CN108864284A - The preparation of rabbit-anti human PD-L 1 protein monoclonal antibody and its immunohistochemistry purposes - Google Patents
The preparation of rabbit-anti human PD-L 1 protein monoclonal antibody and its immunohistochemistry purposes Download PDFInfo
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- CN108864284A CN108864284A CN201810323547.3A CN201810323547A CN108864284A CN 108864284 A CN108864284 A CN 108864284A CN 201810323547 A CN201810323547 A CN 201810323547A CN 108864284 A CN108864284 A CN 108864284A
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- cancer
- albumen
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- monoclonal antibody
- antibody
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N2333/70532—B7 molecules, e.g. CD80, CD86
Abstract
The invention discloses a kind of monoclonal antibodies of rabbit-anti human PD-L 1 albumen, can specifically identify the PD-L1 albumen of cell surface expression, and performance is stablized and potency is high, with intracellular other albumen no cross reactions, is worth with certain medical basic research.The invention further relates to said monoclonal antibodies to prepare the application in the immunohistochemistry detection instrument for detecting PD-L1 albumen.The present invention detects the expression of tumor cell surface PD-L1 using immunohistochemical method, it mainly include melanoma, non-small cell lung cancer, bladder cancer, kidney, gastric cancer, colorectal cancer, prostate cancer, cervical carcinoma, breast cancer, whether to provide scientific reference using the Index for diagnosis that PD-1/PD-L1 inhibitor carries out immunization therapy and tumour, medical expense is effectively reduced, there is important application value.
Description
Technical field
The present invention relates to field of biotechnology, in particular to a kind of monoclonal antibody of rabbit-anti human PD-L 1 albumen, and should
The immunohistochemistry purposes of antibody.
Background technique
Apoptosis receptor-ligand 1 (Programmed cell death-ligand 1, PD-L1), belongs to I
Type transmembrane protein, 290 amino acid of overall length, molecular weight is about 40kDa.In recent years, research finds PD-L1 in kinds of tumor cells
Surface expression up-regulation, this phenomenon are related to the immunologic escape of tumour.The immune system of body would generally tumor cell simultaneously
Reaction is generated to it, promotes the T cell hyperplasia with antigentic specificity, thus tumour cell is killed or excluded.But body pair
Control of the immune excretion of tumour by immunological regulation point, most important one point of adjustment be exactly apoptosis by
Body -1 (Programmed cell death-1, PD-1).As the PD-L1 on the PD-1 tumor cell surface on T cell surface,
The signal that inhibition can be conducted reduces the hyperplasia of T cell, and T cell cannot find tumour cell, cannot send out to tumour cell
Signal to attack out leads to the generation of tumor immune escape.Meanwhile in tumor microenvironment, PD-L1 is also that tumour cell is caused to escape
One of the important factor of ease immunosurveillance.On the one hand, some inflammatory cytokines, such as IFN-α, IFN- existing for tumor microenvironment
β, IFN-γ and TNF-α can promote the PD-L1 expression of tumor cell surface.On the other hand, PD-L1 may additionally facilitate tumour cell
Epithelial and stromal occurs and promotes the transfer and infiltration of tumour.Research and clinical test results are shown, are pressed down using PD-1/PD-L1
Inhibitor Blocks PD-1/PD-L1 signal path restores the immunologic cytotoxicity function of T cell, can play good therapeutic effect.But
This therapy is only for there are the highly expressed tumours of PD-L1 to have ideal curative effect, tumor efficiency highly expressed for no PD-L1
It is little, and there is side effect.Therefore, the PD-L1 antibody that a species specificity is high, performance is stable is developed, for detecting tumour table
The expression of face PD-L1, for it is subsequent whether using PD-1/PD-L1 inhibitor carry out immunization therapy be of great significance.
