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CAR陽性表達率檢測

18808  |  
2018-02-27 13:52

国产视频app CAR陽性表達率檢測是CAR-T療法質量控制的重要一環,常用的檢測方案有利用Protein L、Anti-Fab抗體或靶點蛋白結合流式細胞術進行檢測。其中,靶點蛋白結合流式細胞術的檢測方案因其靶點特異性強的優勢而備受青睞,許多業內人士預期靶點特異性的檢測將會被監管機構納入CAR-T質量控制監管考量的范疇。本文將對CAR陽性表達率檢測相關案例做一匯總以供大家參考。

三種CAR-T陽性率檢測試劑優劣勢對比

檢測試劑 檢測原理 優點 缺點
Protein L 結合抗體κ輕鏈 成本低,且市面有能簡化操作的直標Protein L 背景值高,無靶點特異性,不能檢測輕鏈λ型CAR
Anti-Fab抗體 結合抗體Fab段 成本低,且市面有能簡化操作的直標抗體 背景值高,無靶點特異性
靶點蛋白 特異性結合scFv抗原識別區 具靶點特異性,且可以評估親和力 成本相對高,不同靶點需建立不同方法,市面少有直標靶點蛋白
案例匯總

案例名稱 數據來源
案例一 利用生物素標記hBCMA檢測anti-BCMA CAR陽性表達率 深圳普瑞金 點擊查看
案例二 利用生物素標記hCD19檢測anti-CD19 CAR陽性表達率 ACROBiosystems 點擊查看
案例三 利用生物素標記hCD19檢測anti-CD19 CAR陽性表達率 MacLeod DT 點擊查看
案例四 利用hCD19, His Tag蛋白檢測anti-CD19 CAR陽性表達率 ACROBiosystems 點擊查看
案例五 利用hCD19, Fc tag蛋白檢測anti-CD19 CAR陽性表達率 ACROBiosystems 點擊查看
案例六 不同供應商hCD19, Fc tag蛋白的結合特異性驗證 ACROBiosystems 點擊查看
(點擊對應案例即可跳轉案例詳情)

国产视频app ACROBiosystems積極與CAR-T相關的醫藥研發人員緊密合作, 構建CAR-T相關實驗體系建設,如果您有CAR-T靶點的需求可以聯系我們申請免費試用,或對以下方案分享有什么疑問,可以及時聯系我們,我們將持續分享該領域的成功案例以加速CAR-T藥物的研發進程。

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

分享新案例申請靶點試用 點擊查看CAR-T靶點產品列表

案例詳情

案例一、利用生物素標記hBCMA檢測anti-BCMA CAR陽性表達率

返回案例匯總

應用案例由深圳普瑞金生物藥業公司友情提供!

檢測方法:流式細胞術

檢測儀器:BD FACSCalibur流式細胞分析儀

樣本信息:轉染anti-BCMA CAR的人原代T淋巴細胞

主要試劑:

  • 生物素標記的hBCMA蛋白 (Biotinylated Human BCMA / TNFRSF17 Protein, Fc Tag, Avi Tag (Avitag?), ACROBiosystems, Cat.No. BC7-H82F0);
  • PE標記的鏈霉親和素(PE Streptavidin , Biolegend, Cat.No. 405204);

  • 簡要流程:

    • 1. 將anti-BCMA CAR基因通過慢病毒質粒轉染,整合到來自患者的自體T細胞基因中;
    • 2. 利用anti-BCMA CAR特異性結合BCMA蛋白的特性,將生物素標記的hBCMA蛋白(ACROBiosystems, Cat.No. BC7-H82F0)標記到anti-BCMA CAR-T細胞表面;
    • 3. 利用生物素特異性結合親和素的特性,將PE標記的鏈霉親和素(Biolegend, Cat.No. 405204) 作為熒光二抗,標記到anti-BCMA CAR-T細胞表面;
    • 4. 用BD FACSCalibur流式細胞分析儀進行檢測分析。


    檢測結果:国产视频app結果顯示1號患者anti-BCMA-CAR T細胞陽性率在52.72%,2號患者anti-BCMA-CAR T細胞陽性率在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)

    相關產品:

    BC7-H82F0 (Biotinylated Human BCMA, Fc & Avi Tag) 點擊申請Protocol 點擊咨詢報價 點擊分享新案例

    案例二、利用生物素標記hCD19檢測anti-CD19 CAR陽性表達率

    返回案例匯總

    應用案例由ACRO應用開發團隊自主研發提供!

