of cloning, cell culture 2-3 days, in collection
Clear liquid and obtain a large amount of single cloning antibody.Mice celiac inoculation be select 8-10 week old BALB/c female mice (on
This extra large Lake experimental animal Co., Ltd), incomplete Freund's adjuvant pre-processes after a week, every mouse peritoneal injection 2 × 106It is a
Cell (500 μ l PBS resuspension), 7-10 days or so generation ascites collect mouse ascites, 4 DEG C, 12000g be centrifuged 10min, in receipts
Clear liquid saves or carries out next step purifying.The antibody obtained by the above method, can use ammonium sulfate precipitation, MabSelect
Affinity chromatography and molecular sieve methods purifying (as shown in Figure 1), the purity of antibody are identified with SDS-PAGE.As shown in Fig. 2, by pure
The purity of anti-human CD83 monoclonal antibody CD83-1H10, CD83-11A7 and CD83-14E4 for changing reach 95% or more, anti-human
The molecular weight about 160kDa of CD83 monoclonal antibody (Ig (H+L)), wherein heavy chain Ig (H) about 55kDa, light chain Ig (L) be about
25kDa。
Above-mentioned PBS, full name Phosphate Buffered Saline.Phosphate buffer is indicated in medical vocabulary, is used
In molecular cloning and cell culture, pH=7.4 is isotonic with blood of human body, main component be potassium dihydrogen phosphate, disodium hydrogen phosphate,
Sodium chloride and potassium chloride.
Resuspension refers to " suspending again ", i.e., the solid that buffer or culture solution appropriate obtain the methods of centrifugation or sedimentation
(precipitating, cell, active material etc.) suspends again.
The identification of the anti-human CD83 monoclonal antibody of embodiment 3
1, indirect ELISA measures the affinity costant of anti-human CD83 monoclonal antibody.
Coating buffer dilution recombination sCD83 albumen is to 2 μ g/ml, 1 μ g/ml, 0.5 μ g/ml and 0.25 μ g/ml, 100 holes μ l/,
It is coated with 96 hole elisa plates respectively, 4 DEG C overnight.By S3, S5, S7 (corresponding monoclonal antibody is shown in Table 1) by 2 times of gradient dilutions
For 16 concentration (2000ng/ml-0ng/ml), reacted with the sCD83 of bed board.Use the sheep anti-mouse igg Fc of HRP label as two
It develops the color after anti-.With the absolute affinity costant of both indirect elisa method measurements.
It is mapped with concentration of the OD 450nm to antibody, the calculating affinity using propositions such as J.David Beatty (1987) is normal
Several formula calculates the absolute affinity costant (Kaff) of the two：
Kaff(M-1)=1/2 (2 [monoclonal antibody '] t- [monoclonal antibody] t)
[monoclonal antibody] t is indicated with a certain amount of sCD83 (antigen) bed board, when OD 450nm reaches half absorption value, monoclonal antibody
Total concentration；
[monoclonal antibody '] t is indicated with the antigen bed board of the amount of (antigen)/2, when OD 450nm reaches half absorption value, antibody
Total concentration.
A Kaff can be calculated between every two adjacent sCD83 concentration, four concentration can be calculated 3 altogether
Kaff value, averages, and average value is asked to the absolute affinity costant KD of as sCD83 and monoclonal antibody reciprocal.
2, the subclass measurement of anti-human CD83 monoclonal antibody
The mouse monoclonal type identification quick detection kit IsoQuickTM Kit provided using Sigma-Aldrich
Subclass Test paper in for Mouse Monoclonal Isotyping IS0Q5 is measured.It is mono- to three kinds of anti-human CD83
Clonal antibody is numbered, i.e., anti-human CD83 monoclonal antibody CD83-1H10 number is S3, anti-human CD83 monoclonal antibody
CD83-11A7 number is S5, and anti-human CD83 monoclonal antibody CD83-14E4 number is S7.The monoclonal of above-mentioned S3, S5, S7 are anti-
Body subclass and affinity constant are as shown in table 1.
