Selection Guide

*Anti-scFv antibody Cocktail (Min X) has no cross-reactivity to human IgG, mouse IgG, goat IgG and rabbit IgG.

Product performance

• Versatile Applicability

  ELISA results show that MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, A02282) can specifically recognize scFv from various sources, various connection orders (VH-VL or VL-VH), various linkers, and various forms of scFv-based bispecific antibodies, such as BiTE, IgG(H)-(scFv)2, (scFv)2-(H)IgG, scFv-Fab-Fc, etc.

  

  Figure 1. Binding curves of anti-scFv antibodies with scFvs of different sequences and bispecific antibodies detected by ELISA

  scFv from different sources, different connection orders (VH-VL or VL-VH), different linkers or different forms of bispecific antibodies are coated on the plate at a concentration of 1 μg/mL;

  The concentration of MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, A02282) ranges from 0.017 ng/mL to 1000 ng/mL; Mouse Anti-Rabbit IgG Fc Antibody (14H9H10) [HRP], mAb (GenScript, A01856) is used as the secondary antibody, and the dilution ratio is 1:20000.

• High Affinity

  The results of the Bio-Layer Interferometry (BLI) technology show that MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, A02282) exhibits high affinity to scFvs with various sources, various connection orders (VH-VL or VL-VH), various linkers, and various forms of bispecific antibodies based on scFv.

  

  Figure 2. BLI detects the affinity of Anti-scFv antibodies to different sequence scFv and bispecific antibodies

• High Sensitivity

  Western Blot results show that MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, A02282) exhibits high sensitivity to scFvs from different sources and bispecific antibody BiTE.

  

  Figure 3. Western Blot detects the sensitivity of Anti-scFv antibodies to scFvs from different sources and bispecific antibodies

  Western blot analysis of human scFv, mouse scFv-Fc and bispecific antibody (BiTE) at different 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).

• High Consistency

  Three batches of MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, A02282) showed consistent binding curves to scFv, with CV<10% at each concentration point.

  

  Figure 4. Detection of batch-to-batch stability of MonoRab™ Rabbit Anti-scFv Cocktail by ELISA

  The scFv coating concentration was 1 μg/mL; MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, A02282) was diluted 3-fold starting from 111 ng/mL; Mouse Anti-Rabbit IgG Fc Antibody (14H9H10) [HRP], mAb (GenScript, A01856) was used as a detection antibody at a dilution ratio of 1:20000.

• Non-binding blocking

  Competition ELISA results showed that Anti-scFv antibody did not affect the binding of scFv to its target antigen after binding to scFv.

  

  Figure 5. 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 MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, 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.

Application Data

• Characterization of CAR-T cells by flow cytometry

  GenScript's Anti-scFv antibody exhibits ultra-high specificity for scFv-based CAR-T cells. Experimental results show that Anti-scFv antibodies at different dilution ratios can accurately detect the positive rate of CAR-T cells, and the antibody shows extremely low non-specific binding to untransduced T cells, therefore it can be used for the characterization of CAR-T cells.

  

  Figure 1. Characterization of scFv-based CAR-T cells by Anti-scFv antibodies

  The same CAR-T cells were detected using MonoRab™ Rabbit Anti-scFv Cocktail [iFluor 647] (GenScript, A02288) at different dilutions (1:50-1:500). The untransduced T cells and concentration-matched Rabbit IgG [iFluor 647] (GenScript, A02026) were used as controls.

• Comparison of scFv-based CAR-T cell characterization tools

  Flow cytometry results show that Anti-scFv antibody has better cell clustering effect and higher positive rate than Protein L and Anti-Fab antibodies in CAR-T cell characterization.

  

  Figure 2. Comparison of scFv-based CAR-T cell characterization tools

  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) and concentration-matched FITC-Labeled Recombinant Protein L and Alexa Fluor^® 647 AffiniPure Goat Anti-Human IgG, F(ab‘)2 were used to characterize the CAR-T cells. The signal of MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, A02282) was amplified using Alexa Fluor^® 488 -Labeled Goat Anti-Rabbit IgG.

• Detect the binding of scFv to its target protein by ELISA

  Anti-scFv antibodies can be used to detect the binding ability of human scFv, murine scFv and bispecific antibodies to their target proteins.

  

  Figure 3. 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, MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, 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.

• Detect the binding of scFv to its target cells by flow cytometry

  Flow cytometry results show that the Anti-scFv antibody exhibits a high specific signal for cells bound to scFv and an extremely low non-specific signal for cells not bound to scFv, thus it can be used to detect the binding ability of scFv to its target cells.

  

  Figure 4. Anti-scFv antibody detects the binding activity of scFv to its target cells

  MonoRab™ Rabbit Anti-scFv Cocktail [FITC] (GenScript, A02284) was used to analyze the binding ability of anti-CD20 antibodies to Raji cells. The blue solid line represents unstained cells, the red solid line represents cells with only A02284 added, and the green solid line represents cells with anti-CD20 scFv and A02284 added.

Resources

Anti-VHH Antibodies Flyer

Advancing nanobody discovery and development

Free Download

GenScript’s scFv and VHH Handbook

Comprehensive Applications of Single-Chain Antibodies and Nanobodies for CAR-T Therapy and Antibody Drug Research

Free Download