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CAR阳性表达率检测

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2018-02-27 13:52

CAR阳性表达率检测是CAR-T疗法质量控制的重要一环,常用的检测方案有利用Protein L、Anti-Fab抗体或靶点蛋白结合流式细胞术进行检测。其中,靶点蛋白结合流式细胞术的检测方案因其靶点特异性强的优势而备受青睐,许多业内人士预期靶点特异性的检测将会被监管机构纳入CAR-T质量控制监管考量的范畴。本文将对CAR阳性表达率检测相关案例做一汇总以供大家参考。

三种CAR-T阳性率检测试剂优劣势对比

检测试剂 检测原理 优点 缺点
Protein L 结合抗体κ轻链 成本低,且市面有能简化操作的直标Protein L 背景值高,无靶点特异性,不能检测轻链λ型CAR
Anti-Fab抗体 结合抗体Fab段 成本低,且市面有能简化操作的直标抗体 背景值高,无靶点特异性
靶点蛋白 特异性结合scFv抗原识别区 具靶点特异性,且可以评估亲和力 成本相对高,不同靶点需建立不同方法,市面少有直标靶点蛋白
案例汇总

案例名称 数据来源
案例一 利用生物素标记hBCMA检测anti-BCMA CAR阳性表达率 深圳普瑞金 点击查看
案例二 利用生物素标记hCD19检测anti-CD19 CAR阳性表达率 ACROBiosystems 点击查看
案例三 利用生物素标记hCD19检测anti-CD19 CAR阳性表达率 MacLeod DT 点击查看
案例四 利用hCD19, His Tag蛋白检测anti-CD19 CAR阳性表达率 ACROBiosystems 点击查看
案例五 利用hCD19, Fc tag蛋白检测anti-CD19 CAR阳性表达率 ACROBiosystems 点击查看
案例六 不同供应商hCD19, Fc tag蛋白的结合特异性验证 ACROBiosystems 点击查看
(点击对应案例即可跳转案例详情)

ACROBiosystems积极与CAR-T相关的医药研发人员紧密合作, 构建CAR-T相关实验体系建设,如果您有CAR-T靶点的需求可以联系我们申请免费试用,或对以下方案分享有什么疑问,可以及时联系我们,我们将持续分享该领域的成功案例以加速CAR-T药物的研发进程。

联系方式: 400-682-2521 or cart@acrobiosystems.com

分享新案例申请靶点试用 点击查看CAR-T靶点产品列表

案例详情

案例一、利用生物素标记hBCMA检测anti-BCMA CAR阳性表达率

返回案例汇总

应用案例由深圳普瑞金生物药业公司友情提供!

检测方法:流式细胞术

检测仪器:BD FACSCalibur流式细胞分析仪

样本信息:转染anti-BCMA CAR的人原代T淋巴细胞

主要试剂:

  • 生物素标记的hBCMA蛋白 (Biotinylated Human BCMA / TNFRSF17 Protein, Fc Tag, Avi Tag (Avitag™), ACROBiosystems, Cat.No. BC7-H82F0);
  • PE标记的链霉亲和素(PE Streptavidin , Biolegend, Cat.No. 405204);

  • 简要流程:

    • 1. 将anti-BCMA CAR基因通过慢病毒质粒转染,整合到来自患者的自体T细胞基因中;
    • 2. 利用anti-BCMA CAR特异性结合BCMA蛋白的特性,将生物素标记的hBCMA蛋白(ACROBiosystems, Cat.No. BC7-H82F0)标记到anti-BCMA CAR-T细胞表面;
    • 3. 利用生物素特异性结合亲和素的特性,将PE标记的链霉亲和素(Biolegend, Cat.No. 405204) 作为荧光二抗,标记到anti-BCMA CAR-T细胞表面;
    • 4. 用BD FACSCalibur流式细胞分析仪进行检测分析。


    检测结果:结果显示1号患者anti-BCMA-CAR T细胞阳性率在52.72%,2号患者anti-BCMA-CAR T细胞阳性率在73.49%.

