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HEK293 表达的全长CD20蛋白

FACS验证,免费提供protocol

背景

Lionel Rougé, et al. Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab. Science. 2020
作为B细胞表面抗原,CD20在前B细胞到成熟B细胞阶段均有表达,而不表达在祖B细胞以及成熟的浆细胞上。同时, CD20表达于B细胞来源的淋巴瘤、白血病等肿瘤细胞、涉及炎症和免疫相关疾病的B细胞,因而CD20也成为了淋巴瘤、白血病和某些自身免疫疾病的治疗靶标。
靶向CD20的抗体药物经历了三代创新:第一代是以Rituximab(利妥昔单抗)为代表的人鼠嵌合抗体,人源化程度不高,部分患者会产生耐药或者不良反应;第二代是以Ofatumumab(奥法木单抗)为代表的人源化改造抗体,虽然降低了免疫原性,减少了不良反应,但抗体特异性和抗原结合的亲和力有一定的下降;第三代是以Obinutuzumab(奥妥珠单抗)为代表的Fc片段糖基化修饰抗体,增强抗体的特异性及与抗原结合的亲和力,使得ADCC与ADCP效应增强。
不局限于单抗药物领域,CD20通过结合CD3等靶点进行双特异抗体的开发实现了将T细胞募集到B细胞周围,进而实现T细胞对于B细胞的杀伤作用。
研发靶向CD20的抗体药物,优质抗原必不可少。2020年2月20日Genentech在Science上发表的一篇文章Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab,突破性地解析了全长CD20蛋白以及CD20蛋白与Rituximab Fabs复合物的三维结构。文章证实Rituximab对CD20的识别不止有一级表位ECL2,还识别其二级表位ECL1/ECL2。SPR验证的Rituximab与全长CD20蛋白以及与ECL2多肽表位的亲和力差异,表明二级表位ECL1/ECL2会影响Rituximab和CD20的亲和力。另外文章发现二级表位ECL1/ECL2还会影响Rituximab的CDC效应,揭示了具有完整结构的全长CD20蛋白的重要意义。(页面尾部可下载文献全文)
因为全长CD20蛋白的表达难度高、活性不稳定等因素,目前市面上很难找到合适的CD20蛋白用于抗体活性分析。ACROBiosystems采用独特的设计开发了由HEK293表达的四次跨膜全长CD20蛋白,包括未进行标记的和生物素标记的Nanodisc、DDM、VLP不同版本产品。全长CD20蛋白同时有small loop和big loop,具有完整的天然正确构象,可用于免疫/ ELISA/ SPR/ BLI/ FACS。我们验证了Rituximab、Ofatumumab与全长CD20蛋白的结合活性,全长CD20蛋白展现出纳摩尔级别的高亲和力,可免费提供Protocol。
不同类型的全长CD20蛋白
DDM版本
DDM是一种非离子型去垢剂,能够使脂膜解体释放膜蛋白,并在溶液中为去膜状态下的膜蛋白提供疏水环境,维持和保护膜蛋白的疏水跨膜结构。但DDM的存在会破坏细胞膜的结构,一般不用推荐于细胞实验。
Nanodisc版本(不含去垢剂)
Nanodisc (纳米磷脂盘) 是由膜支架蛋白(membrane scaffold proteins, MSPs)和磷脂分子构成的磷脂双分子层类膜结构,膜蛋白可以整合到Nanodisc的特殊结构中,保持其生物学活性并有很好的水溶性,应用更广泛。ACROBiosystems使用的Nanodisc技术已经获得专利持有方的授权,生物药研发企业在研发过程中可放心使用。

Ilia G. Denisov, et al., Nanodisc in membrane biochemistry and biophysics. Chem Rev. 2017(页面尾部可下载文献全文)

