Figure 1. The specificity of MonoRab™ Rabbit Anti-Camelid VHH Cocktail (GenScript, A02014) to VHH-transduced CAR-T cells was tested by immunocytochemical/immunofluorescence analysis of VHH-transduced CAR-T cells infiltrated NCG mouse spleen.

VHH was labelled with MonoRab™ Rabbit Anti-Camelid VHH Cocktail (GenScript, A02014) at 0.5 µg/mL, and anti-rabbit IgG (H+L) (Alexa Fluor 488) was used as secondary antibody at 1/500 concentration (4 μg/mL; green). APC hCD45 monoclonal antibody was used to define all infused T cells at 1/50 concentration (0.5 μg/mL; red). Confocal images showed cytoplasmic staining in infiltrated VHH-transduced CAR-T cells in the NCG mouse spleen. The untransduced infiltrated T cells showed a negative signal with MonoRab™ Rabbit Anti-Camelid VHH Cocktail (GenScript, A02014).

Figure 2. The specificity of MonoRab™ Rabbit Anti-Camelid VHH Cocktail (GenScript, A02014) to VHH-transduced CAR-T cells was tested by Immunohistochemical analysis of VHH-transduced CAR-T cells infiltrated liver tissue of PDX mouse. VHH was labelled with MonoRab™ Rabbit Anti-Camelid VHH Cocktail (GenScript, A02014) at 1 μg/mL, followed by incubation with biotinylated anti-rabbit IgG (H+L) (2.5 μg/mL). The final signal was amplified by ABC (Avidin-Biotin Complex) Kit.

Positive staining was shown in the membrane or cytoplasm of VHH-transduced CAR-T cells infiltrated into liver tissue. Counterstained with hematoxylin.

Figure 3. The affinity of MonoRab™ Rabbit Anti-Camelid VHH Cocktail (GenScript, A02014) for different VHHs was analyzed by Western blotting using MonoRab™ Rabbit Anti-Camelid VHH Cocktail (GenScript, A02014, 0.5 µg/mL). The signal was developed with Goat Anti-Rabbit IgG Antibody (H&L) [HRP], pAb (Genscript, A00098, 1:5,000).
Lanes 1-3: 100 ng, 50 ng, 25 ng VHH #1
Lanes 4-6: 100 ng, 50 ng, 25 ng VHH #2

Figure 4. The specificity of MonoRab™ Rabbit Anti-Camelid VHH Cocktail (A02014) to camelid IgG1, IgG2, and IgG3 was tested using ELISA. The results show that MonoRab™ Rabbit Anti-Camelid VHH Cocktail (A02014) is specific to camelid heavy chain antibodies (IgG2 & 3), but not to the camelid conventional antibody (IgG1).

Figure 5. The specificity of MonoRab™ Rabbit Anti-Camelid VHH Cocktail (A02014) to non-camelid IgG was tested using ELISA. The results show that MonoRab™ Rabbit Anti-Camelid VHH Cocktail (A02014) is specific to camelid IgG and has no cross-reactivity with mouse, rat, rabbit, goat and human immunoglobulins.

Figure 6. Standard curve for quantitative detection of VHH by sandwich ELISA. The VHH Sandwich ELISA assay is developed by using MonoRab™ Rabbit Anti-Camelid VHH Antibody, mAb (GenScript, A01860) and MonoRab™ Rabbit Anti-Camelid VHH Cocktail [HRP] (GenScript, A02016) as the capture and detection antibodies, respectively. In this ELISA assay, the detection antibody was labeled with horseradish peroxidase (HRP). However, of an unconjugated detection antibody is desired, MonoRab™ Rabbit Anti-Camelid VHH Cocktail (A02014) can be used for this assay.
Note: GenScript can provide customized conjugation services for this product as per customer's request.

Figure 7. The affinity of MonoRab™ Rabbit Anti-Camelid VHH Cocktail (Cat.no A02014) with 15 random VHHs was measured by Biacore. The cocktail antibody shows comprehensive binding activity and high affinity with all of the 15 VHHs.