    FACS Analysis of anti-BCMA CAR expression

    Human T cells were transfected with anti-BCMA CAR and cultured for 3 days. Three days post-transfection, 1x10 6 cells were first incubated with 50ul biotinylated human BCMA protein (Cat.No. BC7-H82F0, 8ug/ml ), washed and then stained with PE Streptavidin and analyzed by flow cytometry. (Data are kindly provided by PREGENE Biopharma)

    相关产品:

    BC7-H82F0 (Biotinylated Human BCMA, Fc & Avi Tag) 点击申请Protocol 点击咨询报价 点击分享新案例

    案例二、利用生物素标记hCD19检测anti-CD19 CAR阳性表达率

    返回案例汇总

    应用案例由ACRO应用开发团队自主研发提供!

    检测方法:流式细胞术

    检测仪器:NovoCyteTM Flow Cytometer, ACEA Biosciences

    样本信息:R1013-C6细胞(过表达Anti-CD19[FMC63] scFv & RFP的Expi 293细胞)

    主要试剂:

  • 生物素标记的hCD19蛋白 (Biotinylated Human CD19, Fc Tag, ultra sensitivity (primary amine labeling), ACROBiosystems, Cat.No. CD9-H8259);
  • FITC标记的链霉亲和素(FITC Streptavidin, Biolegend, Cat.No. 405201);

  • 简要流程:

    • 1. 将Anti-CD19[FMC63] scFv & RFP基因通过质粒转染,整合到Expi 293细胞基因中,命名为R1013-C6细胞;
    • 2. 利用anti-CD19[FMC63] scFv特异性结合CD19蛋白的特性,将生物素标记的hCD19蛋白(ACROBiosystems, Cat.No. CD9-H8259)标记到R1013-C6细胞表面;
    • 3. 利用生物素特异性结合亲和素的特性,将FITC标记的链霉亲和素(FITC Streptavidin, Biolegend, Cat.No. 405201)作为荧光二抗,标记到R1013-C6细胞表面;
    • 4. 用NovoCyteTM Flow Cytometer流式细胞分析仪进行检测分析。


    检测结果:结果显示生物素标记的CD19蛋白能够与 R1013-C6细胞膜表面的anti-CD19 [FMC63] scFv特异性结合,这种结合能够被游离的抗CD19的抗体FMC63拮抗。

    FACS Analysis of anti-CD19 CAR expression

    293 cells were transfected with FMC63-scFv and RFP tag . 2x105 of the cells were first incubated with A. Biotinylated protein control. B. Recombinant biotinylated human CD19 (Cat.No. CD9-H8259, 10ug/ml ) . C. Recombinant biotinylated human CD19 (Cat.No. CD9-H8259, 10ug/ml) and FMC63(Mouse anti-CD19 antibody). FITC Streptavidin was used to analyse with FACS. RFP was used to evaluate CAR(FMC63-scFv) expression and FITC was used to evaluate the binding activity of recombinant biotinylated human CD19 (Cat.No. CD9-H8259) .

    相关产品:

    CD9-H8259 Biotinylated Human CD19, Fc Tag, ultra sensitivity (primary amine labeling) 点击申请Protocol 点击咨询报价 点击分享新案例

    案例三、利用生物素标记hCD19检测anti-CD19 CAR阳性表达率

    返回案例汇总

    应用案例来自 MacLeod DT, et al., 2017, Mol Ther. 25(4):949-961.doi: 10.1016/j.ymthe.2017.02.005.

    检测方法:流式细胞术

    检测仪器:BD Fortessa flow cytometer (BD Biosciences)

    样本信息:TRC1-2-treated, AAV:TRAC:CAR-transduced T cells

    主要试剂:

  • 生物素标记的hCD19蛋白 (Biotinylated Human CD19, Fc Tag, ultra sensitivity (primary amine labeling), ACROBiosystems, Cat.No. CD9-H8259);
  • PE标记的链霉亲和素(PE Streptavidin, Biolegend);
  • Anti-CD3-BV711抗体(Brilliant Violet 711™ anti-human CD3 Antibody, BioLegend).