    檢測方法:流式細胞術

    檢測儀器:NovoCyteTM Flow Cytometer, ACEA Biosciences

    樣本信息:R1013-C6細胞(過表達Anti-CD19[FMC63] scFv & RFP的Expi 293細胞)

    主要試劑:

  • 生物素標記的hCD19蛋白 (Biotinylated Human CD19, Fc Tag, ultra sensitivity (primary amine labeling), ACROBiosystems, Cat.No. CD9-H8259);
  • FITC標記的鏈霉親和素(FITC Streptavidin, Biolegend, Cat.No. 405201);

  • 簡要流程:

    • 1. 將Anti-CD19[FMC63] scFv & RFP基因通過質粒轉染,整合到Expi 293細胞基因中,命名為R1013-C6細胞;
    • 2. 利用anti-CD19[FMC63] scFv特異性結合CD19蛋白的特性,將生物素標記的hCD19蛋白(ACROBiosystems, Cat.No. CD9-H8259)標記到R1013-C6細胞表面;
    • 3. 利用生物素特異性結合親和素的特性,將FITC標記的鏈霉親和素(FITC Streptavidin, Biolegend, Cat.No. 405201)作為熒光二抗,標記到R1013-C6細胞表面;
    • 4. 用NovoCyteTM Flow Cytometer流式細胞分析儀進行檢測分析。


    檢測結果:結果顯示生物素標記的CD19蛋白能夠與 R1013-C6細胞膜表面的anti-CD19 [FMC63] scFv特異性結合,這種結合能夠被游離的抗CD19的抗體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) .

    相關產品:

    CD9-H8259 Biotinylated Human CD19, Fc Tag, ultra sensitivity (primary amine labeling) 點擊申請Protocol 點擊咨詢報價 點擊分享新案例

    案例三、利用生物素標記hCD19檢測anti-CD19 CAR陽性表達率

    返回案例匯總

    国产视频app 應用案例來自 MacLeod DT, et al., 2017, Mol Ther. 25(4):949-961.doi: 10.1016/j.ymthe.2017.02.005.

    檢測方法:流式細胞術

    檢測儀器:BD Fortessa flow cytometer (BD Biosciences)

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

    主要試劑:

  • 生物素標記的hCD19蛋白 (Biotinylated Human CD19, Fc Tag, ultra sensitivity (primary amine labeling), ACROBiosystems, Cat.No. CD9-H8259);
  • PE標記的鏈霉親和素(PE Streptavidin, Biolegend);
  • Anti-CD3-BV711抗體(Brilliant Violet 711? anti-human CD3 Antibody, BioLegend).

  • 簡要流程:

    • 1. 利用CD19 CAR能特異性結合CD19蛋白的特性,如果待檢T細胞正常表達了CD19 CAR,生物素標記的CD19-Fc蛋白就能被標記到待檢T細胞表面;
    • 2. 利用生物素能特異性結合親和素(PE Streptavidin)和Anti-CD3-BV711抗體能特異性結合CD3分子的特性,如果待檢T細胞表面有生物素和CD3分子,鏈霉親和素上的PE和Anti-CD3抗體上的BV711熒光標記就能被標記到待檢T細胞表面;
    • 3. 將制備好的單細胞懸液上機進行流式細胞分析。


    檢測結果:国产视频app結果顯示CD3陰性T細胞群中CD19 CAR表達的比例要高于CD3陽性T細胞群。

    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.

    相關產品:

    CD9-H8259 Biotinylated Human CD19, Fc Tag, ultra sensitivity (primary amine labeling) 點擊咨詢報價 點擊分享新案例

    案例四、利用hCD19, His Tag蛋白檢測anti-CD19 CAR陽性表達率

    返回案例匯總

    驗證數據由ACRO應用開發團隊自主研發提供!