VLP版本
 VLP (Virus like particles) 技术平台将构象完整的膜蛋白直接聚集在细胞表面,使这些复杂的膜蛋白转变为可溶性的、高浓度的蛋白用于抗体免疫和筛选,所制备的包膜VLPs在其固有的细胞膜上显示正确折叠的多次跨膜蛋白,从而能够诱导和筛选识别靶标天然构象的功能性抗体。
产品列表
  • 膜蛋白-去垢剂平台
  • Nanodisc平台
  • VLP平台
分子 货号 产品描述 应用 订购/预购
CD20 CD0-H52H3 Human CD20 / MS4A1 Full Length Protein, His Tag (HEK293) (SPR verified) 免疫
ELISA
SPR
BLI

订购

CD20 CD0-H82E5 Biotinylated Human CD20 / MS4A1 Full Length Protein, His,Avitag™ (HEK293)

订购

CD20 CD0-C52H8 Cynomolgus CD20 / MS4A1 Full Length Protein, His Tag

订购

CD20 CD0-C52H5 Canine CD20 / MS4A1 Full Length Protein, His Tag

订购

Buffer DC-11 200x DDM CHS buffer 维持膜蛋白溶解性和活性
稀释膜蛋白或抗体
在SPR或BLI检测中制备运行缓冲液

订购

分子 货号 产品描述 应用 订购/预购
CD20 CD0-H52H1 Human CD20 / MS4A1 Full Length Protein, His Tag (Nanodisc) (HEK293) 免疫
ELISA
SPR
BLI
细胞实验
CAR阳性率检测

订购

CD20 CD0-H82E3 Biotinylated Human CD20 / MS4A1 Full Length Protein, His,Avitag™ (Nanodisc) (HEK293)

订购

CD20 CD0-C52H9 Canine CD20 / MS4A1 Full Length Protein, His Tag (Nanodisc) (HEK293)

订购

MSP1D1 APO-H51H3 Human MSP1D1 Protein, His Tag (Nanodisc) 同型对照

订购

MSP1D1 APO-H81Q5 Biotinylated Human MSP1D1 Protein, His,Avitag™ (Nanodisc)

订购

分子 货号 产品描述 应用 订购/预购
CD20 CDP-H52P6 Human CD20 Full Length Protein-VLP (HEK293) 免疫
ELISA
SPR/BLI
细胞实验
CAR阳性率检测

订购

VLP VLP-N5213 Virus-Like Particle (VLP) isotype control VLP同型对照

订购

VLP VLP-NF2P4 Fluorescent Virus-Like Particle (VLP) isotype control

订购

产品特点
全长CD20蛋白由真核系统表达具有完整的天然构象(同时具有small loop和big loop)
全长CD20蛋白能提供更好的活性

A) HEK293 Cell (Full Length)

Immobilized Rituximab at 2 μg/mL (100 μL/well) can bind Human CD20 Full Length, His Tag, HEK293 (SPR verified) (Cat. No. CD0-H52H3) with a linear range of 0.4-3 ng/mL (in presence of DDM and CHS).

Protocol

B) E.coli (Extracellular)

Immobilized Rituximab at 5 μg/mL (100 μL/well) can bind Human CD20, TrxA Tag, His Tag (E.coli) with a linear range of 78-2500 ng/mL.

ACRO的全长CD20蛋白活性经FACS/ELISA/ SPR验证,可免费提供相应的Protocol
FACS验证
Cat. No. CD0-H82E3 (HEK293表达)
FACS Analysis of anti-CD20 CAR expression

2e5 of CD20-CAR-293 cells transfected with anti-CD20-scFv were stained with 100 μL of 3 μg/mL of Biotinylated Human CD20 Full Length, His,Avitag (Cat. No. CD0-H82E3) and negative control protein respectively, washed and then followed by PE-SA and analyzed with FACS (QC tested).

Protocol

Cat. No. CD0-H52H1 (HEK293表达)
FACS Analysis of anti-CD20 CAR expression

2e5 of CD20-CAR-293 cells transfected with anti-CD20-scFv were stained with 100 μL of 3 μg/mL of Human CD20 / MS4A1 Full Length Protein, His Tag (Nanodisc) (HEK293)(Cat. No. CD0-H52H1) and negative control protein respectively, washed and then followed by PE anti-His antibody and analyzed with FACS (QC tested).

