A novel staining protocol based on protein L for detection of expression of chimeric antigen receptor by flow cytometry / 临床检验杂志

ABSTRACT

Objective To evaluate the feasibility of the novel staining protocol based on protein L in flow cytometry to detect the expression of chimeric antigen receptor (CAR) on the surface of T cells from patients.Methods The peripheral blood mononuclear cells(PBMCs) were collected from 2 patients with CD19 + lymphoma by hemapheresis and T cells were purified by magnetic bead selection.CAR was transfected with retroviruses targeting CD19 and CD22 into T cells to induce chimeric antigen receptor-modified T cells (CAR-T cells).The single-chain fragments of antibody molecules on the surface of CAR-T cells were labeled by biotinylated protein L-streptavidin-PE system.The proportions of activated CD19-CAR and CD22-CAR positive T cells in the culture were detected by flow cytometry.The results were compared with those detected by conventional flow cytometry method based on Anti-IgG staining.Results The expression of CAR on the surface of CAR-T cells was successfully detected by flow cytometry protocol based on the staining of single-chain variable fragment (scFv)-biotinylated protein L-streptavidin-PE.The percentages of CD19-CAR-T cells from the 2 patients were 71.6％ vs 64.2％ and 49.3％ vs 43.8％ in the protein L group and the Anti-IgG control group respectively,and the percentages of CD22-CAR-T cells were 53.1％ vs 46.3％ and 56.5％ vs 64.0％.The results of the both groups were similar.Conclusion The staining protocol based on protein L could be used as a routine staining method in flow cytometry for the detection of CAR-T cells.