CN109369807B - Anti-human CD83 monoclonal antibody and preparation, identification and application thereof - Google Patents
Anti-human CD83 monoclonal antibody and preparation, identification and application thereof Download PDFInfo
- Publication number
- CN109369807B CN109369807B CN201810970426.8A CN201810970426A CN109369807B CN 109369807 B CN109369807 B CN 109369807B CN 201810970426 A CN201810970426 A CN 201810970426A CN 109369807 B CN109369807 B CN 109369807B
- Authority
- CN
- China
- Prior art keywords
- human
- monoclonal antibody
- antibody
- scd83
- elisa
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Abstract
The invention discloses an anti-human CD83 monoclonal antibody, the specificity of which is directed against the amino acid sequence of 20 th to 145 th of extracellular domain of human CD 83. The invention also discloses a hybridoma cell strain for producing the monoclonal antibody. The invention also discloses a preparation method and an identification method of the monoclonal antibody. The invention also discloses the application of the monoclonal antibody and the development of a detection kit thereof, and the antihuman CD83 monoclonal antibody can be used for immunological experiments such as immunoblotting, immunoprecipitation, immunohistochemistry, ELISA, flow cytometry and the like, can be used for quantitative and qualitative detection of CD83 or sCD83 in human serum, human histiocytes, human tissue specimens, human secretion and human cell culture, and can also be used for activation or inhibition of human histiocyte expressing CD83 and antagonism and blocking of CD 83.
Description
Technical Field
The invention relates to the technical field of monoclonal antibodies, in particular to an anti-human CD83 monoclonal antibody, and specifically relates to a monoclonal antibody with reactivity aiming at amino acid sequences from 20 th to 145 th positions of extracellular domains of human CD 83. The invention also relates to a hybridoma cell strain for producing the monoclonal antibody. The invention also relates to a preparation method and an identification method of the monoclonal antibody. The invention also relates to application of the monoclonal antibody and a kit thereof.
Background
CD83 is a surface glycoprotein with a molecular weight of 45kDa and belongs to the immunoglobulin superfamily. CD83 exists in two molecular forms, membrane bound (membrane CD83, mCD83) and soluble (soluble CD83, sCD83), both of which have different biological functions. sCD83 contains only the extracellular segment of mCD83 and has immunosuppressive functions. A large number of in vitro experiments prove that the sCD83 shed from the cell surface or recombined in vitro has obvious immunosuppressive action on the proliferation of the T cells mediated by DC, and animal in vivo experiments also find that the recombined sCD83 protein has good prevention or treatment action on Experimental Autoimmune Encephalomyelitis (EAE) of mice and allogeneic skin transplantation acute rejection. These results suggest that recombinant sCD83 is likely to play an important role in immune rejection in organ transplantation surgery. In addition, high levels of sCD83 were found in patients with hematologic malignancies and rheumatoid arthritis. However, the mechanism behind sCD83 release has not yet been elucidated.
In order to study the functions of mCD83 and sCD83 in more detail, it is of great clinical significance to develop a tool for accurately and rapidly detecting protein expression of mCD83 and sCD83 locally in blood or tissues and further develop biological therapies targeting CD 83.
Disclosure of Invention
It is an object of the present invention to provide an anti-human CD83 monoclonal antibody, specifically a monoclonal antibody reactive with the amino acid sequence at positions 20 to 145 of the extracellular domain of human CD 83.
Still another object of the present invention is to provide a hybridoma cell line producing the above-mentioned anti-human CD83 monoclonal antibody.
Still another object of the present invention is to provide a method for preparing and identifying the above-mentioned anti-human CD83 monoclonal antibody.
Still another objective of the present invention is to provide the use of the above anti-human CD83 monoclonal antibody in immunoblotting, immunoprecipitation, immunohistochemistry, ELISA or flow cytometry assays for detecting CD 83.
After 8-10 weeks-old BALB/c female mice are immunized by using pichia pastoris to express CD83 extracellular recombinant protein, spleen cells of the mice are taken to be fused with myeloma SP2/0 cells in vitro under the action of polyethylene glycol, and positive hybridoma cell strains are screened by a limiting dilution method and an indirect enzyme-linked immunosorbent assay (ELISA). Ascites fluid was prepared conventionally and the antibody was purified by MabSelect affinity chromatography. And titer determination, determination of affinity constant, determination of Ig subclasses and identification of specificity were performed against the human CD83 monoclonal antibody. The preparation of the anti-human CD83 monoclonal antibody provides a basis for the detection of CD83 molecules and the research of CD83 functions, and the anti-human CD83 monoclonal antibody can be widely used in the research of molecular immunology.
In order to solve the above technical problems, the present invention provides the following:
the invention provides an anti-human CD83 monoclonal antibody, which has reactivity to the amino acid sequence from 20 th to 145 th of the extracellular domain of human CD 83.
Specifically, the anti-human CD83 monoclonal antibody is IgG1, kappa subclass.
Specifically, the anti-human CD83 monoclonal antibody is prepared from a monoclonal antibody with the preservation number of CCTCC NO: the hybridoma cell strain 1H10 of C2014186 secretes and produces the monoclonal antibody CD83-1H 10.
Specifically, the anti-human CD83 monoclonal antibody is prepared from a monoclonal antibody with the preservation number of CCTCC NO: the hybridoma cell strain 11A7 of C2014187 secretes and produces the monoclonal antibody CD83-11A 7.
Specifically, the anti-human CD83 monoclonal antibody is prepared from a monoclonal antibody with the preservation number of CCTCC NO: the hybridoma cell strain 14E4 of C2014188 secretes the produced monoclonal antibody CD83-14E 4.