The anti-human CD83 monoclonal antibody subgroup identification of table 1 and affinity constant
| Monoclonal antibody number | S3 | S5 | S7 |
| Monoclonal antibody title | CD83-1H10 | CD83-11A7 | CD83-14E4 |
| Monoclonal antibody subclass | LgG1,κ | LgG1,κ | LgG1,κ |
| Affinity constant KD | 6.7×109M-1 | 3.3×109M-1 | 1010M-1 |
3, the specificity identification of anti-human CD83 monoclonal antibody
Identify the specificity of anti-human CD83 monoclonal antibody using two methods：Western blot and ELISA method.
The experiment flow of immunoblotting：Taking 20 μ l Yeast Cultivation liquid respectively, (negative control 1, unconverted yeast GS115 is in phase
With the culture supernatant under condition of culture), the recombination IL-33-His of 1 μ g (negative control 2, (detailed preparation method referring to
Lingel, A, et al., Structure of IL-33and its interaction with the ST2and IL-
1RAcP receptors--insight into heterotrimeric IL-1signaling
complexes.Structure 17:1398-1410,2009)) and recombination 15kDa GNLY-His albumen (negative control 3, in detail
Thin preparation method can be found in Chinese invention patent《The expression and preparation method of granulysin》, application No. is
201210250501.6, publication date is on October 31st, 2012, Publication No. 102757977A；Or referring to research paper Yugang
Guo, et al., Production and characterization of recombinant 9and 15kDa
granulysin by fed-batch fermentation in Pichia pastoris.Appl Microbiol
Biotechnol 97(17):7669-7677,2013) and sCD83-His albumen is recombinated as test sample, SDS-PAGE electrophoresis
Protein isolate.Destination protein is transferred to by pvdf membrane (BioRad company) using half-dried transferring film instrument.Pvdf membrane is put into 5% degreasing ox
Milk room temperature closes lh.The anti-human CD83 monoclonal antibody (1 of lmg/ml:5000 dilutions) incubation at room temperature 2-3h, TBST (50mM
Tris, 0.9%NaCl, 0.1%Tween 20, pH=7.4) it washes 3 times.The sheep anti mouse of horseradish peroxidase (HRP) label is anti-
Body (1:5000 dilutions) it is incubated at room temperature lh, TBST is washed 3 times.Chemiluminescence colour developing (Thermo scientific company, article No.
34080) it detects.Through western blotting qualification, as a result as shown in Fig. 3-A, have at relative molecular mass 20kDa (sCD83-His)
One specific band, and there is no specific band at 18kDa (recombination IL-33-His) and 15kDa (15kDa GNLY-his).Card
Bright S3 and S7 is specificity for CD83, and to His-Tag without reactivity.S5 cannot effectively identify the recombination of denaturation
SCD83, but can identify unmodified sCD83 (seeing below ELISA method detection), it prompts, what S5 may be identified is the sky of sCD83
Between configuration.
For ELISA method measurement S3, S5 and S7 specificity experiments steps are as follows：Coating buffer dilution recombination IL-33-His,
15kDa GNLY-His (negative control 2) and recombination sCD83-His albumen are recombinated to 2 μ g/ml, 100 holes μ l/ are coated with 96 holes
Elisa plate, 4 DEG C overnight.Second day abandoning supernatant, PBST are washed 2 times, 1%BSA closing, 37 DEG C of incubation 2h.Prepare each anti-human CD83
Monoclonal antibody dilution (concentration be 1 μ g/ml), PBST be added after washing 2 times the anti-human CD83 monoclonal antibody dilution of 100 μ l in
In 96 orifice plates, if PBS is zeroing hole, 37 DEG C of incubation 2h.PBST is washed 5 times, and 100 μ l horseradish peroxidases (HRP) are added in every hole
The sheep anti-mouse antibody (1 of label:10000 dilutions), 37 DEG C of incubation lh.PBST is washed 5 times, tmb substrate liquid is added, 100 holes μ l/ are kept away
Light colour developing 10-15min, is added 1/ hole terminate liquid (1M sulfuric acid) 100 μ, is measured immediately with microplate reader, read wavelength 450nm
Light absorption value (OD 450nm).As a result as shown in Fig. 3-B, S3, S5 and S7 have specific binding to sCD83, and to recombination IL-
33-His and recombination 15kDa GNLY-His illustrate that S3, S5 and S7 have good specificity to CD83 without combination.
