Purification method of anti-IL-17 RA monoclonal antibody
阅读说明：本技术 一种抗il-17ra单克隆抗体的纯化方法 (Purification method of anti-IL-17 RA monoclonal antibody ) 是由 白义 刘晓航 孟艳敏 于 2020-06-19 设计创作，主要内容包括：本发明涉及抗体工程技术领域,具体提供了一种抗IL-17RA单克隆抗体的纯化方法,该方法包括亲和层析、阳离子交换层析和阴离子交换层析。本发明提供的纯化方法用于纯化的抗IL-17RA单克隆抗体具有较强的亲和力和良好的生物活性,能够与IL-17RA抗原特异性结合,是具有巨大潜力的自身免疫性疾病治疗药物,其次,本发明采用三步即可完成对抗IL-17RA单克隆抗体的纯化,亲和层析用于捕获目的蛋白,阳离子交换层析去除酸性峰,阴离子交换层析去除聚集体,最终获得质量合格的抗IL17-RA单克隆抗体,该方法易于中试放大生产,没有中间样品的调整步骤,各步工艺衔接顺畅,缩短工艺周期。(The invention relates to the technical field of antibody engineering, and particularly provides a purification method of an anti-IL-17 RA monoclonal antibody, which comprises affinity chromatography, cation exchange chromatography and anion exchange chromatography. The anti-IL-17 RA monoclonal antibody purified by the purification method provided by the invention has stronger affinity and good bioactivity, can be specifically combined with an IL-17RA antigen, and is an autoimmune disease treatment drug with great potential.)
1. A method for purifying an anti-IL-17 RA monoclonal antibody, comprising the steps of:
s1, affinity chromatography;
s11, firstly, using 3-5CV buffer solution A to balance the affinity chromatography column at the flow rate of 100-200cm/h, and loading the cell supernatant containing the anti-IL-17 RA monoclonal antibody at the flow rate of 100-200 cm/h;
s12, secondly, after the sample loading is finished, the buffer solution A is used for carrying out rebalancing for the first time, after the ultraviolet signal is stable, the buffer solution B is used for carrying out leaching for the second time, and the rinsing is stopped until the curve of the ultraviolet absorption value is reduced to be stable;
s13, finally, eluting the affinity chromatography column by using a buffer solution C at the flow rate of 100-200cm/h, collecting the affinity eluent when the ultraviolet absorption value rises to 100mAU, and ending the collection when the ultraviolet absorption value falls to 100mAU for later use;
s2, cation exchange chromatography;
s21, firstly, using 3-5CV buffer solution D to balance the cation exchange chromatography column at the flow rate of 100-;
s22, secondly, after the sample loading is finished, rebalancing is carried out by using the buffer solution D, after the ultraviolet absorption value is stable, the buffer solution E is used for leaching the cation exchange chromatography column at the flow rate of 100-200cm/h, and the washing volume is 5-15 CV;
s23, finally, eluting the cation exchange chromatography column by using a buffer solution F at the flow rate of 100-200cm/h, collecting cation eluent when the ultraviolet absorption value begins to rise, and finishing collection when the ultraviolet absorption value is reduced to 300mAU for later use;
s3, anion exchange chromatography;
s31, firstly, balancing the anion exchange chromatography column with a buffer solution G with the concentration of 7-10CV at the flow rate of 100-;
and S32, after the sample loading is finished, washing by using the buffer solution G again, collecting the flow-through liquid when the ultraviolet absorption value is increased to 100mAU, and finishing the collection when the ultraviolet absorption value is decreased to 200 mAU.