Protocol

ELISA验证
Cat. No. CD0-H82E3 (HEK293表达)

Immobilized Rituximab at 5 μg/mL (100 μL/well) can bind Biotinylated Human CD20 Full Length, His,Avitag (Cat. No. CD0-H82E3) with a linear range of 0.008-0.125 μg/mL

Protocol

Cat. No. CD0-H52H1 (HEK293表达)

Immobilized Rituximab at 2 μg/mL (100 μL/well) can bind Human CD20 Full Length, His Tag (Cat. No. CD0-H52H1) with a linear range of 1-63 ng/mL

Protocol

Cat. No. CD0-H82E5 (HEK293表达)

Immobilized Rituximab at 2 μg/mL (100 μL/well) can bind Biotinylated Human CD20 Full Length, His,Avitag (HEK293) (Cat. No. CD0-H82E5) with a linear range of 4-63 ng/mL (in presence of DDM and CHS).

Protocol

Cat. No. CD0-H82E5 (HEK293表达)

Immobilized Ofatumumab at 2 μg/mL (100 μL/well) can bind Biotinylated Human CD20 Full Length, His,Avitag (HEK293) (Cat. No. CD0-H82E5) with a linear range of 4-63 ng/mL (in presence of DDM and CHS).

Protocol

Cat. No. CD0-H52H3 (HEK293表达)

Immobilized Rituximab at 2 μg/mL (100 μL/well) can bind Human CD20 Full Length, His Tag, HEK293 (SPR verified) (Cat. No. CD0-H52H3) with a linear range of 0.4-3 ng/mL (in presence of DDM and CHS).

Protocol

Cat. No. CD0-H52H3 (HEK293表达)

Immobilized Ofatumumab at 2 μg/mL (100 μL/well) can bind Human CD20 Full Length, His Tag, HEK293 (SPR verified) (Cat. No. CD0-H52H3) with a linear range of 0.4-6 ng/mL (in presence of DDM and CHS).

Protocol

SPR验证
Cat. No. CD0-H82E5 (HEK293表达)

Biotinylated Human CD20, His,Avitag (HEK293) (Cat. No. CD0-H82E5) captured on Biotin CAP-Series S Sensor Chip can bind Rituximab with an affinity constant of 1.73 nM as determined in a SPR assay (in presence of DDM and CHS) (Biacore T200).

Protocol

Cat. No. CD0-H52H3 (HEK293表达)

Human CD20 Full Length, His Tag, HEK293 (SPR verified) (Cat. No. CD0-H52H3) captured on CM5 chip via Anti-human IgG Fc antibodies surface can bind MabThera® (Rituximab) with an affinity constant of 6.21 nM as determined in a SPR assay (in presence of DDM and CHS) (Biacore 8K).

Protocol

产品评论
 
评论(3)
  1. 177XXXXXXX3
  2. 25人赞
  3. 重组抗CD20单克隆抗体的靶点受体是跨膜蛋白,且体外表达的CD20受体很难维持其生物活性,我们常采用细胞法或流式方法进行活性评价,其涉及细胞制备,周期比较长,重复性不是很好。因此我们迫切需要建立SPR及ELISA分析方法对重组抗CD20单克隆抗体结合活性进行评价,而这个体外表达的CD20受体蛋白正好满足我们需求,并且已对其实用性进行验证。对加快我门产品研发进度,非常有帮助!
  4. 2019-10-11
  1. 158XXXXXXX9
  2. 40人赞
  3. BLI实验。相比之前在另一家公司买的CD20蛋白,这个蛋白结合活性更强,CD20这种特殊膜蛋白,能够全长表达还具有生物活性,还是可以的。就是价格能稍微便宜点就更好了
  4. 2019-10-23
  1. 150XXXXXXX0
  2. 26人赞
  3. CD20因为是多次跨膜蛋白,找了很多家公司都表达不好。 抗体筛选和Elisa都定了很多次,实验效果不错。 除了有点小贵没有别的缺点啦!
  4. 2019-10-11
 
相关参考文献

Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab

Nanodisc in membrane biochemistry and biophysics.

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