The application of the anti-human CD83 monoclonal antibody of embodiment 4
1, sCD83 is detected for immune-blotting method sCD83 and immunoprecipitation
Experiment flow is the same as the immunoblotting process in the specificity identification of above-mentioned anti-human CD83 monoclonal antibody.Such as Fig. 3-A
Shown, S3 and S7 can specifically detect recombination sCD83 albumen, and S5 cannot effectively identify the recombination sCD83 of denaturation, but can
It identifies unmodified sCD83, prompts, what S5 may be identified is the steric configuration of sCD83.In another immunoblotting application case
In, the S3 and S7 are enough in the fermentation expression detection of sCD83.As shown in Fig. 4-A, S3 and S7 can specifically detect recombination
SCD83 albumen, S5 cannot effectively identify the recombination sCD83 of denaturation, almost the same with the above results.Those skilled in the art are easy to
It associates, the anti-human CD83 monoclonal antibody can also be applied to the immune-blotting method of the CD83 of endogenous cellular.
Anti-human CD83 monoclonal antibody of the present invention in addition to the sCD83 in immune-blotting method fermentation liquid can be used,
Can be also used for immunoprecipitation detected.4ml sCD83 fermentation supernatant is taken, pH is adjusted to alkalescent (pH 7.5), 20 μ is added
G S3, S5, S7 and the normal lgG of control antibodies mouse (mlgG) mildly shake 2 hours on 4 DEG C of shaking tables, and 50 μ l protein are added
A/G Plus-Agarose shakes 2 hours, 10000g, 4 DEG C and is centrifuged 10 minutes, abandons supernatant, and PBST is washed three times, each 10000g, and 4
DEG C centrifugation 10 minutes, abandon supernatant.It being resuspended with 50 μ lPBS, protein electrophoresis sample-loading buffer is added, boiling boils 10 minutes, 10000g, and 4
DEG C centrifugation 10 minutes, supernatant is taken to carry out SDS-PAGE electrophoresis and western blotting qualification.As shown in Fig. 4-B, S3, S5 and S7 can be with
Specifically by sCD83, immunoprecipitation is come out from fermentation liquid, and negative control antibody mlgG then cannot.Prove S3, S5 and S7
It can be used for the immunoprecipitation of sCD83.Since S3 and S7 can identify that the CD83 of membranous type, those skilled in the art are easy to join
Expect, S3 of the invention and S7 can be also used for the immunoprecipitation of the CD83 in cell pyrolysis liquid.
Further, since antibody of the present invention can identify in immunoblotting identifies non-change in denatured antigen and ELISA
Property antigen, therefore those skilled in the art are easy to associate, anti-human CD83 monoclonal antibody of the present invention can be also used for
The mCD83 and sCD83 in the samples such as tissue, cell are detected in the experiment such as immunohistochemistry, immunofluorescence, this specification is no longer one by one
Citing.
2, it is used for Flow cytometry cell membrane surface CD83
It may refer to China with the specific method of the CD83 of Flow cytometry cell membrane surface and antibody competition experiment
Patent of invention《The expression and preparation method of Soluble CD83》, application No. is 201310187915.3, publication date is in August, 2013
14 days, Publication No. 103243119A；Or refer to research paper：Yugang Guo, et al., The expression and
characterization of functionally active soluble CD83by Pichia pastoris using
High-density fermentation, PLOS ONE 9 (2):E89264,2014.