Currently, in the world for selecting suitable PD-1/PD-L1 inhibitor to need corresponding PD-L1 detection method, in view of
PD-L1 antibody used in every kind of detection and technology are all different, costly when carrying out auxiliary diagnosis using these antibody, together
When limited in necessary instrument and application aspect by international vendor.And the PD-L1 antibody of domestic research and development is rare, it is specific and affine
It is to be improved in terms of power, therefore the urgently new anti-PD-L1 monoclonal antibody haveing excellent performance of research and development, fill up the domestic industry
Blank reduces medical expense, mitigates patient's burden.The present invention successfully develops species specificity height, performance is stablized and potency is high
Rabbit source PD-L1 albumen monoclonal antibody, can be used for the expression research of different immunocytes and tumour cell PD-L1,
Disease occurs, progress, the relevant pathologic, physiologic of prognosis are studied and the mechanism study of PD-L1 adjusting body's immunity, has
Certain medical basic research value.The present invention is using immunohistochemical method (IHC) detection tumor cell surface PD-L1's
Expression, mainly include melanoma, non-small cell lung cancer, bladder cancer, kidney, gastric cancer, colorectal cancer, prostate cancer, cervical carcinoma,
Breast cancer has whether to provide scientific reference using the Index for diagnosis that PD-1/PD-L1 inhibitor carries out immunization therapy and tumour
Effect reduces medical expense, has important application value.
Summary of the invention
The technical problem to be solved in the present invention is to provide the anti-PD-L1 egg that a species specificity is good, performance is stablized and potency is high
White monoclonal antibody and the antibody are preparing the application in the Immunohistochemical detection tool for detecting PD-L1 albumen.
In order to solve the above technical problems, the present invention provides a kind of monoclonal antibodies for specifically binding human PD-L 1 albumen.
The preparation method of monoclonal antibody of the present invention is as follows：
(1) preparation of immunogen：According to PD-L1 gene (BC113734) sequence information, synthesis N-terminal band BSA sequence is ordered
(Canada applies biomaterial Co., Ltd, Applied to the mankind PD-L1 polypeptide of column (immunogenicity for improving polypeptide)
Biological Materials Inc.), it is used for immunization experiment animal and ELISA screening positive clone.
(2) blood sampling before being immunized：Choose purebred New Zealand White Rabbit (New Zealand white rabbit), alcohol wipe rabbit
Ear, taking auricular vein blood is immune preceding blood sample.
(3) screening and preparation of monoclonal antibody：It is immunized using the N-terminal of synthesis with mankind's PD-L1 polypeptide of BSA sequence purebred new
Western orchid white rabbit takes rabbit auricular vein blood using PD-L1 antibody titer in enzyme-linked immunosorbent assay (ELISA) test serum.It is right
Animals showing positive serum prepares peripheral blood mononuclear cells suspension, is dyed using the aforementioned polypeptides antigen of fluorescent marker, using stream
The individual cells that formula cell instrument chooses the expression of PD-L1 antibody positive are added in 96 holes.To the individual cells of each above-mentioned selection
Rabbit antibody light and heavy chain is expanded using RT-PCR.It is cloned into respectively accordingly by the mating light and heavy chain amplified production that same cell obtains
Expression vector.After Sanger is sequenced and carries out functional nucleotide sequence analysis, the vector plasmid with potent antibodies systematic function is selected
Transfect 293T cell.It collects cell culture fluid application ELISA and Western Blot (the result is shown in Figure 1) and screens positive antibody gram
It is grand.Further pass through proteinA column chromatographic purifying PD-L1 monoclonal antibody.The list is verified by immunohistochemical experiment (result is shown in Fig. 2)
Anti- sensitivity and specificity.
It is another object of the present invention to provide a kind of said monoclonal antibody in preparation for detecting PD-L1 albumen
Application in immunohistochemistry tool.
The immune detection tool is reagent, kit, chip or test strips.
The present invention also provides application said monoclonal antibodies to express in preparation for detecting PD-L1 in a variety of entity tumors
Application.Since PD-L1 is one of the important factor of tumour cell escape immunosurveillance, PD-L1 is detected in tumour cell
The expression on surface is for selecting immunologic test point inhibitor for treating appropriate to have important directive significance.
By taking the immunohistochemistry automatic staining machine BondMax for using Leica as an example, carried out using the PD-L1 monoclonal antibody
The condition of immunohistochemical staining is：
(1) the IHC protocol F carried using machine, by peroxidase closing step by using primary antibody to move forward to
Before DAB colour developing；
(2) antibody uses final concentration of 1.0 μ g/ml, is incubated at room temperature 30min；
(3) antigen retrieval uses the antigen retrieval buffers (ER2) of pH 9.0,100 DEG C of incubation with heat 30min.
When using other IHC automatic staining machines or carrying out hand dyeing, above-mentioned condition progress is please referred to.