2. The method for purifying an anti-IL-17 RA monoclonal antibody as claimed in claim 1, wherein in step S1, the anti-IL-17 RA monoclonal antibody is selected from any one of the following:
(mAb-1) the amino acid sequence of the heavy chain variable region is SEQ ID NO:1, and the amino acid sequence of the light chain variable region is SEQ ID NO: 11;
(mAb-2) the amino acid sequence of the heavy chain variable region is SEQ ID NO:2, and the amino acid sequence of the light chain variable region is SEQ ID NO: 11;
(mAb-3) the amino acid sequence of the heavy chain variable region is SEQ ID NO:3, and the amino acid sequence of the light chain variable region is SEQ ID NO: 11;
(mAb-4) the amino acid sequence of the heavy chain variable region is SEQ ID NO:4, and the amino acid sequence of the light chain variable region is SEQ ID NO: 11;
(mAb-5) the amino acid sequence of the heavy chain variable region is SEQ ID NO:5, and the amino acid sequence of the light chain variable region is SEQ ID NO: 11;
(mAb-6) the amino acid sequence of the heavy chain variable region is SEQ ID NO:6, and the amino acid sequence of the light chain variable region is SEQ ID NO: 12;
(mAb-7) the amino acid sequence of the heavy chain variable region is SEQ ID NO:7, and the amino acid sequence of the light chain variable region is SEQ ID NO: 12;
(mAb-8) the amino acid sequence of the heavy chain variable region is SEQ ID NO:8, and the amino acid sequence of the light chain variable region is SEQ ID NO: 13;
(mAb-9) the amino acid sequence of the heavy chain variable region is SEQ ID NO:9, and the amino acid sequence of the light chain variable region is SEQ ID NO: 13;
(mAb-10) the amino acid sequence of the heavy chain variable region is SEQ ID NO:10, and the amino acid sequence of the light chain variable region is SEQ ID NO: 13;
(mAb-11) the amino acid sequence of the heavy chain variable region is SEQ ID NO:2 and the amino acid sequence of the light chain variable region is SEQ ID NO: 13.
3. The method of claim 2, wherein the monoclonal antibody further comprises a heavy chain constant region that is one of human IgG1, IgG2, IgG3, IgG4 and a light chain constant region that is human ck;
preferably, the heavy chain constant region is human IgG 1.
4. The method for purifying an anti-IL-17 RA monoclonal antibody of claim 1, wherein in step S1, the medium of the affinity chromatography column is MabSelect SurE, MabSelect Sure LX or Eshmuno-A.
5. The method for purifying an anti-IL-17 RA monoclonal antibody of claim 1, wherein in step S1, the buffer A comprises 50mM Tris-HCl and 150-160 mM NaCl, the pH of the buffer A is 7.2-7.6, and the conductivity of the buffer A is 15-20 mS/cm;
the buffer solution B comprises 50mM sodium acetate, the pH value of the buffer solution B is 5.0-6.0, and the conductivity of the buffer solution B is 2-5 mS/cm;
the buffer solution C comprises sodium acetate, sodium citrate or glycine, and the pH value of the buffer solution C is 3.5-3.7.
6. The method for purifying an anti-IL-17 RA monoclonal antibody of claim 5, wherein in step S1, buffer A consists of 50mM Tris-HCl and 150mM sodium chloride, and the pH of buffer A is 7.4; the pH of the buffer solution B is 5.0; the buffer C was 50mM sodium acetate and the pH of the buffer C was 3.6.
7. The method for purifying an anti-IL-17 RA monoclonal antibody as claimed in claim 1, wherein in step S2, the medium of the cation exchange chromatography column comprises Capto SP, Eshmuno CPX or SepHarose DEAE;
preferably, the medium of the cation exchange chromatography column is Eshmuno CPX.
8. The method for purifying an anti-IL-17 RA monoclonal antibody according to claim 1, wherein the buffer D comprises 20mmol/L sodium phosphate, 20mmol/L sodium citrate or 20mmol/L MES buffer, the pH of the buffer D is 6.0, and the conductivity of the buffer D is 2-4 mS/cm;
the buffer solution E comprises 20mmol/L sodium phosphate and 10-20 mmol/L sodium chloride, the pH value of the buffer solution E is 7.2-7.4, and the conductivity of the buffer solution E is 4-6 mS/cm; the buffer solution F comprises 20mmol/L sodium phosphate and 70-100 mmol/L sodium chloride, the pH value of the buffer solution F is 7.2-7.4, and the conductivity of the buffer solution F is 7-15 mS/cm;
preferably, the buffer D is 20mmol/L sodium phosphate.
9. The method for purifying an anti-IL-17 RA monoclonal antibody as claimed in claim 1, wherein the medium of the anion exchange chromatography column is POROS XQ, Capto Q, or eshumno Q;
preferably, the medium of the anion exchange chromatography column is POROS XQ.
10. The method for purifying an anti-IL-17 RA monoclonal antibody according to claim 1, wherein the buffer G comprises 20mmol/L sodium phosphate and 50-80 mmol/L sodium chloride, the pH of the buffer G is 7.2-7.4, and the conductivity of the buffer G is 6-12 mS/cm.
Technical Field
The invention relates to the technical field of antibody engineering, in particular to a purification method of an anti-IL-17 RA monoclonal antibody.