As shown in fig. 5-A, S3 and S7 is preferable with cell membrane surface mCD83 protein binding effect, basic with commercial antibody
Quite, but S5 is without significant binding ability, prompts the binding site of S5 close to the transmembrane region of CD83, since steric hindrance combines not
Up.But since S5 (can be confirmed) in conjunction with free sCD83 in ELISA detection, measuring samples are distinguished using S5
In membranous type mCD83 and soluble sCD83.
As shown in fig. 5-b, in three antibody of the present invention, S3 and S7 have apparent competition effect to commercial antibody
Fruit prompts S3 and S7 can be with specific recognition Cell model CD83, and is closer to the epitope of commercial antibody identification.
3, it is used to prepare the ELISA kit of detection CD83
Double crush syndrome (Sandwich ELISA) is a kind of High sensitivity, special immunoassay technology.The present invention
It is assembled respectively using anti-human CD83 monoclonal antibody and polyclonal antibody, two different anti-human CD83 monoclonal antibodies for examining
Survey the ELISA kit of CD83.
1) anti-human CD83 monoclonal antibody and polyclonal antibody assemble ELISA kit
Experiment flow：Corresponding anti-human CD83 monoclonal antibody is coated with 96 hole ELISA by the design concentration of table 2 and table 3
Plate, 4 DEG C overnight.PBST is washed 2 times, 1%BSA closing, 37 DEG C of incubation 1h.PBST is washed 3 times, and every hole is separately added into 100 μ l tables and sets
The sCD83 standard items of concentration are counted, if standard dilutions are zeroing hole, 37 DEG C of incubation lh.PBST is washed 3 times, and 100 μ l are added in every hole
(it is special that the specific preparation method of polyclonal antibody may refer to Chinese invention to the rabbit-anti sCD83 polyclonal antibody of Table Design concentration
Benefit《The expression and preparation method of Soluble CD83》, application No. is 201310187915.3, publication date is on August 14th, 2013,
Publication No. 103243119A；Or refer to research paper：Yugang Guo, et al., The expression and
characterization of functionally active soluble CD83by Pichia pastoris using
High-density fermentation, PLOS ONE 9 (2):E89264,2014), 37 DEG C of incubation lh.PBST washes 3 times, often
The goat anti-rabbit antibody (1 of horseradish peroxidase (HRP) label is added in hole:10000 dilutions), 37 DEG C of incubation lh.PBST is washed 5 times,
Tmb substrate liquid is added, 100 holes μ l/ are protected from light colour developing 10-15min, 1/ hole terminate liquid (1M sulfuric acid) 100 μ are added, immediately with enzyme mark
Instrument is measured, and reads the light absorption value (OD 450nm) of wavelength 450nm.
The coated antibody type of the anti-human CD83 monoclonal antibody of table 2 and polyclonal antibody assembling ELISA kit optimizes letter
Cease table
The coated antibody type of the anti-human CD83 monoclonal antibody of table 3 and polyclonal antibody assembling ELISA kit optimizes letter
Cease table
| Primary antibody type | S7 |
| Primary antibody diluted concentration | Antibody concentration 1mg/ml, by 1:500-1:160002 times of gradient dilutions, totally 6 gradients |
| SCD83 sample diluted concentration | 400ng/ml-25pg/ml, 2 times of gradient dilutions, totally 15 gradients |
| Secondary antibody diluted concentration | 10 μ g/ml of Rabbit anti CD83 polyclonal antibody |
| HRP antibody diluted concentration | Goat anti rabbit-HRP 0.75μg/ml |
As shown in Fig. 6-A, S3 and S7 can be assembled into the relatively high ELISA kit of sensitivity with polyclonal antibody,
Sensitivity reaches 0.5ng/ml, range of linearity 0.5ng/ml-50ng/ml, suitable for cell culture supernatant, patients serum
Detect CD83 content.S5 can be assembled into the relatively low ELISA kit of sensitivity with polyclonal antibody, and sensitivity reaches
10ng/ml, range of linearity 10ng/ml-1000ng/ml are suitable for Concentration Testing in sCD83 preparation process.
