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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) (SPR verified)

订购

CD20 CD0-M36 Rituximab biosimilar–Research Grade

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

CD20 CDP-HF2P8 Fluorescent Human CD20 Full Length Protein-VLP (HEK293)

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(4)
  1. 158XXXXXXX9
  2. 43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. 177XXXXXXX3
  2. 30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. 150XXXXXXX0
  2. 29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
  1. 425XXXXXXX
  2. 1人赞
  3. I purchased this CD20 protein in order to test BLI binding of anti-CD20 antibodies. At first, I left out the CHS and DDM, even though Acro's directions were clear about how, when, and why to include these, and the curves did not look good. But then I included the CHS and DDM and the BLI curves were clear and of high quality. It is great to be able to test binding to this tricky membrane protein. Because of Acro's CD20, I was able to compare antibodies and enable decision making.
  4. 2021-11-30
 
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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