The invention also provides a mouse hybridoma cell strain 1H10 with the preservation number of CCTCC NO: C2014186.
the invention also provides a mouse hybridoma cell strain 11A7 with the preservation number of CCTCC NO: C2014187.
the invention also provides a mouse hybridoma cell strain 14E4 with the preservation number of CCTCC NO: C2014188.
the invention also provides the application of the anti-human CD83 monoclonal antibody in detecting CD83, wherein the detection is carried out by immunoblotting, immunoprecipitation, immunohistochemistry, ELISA or flow cytometry.
The invention also provides a kit for detecting CD83, which comprises the above-mentioned anti-human CD83 monoclonal antibody.
Specifically, the kit is used for detecting CD83 on immune cells or sCD83 in a blood sample.
The present invention also provides a method of detecting CD83 on a cell, the method comprising: (1) contacting the anti-human CD83 monoclonal antibody with an isolated cell to be detected or a lysate of the cell; and (2) judging whether a positive reaction occurs.
Specifically, the positive reaction is judged by immunoblotting, immunoprecipitation, ELISA or flow cytometry detection.
The invention also provides a method of detecting sCD83 in a liquid sample, the method comprising: (1) contacting the anti-human CD83 monoclonal antibody with a blood sample to be detected; and (2) judging whether a positive reaction occurs.
Specifically, the positive reaction is judged by immunoblotting, immunoprecipitation, ELISA or flow cytometry detection.
The invention content is detailed as follows:
one aspect of the present invention relates to a monoclonal antibody specific for human CD83, which is reactive with a sequence of amino acids 20 to 145 of CD83(Genbank accession number CAG 33300.1); preferably, the anti-human CD83 monoclonal antibody is CD83-1H10, CD83-11A7 and CD83-14E 4.
The invention also relates to mouse hybridoma cell strains 1H10, 11A7 and 14E4 which stably secrete anti-human CD83 monoclonal antibodies, wherein the preservation numbers are CCTCC NO: c2014186, CCTCC NO: c2014187 and CCTCC NO: C2014188.
the invention also relates to a preparation method of the anti-human CD83 monoclonal antibody, which comprises the steps of immunizing a BALB/c female mouse with age of 8-10 weeks by using recombinant CD83 protein expressed by pichia pastoris, taking spleen cells of the mouse and myeloma SP2/0 cells to perform in-vitro fusion under the action of polyethylene glycol, and screening a positive hybridoma cell strain by a limiting dilution method and an indirect ELISA method. Ascites fluid was prepared conventionally and the antibody was purified by MabSelect affinity chromatography.
Another aspect of the present invention relates to a method for identifying said anti-human CD83 monoclonal antibody, comprising: measuring titer, measuring affinity constant, measuring antibody subclass and identifying specificity.
Another aspect of the invention relates to the use of anti-human CD83 monoclonal antibodies in an immunoblot assay for detecting CD 83. The anti-human CD83 monoclonal antibody is used for detecting CD83 by an immunoblotting test, and the result shows that the anti-human CD83 monoclonal antibody can be used for detecting recombinant CD83 protein and cell endogenously expressed CD83 protein in the immunoblotting test.
Another aspect of the invention relates to the use of anti-human CD83 monoclonal antibodies in an ELISA assay for the detection of CD 83. The results of detecting CD83 with different concentrations by using the anti-human CD83 monoclonal antibody show that the anti-human CD83 monoclonal antibody can be used for detecting CD83 by ELISA and can be used for detecting sCD83 in blood samples of tumor patients.
Another aspect of the invention relates to the use of anti-human CD83 monoclonal antibodies in assays for detecting CD83 by flow cytometry. The monoclonal antibody against human CD83 of the present invention can bind to CD83 positive cells, but not to CD83 negative cells. The anti-human CD83 monoclonal antibody was shown to be useful for flow cytometry detection of cell surface CD 83.
In the present invention, the term "specificity of a monoclonal antibody" refers to the property of a monoclonal antibody to recognize and bind to a specific epitope or antigenic determinant on an antigen.
The term "reactivity of a monoclonal antibody" refers to the ability of a monoclonal antibody to bind to an antigen under suitable reaction conditions.
The term "cell line" refers to a single cell culture obtained from a primary culture or cell line by screening or limiting dilution methods.
The invention provides an anti-human CD83 monoclonal antibody and preparation, identification and application thereof. The monoclonal antibody has the characteristics of high titer, high specificity and the like, and is widely applied. The monoclonal antibodies with clone numbers 1H10, 11A7 and 14E4 are directed against the epitope with spatial conformation of CD83 and can be well used for detection by flow cytometry and ELISA. In particular, the monoclonal antibody with the clone number 11A7 can recognize sCD83 but cannot recognize mCD83, so that the monoclonal antibody can be used for flow detection to distinguish two different types of CD 83.
Different from the existing monoclonal antibody, the immunogen used by the anti-human CD83 monoclonal antibody prepared by the invention is CD83 recombinant protein expressed by pichia pastoris, and aims at CD83 extracellular segment containing a complete functional region, namely amino acid sequences from 20 th site to 145 th site of CD83, and hybridoma cell strains with high reactivity to the CD83 recombinant protein are screened by ELISA.
The titer, specificity and application of the anti-human CD83 monoclonal antibody of the invention are changed with the difference from the existing CD83 region or site aimed by the antibody.
The anti-human CD83 monoclonal antibody has the advantages of high titer, high specificity, wide application range and the like, and the anti-human CD83 monoclonal antibody provided by the invention can be widely used for detecting CD83 protein by different means such as immunoblotting, immunoprecipitation, ELISA, flow cytometry and the like, and provides a basis for researching the function of CD 83.
Drawings
FIG. 1 is a typical chromatographic peak pattern during the purification process of the anti-human CD83 monoclonal antibody provided by the present invention.
FIG. 2 shows the result of the purity determination of the anti-human CD83 monoclonal antibody according to the present invention.
FIGS. 3-A and 3-B are the results of the specificity identification of the anti-human CD83 monoclonal antibody according to the present invention.
FIG. 4 shows the detection of sCD83 in fermentation broth by the monoclonal antibody against human CD 83.