Coated antibody concentration is advanced optimized, as shown in figure 6-b, increase of the discovery with coated antibody concentration, detection letter
It number gradually increases, sensitivity also increased, however the range of linearity is opposite becomes smaller, therefore comprehensively consider detection signal strength or weakness, spirit
The factors such as sensitivity and the range of linearity, 5 μ g/ml are preferably peridium concentrations.
2) two anti-human CD83 monoclonal antibodies assemble ELISA kit
Experiment flow：According to biotin labeling reagent box (Thermo Scientific, article No.：21435EZ-Link) grasp
Make handbook and biotinylation label is carried out to S3, S5 and S7, and detects biotinylation labeling effciency.It is computed as shown in table 4, three
The biotin labeling rate of antibody can be used normally between 3-7.
The control of the anti-human CD83 monoclonal antibody ubiquitination mark quality of table 4
| S3 | S5 | S7 | |
| MAb concentration (mg/mL) after label | 1.609 | 1.116 | 0.764 |
| Biotinylated MAb concentration (mmol/mL) | 1.07E-05 | 7.44E-06 | 5.09E-06 |
| The concentration (mmol/mL) of biotin in analysis of mixtures | 5.82E-06 | 4.35E-06 | 1.82E-06 |
| The concentration (mmol/mL) of biotin in monoclonal antibody | 5.82E-05 | 4.35E-05 | 1.82E-05 |
| Biotin labeling rate (the biotin number on each antibody molecule) | 5.43 | 5.85 | 3.58 |
Next corresponding anti-human CD83 monoclonal antibody is coated with 96 hole elisa plates, 4 DEG C of mistakes by the design concentration of table 5
Night.PBST is washed 2 times, 1%BSA closing, 37 DEG C of incubation 1h.PBST is washed 3 times, and every hole is separately added into 100 μ l Table Design concentration
SCD83 standard items, if standard dilutions are zeroing hole, 37 DEG C of incubation lh.PBST is washed 3 times, and corresponding 100 μ l table is added in every hole
Biontinylated anti-human's CD83 monoclonal antibody of design concentration, 37 DEG C of incubation lh.PBST is washed 3 times, and Avidin label is added in every hole
Horseradish peroxidase (R&D, article No.：AEM771012, Part No.890803), 37 DEG C of incubation lh.PBST is washed 5 times, is added
Tmb substrate liquid, 100 holes μ l/, be protected from light colour developing 10-15min, be added 1/ hole terminate liquid (1M sulfuric acid) 100 μ, immediately with microplate reader into
Row measurement, reads the light absorption value (OD450nm) of wavelength 450nm.
5 two, table anti-human CD83 monoclonal antibody assembling ELISA kit antibody combination optimizations
As shown in fig. 7, S3 is most strong as combination (S3/S7) the detection signal of detection antibody as coated antibody, S7, it is sensitive
Highest is spent, S7 takes second place as coated antibody, S3 as the combination (S7/S3) of detection antibody, and S5 is as coated antibody, S3 as inspection
The combination (S5/S3) for surveying antibody is most weak.However S7/S3 is linear (0.5ng/ml-25ng/ml) widest in area in these combinations
One group.
Next highly sensitive ELISA kit and table 8 resist muting sensitivity ELISA kit by table 6,7 respectively
The concentration of body optimizes.As a result as shown in Fig. 8-A, Fig. 8-B, Fig. 9-A and Fig. 9-B, the antibody combination of low concentration (5 μ g/ml)
Good detection effect is had reached, detection effect can't be promoted by providing concentration, be decreased instead sometimes.