Background
Autoimmune diseases refer to diseases caused by the body's immune reaction to autoantigens, which results in damage to the tissues. Many diseases are subsequently classified as autoimmune diseases, such as psoriasis, rheumatoid arthritis, ankylosing spondylitis, scleroderma and the like. Among them, Psoriasis (Psoriasis) is also called Psoriasis and is characterized in that the skin has erythema, scaling plaques with different sizes and clear boundaries, and is covered with a large amount of dry silvery white scales. The histological features of psoriatic skin are epidermal keratinocyte hyperproliferation, vascular proliferation, and infiltration of dendritic cells, macrophages, neutrophils, T cells, the pathogenesis of psoriasis involving a complex inflammatory response and immune system. The prevalence rate of China is about 0.47%, and more than 600 million psoriasis patients are calculated at present in China. Psoriasis, once it develops, often suffers from lifelong, recurrent episodes, with the majority of patients presenting with a course of alternating relapses and remissions. Rheumatoid Arthritis (RA) is a systemic autoimmune disease characterized by chronic erosive arthritis, and is characterized by synovitis, and the resulting destruction of articular cartilage and bone mass, ultimately leading to joint deformity and loss of function. The worldwide morbidity is about 0.5-1.0%, the national morbidity is 0.3-0.4%, and the morbidity of women is 3 times that of men generally. Ankylosing Spondylitis (AS) is a chronic inflammatory disease with inflammation of sacroiliac joints, spinal attachment points and axial bones AS main symptoms, and fibrosis and ossification of connective tissues around the annulus fibrosus and intervertebral disc and ankylosis AS pathological features, belonging to the category of rheumatism, and the cause of the disease is unknown. It is often associated with infection, genetic factors, environmental factors (e.g., long term exposure to cold, humid environments), etc.
With the continuous and intensive research of biological antibody drugs, interleukin-17 (IL-17) is a characteristic cytokine secreted by helper T cell 17(Th17 cell), which can promote the local production of chemokines, recruit neutrophils, promote the proliferation and differentiation of cells; however, it may also bind to receptors to produce a cascade-like inflammatory effect, causing tissue damage, which has been shown to be closely related to the pathogenesis of various autoimmune diseases. Antagonizing and blocking IL-17/IL-17R signal channel is an autoimmune disease treatment target with great potential, and is expected to effectively relieve the symptoms of autoimmune diseases and prevent the progress of diseases.
The IL-17 receptor (IL-17R) family consists of 5 members, IL-17RA, IL-17RB, IL-17RC, IL-17RD, and IL-17RE, all of which are type I single-transmembrane glycoproteins with conserved structural motifs, including an extracellular fibronectin III-like domain and an intracellular domain. The human IL-17RA gene is located on chromosome P22, and the human IL-17RA protein has 866 amino acids in total length, multiple disulfide bonds and glycosylation sites, two modification sites, an extracellular region, a transmembrane region and an intracellular region.
IL-17RA is a receptor for IL-17A and also a receptor for IL-17F, and IL-17RA binds IL-17A with greater affinity than IL-17F. Activation of IL-17RA reduces the expression of inflammatory factors such as CXCL1, CXCL8/IL-8 and IL-6. IL-17RA is widely expressed, especially in hematopoietic tissues at higher levels. IL-17RA comprises two distinct domains, a toll/
At present, many problems are often encountered during the process development process for anti-IL-17 RA monoclonal antibody, the dosage of monoclonal antibody products is generally high, about 200 mg/time, but the administration volume of subcutaneous injection is limited, and 1.0-1.5ml is already a limit, which indicates that the concentration of anti-IL-17 RA monoclonal antibody is as high as 100-150mg/ml, which is more demanding for the process purification. brodalumab is the first and only fully humanized monoclonal antibody selectively targeting IL-17 receptor a (IL-17RA), but the purification process of brodalumab is complex, and in the conventional purification process, after each step is completed, the pH condition of the next step needs to be adapted by replacing or debugging buffer solution, so that a lot of time is wasted in the batch production process, and impurities are likely to be introduced in the debugging process, so that the intermediate operation steps are more, the recovery rate is low, and the cost is increased.
Disclosure of Invention
In order to meet the requirements of domestic markets, the invention utilizes a phage antibody library display technology and a high-throughput screening method to screen an anti-IL-17 RA monoclonal antibody with higher affinity and better activity, and meanwhile, the invention can overcome the defects existing in the purification process of the existing anti-IL-17 RA monoclonal antibody according to the characteristics of the monoclonal antibody, and finally provides a purification method suitable for the anti-IL-17 RA monoclonal antibody provided by the invention through a large number of experiments.