  • 简要流程:

    • 1. 利用CD19 CAR能特异性结合CD19蛋白的特性,如果待检T细胞正常表达了CD19 CAR,生物素标记的CD19-Fc蛋白就能被标记到待检T细胞表面;
    • 2. 利用生物素能特异性结合亲和素(PE Streptavidin)和Anti-CD3-BV711抗体能特异性结合CD3分子的特性,如果待检T细胞表面有生物素和CD3分子,链霉亲和素上的PE和Anti-CD3抗体上的BV711荧光标记就能被标记到待检T细胞表面;
    • 3. 将制备好的单细胞悬液上机进行流式细胞分析。


    检测结果:结果显示CD3阴性T细胞群中CD19 CAR表达的比例要高于CD3阳性T细胞群。

    FACS Analysis of anti-CD19 CAR expression

    Activated T cells were electroporated with TRC1-2 mRNA and transduced with AAV:TRAC:CAR at an MOI of 400,000 vg/cell and cultured for 5 days in the presence of IL-2. Five days post-transduction, cells were stained for expression of the CAR using abiotinylated CD19-Fc reagent and CD3, with TRC1-2-treated, mock-transduced cells used as a control for gating of CAR expression. CD3+ cells were then depleted. Enriched CD3 cells were cultured for 3 additional days in the presence of IL-15 and IL-21 and then analyzed again by flow cytometry for CD3 and CAR expression.

    相关产品:

    CD9-H8259 Biotinylated Human CD19, Fc Tag, ultra sensitivity (primary amine labeling) 点击咨询报价 点击分享新案例

    案例四、利用hCD19, His Tag蛋白检测anti-CD19 CAR阳性表达率

    返回案例汇总

    验证数据由ACRO应用开发团队自主研发提供!

    检测方法:流式细胞术

    细胞样本:R1013-C6 cells (过表达Anti-CD19[FMC63] scFv & RFP的Expi 293细胞)

    主要试剂:

  • Human CD19, His Tag蛋白(ACROBiosystems, Cat.No. CD9-H52H2);
  • FITC anti-6xHis tag antibody(Abcam, Cat.No. ab1206).

  • 简要流程:

    • 1. 将anti-CD19[FMC63] scFv & RFP基因通过质粒转染,整合到Expi 293细胞基因中,命名为R1013-C6细胞;
    • 2. 利用anti-CD19[FMC63] scFv特异性结合CD19蛋白的特性,将hCD19, His Tag蛋白(ACROBiosystems, Cat.No. CD9-H52H2)标记到R1013-C6细胞表面;
    • 3. 将荧光二抗FITC anti-6xHis tag antibody (Abcam, Cat.No. ab1206)标记到R1013-C6细胞表面;
    • 4. 用Accuri C6 Flow Cytometer流式细胞分析仪进行检测分析。


    检测结果:结果显示hCD19, His Tag蛋白能够与 R1013-C6细胞膜表面的anti-CD19 [FMC63] scFv特异性结合,这种结合能够被游离的抗CD19的抗体FMC63拮抗。

    FACS Analysis of anti-CD19 CAR expression

    293 cells were transfected with FMC63-scFv and RFP tag . 2x105 of the cells were first incubated with A. His Tag-protein control. B. Recombinant His Tag-human CD19 (Cat.No. CD9-H52H2, 10ug/ml). C. The mixture of recombinant His Tag-human CD19 (Cat.No. CD9-H52H2, 10ug/ml) and FMC63(Mouse anti-CD19 antibody), followed by FITC anti-6xHis tag antibody, and then analyzed using Accuri C6 Flow Cytometer. RFP was used to evaluate CAR(FMC63-scFv) expression and FITC was used to evaluate the binding activity of recombinant His Tag-human CD19 (Cat.No. CD9-H52H2).

    相关产品:

    CD9-H52H2 (Human CD19 Protein, His Tag) 点击申请Protocol 点击咨询报价 点击分享新案例

    案例五、利用hCD19, Fc tag蛋白检测anti-CD19 CAR阳性表达率

    返回案例汇总

    验证数据由ACRO应用开发团队自主研发提供!

    检测方法:流式细胞术

    细胞样本:R1013-C6 cells (过表达Anti-CD19[FMC63] scFv & RFP的Expi 293细胞)

    主要试剂:

  • Human CD19, Fc tag蛋白 (Acrobiosystems, Cat.No. CD9-H5259);
  • FITC anti-human IgG Fc antibody ( Biolegend, Cat.No. 409310).