The highly sensitive ELISA kit primary antibody concentration optimization of table 6
The highly sensitive ELISA kit secondary antibody concentration optimization of table 7
8 muting sensitivity ELISA kit secondary antibody concentration optimization of table
3) using the sCD83 content in above-mentioned assembling ELISA kit detection patients serum
SCD83's contains in the highly sensitive ELISA kit detection clinical tumor patient serum combined using above-mentioned S3/S7
Amount, standard curve result is as shown in Figure 10-A, and (wherein No. 1 is healthy volunteer's blood serum sample to sample to be tested, and No. 2-47 is tumour
Patient serum sample) result as shown in figure 10-b, the higher (tumor patient of sCD83 content is detected in Partial tumors patients serum
Blood serum sample is 46 total), and almost all is without containing sCD83 (the total inspection of healthy volunteer in healthy volunteer's blood serum sample
40 are surveyed, wherein No. 1 is representative sample), among sCD83 content and tumor development correlation are being analyzed at present.This reality
It tests and shows that above-mentioned assembling ELISA kit can be used for the sCD83 content detection in Serum of Cancer Patients.
Sequence table description
SEQ ID No:Amino acid sequence (the GenBank Accession No. of 1 people's overall length CD83:CAG33300.1)；
SEQ ID No:The sCD83 amino acid sequence of 2 Pichia pastoris recombinant expression.
Above-mentioned 1M hydrochloric acid is 1mol/L hydrochloric acid, and 1M sulfuric acid is 1mol/L sulfuric acid, and 50mM Tris is that the Tris of 50mmol/L is molten
Liquid.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (5)
1. anti-human CD83 monoclonal antibody has reactivity for the 20th to 145 amino acids sequence of people CD83 extracellular fragment；Institute
The anti-human CD83 monoclonal antibody stated is IgG1, and κ subclass, it is CCTCC that the anti-human CD83 monoclonal antibody, which is by deposit number,
NO：The monoclonal antibody that the hybridoma cell strain 1H10 secretion of C2014186 generates.
2. secreting the mouse hybridoma cell strain 1H10 of anti-human CD83 monoclonal antibody as described in claim 1, deposit number is
CCTCC NO：C2014186.
3. application of the anti-human CD83 monoclonal antibody according to claim 1 in preparation detection CD83 kit, wherein
The detection is carried out by immunoblotting, immunoprecipitation, immunohistochemistry, ELISA or flow cytometry.
4. the kit for detecting CD83, it includes anti-human CD83 monoclonal antibodies described in claim 1.
5. according to claim 4 for detecting the kit of CD83, the kit is for detecting on immunocyte
SCD83 in CD83 or blood sample.
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| CN103906766A (en) * | 2011-07-01 | 2014-07-02 | 弗·哈夫曼-拉罗切有限公司 | Use of anti-cd83 agonist antibodies for treating autoimmune diseases |
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| US6479247B1 (en) * | 1996-10-09 | 2002-11-12 | The Corporation Of The Trustees Of The Order Of The Sisters Of Mercy In Queensland | Dendritic cell-specific antibodies |
| EP1422241A1 (en) * | 2002-11-19 | 2004-05-26 | Alexander Steinkasserer | Use of soluble forms of CD83 and nucleic acids encoding them for the treatment or prevention of diseases |
| EP2258724A1 (en) * | 2002-11-21 | 2010-12-08 | Celltech R & D, Inc. | Modulating immune responses using multimerized anti-CD83 antibodies |
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| CN103243119A (en) * | 2013-05-20 | 2013-08-14 | 中国科学技术大学 | Expression and preparation method of soluble CD83 |
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| Lin Zhang等.Structural identification of recombinant human CD83 mutant variant as a potent therapeutic protein.《Protein Expression and Purification》.2010,(第73期),第140页左栏第1段、第141页材料与方法. * |
| Structural identification of recombinant human CD83 mutant variant as a potent therapeutic protein;Lin Zhang等;《Protein Expression and Purification》;20100608(第73期);第140页左栏第1段、第141页材料与方法 * |
| The Expression and Characterization of Functionally Active Soluble CD83 by Pichia pastoris Using High-Density Fermentation;Yugang Guo等;《PLOS ONE》;20140220;第9卷(第2期);全文 * |
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