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荧光标记靶点蛋白带领生物人玩转CAR表达检测和抗体药筛选!

2020-04-28 11:54

近年来,流式细胞术被广泛应用于CAR转染阳性率检测和抗体药筛选中。尤其在CAR-T细胞治疗领域, CAR转染阳性率是CAR-T质量控制的必检项,监管部门建议应采用流式细胞法检测CAR转染阳性率。目前有针对CAR不同结构区域的检测方法,包括针对CAR抗原结合位点的,比如CD19抗原,或针对轻链或铰链区的抗Fab抗体或Protein L蛋白。监管部门推荐使用具有更好专属性的针对抗原结合部位的抗原蛋白或抗独特型抗体进行检测。


荧光标记蛋白作为流式细胞术的首选试剂,因其预先标记了荧光素基团,可通过一步染色完成检测,既简化了检测流程、节省了检测时间,又避免了因使用二抗而产生的非特异背景。然而,高质量荧光标记蛋白的生产面临着诸多难题,如标记后蛋白活性难以维持、产品的批间一致性难以控制等,目前市场上少有高质量荧光标记蛋白可供购买。


ACROBiosystems作为专注于生物药研发领域的蛋白供应商,利用专业的蛋白研发及标记平台、稳定株开发平台和流式细胞分析平台,开发了一系列包括FITC标记和PE标记的靶点蛋白,适用于基于流式细胞术的分析试验,如CAR阳性表达率的检测和流式细胞术筛选抗体候选药物等。




应用案例



流式细胞术检测Anti-CD19 CAR阳性表达率


293 cells were transfected with anti-CD19-scFv and RFP tag. 2e5 of the cells were stained with B. FITC-Labeled Human CD19 (20-291), Fc Tag (Cat. No. CD9-HF251, 10 μg/ml) and C. FITC-labeled protein control. A. Nin-transfected 293 cells and C. FITC-labeled protein control were used as negative control. RFP was used to evaluate CAR (anti-CD19-scFv) expression and FITC was used to evaluate the binding activity of FITC-Labeled Human CD19 (20-291), Fc Tag (Cat. No. CD9-HF251) (QC tested).

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基于流式细胞术的中和抗体筛选试验


FACS analysis shows that the binding of PE-Labeled Human PD-1, Fc Tag, His Tag (recommended for neutralizing assay) (Cat.No.PD1-HP2F2) to 293T overexpressing PD-L1 was inhibited by increasing concentration of neutralizing antihuman PD-L1 antibody. The IC50 is 0.29 μg/ml (QC tested).

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产品特点

ELISA和FACS验证结果显示不同批次间结合活性高度一致


Binding activity of three different lots of FITC-labeled Human MSLN, Fc Tag was evaluated in the above FACS analysis against Anti-MSLN CAR-293 cells. The result shows very high batch-to-batch consistency.



Binding activity of three different lots of FITC-labeled Human MSLN, Fc Tag was evaluated in the above ELISA analysis against Anti-MSLN Antibody. The result shows very high batch-to-batch consistency.



ELISA验证结果显示标记前后结合活性保持一致


Binding activity of the Human MSLN, Fc Tag before and after FITC labeling was evaluated in the above ELISA analysis. The result shows that FITC-Labeled and Unconjugated Human MSLN, Fc Tag have almost the same level of binding activity.



加速稳定性验证结果显示37℃放置24h结合活性保持不变


Binding activity of FITC-labeled Human MSLN, Fc Tag was evaluated in the above FACS analysis against Anti-MSLN CAR-293 cells. The result shows that the FITC-labeled Human MSLN is stable at 37℃ for 24 hours without performance reduction.



冻融稳定性验证结果显示3次冻融结合活性保持不变


Binding activity of FITC-labeled Human MSLN, Fc Tag was evaluated in the above FACS analysis against Anti-MSLN CAR-293 cells. The result shows that the FITC-labeled Human MSLN is stable within three freeze-thaw cycles without performance reduction.