FIGS. 5-A and 5-B show the detection of CD83 on the surface of IM9 cell membrane by the anti-human CD83 monoclonal antibody provided by the invention.
FIGS. 6-A and 6-B are the ELISA kit optimization experiments for the assembly of anti-human CD83 monoclonal antibody and polyclonal antibody according to the present invention.
FIG. 7 shows the optimization of the antibody combination of the ELISA kit assembled by two anti-human CD83 monoclonal antibodies.
FIGS. 8-A and 8-B are high sensitivity ELISA kit antibody concentration optimization.
FIGS. 9-A and 9-B are antibody concentration optimization for low sensitivity ELISA kits.
FIGS. 10-A and 10-B show the content of sCD83 in the serum of tumor patients detected by CD83 ELISA kit.
Detailed Description
The technical solution of the present invention will be described in detail below with reference to specific examples.
Example 1 preparation of sCD83 recombinant protein
In the embodiment of the invention, for the preparation of sCD83 recombinant protein, please refer to the chinese invention patent expression and preparation method of soluble CD83, with application number 201310187915.3, publication number of 14/8/2013, publication number of 103243119 a; or reference to research papers: yugang Guo et al, The expression and characterization of functional active solution CD83 by Pichia pastoris using high-sensitivity transfer, PLOS ONE 9(2) e89264, 2014.
EXAMPLE 2 preparation of anti-human CD83 monoclonal antibody
The specific implementation of the anti-human CD83 monoclonal antibody hybridoma was made by Beijing Aibo Biotechnology Co., Ltd. (Beijing, China, postal code 100085). The general description of the implementation process can be found in Chinese patent of preparation, identification and application of anti-human NKp30 monoclonal antibody, with application number of 201010531489.7, publication number of 3/16/2011 and publication number of 101985476A.
Briefly described as follows:
1. preparation before cell fusion
The purified sCD83 recombinant protein was mixed with an equal volume of complete Freund's adjuvant and 8-10 week old BALB/c female mice were immunized. After a total of 5 booster immunizations every 2 weeks, the serum titer of the ELISA-tested mice reached 1:105The method comprises the following steps of carrying out impact immunization on a mouse once, taking blood from an orbit of the mouse after the impact immunization after 3 days, dislocating and killing the mouse, disinfecting the mouse by 75% alcohol, taking a spleen, removing connective tissues, collecting spleen cell suspension, and preparing the spleen single cell suspension for later use. One day before cell fusion, mouse peritoneal macrophages were prepared as feeder cells and cultured in a cell culture chamber at 37 ℃ under 5% C02. SP2/0 myeloma cells (ATCC accession number CRL-1581) were revived to ensure that the cells were in logarithmic growth phase at the time of fusion. SP2/0 cells were washed 2 times with 30-40ml incomplete RPMI-1640 medium prior to cell fusion. Cells were resuspended in incomplete RPMI-1640 medium.
2. Cell fusion and preparation of hybridoma cells
The spleen cell suspension and SP2/0 cells were subjected to fusion induction using 50% PEG4000(pH 8.0-8.2). The fused cells are cultured in HAT culture solution for 1 week, and the supernatant is absorbed to detect the antibody by an indirect ELISA method. Screening positive hybridoma cell strain, and subcloning by limiting dilution method. After multiple screening, hybridoma cell strains 1H10, 11A7 and 14E4 are obtained, and after continuous in vitro culture for more than 2 months or liquid nitrogen freezing storage for 6 months, the cell strains can still stably secrete a large amount of anti-human CD83 antibodies, and the monoclonal antibodies are named as CD83-1H10, CD83-11A7 and CD83-14E 4.
Hybridoma cell lines 1H10, 11A7 and 14E4 have been deposited in China center for type culture Collection (CCTCC, Wuhan university, zip code: 430072) in 11 months and 25 days 2014, with respective deposition numbers of CCTCC NO: c2014186, CCTCC NO: c2014187 and CCTCC NO: C2014188.
3. mass preparation of anti-human CD83 monoclonal antibody
The anti-human CD83 monoclonal antibody can be prepared by two methods, namely an incremental culture method and a mouse intraperitoneal inoculation method. The incremental culture method is to culture the cloned cells in a large amount in a low serum concentration culture medium in vitro for 2 to 3 days, and collect the supernatant to obtain a large amount of single cloned antibody. The mouse intraperitoneal inoculation method is to select BALB/c female mice (Shanghai Leike laboratory animal Co., Ltd.) with age of 8-10 weeks, and inject 2 × 10 into the abdominal cavity of each mouse after one week of incomplete Freund's adjuvant pretreatment6Each cell (500. mu.l PBS for heavy suspension), ascites are generated in about 7-10 days, the ascites of the mouse is collected, centrifuged at 12000g for 10min at 4 ℃, and the supernatant is collected for storage or further purification. The antibody obtained by the above method can be purified by ammonium sulfate precipitation, MabSelect affinity chromatography and molecular sieve method (as shown in FIG. 1), and the purity of the antibody can be identified by SDS-PAGE. As shown in FIG. 2, the purity of purified anti-human CD83 monoclonal antibody CD83-1H10, CD83-11A7 and CD83-14E4 reaches more than 95%, and the molecular weight of anti-human CD83 monoclonal antibody (Ig (H + L)) is about 160kDa, wherein the heavy chain Ig (H) is about 55kDa, and the light chain Ig (L) is about 25 kDa.
The PBS is called Phosphonate Buffered Saline. In the medical vocabulary, phosphate buffer is used for molecular cloning and cell culture, has a pH of 7.4, is isotonic with human blood, and contains potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and potassium chloride as main components.
Resuspension refers to "resuspension", i.e., the resuspension of solids (pellets, cells, actives, etc.) obtained by centrifugation or sedimentation in an appropriate buffer or culture medium.
