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Product Details
Source
Monoclonal Anti-FMC63 Antibody, Rabbit IgG (1G10) is a neutralizing rabbit monoclonal antibody recombinantly expressed from human 293 cells (HEK293), which provides higher batch consistency and long term security of supply. It shows superior performance in flow cytometry assays with higher affinity and better specificity.
Application
Flow Cytometry (Evaluation of Anti-CD19 (FMC63 scFv) CAR Expression).
Clone
1G10
Species
Rabbit
Isotype
Rabbit IgG | Rabbit Kappa
Specificity
Specifically recognizes the antigen-recognition domain of FMC63 derived CARs.
Immunogen
Recombinant FMC63 scFv derived from HEK293 cells
Conjugate
PE
Excitation Wavelength: 488 nm / 561 nm
Emission Wavelength: 575 nm
Recommended Dilution
1:50
Formulation
Lyophilized from a 0.22 μm-filtered solution in PBS (pH 7.4) containing 0.03% ProClin 300 and 0.2% BSA, with trehalose as protectant.
Please contact us for customized product forms or formulations.
Reconstitution
Please refer to the Certificate of Analysis (CoA) for specific instructions.
For best performance, we strongly recommend following the reconstitution protocol provided in the CoA.
Storage
For long term storage, the product should be stored in a lyophilized state at -20°C or lower.
Please protect from light and avoid repeated freeze-thaw cycles.
This product is stable after storage at:
- -20°C to -70°C for 24 months in lyophilized state;
- -70°C for 12 months after reconstitution.
- 2-8°C for 12 months after reconstitution.
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Performance Data
Bioactivity-FACS
Flow cytometric analysis of Anti-CD19 CAR-293 cells staining with PE-Labeled Monoclonal Anti-FMC63 Antibody, Rabbit IgG1 (1G10) (Cat. No. FM3-PFM721) at 1:50 dilution (2 μL of the antibody stock solution corresponds to labeling of 1e6 cells in a final volume of 100 µL), compared with isotype control antibody. PE signal was used to evaluate the binding activity (QC tested).
Protocol
Non-specificity of PE-Labeled Monoclonal Anti-FMC63 Antibody, Rabbit IgG1 (1G10) (Cat. No. FM3-PFM721) binding to CD3+ cells present in human PBMC. Human PBMCs were simultaneously stained with FITC-labeled anti-CD3 antibody and PE-Labeled Monoclonal Anti-FMC63 Antibody, Rabbit IgG1 (1G10) (2 μL of the antibody stock solution corresponds to labeling of 5e5 cells in a final volume of 100 µL), washed and then analyzed with FACS. Both FITC and PE positive signals was used to evaluate the non-specific binding activity to human CD3+ cells (QC tested).
Protocol
FACS Analysis of Blood Samples
After Fc receptor blockade, whole blood only control (Figure A) and whole blood spiked with CAR-T cells (Figure B) were first stained with PE-Labeled Monoclonal Anti-FMC63 Antibody, Rabbit IgG (1G10) (Cat. FM3-PFM721), following 7-AAD staining. The data were analyzed using FCS Express 7 software (Routinely tested). CAR-T cells were identified as PE-positive events within the live CD45+CD3+ (or CD45+) lymphocyte population. The proportion of CD45+CAR+ cells is around 20%, which is largely consistent with the theoretical spike-in value.
Evaluation of CAR expression
FACS Analysis of Non-specific binding to Human whole blood
Non-specificity of PE-Labeled Monoclonal Anti-FMC63 Antibody, Rabbit IgG (1G10) (0.03% Proclin) (Cat. No. FM3-PFM721) binding to CD3+ cells present in human whole blood. 100 μl of human whole blood were simultaneously stained with APC/Cyanine7 anti-human CD3 Antibody and PE-Labeled Monoclonal Anti-FMC63 Antibody, Rabbit IgG (1G10) (2 μL of the antibody stock solution in a final volume of 100 µL), compared with isotype control antibody and positive control antibody. Both APC/Cyanine7 and PE positive signals was used to evaluate the non-specific binding activity to human CD3+ cells.
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