Specific monoclonal antibody of the present invention for PD-L1 can improve the spy of experiment with PD-L1 albumen specific bond
Anisotropic and reliability, and establish the immunohistochemistry technology based on the monoclonal antibody is mainly used for melanoma, non-
Small Cell Lung Cancer, bladder cancer, kidney, gastric cancer, colorectal cancer, prostate cancer, cervical carcinoma, breast cancer or related neoplasms inhibitor
The Index for diagnosis of therapeutic scheme selection and tumour provides science reference.
Detailed description of the invention
Present invention will be further explained below with reference to the attached drawings and examples.
Fig. 1 is the Western Blot result of monoclonal antibody of the present invention：Loading sample is same transfection PD-L1 gene
293T cell pyrolysis liquid, be followed successively by from left to right：M swimming lane be Marker, 1 swimming lane be screen PD-L1 monoclonal sample, 2
Swimming lane be using β-Actin antibody test positive control as a result, 3 swimming lanes are the result of no primary antibody negative control.
Fig. 2 is using monoclonal antibody of the present invention as primary antibody, and Immunohistochemical Method detects PD-L1 egg in Non-Small Cell Lung Carcinoma
White expression colored graph.
Specific embodiment
It is right combined with specific embodiments below in order to make those skilled in the art more fully understand technical solution of the present invention
The present invention is described in further detail.
The preparation of the anti-PD-L1 monoclonal antibody of embodiment 1
One, the preparation of immunogen：According to PD-L1 gene (BC113734) sequence information, synthesis N-terminal band BSA sequence is ordered
Mankind's PD-L1 polypeptide of column is used for immunization experiment animal and ELISA screening positive clone.
Two, blood sampling before being immunized：Purebred New Zealand White Rabbit is chosen, taking auricular vein blood is immune preceding blood sample.
Three, the screening and preparation of monoclonal antibody
1, animal immune：Mankind PD-L1 polypeptide of the N-terminal of above-mentioned synthesis with BSA sequence and isometric Freund are complete
Full adjuvant is sufficiently mixed, and is prepared the immunogen emulsified completely and is exempted from using the purebred New Zealand White Rabbit of subcutaneous injection method initial immunity
Epidemic disease dosage is 500 μ g/.After two weeks, it carries out second to be immunized, be emulsified using incomplete Freund's adjuvant, immunizing dose is 500 μ
G/ is only.After two weeks, it takes rabbit auricular vein blood enzyme-linked immunosorbent assay (ELISA) to measure serum titer, is determined according to result
Whether booster immunization.
2, it screens and clones：After a week, rabbit auricular vein blood is taken to prepare peripheral blood mononuclear cells suspension, PD-L1 polypeptide
Antigen is marked using 488 NHS-Ester of Alexa Fluor, is dyed using the polypeptide antigen of fluorescent marker, uses streaming
Cell technology (FACS) squeezes into single antibody cellulation in pre- 96 orifice plates added with 10 μ l of reverse transcription reaction reagent respectively, into
Row RT-PCR reaction amplification rabbit antibody light and heavy chain.Phase is cloned by the mating light and heavy chain amplified production that same cell obtains respectively
The expression vector answered.After Sanger is sequenced and carries out functional nucleotide sequence analysis, the carrier matter with potent antibodies systematic function is selected
Grain transfection 293T cell.It collects cell culture fluid and carries out antibody cloning screening.Using ELISA and Western Blot, (result is shown in
Fig. 1) screening positive antibody clone.Mark cell strain number.Limiting dilution, 5-6 after each limiting dilution are carried out to positive hole cell
Its measurement ELISA value, the picking positive are worth higher monoclonal hole and carry out limiting dilution, until ELISA 96 orifice plates of measurement are entirely hardened
Fruit is the positive.
3, the preparation and purification of monoclonal antibody：The cell culture fluid 20ml progress protein A column chromatography for collecting the ELISA positive is pure
Change, monoclonal antibody after purification first carries out concentration mensuration, dispenses, frozen at -20 DEG C afterwards.