Example 3 identification of anti-human CD83 monoclonal antibodies
1. The indirect ELISA measures the affinity constant of the anti-human CD83 monoclonal antibody.
The coating solution diluted the recombinant sCD83 protein to 2. mu.g/ml, 1. mu.g/ml, 0.5. mu.g/ml and 0.25. mu.g/ml, 100. mu.l/well, and coated separately on 96-well ELISA plates overnight at 4 ℃. S3, S5, S7 (corresponding monoclonal antibodies see Table 1) were diluted from a 2-fold gradient to 16 concentrations (2000ng/ml-0ng/ml) and reacted with plated sCD 83. The secondary antibody was HRP-labeled goat anti-mouse IgG Fc and developed. The absolute affinity constants of the two were determined by indirect ELISA.
The absolute affinity constants (Kaff) were calculated by plotting OD 450nm against the antibody concentration using the formula for calculating affinity constants proposed by J.David Beatty (1987) et al:
Kaff(M-1) 1/2(2[ monoclonal antibody']t- [ monoclonal antibody]t)
[ mab ] t represents the total concentration of mab plated with an amount of sCD83 (antigen) when OD 450nm reached the median absorbance;
[ mab' ] t represents the total concentration of antibody when OD 450nm reaches the median absorbance value, plated with antigen in an amount of (antigen)/2.
One Kaff can be calculated between every two adjacent concentrations of sCD83, 3 Kaff values can be calculated for the four concentrations, the average value is calculated, and the reciprocal of the average value is the absolute affinity constant KD of the sCD83 and the monoclonal antibody.
2. Subclass determination of anti-human CD83 monoclonal antibody
The determination was carried out using a subclass test paper of the mouse Monoclonal typing rapid detection Kit IsoQuickTM Kit for mouse Monoclonal typing IS0Q5 supplied by Sigma-Aldrich. Three anti-human CD83 monoclonal antibodies were numbered, namely anti-human CD83 monoclonal antibody CD83-1H10 was numbered S3, anti-human CD83 monoclonal antibody CD83-11A7 was numbered S5, and anti-human CD83 monoclonal antibody CD83-14E4 was numbered S7. The subclasses and affinity constants of the monoclonal antibodies of S3, S5 and S7 are shown in Table 1.
TABLE 1 subclass identification and affinity constants of anti-human CD83 monoclonal antibody
3. Specificity identification of anti-human CD83 monoclonal antibody
Two methods were used to identify the specificity of the anti-human CD83 monoclonal antibody: immunoblotting and ELISA.
Experimental procedure for immunoblotting: 20. mu.l of yeast culture broth (negative control 1, culture supernatant of untransformed yeast GS115 under the same culture conditions), 1. mu.g of recombinant IL-33-His (negative control 2, (detailed preparation method see Lingel, A, et al, Structure of IL-33and its interaction with the ST2 and IL-1RAcP receptors- -detailed preparation method see expression and preparation method of granulysin in China patent application No. 201210250501.6, published as 10.31.2012, published as 102757977A; or study paper Yongang Guo, et al, Production method of polysaccharide and polysaccharide 9-7697, 9 kDa 7697, 97, 9. sup. Biochemical engineering and 9. sup. 9. Biochemical engineering of polysaccharide), 2013) and recombinant sCD83-His protein as a detection sample, and SDS-PAGE electrophoretically separates the proteins. The target protein was transferred to PVDF membrane (BioRad) using a semi-dry transfer membrane apparatus. The PVDF membrane is placed in 5% skim milk and sealed lh at room temperature. lmg/ml anti-human CD83 monoclonal antibody (1:5000 dilution) was incubated at room temperature for 2-3h and washed 3 times with TBST (50mM Tris, 0.9% NaCl, 0.1% Tween 20, pH 7.4). Horse Radish Peroxidase (HRP) -labeled goat anti-mouse antibody (1:5000 dilution) was incubated lh at room temperature and washed 3 times with TBST. Chemiluminescence detection (Thermo scientific, cat # 34080). The result of immunoblotting is shown in FIG. 3-A, where there is a specific band at a relative molecular mass of 20kDa (sCD83-His) and no specific bands at 18kDa (recombinant IL-33-His) and 15kDa (15kDa GNLY-His). It was demonstrated that S3 and S7 are specific for CD83 and not reactive to His-Tag. S5 was not able to efficiently recognize denatured recombinant sCD83, but was able to recognize native sCD83 (see below for ELISA assay), suggesting that S5 may recognize the spatial configuration of sCD 83.
The specific experimental procedures for the ELISA assay of S3, S5 and S7 were as follows: the coating solution diluted the recombinant IL-33-His, recombinant 15kDa GNLY-His (negative control 2) and recombinant sCD83-His protein to 2. mu.g/ml, 100. mu.l/well, coated 96-well ELISA plates, 4 ℃ overnight. The next day the supernatant was discarded, PBST washed 2 times, blocked with 1% BSA and incubated at 37 ℃ for 2 h. Each anti-human CD83 monoclonal antibody dilution (concentration 1. mu.g/ml) was prepared, PBST was washed 2 times, 100. mu.l of anti-human CD83 monoclonal antibody dilution was added to a 96-well plate, and the plate was incubated at 37 ℃ for 2 hours with PBS as a zero-setting well. PBST was washed 5 times, 100. mu.l of Horse Radish Peroxidase (HRP) -labeled goat anti-mouse antibody (1:10000 dilution) was added to each well, and lh was incubated at 37 ℃. PBST was washed 5 times, TMB substrate solution was added, 100. mu.l/well was developed in the dark for 10-15min, stop solution (1M sulfuric acid) was added at 100. mu.1/well, measurement was immediately carried out with a microplate reader, and the absorbance (OD 450nm) at a wavelength of 450nm was read. The results are shown in FIG. 3-B, where S3, S5, and S7 all specifically bound to sCD83, but not to recombinant IL-33-His and recombinant 15kDa GNLY-His, indicating that S3, S5, and S7 have good specificity for CD 83.