  • 简要流程:

    • 1. 将anti-CD19[FMC63] scFv & RFP基因通过质粒转染,整合到Expi 293细胞基因中,命名为R1013-C6细胞;
    • 2. 利用anti-CD19[FMC63] scFv特异性结合CD19蛋白的特性,将hCD19, Fc tag蛋白(Acrobiosystems, Cat.No. CD9-H5259)标记到R1013-C6细胞表面;
    • 3. 将荧光二抗FITC anti-human IgG Fc antibody ( Biolegend, Cat.No. 409310)标记到R1013-C6细胞表面;
    • 4. 用NovoCyteTM Flow Cytometer流式细胞分析仪进行检测分析。


    检测结果:结果显示hCD19, Fc tag蛋白能够与 R1013-C6细胞膜表面的anti-CD19 [FMC63] scFv特异性结合,这种结合能够被游离的抗CD19的抗体FMC63拮抗。

    FACS Analysis of anti-CD19 CAR expression

    293 cells were transfected with FMC63-scFv and RFP tag . 2x105 of the cells were first incubated with A. Human Fc tag control. B. Recombinant human CD19,Fc Tag (Cat.No. CD9-H5259, 10ug/ml ). C. Recombinant human CD19,Fc Tag(Cat.No. CD9-H5259, 10ug/ml ) and FMC63(Mouse anti-CD19 antibody) . The FITC anti-human IgG Fc was used to analyse with FACS. RFP was used to evaluate CAR(FMC63-scFv) expression and FITC was used to evaluate the binding activity of recombinant human CD19,Fc Tag .

    相关产品:

    CD9-H5259 (Human CD19 Protein, Fc Tag) 点击申请Protocol 点击咨询报价 点击分享新案例

    案例六、不同供应商hCD19, Fc tag蛋白的结合特异性验证

    返回案例汇总

    验证数据由ACRO应用开发团队自主研发提供!

    检测方法:流式细胞术

    细胞样本: R1013-C6 cells (过表达Anti-CD19[FMC63] scFv & RFP的Expi 293细胞);Expi 293 cells;Jurkat E6.1 cells.

    主要试剂:

  • 蛋白样本1:Human CD19 Protein, Fc Tag (ACROBiosystems, Cat.No. CD9-H5259);
  • 蛋白样本2:Human CD19 Protein, Fc Tag (Company N);
  • 阴性对照蛋白:Human PD-L1 Protein, Fc Tag (ACROBiosystems, Cat.No. PD1-H5258);
  • 荧光二抗:FITC anti-human IgG Fc antibody (Biolegend, Cat.No. 409310).

  • 简要流程:

    • 1. 用Human CD19 Fc tag蛋白分别标记R1013-C6 细胞、Expi 293细胞和Jurkat E6.1细胞;
    • 2. 将FITC anti-human IgG Fc antibody作为荧光二抗,对以上细胞进行二次标记;
    • 3. 用NovoCyteTM Flow Cytometer流式细胞分析仪进行检测分析。


    检测结果:结果显示ACROBiosystems的Human CD19 Protein, Fc Tag蛋白仅能特异性识别R1013-C6细胞表面的Anti-CD19[FMC63] scFv,不能与Expi 293细胞和Jurkat E6.1细胞发生非特异性结合反应;而同等实验条件下,Company N的Human CD19 Protein, Fc Tag蛋白与Expi 293细胞和Jurkat E6.1细胞发生了较强的非特异性结合。

    Binding specificity analysis of ACROBiosystems hCD19(C-Fc tag) protein

    FACS analysis of human CD19 protein, Fc Tag (ACROBiosystems, Cat.No. CD9-H5259) binding to A. R1013-C6 cells, B. Expi 293 cells, C. Jurkat E6.1 cells. Cells were first stained with human CD19 protein, Fc Tag (ACROBiosystems, Cat.No. CD9-H5259) followed by FITC anti-human IgG Fc antibody, and then analyzed using NovoCyteTM Flow Cytometer. The data were analyzed with FCS Express 6Plus and GraphPad Prism 5 software.

    Binding specificity analysis of Company N hCD19(C-Fc tag) protein

    FACS analysis of human CD19 protein, Fc Tag (Company N) binding to A. R1013-C6 cells, B. Expi 293 cells, C. Jurkat E6.1 cells. Cells were first stained with human CD19 protein, Fc Tag (Company N) followed by FITC anti-human IgG Fc antibody, and then analyzed using NovoCyteTM Flow Cytometer. The data were analyzed with FCS Express 6Plus and GraphPad Prism 5 software.

    相关产品:

    CD9-H5259 (Human CD19 Protein, Fc Tag) 点击申请Protocol 点击咨询报价 点击分享新案例
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