    檢測方法:流式細胞術

    細胞樣本:R1013-C6 cells (過表達Anti-CD19[FMC63] scFv & RFP的Expi 293細胞)

    主要試劑:

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

  • 簡要流程:

    • 1. 將anti-CD19[FMC63] scFv & RFP基因通過質粒轉染,整合到Expi 293細胞基因中,命名為R1013-C6細胞;
    • 2. 利用anti-CD19[FMC63] scFv特異性結合CD19蛋白的特性,將hCD19, His Tag蛋白(ACROBiosystems, Cat.No. CD9-H52H2)標記到R1013-C6細胞表面;
    • 3. 將熒光二抗FITC anti-6xHis tag antibody (Abcam, Cat.No. ab1206)標記到R1013-C6細胞表面;
    • 4. 用Accuri C6 Flow Cytometer流式細胞分析儀進行檢測分析。


    檢測結果:結果顯示hCD19, His Tag蛋白能夠與 R1013-C6細胞膜表面的anti-CD19 [FMC63] scFv特異性結合,這種結合能夠被游離的抗CD19的抗體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).

    相關產品:

    CD9-H52H2 (Human CD19 Protein, His Tag) 點擊申請Protocol 點擊咨詢報價 點擊分享新案例

    案例五、利用hCD19, Fc tag蛋白檢測anti-CD19 CAR陽性表達率

    返回案例匯總

    国产视频app 驗證數據由ACRO應用開發團隊自主研發提供!

    檢測方法:流式細胞術

    細胞樣本:R1013-C6 cells (過表達Anti-CD19[FMC63] scFv & RFP的Expi 293細胞)

    主要試劑:

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

  • 簡要流程:

    • 1. 將anti-CD19[FMC63] scFv & RFP基因通過質粒轉染,整合到Expi 293細胞基因中,命名為R1013-C6細胞;
    • 2. 利用anti-CD19[FMC63] scFv特異性結合CD19蛋白的特性,將hCD19, Fc tag蛋白(Acrobiosystems, Cat.No. CD9-H5259)標記到R1013-C6細胞表面;
    • 3. 將熒光二抗FITC anti-human IgG Fc antibody ( Biolegend, Cat.No. 409310)標記到R1013-C6細胞表面;
    • 4. 用NovoCyteTM Flow Cytometer流式細胞分析儀進行檢測分析。


    檢測結果:国产视频app結果顯示hCD19, Fc tag蛋白能夠與 R1013-C6細胞膜表面的anti-CD19 [FMC63] scFv特異性結合,這種結合能夠被游離的抗CD19的抗體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 .

    相關產品:

    CD9-H5259 (Human CD19 Protein, Fc Tag) 點擊申請Protocol 點擊咨詢報價 點擊分享新案例

    案例六、不同供應商hCD19, Fc tag蛋白的結合特異性驗證

    返回案例匯總

    驗證數據由ACRO應用開發團隊自主研發提供!

    檢測方法:流式細胞術

    細胞樣本: R1013-C6 cells (過表達Anti-CD19[FMC63] scFv & RFP的Expi 293細胞);Expi 293 cells;Jurkat E6.1 cells.

    主要試劑:

  • 蛋白樣本1:Human CD19 Protein, Fc Tag (ACROBiosystems, Cat.No. CD9-H5259);
  • 蛋白樣本2:Human CD19 Protein, Fc Tag (Company N);
  • 陰性對照蛋白:Human PD-L1 Protein, Fc Tag (ACROBiosystems, Cat.No. PD1-H5258);
  • 熒光二抗:FITC anti-human IgG Fc antibody (Biolegend, Cat.No. 409310).

  • 簡要流程:

    • 1. 用Human CD19 Fc tag蛋白分別標記R1013-C6 細胞、Expi 293細胞和Jurkat E6.1細胞;
    • 2. 將FITC anti-human IgG Fc antibody作為熒光二抗,對以上細胞進行二次標記;
    • 3. 用NovoCyteTM Flow Cytometer流式細胞分析儀進行檢測分析。


    檢測結果:結果顯示ACROBiosystems的Human CD19 Protein, Fc Tag蛋白僅能特異性識別R1013-C6細胞表面的Anti-CD19[FMC63] scFv,不能與Expi 293細胞和Jurkat E6.1細胞發生非特異性結合反應;而同等實驗條件下,Company N的Human CD19 Protein, Fc Tag蛋白與Expi 293細胞和Jurkat E6.1細胞發生了較強的非特異性結合。

    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.

    相關產品:

    CD9-H5259 (Human CD19 Protein, Fc Tag) 點擊申請Protocol 點擊咨詢報價 點擊分享新案例

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