EXAMPLE 4 use of anti-human CD83 monoclonal antibody
1. For immunoblot detection of sCD83 and immunoprecipitation detection of sCD83
The experimental procedure is the same as the immunoblotting procedure in the above-mentioned specificity identification of anti-human CD83 monoclonal antibody. As shown in fig. 3-a, S3 and S7 were able to specifically detect recombinant sCD83 protein, S5 was not able to efficiently recognize denatured recombinant sCD83, but was able to recognize native sCD83, suggesting that S5 may recognize the spatial configuration of sCD 83. In another immunoblotting application case, the S3 and S7 can be used for fermentation expression detection of sCD 83. As shown in fig. 4-a, S3 and S7 were able to specifically detect recombinant sCD83 protein, and S5 was unable to efficiently recognize denatured recombinant sCD83, in substantial agreement with the above results. As will be readily appreciated by those skilled in the art, the anti-human CD83 monoclonal antibody can also be used for immunoblot detection of CD83 endogenous to cells.
The anti-human CD83 monoclonal antibody can be used for detecting sCD83 in fermentation liquor by using immunoblotting and can also be used for immunoprecipitation detection. 4ml of sCD83 fermentation supernatant was taken, pH was adjusted to weak alkalinity (pH 7.5), 20. mu. G S3, S5, S7 and the control antibody mouse normal IgG (mIgG) were added, shaking was carried out gently on a shaker at 4 ℃ for 2 hours, 50. mu.l of protein A/G Plus-Agarose was added and shaking was carried out for 2 hours, 10000G was carried out, centrifugation was carried out at 4 ℃ for 10 minutes, the supernatant was discarded, PBST was washed three times, 10000G each time, centrifugation was carried out at 4 ℃ for 10 minutes, and the supernatant was discarded. Resuspend with 50 μ l PBS, add protein electrophoresis loading buffer, boil for 10 minutes, 10000g, centrifuge for 10 minutes at 4 ℃, take the supernatant to do SDS-PAGE electrophoresis and immunoblotting identification. As shown in FIG. 4-B, S3, S5, and S7 all specifically immunoprecipitated sCD83 from the fermentation broth, whereas the negative control antibody mIgG did not. It was demonstrated that S3, S5 and S7 could be used for immunoprecipitation of sCD 83. Since S3 and S7 can recognize membrane-type CD83, it is easily suggested to those skilled in the art that S3 and S7 of the present invention can also be used for immunoprecipitation of CD83 in cell lysates.
In addition, since the antibody of the present invention can recognize denatured antigen in immunoblotting and non-denatured antigen in ELISA, it is easy for those skilled in the art to think that the anti-human CD83 monoclonal antibody of the present invention can also be used for detecting mCD83 and sCD83 in samples such as tissues and cells in experiments such as immunohistochemistry and immunofluorescence, and the description is not given by way of example.
2. Detection of cell membrane surface CD83 by flow cytometry
The specific method for detecting the CD83 on the surface of the cell membrane and antibody competition experiment by using flow cytometry can be seen in Chinese invention patent 'expression and preparation method of soluble CD 83', with application number of 201310187915.3, publication number of 2013, 8 and 14 months, and publication number of 103243119A; or reference to research papers: yugang Guo, et al, The expression and characterization of functional active solution CD83 by Pichia pastoris using high-sensitivity transfer, PLOS ONE 9(2) e89264, 2014.
As shown in FIG. 5-A, S3 and S7 have better binding effect with mCD83 protein on the surface of cell membrane, and are basically equivalent to commercial antibody, but S5 has no significant binding capacity, which indicates that the binding site of S5 is close to the transmembrane region of CD83, and the binding cannot be achieved due to steric hindrance. However, since S5 can bind to free sCD83 (as demonstrated in ELISA assays), S5 was used to distinguish between membrane-type mCD83 and soluble sCD83 in the test sample.
As shown in FIG. 5-B, among the three antibodies of the present invention, S3 and S7 have significant competitive effect on the commercial antibody, suggesting that S3 and S7 can specifically recognize cell membrane type CD83 and are closer to the epitope recognized by the commercial antibody.
3. ELISA kit for preparing CD83
Double antibody sandwich ELISA (Sandwich ELISA) is a highly sensitive, specific immunoassay technique. The invention applies anti-human CD83 monoclonal antibody and polyclonal antibody, two different anti-human CD83 monoclonal antibodies to respectively assemble ELISA kits for detecting CD 83.
1) Anti-human CD83 monoclonal antibody and polyclonal antibody assembly ELISA kit
The experimental process comprises the following steps: the corresponding anti-human CD83 monoclonal antibody was coated on 96-well ELISA plates at the designed concentrations in tables 2 and 3 overnight at 4 ℃. PBST was washed 2 times, blocked with 1% BSA and incubated at 37 ℃ for 1 h. PBST was washed 3 times, 100. mu.l sCD83 standard of the design concentration were added to each well, the standard dilution was set as a zero-adjustment well, and the incubation was carried out at 37 ℃ for lh. PBST was washed 3 times, and 100. mu.l of rabbit anti-sCD 83 polyclonal antibody (The specific preparation method of polyclonal antibody can be found in Chinese patent "expression and preparation method of soluble CD 83", application No. 201310187915.3, published as 2013, 8/14, and publication No. 103243119A; or reference research papers: Yugang Guo, et al, The expression and characterization of functional active solution CD83 by Pichia pastoris using high-sensitivity transformation, PLOS E9 (2): e89264, 2014) was added to each well and incubated at 37 ℃ for l. PBST was washed 3 times, and horseradish peroxidase (HRP) -labeled goat anti-rabbit antibody (1:10000 dilution) was added to each well and incubated at 37 ℃ for lh. PBST was washed 5 times, TMB substrate solution was added, 100. mu.l/well was developed in the dark for 10-15min, stop solution (1M sulfuric acid) was added at 100. mu.1/well, measurement was immediately carried out with a microplate reader, and the absorbance (OD 450nm) at a wavelength of 450nm was read.