The specific technical scheme of the invention is as follows:
the invention provides a purification method of an anti-IL-17 RA monoclonal antibody, which comprises the following steps:
s1, affinity chromatography;
s11, firstly, using 3-5CV buffer solution A to balance the affinity chromatography column at the flow rate of 100-200cm/h, and loading the cell supernatant containing the anti-IL-17 RA monoclonal antibody at the flow rate of 100-200 cm/h;
s12, secondly, after the sample loading is finished, the buffer solution A is used for carrying out rebalancing for the first time, after the ultraviolet signal is stable, the buffer solution B is used for carrying out leaching for the second time, and the rinsing is stopped until the curve of the ultraviolet absorption value is reduced to be stable;
s13, finally, eluting the affinity chromatography column by using a buffer solution C at the flow rate of 100-200cm/h, collecting the affinity eluent when the ultraviolet absorption value rises to 100mAU, and ending the collection when the ultraviolet absorption value falls to 100mAU for later use;
s2, cation exchange chromatography;
s21, firstly, using 3-5CV buffer solution D to balance the cation exchange chromatography column at the flow rate of 100-;
s22, secondly, after the sample loading is finished, rebalancing is carried out by using the buffer solution D, after the ultraviolet absorption value is stable, the buffer solution E is used for leaching the cation exchange chromatography column at the flow rate of 100-200cm/h, and the washing volume is 5-15 CV;
s23, finally, eluting the cation exchange chromatography column by using a buffer solution F at the flow rate of 100-200cm/h, collecting cation eluent when the ultraviolet absorption value begins to rise, and finishing collection when the ultraviolet absorption value is reduced to 300mAU for later use;
s3, anion exchange chromatography;
s31, firstly, balancing the anion exchange chromatography column with a buffer solution G with the concentration of 7-10CV at the flow rate of 100-;
and S32, after the sample loading is finished, washing by using the buffer solution G again, collecting the flow-through liquid when the ultraviolet absorption value is increased to 100mAU, and finishing the collection when the ultraviolet absorption value is decreased to 200 mAU.
The invention provides a purification method special for an anti-IL-17 RA monoclonal antibody, which not only reduces the process impurities such as host protein (HCP), protein-A, DNA and the like in antibody protein on the basis of ensuring the protein quality by a three-step chromatography method, but also simplifies the operation process, does not need to independently regulate proper pH value again in the transition process from cation exchange chromatography to anion exchange chromatography, has smooth two-step connection, avoids introducing impurities in the process of regulating the pH value, shortens the purification period, improves the purification efficiency, saves time for the pilot-scale production in the later period, and effectively removes impurities related to various products such as polymers, acid and alkali isomers and the like, thereby achieving the effect of obviously improving the purity of target protein. In the invention, affinity chromatography is used for capturing target protein, anion-cation exchange chromatography is used for finely purifying protein, product impurities are further removed, and finally the anti-IL-17 RA monoclonal antibody with qualified quality is obtained; compared with the prior art, the method can obtain high-yield active protein in the shortest time, has simple operation and lower cost, and can meet the purification requirement of pilot scale amplification on the anti-IL-17 RA monoclonal antibody.
Further, in step S1 of the above method provided by the present invention, the anti-IL-17 RA monoclonal antibody is selected from any one of the following:
(mAb-1) the amino acid sequence of the heavy chain variable region is SEQ ID NO:1, and the amino acid sequence of the light chain variable region is SEQ ID NO: 11;
(mAb-2) the amino acid sequence of the heavy chain variable region is SEQ ID NO:2, and the amino acid sequence of the light chain variable region is SEQ ID NO: 11;
(mAb-3) the amino acid sequence of the heavy chain variable region is SEQ ID NO:3, and the amino acid sequence of the light chain variable region is SEQ ID NO: 11;
(mAb-4) the amino acid sequence of the heavy chain variable region is SEQ ID NO:4, and the amino acid sequence of the light chain variable region is SEQ ID NO: 11;
(mAb-5) the amino acid sequence of the heavy chain variable region is SEQ ID NO:5, and the amino acid sequence of the light chain variable region is SEQ ID NO: 11;
(mAb-6) the amino acid sequence of the heavy chain variable region is SEQ ID NO:6, and the amino acid sequence of the light chain variable region is SEQ ID NO: 12;
(mAb-7) the amino acid sequence of the heavy chain variable region is SEQ ID NO:7, and the amino acid sequence of the light chain variable region is SEQ ID NO: 12;
(mAb-8) the amino acid sequence of the heavy chain variable region is SEQ ID NO:8, and the amino acid sequence of the light chain variable region is SEQ ID NO: 13;
(mAb-9) the amino acid sequence of the heavy chain variable region is SEQ ID NO:9, and the amino acid sequence of the light chain variable region is SEQ ID NO: 13;
(mAb-10) the amino acid sequence of the heavy chain variable region is SEQ ID NO:10, and the amino acid sequence of the light chain variable region is SEQ ID NO: 13;
(mAb-11) the amino acid sequence of the heavy chain variable region is SEQ ID NO:2 and the amino acid sequence of the light chain variable region is SEQ ID NO: 13.
