Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (12): 265-273.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1511

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Comparison of 5 Methods of Evaluating the Expressions of Chimeric Antigen Receptors

WANG Huan-yu(), CHANG Hao-wan, ZHANG Chong-qi, JIN Wei-lin, WEI Fang()   

  1. Life Science and Technology,Shanghai Jiao Tong University,Shanghai 200240
  • Received:2020-12-13 Online:2021-12-26 Published:2022-01-19
  • Contact: WEI Fang E-mail:Huanyu_Wang@sjtu.edu.cn;fangwei@sjtu.edu.cn

Abstract:

This work is to compare 5 different methods for detection of novel chimeric antigen receptor(CAR). Stably expressing CAR cells were generated through lentiviral infection. Five methods,immunoglobulin G antibody staining,protein L staining,green fluorescent protein(GFP)co-expression,real-time fluorescent quantitative PCR and relative quantitative qPCR,were used to detect different novel CAR in Jurkat cells. Six lentiviral vectors expressing different CARs of Meso-CAR,Met-CAR,R132H-CAR1-2-GFP,R132H-CAR2-4-GFP,Dupixent-HL-CAR-GFP,and Dupixent-LH-CAR-GFP were successfully constructed,and their corresponding CAR stably-expressed Jurkat cells were obtained. The expressions of Meso CAR and Met CAR were detected by both immunoglobulin G antibody and protein L staining(better result with immunoglobulin G antibody staining),but the detection of the rest 4 CARs failed. GFP co-expression,absolute quantitative and relative quantitative methods of real-time fluorescent quantitative PCR can be utilized to indirectly detect the expression of CAR that was impossible with fluorescent staining,and there was certain correspondence among 3 methods. In conclusion,different approaches should be used to determine the expressions of CAR molecules for different novel CAR molecules,.

Key words: chimeric antigen receptor, anti-IgG antibody, protein L, green fluorescent protein, real-time fluorescent quantitative PCR