TABLE 2 optimization information table of coated antibody species for ELISA kit assembled by anti-human CD83 monoclonal antibody and polyclonal antibody
TABLE 3 optimization information table of coated antibody species for ELISA kit assembled by anti-human CD83 monoclonal antibody and polyclonal antibody
| First antibody type | S7 |
| Primary dilution resisting concentration | The antibody concentration is 1mg/ml, and the antibody is diluted according to 1:500-1:160002 times of gradient, and the total number of 6 gradients is |
| sCD83 sample dilution concentration | 400ng/ml-25pg/ml, 2-fold gradient dilution, total 15 gradients |
| Concentration of secondary antibody dilution | Rabbit anti CD83 |
| HRP antibody dilution concentration | Goat anti rabbit-HRP 0.75μg/ml |
As shown in FIG. 6-A, S3 and S7 can be assembled with polyclonal antibodies into an ELISA kit with relatively high sensitivity, the sensitivity reaches 0.5ng/ml, the linear range is 0.5ng/ml-50ng/ml, and the kit is suitable for detecting the content of CD83 in cell culture supernatants and patient sera. S5 can be assembled with polyclonal antibodies into an ELISA kit with relatively low sensitivity, the sensitivity reaches 10ng/ml, the linear range is 10ng/ml-1000ng/ml, and the kit is suitable for concentration detection in the preparation process of sCD 83.
Further optimizing the concentration of the coated antibody, as shown in fig. 6-B, it is found that the detection signal is gradually enhanced and the sensitivity is increased with the increase of the concentration of the coated antibody, however, the linear range is relatively reduced, so that the factors such as the strength of the detection signal, the sensitivity and the linear range are comprehensively considered, and 5 μ g/ml is the better coating concentration.
2) ELISA kit assembled by two anti-human CD83 monoclonal antibodies
The experimental process comprises the following steps: the biotinylation labeling was performed on S3, S5 and S7 according to the manual of Biotin labeling kit (Thermo Scientific, cat. No.: 21435EZ-Link), and the biotinylation labeling efficiency was examined. The calculated biotin labeling rate of the three antibodies is between 3and 7, as shown in Table 4, and can be normally used.
TABLE 4 quality control of anti-human CD83 monoclonal antibody ubiquitination marker
| S3 | S5 | S7 | |
| Concentration of labeled monoclonal antibody (mg/mL) | 1.609 | 1.116 | 0.764 |
| Biotinylated monoclonal antibody concentration (mmol/mL) | 1.07E-05 | 7.44E-06 | 5.09E-06 |
| The mixture was analyzed for biotin concentration (mmol/mL) | 5.82E-06 | 4.35E-06 | 1.82E-06 |
| Concentration of Biotin in monoclonal antibody (mmol/mL) | 5.82E-05 | 4.35E-05 | 1.82E-05 |
| Biotin labeling Rate (number of biotins per antibody molecule) | 5.43 | 5.85 | 3.58 |
The corresponding anti-human CD83 monoclonal antibody was then coated onto 96-well ELISA plates at the designed concentrations of Table 5 overnight at 4 ℃. PBST was washed 2 times, blocked with 1% BSA and incubated at 37 ℃ for 1 h. PBST was washed 3 times, 100. mu.l sCD83 standard of the design concentration were added to each well, the standard dilution was set as a zero-adjustment well, and the incubation was carried out at 37 ℃ for lh. PBST was washed 3 times, and 100. mu.l of biotinylated anti-human CD83 monoclonal antibody was added to each well at the indicated concentrations and incubated at 37 ℃ for lh. PBST was washed 3 times, and avidin-labeled horseradish peroxidase (R & D, cat # AEM771012, PartNo.890803) was added to each well and incubated at 37 ℃ for lh. PBST was washed 5 times, TMB substrate solution was added, 100. mu.l/well was developed in the dark for 10-15min, stop solution (1M sulfuric acid) was added at 100. mu.1/well, measurement was immediately carried out with a microplate reader, and the absorbance (OD 450nm) at a wavelength of 450nm was read.
TABLE 5 optimization of antibody combination of ELISA kit assembled by two anti-human CD83 monoclonal antibodies
As shown in fig. 7, the combination of S3 as the coating antibody and S7 as the detection antibody (S3/S7) showed the strongest detection signal and the highest sensitivity, the combination of S7 as the coating antibody and S3 as the detection antibody (S7/S3) was the second order, and the combination of S5 as the coating antibody and S3 as the detection antibody (S5/S3) was the weakest. However, S7/S3 is the group of these combinations with the widest linear range (0.5ng/ml-25 ng/ml).
The antibody concentrations of the high sensitivity ELISA kits and the low sensitivity ELISA kits were then optimized according to tables 6, 7 and 8, respectively. As a result, as shown in FIGS. 8-A, 8-B, 9-A and 9-B, the combination of the antibodies at a low concentration (5. mu.g/ml) achieved a good detection effect, and the concentration provided did not increase the detection effect but rather decreased the detection effect.
TABLE 6 optimization of primary antibody concentration of high sensitivity ELISA kit
TABLE 7 optimization of concentration of second antibody in high-sensitivity ELISA kit
TABLE 8 optimization of the concentration of the second antibody in the low-sensitivity ELISA kit
3) The assembled ELISA kit is used for detecting the sCD83 content in the serum of a patient
The S3/S7 combined high-sensitivity ELISA kit is used for detecting the content of sCD83 in serum of clinical tumor patients, the result of a standard curve is shown in figure 10-A, the result of a sample to be detected (wherein the No. 1 is a serum sample of a healthy volunteer, and the No. 2-47 is a serum sample of a tumor patient) is shown in figure 10-B, the content of sCD83 in serum of part of tumor patients is higher (the total number of the serum samples of the tumor patients is 46), the serum samples of the healthy volunteer hardly contain sCD83 (the total number of the serum samples of the healthy volunteer is 40, wherein the No. 1 is a representative sample), and the correlation between the content of sCD83 and the tumor occurrence and development is analyzed at present. The experiment shows that the assembled ELISA kit can be used for detecting the sCD83 content in the serum of a tumor patient.