The 11 anti-IL-17 RA monoclonal antibodies provided by the invention have stronger affinity and good biological activity, can be specifically combined with IL-17RA antigens, block the combination of IL-17A and IL-17RA, antagonize and block IL-17/IL-17R signal pathways, are autoimmune disease treatment drugs with great potential, effectively relieve the symptoms of autoimmune diseases, prevent the progression of diseases, and the autoimmune diseases include but are not limited to the following: psoriasis, rheumatoid arthritis, ankylosing spondylitis, scleroderma, or the like.
Further, the monoclonal antibody further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is one of human IgG1, IgG2, IgG3 and IgG4, and the light chain constant region is human C kappa;
preferably, the heavy chain constant region is
Further, in step S1, the medium of the affinity chromatography column is MabSelect Sure, MabSelect SureLX or Eshmuno-A. Preferably, the MabSelect Sure LX is selected as a medium in the affinity chromatography column to capture the antibody in the cell supernatant, remove most impurities and reduce the volume of the sample, and the MabSelect Sure LX has the advantages of stable base frame, long service life, less ligand shedding and the like.
Further, in step S1, the buffer solution A comprises 50mM Tris-HCl and 150-160 mM sodium chloride, the pH of the buffer solution A is 7.2-7.6, and the conductivity of the buffer solution A is 15-20 mS/cm;
the buffer solution B comprises 50mM sodium acetate, the pH value of the buffer solution B is 5.0-6.0, and the conductivity of the buffer solution B is 2-5 mS/cm;
the buffer solution C comprises sodium acetate, sodium citrate or glycine, and the pH value of the buffer solution C is 3.5-3.7.
Further, in step S1, the buffer A is composed of 50mM Tris-HCl and 150mM NaCl, and the pH of the buffer A is 7.4; the pH of the buffer solution B is 5.0; the buffer C was 50mM sodium acetate and the pH of the buffer C was 3.6.
The buffer solution A is mainly used for reducing non-specifically bound impurities, and Tris and sodium chloride contained in the buffer solution A provide a proper buffer range to ensure the stability of the protein, so that the column can be bound with the target protein to elute and remove the impurity protein. In addition, in order to adapt to the isoelectric point of the anti-IL-17 RA monoclonal antibody, a large number of experiments prove that the buffer solution A with the pH value of 7.2-7.6 can meet the purification requirement, the buffer solution has stronger buffer capacity in the pH range, and the purification efficiency can be improved by setting the conductivity to be 15-20mS/cm according to the pH value. The buffer solution B is mainly used for leaching and removing host cells, and in addition, sodium acetate contained in the buffer solution B can improve the stability of the target protein and provides a basis for protein purification.
The affinity chromatography selects low pH solution for leaching, has low electrical conductivity, is suitable for the cation exchange chromatography of the second step, avoids the process that the existing affinity chromatography selects high salt solution for leaching and then can enter the cation exchange chromatography after desalting, greatly shortens the time of the purification process and improves the purification efficiency.
Further, in step S2, the medium of the cation exchange chromatography column is Capto SP, Eshmuno CPX or Sepharose CM, and preferably, the medium of the cation exchange chromatography column is Eshmuno CPX. The cation exchange chromatography selects Eshmuno CPX as a medium, can be used for finely purifying antibody protein, further removes impurities in the antibody protein, can separate acid and alkali isomers, improves the content of a main peak, and finally obtains the anti-IL-17 RA monoclonal antibody with qualified quality.
