Flow cytometric analysis of scFv-based CAR-T cells using different characterization methods.

Live pan-CD3+ T cells were isolated from human PBMCs and engineered to express an scFv-based CAR. MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, A02282, 10μg/mL) demonstrated the highest positivity rate and the best cell clustering compared to concentration-matched FITC-Labeled Recombinant Protein L and Alexa Fluor® 647 AffiniPure Goat Anti-Human IgG, F(ab‘)2.The signal was amplified using Alexa Fluor® 488 -Labeled Goat Anti-Rabbit IgG.

Flow cytometric analysis of scFv-based CAR-T cells using MonoRab™ Rabbit Anti-scFv Cocktail [iFluor 647] (GenScript, A02288). The untransduced T cells and concentration-matched Rabbit IgG [iFluor 647] (GenScript, A02026) were used as controls.

The results show that MonoRab™ Rabbit Anti-scFv Cocktail can achieve good cell clustering for CAR-transduced T cells and show minimal non-specificity for untransduced T cells.

The binding compatibility of MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, A02282) with different sequences of scFv or scFv-based bispecific antibodies was detected by ELISA.

The results show that MonoRab™ Rabbit Anti-scFv Cocktail is specific for scFvs in different species (humanized, mouse), different linker (GS linker or whitlow/218 linker) and different orders (VH-linker-VL, VL-linker-VH). It can also recognize various forms of scFv-based bispecific antibodies, such as BiTE, IgG(H)-(scFv)2, (scFv)2-(H)IgG, scFv-Fab-Fc, etc.

Humanized scFv#1: VL-(G4S)3 linker-VH; Humanized scFv#2: VH-Whitlow/218 linker-VL; Mouse scFv#9: VL-Whitlow/218 linker-VH; Mouse scFv#10: VH-(G4S)3 linker-VL.

The affinity of MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, A02282) with different sequences of scFv was measured by BLI (Biolayer Interferometry).

MonoRab™ Rabbit Anti-scFv Cocktail shows high affinity with scFv in different species (humanized, mouse), different linker (GS linker or whitlow/218 linker) and different orders (VH-linker-VL, VL-linker-VH). It also shows high affinity with various forms of scFv-based bispecific antibodies, such as BiTE, IgG(H)-(scFv)2, (scFv)2-(H)IgG, scFv-Fab-Fc, etc.

Humanized scFv#1: VL-(G4S)3 linker-VH; Humanized scFv#2: VH-Whitlow/218 linker-VL; Mouse scFv#9: VL-Whitlow/218 linker-VH; Mouse scFv#10: VH-(G4S)3 linker-VL.

Western blot analysis of humanized scFv, mouse scFv-Fc and bispecific antibody (BiTE) at various concentrations was performed using MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, A02282, 0.1 µg/mL). The signal was detected using Mouse Anti-Rabbit IgG Fc Antibody (14H9H10)[HRP], mAb, (GenScript, A01856, 1:20,000).

MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, A02282) exhibited non-inhibitory properties for the binding between scFv/bispecific antibody and its target antigen.

In this ELISA assay, the target antigen was coated at a concentration of 1 μg/mL, and scFv was mixed with A02282 (with dilutions starting from 3.33 μg/mL) before being added to the antigen-coated plates. Subsequently, THE™ His Tag Antibody [HRP], mAb, Mouse (GenScript, A00612) was utilized as the detection antibody at a dilution of 1:5000.

Humanized scFv#1: anti-VEGF scFv; Antigen#1: VEGF; Mouse scFv#2: anti-BCMA scFv; Antigen#2: BCMA; Bispecific Antibody#3: scFv based anti-CD19×anti-CD3 bispecific antibody; Antigen#3: CD3.

The binding curve between scFv/bispecific antibody and its target antigen was detected using MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, A02282) in an ELISA assay.

In this ELISA assay, the target antigen was coated at a concentration of 1 μg/mL, and scFv was utilized within a concentration range from 1.37 ng/mL to 1000 ng/mL. Subsequently, A02282 was used as a detection antibody at a concentration of 1 μg/mL, and the signal was amplified using Mouse Anti-Rabbit IgG Fc Antibody (14H9H10) [HRP], mAb (GenScript, A01856) at a dilution of 1:20,000.

Humanized scFv#1: anti-VEGF scFv; Antigen#1: VEGF; Mouse scFv#2: anti-BCMA scFv; Antigen#2: BCMA; Bispecific Antibody#3: scFv based anti-CD19×anti-CD3 bispecific antibody; Antigen#3: CD3.