产品列表
PE Labeled Protein
Molecule Cat. No. Species Product Description Host Structure
CD19 CD9-HP2H3 Human PE-Labeled Human CD19 (20-291) Protein, His Tag HEK293
LAG-3 LA3-HP2H3 Human PE-Labeled Human LAG-3 / CD223 Protein, His Tag
(recommended for neutralizing assay)
HEK293
Mesothelin MSN-HP223 Human PE-Labeled Human Mesothelin (296-580) HEK293
PD-1 PD1-HP2F2 Human PE-Labeled Human PD-1, Fc Tag, His Tag
(recommended for neutralizing assay)
HEK293
SIRP alpha SIA-HP252 Human PE-Labeled Human SIRP alpha, Fc Tag
(recommended for neutralizing assay)
HEK293
FITC Labeled Protein
Molecule Cat. No. Species Product Description Host Structure
BAFF BAF-HF268 Human FITC-Labeled Human BAFF / TNFSF13B / CD257 Protein, Fc Tag HEK293
BCMA BCA-HF254 Human FITC-Labeled Human BCMA, Fc Tag HEK293
BCA-HF2H1 Human FITC-Labeled Human BCMA, His Tag HEK293
CD19 CD9-HF251 Human FITC-Labeled Human CD19 (20-291), Fc Tag HEK293
CD9-HF2H2 Human FITC-Labeled Human CD19 (20-291), His Tag HEK293
CD22 SI2-HF2H6 Human FITC-Labeled Human Siglec-2, His Tag HEK293
CD30 CD0-HF2H3 Human FITC-Labeled Human CD30, His Tag HEK293
CD4 CD4-HF255 Human FITC-Labeled Human CD4 Protein, Fc Tag HEK293
CD4-HF2H7 Human FITC-Labeled Human CD4 Protein, His Tag HEK293
CD8A&CD8B CDA-HF2W4 Human FITC-Labeled Human CD8 alpha&beta (CD8A&CD8B) Heterodimer, His Tag&Tag Free HEK293
CLL-1 CLA-HF247 Human FITC-Labeled Human CLEC12A / MICL / CLL-1, His Tag HEK293
CLA-HF268 Human FITC-Labeled Human CLEC12A / MICL / CLL-1, Fc Tag HEK293
EGF R EGR-HF2H5 Human FITC-Labeled Human EGF R Protein, His Tag HEK293
EGFRvIII EGI-HF2H7 Human FITC-Labeled Human EGFRvIII Protein, His Tag HEK293
ErbB3 ER3-HF2H5 Human FITC-Labeled Human ErbB3 / Her3 Protein, His Tag HEK293
FOLR1 FO1-HF2H8 Human FITC-Labeled Human FOLR1, His Tag HEK293
Glypican 3 GP3-HF258 Human FITC-Labeled Human Glypican 3, Fc Tag HEK293
GP3-HF2H1 Human FITC-Labeled Human Glypican 3, His Tag HEK293
GUCY2C GUC-HF2H8 Human FITC-Labeled Human GUCY2C / Guanylyl cyclase C, His Tag HEK293
Her2 HE2-HF224 Human FITC-Labeled Human Her2 / ErbB2 Protein, His Tag HEK293
IL13RA2 IL2-HF2H3 Human FITC-Labeled Human IL-13 R alpha 2 Protein, His Tag HEK293
Mesothelin MSN-HF223 Human FITC-Labeled Human Mesothelin (296-580), His Tag HEK293
MSN-HF253 Human FITC-Labeled Human Mesothelin (296-580), Fc Tag HEK293
MSN-HF25x Human FITC-Labeled Human Mesothelin (296-580), Fc Tag HEK293
PSMA PSA-HF244 Human FITC-Labeled Human PSMA / FOLH1 Protein, His Tag HEK293
SLAMF7 SL7-HF2H7 Human FITC-Labeled Human SLAMF7, His Tag HEK293

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