Sequence listing description
1 amino acid sequence of human full-length CD83(GenBank Accession No.: CAG 33300.1);
SEQ ID No. 2 Pichia pastoris recombinant expression sCD83 amino acid sequence.
The 1M hydrochloric acid is 1mol/L hydrochloric acid, the 1M sulfuric acid is 1mol/L sulfuric acid, and the 50mM Tris is 50mmol/L Tris solution.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Sequence listing
<110> advanced technology research institute of Chinese university of science and technology
<120> anti-human CD83 monoclonal antibody and preparation, identification and application thereof
<130> ZL201310187915
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 205
<212> PRT
<213> amino acid sequence of human full-length CD83 (1)
<400> 1
Met Ser Arg Gly Leu Gln Leu Leu Leu Leu Ser Cys Ala Tyr Ser Leu
1 5 10 15
Ala Pro Ala Thr Pro Glu Val Lys Val Ala Cys Ser Glu Asp Val Asp
20 25 30
Leu Pro Cys Thr Ala Pro Trp Asp Pro Gln Val Pro Tyr Thr Val Ser
35 40 45
Trp Val Lys Leu Leu Glu Gly Gly Glu Glu Arg Met Glu Thr Pro Gln
50 55 60
Glu Asp His Leu Arg Gly Gln His Tyr His Gln Lys Gly Gln Asn Gly
65 70 75 80
Ser Phe Asp Ala Pro Asn Glu Arg Pro Tyr Ser Leu Lys Ile Arg Asn
85 90 95
Thr Thr Ser Cys Asn Ser Gly Thr Tyr Arg Cys Thr Leu Gln Asp Pro
100 105 110
Asp Gly Gln Arg Asn Leu Ser Gly Lys Val Ile Leu Arg Val Thr Gly
115 120 125
Cys Pro Ala Gln Arg Lys Glu Glu Thr Phe Lys Lys Tyr Arg Ala Glu
130 135 140
Ile Val Leu Leu Leu Ala Leu Val Ile Phe Tyr Leu Thr Leu Ile Ile
145 150 155 160
Phe Thr Cys Lys Phe Ala Arg Leu Gln Ser Ile Phe Pro Asp Phe Ser
165 170 175
Lys Ala Gly Met Glu Arg Ala Phe Leu Pro Val Thr Ser Pro Asn Lys
180 185 190
His Leu Gly Leu Val Thr Pro His Lys Thr Glu Leu Val
195 200 205
<210> 2
<211> 126
<212> PRT
<213> amino acid sequence of sCD83 recombinantly expressed in Pichia pastoris (2)
<400> 2
Thr Pro Glu Val Lys Val Ala Cys Ser Glu Asp Val Asp Leu Pro Cys
1 5 10 15
Thr Ala Pro Trp Asp Pro Gln Val Pro Tyr Thr Val Ser Trp Val Lys
20 25 30
Leu Leu Glu Gly Gly Glu Glu Arg Met Glu Thr Pro Gln Glu Asp His
35 40 45
Leu Arg Gly Gln His Tyr His Gln Lys Gly Gln Asn Gly Ser Phe Asp
50 55 60
Ala Pro Asn Glu Arg Pro Tyr Ser Leu Lys Ile Arg Asn Thr Thr Ser
65 70 75 80
Cys Asn Ser Gly Thr Tyr Arg Cys Thr Leu Gln Asp Pro Asp Gly Gln
85 90 95
Arg Asn Leu Ser Gly Lys Val Ile Leu Arg Val Thr Gly Cys Pro Ala
100 105 110
Gln Arg Lys Glu Glu Thr Phe Lys Lys Tyr Arg Ala Glu Ile
115 120 125
Claims (5)
1. An anti-human CD83 monoclonal antibody reactive against the amino acid sequence at positions 20 to 145 of the extracellular domain of human CD 83; the anti-human CD83 monoclonal antibody is IgG1, kappa subclass; the anti-human CD83 monoclonal antibody is prepared from the following components in percentage by weight, wherein the preservation number is CCTCC NO: the hybridoma cell line 11A7 of C2014187 secretes the produced monoclonal antibody.
2. The mouse hybridoma cell strain 11A7 secreting the anti-human CD83 monoclonal antibody of claim 1, wherein the preservation number is CCTCC NO: C2014187.
3. use of the anti-human CD83 monoclonal antibody according to claim 1 for the preparation of a kit for the detection of CD83, wherein the detection is performed by immunoprecipitation, immunohistochemistry, ELISA or flow cytometry.