Anion-cation exchange chromatography is used for fine purification of protein, further removal of product impurities and final obtaining of qualified anti-IL-17 RA monoclonal antibody
Further, the buffer solution D comprises 20mmol/L sodium phosphate, 20mmol/L sodium citrate or 20mmol/L MES buffer solution, the pH value of the buffer solution D is 6.0, and the conductivity of the buffer solution D is 2-4 mS/cm;
the buffer solution E comprises 20mmol/L sodium phosphate and 10-20 mmol/L sodium chloride, the pH value of the buffer solution E is 7.2-7.4, and the conductivity of the buffer solution E is 4-6 mS/cm;
the buffer solution F comprises 20mmol/L sodium phosphate and 70-100 mmol/L sodium chloride, the pH value of the buffer solution F is 7.2-7.4, and the conductivity of the buffer solution F is 7-15 mS/cm;
preferably, the buffer D is 20mmol/L sodium phosphate.
The invention screens the buffer solution components required in the cation exchange chromatography process through a large number of experiments, the screened buffer solution D, buffer solution E and buffer solution F can meet the fine purification requirement of the anti-IL-17 RA monoclonal antibody in the cation exchange chromatography, and simultaneously, the buffer solution D, buffer solution E and buffer solution F can directly enter anion exchange chromatography through the exploration of the preparation components and the pH value without regulating the pH value again, thereby greatly shortening the purification time and saving the materials.
Further, the medium of the anion exchange chromatographic column is POROS XQ, Capto Q or Eshmuno Q;
preferably, the medium of the anion exchange chromatography column is POROS XQ.
Further, the buffer solution G comprises 20mmol/L sodium phosphate and 50-80 mmol/L sodium chloride, the pH value of the buffer solution G is 7.2-7.4, and the conductivity of the buffer solution G is 6-12 mS/cm.
The invention has the following beneficial effects: firstly, the anti-IL-17 RA monoclonal antibody used for purification in the invention has stronger affinity and good biological activity, can be specifically combined with IL-17RA antigen, can block the combination of IL-17A and IL-17RA, antagonize and block IL-17/IL-17R signal channel, is an autoimmune disease treatment drug with huge potential, effectively relieves the symptoms of autoimmune diseases, prevents the progression of the diseases, and the autoimmune diseases comprise but are not limited to the following: psoriasis, rheumatoid arthritis, ankylosing spondylitis, scleroderma, or the like; secondly, the method provided by the invention adopts three steps of affinity chromatography and anion-cation exchange chromatography to complete the purification of the anti-IL-17 RA monoclonal antibody, the affinity chromatography is used for capturing target protein, the cation exchange chromatography is used for removing an acid peak, the anion exchange chromatography is used for removing aggregates, and finally the anti-IL-17 RA monoclonal antibody with qualified quality is obtained, compared with the purification method provided by the prior art, the method is easy for pilot scale production, does not have the adjustment step of an intermediate sample, has smooth process connection in each step, shortens the process period, shortens the purification time of the existing anti-IL-17 RA monoclonal antibody from 3 days to 10-12 hours, greatly improves the purification efficiency, in addition, the method is suitable for continuous flow process production, the sample in the previous process step can directly enter the next process step without adjusting the product, therefore, the invention can realize the whole process in a closed system, reduce the risk of pollution and ensure the safety of the antibody.
Drawings
FIG. 1 is a plasmid map of pScFvDisb-s in example 1 of the present invention;
FIG. 2 is a graph of Elisa experimental data for identification of monoclonal antibody phage in example 1 of the present invention;
FIG. 3 is a graph of gradient dilution Elisa experimental data of phage purified monoclonal antibody in example 2 of the present invention;
FIG. 4 is a plasmid map of pTSE in example 3 of the present invention;
FIG. 5 is a graph showing a comparison of the binding ability of anti-IL-17 RA whole antibody to IL-17RA at the molecular level in example 4 of the present invention;
FIG. 6 is a graph showing a comparison of the ability of anti-IL-17 RA whole antibody in example 5 of the present invention to competitively inhibit the binding between IL-17A and IL-17 RA;
FIG. 7 is a graph comparing the binding ability of anti-IL-17 RA whole antibody to IL-17RA at the cellular level in example 7 of the present invention;
FIG. 8 is a graph showing experimental comparison of the biological activities of the anti-IL-17 RA antibody of example 8 of the present invention in inhibiting IL-6 production in HDF cells.
Detailed Description
The present invention will be described in further detail with reference to the following examples.
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