Embodiment 2 is using monoclonal antibody of the present invention as the immunohistochemical experiment of primary antibody
1,24 kinds of different types of cancerous tissue sampling production organization chips are taken respectively, are cut using Leica RM2235 type tissue
Piece machine is sliced, and slice thickness is 4 μm；
2, immunohistochemical staining survey is carried out to antibody of the present invention using Leica BondMax immunohistochemistry automatic staining machine
Examination, the dewaxing carried using machine and hydrating condition, the specific steps are：60 DEG C of incubation 30min, using dewaxed solution (Leica)
It washes 3 times.Antigen retrieval uses antigen retrieval buffers 2 (ER2, Leica), 100 DEG C of incubation 30min.Primary antibody uses antibody of the present invention, adopts
Final concentration of 1.0 μ g/ml, 150 μ l are diluted to antibody diluent (Leica).Antibody at room temperature is incubated for 30min.Using matched
150 μ l of secondary antibody (Leica) is incubated at room temperature 8min.Using 150 μ l of poly analyte detection liquid (Leica), it is incubated at room temperature 8min.It uses
150 μ l of endogenous peroxydase confining liquid is incubated at room temperature 5min.Use 150 μ l of DAB developing solution (Leica), incubation at room temperature
10min.Haematoxylin is redyed, and 5min is incubated at room temperature；
3, it is dehydrated and transparent：Deionized water cleans 1min, 95% ethyl alcohol 1min, 100% ethyl alcohol 2min x 2 times, dimethylbenzene
2min x 3 times, neutral gum mounting；
4, microscopy, as a result：Partial tumors show positive staining, including lung cancer, melanoma, oophoroma, colon cancer, bladder
Cancer etc., Partial tumors display is negative to be dyed without PD-L1, including the cancer of the brain, breast cancer, liver cancer etc..Wherein Non-Small Cell Lung Carcinoma
Dyeing is as shown in Figure 2, it is seen that obvious cell membrane dyeing, staining cell includes that Partial tumors lymphocyte infiltration and Partial tumors are thin
Born of the same parents.Staining pattern is correct, and signal is stronger, has no obvious non-specific dyeing.
The specific detection of the monoclonal antibody of the present invention of embodiment 3
Any positive band is had no without the 293T cell of transfection using antibody test of the present invention.
Using 96 orifice plates (Her-2) that antibody of the present invention is overlay using ELISA detection irrelevant antigen, result is feminine gender.
The above is only a preferred embodiment of the present invention, it is noted that come for those of ordinary skill in the art
It says, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (6)
1. a kind of rabbit-anti human monoclonal antibodies, it is characterised in that human PD-L 1 albumen can be specifically bound.
2. monoclonal antibody described in claim 1 is in preparing the immunohistochemistry detection instrument for detecting PD-L1 albumen
Using.
3. application according to claim 2, which is characterized in that the immunohistochemistry detection instrument is reagent, kit, core
Piece or test strips.
4. application according to claim 2, which is characterized in that the immunohistochemistry detection instrument can be used for the side of adjuvant treatment
Case selection, tumour Index for diagnosis and medical basic research.
5. application according to claim 4, which is characterized in that the supplemental treatment regimens selection can be used for including black
Plain tumor, non-small cell lung cancer, bladder cancer, kidney, gastric cancer, colorectal cancer, prostate cancer, cervical carcinoma, breast cancer or related neoplasms.
6. application according to claim 4, which is characterized in that the medical basic research can be used for PD-L1 not
With the expression of immunocyte and tumour cell, disease occurs, progress, the relevant pathologic, physiologic of prognosis is studied and PD-L1 tune
Save the mechanism study of body's immunity.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109627338A (en) * | 2019-01-25 | 2019-04-16 | 苏州药明泽康生物科技有限公司 | A kind of novel anti human PD-L 1 antibody and its application |
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| CN107266572A (en) * | 2017-07-13 | 2017-10-20 | 无锡傲锐东源生物科技有限公司 | Anti- PD L1 protein monoclonal antibodies and application thereof |
| CN107298713A (en) * | 2017-08-15 | 2017-10-27 | 联合益康（北京）生物科技有限公司 | A kind of anti-PD L1 antibody and application, preparation method, kit and medicine |
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2018
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| CN106604933A (en) * | 2014-07-11 | 2017-04-26 | 基因泰克公司 | Anti-pd-l1 antibodies and diagnostic uses thereof |
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| CN107266572A (en) * | 2017-07-13 | 2017-10-20 | 无锡傲锐东源生物科技有限公司 | Anti- PD L1 protein monoclonal antibodies and application thereof |
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Cited By (2)
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| CN109627338A (en) * | 2019-01-25 | 2019-04-16 | 苏州药明泽康生物科技有限公司 | A kind of novel anti human PD-L 1 antibody and its application |
| CN109627338B (en) * | 2019-01-25 | 2022-10-18 | 苏州药明泽康生物科技有限公司 | Novel anti-human PD-L1 antibody and application thereof |
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Application publication date: 20181123 |