4. A kit for detecting CD83, comprising the anti-human CD83 monoclonal antibody of claim 1.
5. The kit for detecting CD83 according to claim 4, for detecting sCD83 in a blood sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810970426.8A CN109369807B (en) | 2015-01-27 | 2015-01-27 | Anti-human CD83 monoclonal antibody and preparation, identification and application thereof |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510041930.6A CN104610452B (en) | 2015-01-27 | 2015-01-27 | Anti-human CD83 monoclonal antibody and its preparation, identification and application |
| CN201810970426.8A CN109369807B (en) | 2015-01-27 | 2015-01-27 | Anti-human CD83 monoclonal antibody and preparation, identification and application thereof |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510041930.6A Division CN104610452B (en) | 2015-01-27 | 2015-01-27 | Anti-human CD83 monoclonal antibody and its preparation, identification and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109369807A CN109369807A (en) | 2019-02-22 |
| CN109369807B true CN109369807B (en) | 2021-07-27 |
Family
ID=53145126
Family Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810970427.2A Active CN109320610B (en) | 2015-01-27 | 2015-01-27 | Anti-human CD83 monoclonal antibody and preparation, identification and application thereof |
| CN201810970426.8A Active CN109369807B (en) | 2015-01-27 | 2015-01-27 | Anti-human CD83 monoclonal antibody and preparation, identification and application thereof |
| CN201510041930.6A Active CN104610452B (en) | 2015-01-27 | 2015-01-27 | Anti-human CD83 monoclonal antibody and its preparation, identification and application |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810970427.2A Active CN109320610B (en) | 2015-01-27 | 2015-01-27 | Anti-human CD83 monoclonal antibody and preparation, identification and application thereof |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510041930.6A Active CN104610452B (en) | 2015-01-27 | 2015-01-27 | Anti-human CD83 monoclonal antibody and its preparation, identification and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (3) | CN109320610B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997029781A1 (en) * | 1996-02-15 | 1997-08-21 | Immunex Corporation | Methods and compositions for modulating an immune response |
| 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 |
| WO2004048552A2 (en) * | 2002-11-21 | 2004-06-10 | Celltech R & D, Inc. | Modulating immune responses |
| CN103243119A (en) * | 2013-05-20 | 2013-08-14 | 中国科学技术大学 | Expression and preparation method of soluble CD83 |
| 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 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE69731977T2 (en) * | 1996-10-09 | 2005-12-08 | Canterbury Health Ltd. | SPECIFIC ANTIBODIES FOR DENDRITIC CELLS |
-
2015
- 2015-01-27 CN CN201810970427.2A patent/CN109320610B/en active Active
- 2015-01-27 CN CN201810970426.8A patent/CN109369807B/en active Active
- 2015-01-27 CN CN201510041930.6A patent/CN104610452B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997029781A1 (en) * | 1996-02-15 | 1997-08-21 | Immunex Corporation | Methods and compositions for modulating an immune response |
| 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 |
| WO2004048552A2 (en) * | 2002-11-21 | 2004-06-10 | Celltech R & D, Inc. | Modulating immune responses |
| 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 |
| CN103243119A (en) * | 2013-05-20 | 2013-08-14 | 中国科学技术大学 | Expression and preparation method of soluble CD83 |
Non-Patent Citations (2)
| Title |
|---|
| A New Monoclonal Anti-human CD83 Antibody for Flow Cytometry, Western Blot Analysis, and ELISA;Stefanie Papp等;《Monoclonal Antibodies in Immunodiagnosis and Immunotherapy》;20130422;第32卷(第2期);第98-104页 * |
| 一株识别CD83单克隆抗体的研制及其生物学特性鉴定;高超等;《细胞与分子免疫学杂志》;20091031;第25卷(第10期);第914-916页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109369807A (en) | 2019-02-22 |
| CN109320610A (en) | 2019-02-12 |
| CN104610452B (en) | 2018-11-20 |
| CN104610452A (en) | 2015-05-13 |
| CN109320610B (en) | 2021-07-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112194724B (en) | anti-MPO protein monoclonal antibody, cell line, preparation method and application thereof | |
| CN112457400B (en) | Anti-beta-catenin protein monoclonal antibody, cell line, preparation method and application thereof | |
| CN113045656B (en) | Monoclonal antibodies against HLA-G isoform molecules HLA-G5 and HLA-G6 and uses thereof | |
| CN112442124B (en) | anti-CD 23 protein monoclonal antibody, cell line, preparation method and application thereof | |
| CN109517802B (en) | Hybridoma cell strain secreting salmonella pullorum IpaJ monoclonal antibody, monoclonal antibody thereof and application of monoclonal antibody | |
| CA2711863C (en) | Antibody directed against pcrv | |
| CN109485724B (en) | anti-Desmin protein monoclonal antibody, cell line, preparation method and application thereof | |
| CN114480298A (en) | Hybridoma cell strain secreting anti-TIGIT monoclonal antibody and application thereof | |
| CN114605543A (en) | Monoclonal antibody for resisting HLA-G isomer molecules HLA-G5 and HLA-G6 and application thereof | |
| CN111705066B (en) | Genetically modified TIGIT protein, monoclonal antibody and application thereof | |
| CN114075552A (en) | Hybridoma cell strain secreting anti-FGL 1 monoclonal antibody and application thereof | |
| KR101263913B1 (en) | Bovine tuberculosis diagnosing kit and diagnosing method using the same | |
| CN109369807B (en) | Anti-human CD83 monoclonal antibody and preparation, identification and application thereof | |
| CN113831410B (en) | anti-CD 56 protein monoclonal antibody and cell strain, preparation method and application thereof | |
| EP3656794A1 (en) | Composition and methods for detecting cancer | |
| CN113845592B (en) | anti-CK 5/6 protein monoclonal antibody, cell strain thereof, preparation method and application | |
| CN112646037B (en) | anti-CD 105 specific monoclonal antibody and application thereof | |
| CN100462432C (en) | Hybridoma cell strain, and anti-human heparin enzyme monoclonal anti-body produced by it | |
| JP5920761B2 (en) | Anti-DCD monoclonal antibody | |
| CN112684185A (en) | Soluble B7-H4 quantitative detection kit and application thereof | |
| JP5565723B2 (en) | Anti-DCD monoclonal antibody | |
| JP2018080114A (en) | Anti-human VAP-1 monoclonal antibody | |
| CN113307875B (en) | Monoclonal antibody for resisting TCR beta F1 protein, cell strain, preparation method and application thereof | |
| CN111434686A (en) | Anti-human PBX1 monoclonal antibody, preparation method thereof and application thereof in clinical diagnosis of recurrent abortion | |
| CN110922485B (en) | anti-Ep-cam protein monoclonal antibody, cell line, preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |