The rabbit monoclonal antibody M5-B6 specifically binds to the scFv region of a M5-specific human monoclonal antibody (mAb, clone M5). Mesothelin, also known as MSLN, is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein, Because of its limited expression in normal tissues and its overexpression in several solid tumors such as ovarian cancer, breast cancer, colorectal cancer and pancreatic cancer, which is associated with a lower survival rate, it is an ideal target for tumor-specific therapy. The scFv region of M5 has been used to develop MSLN specific chimeric antigen receptor (CAR) T cells utilized in clinical trials.
Shipping and StorageÂ
⚫ Store undiluted at 2-8°C.
âš« Avoid prolonged exposure to light.
âš« Avoid freeze/thaw cycle of the reagent.
âš« The monoclonal antibody was purified by Protein A.
âš« The antibody was conjugated with Alexa Fluor 647 under optimum conditions, and unincorporated dye was removed.
(Optional) For Whole Blood Sample
1. Pipette 1 μL Anti-MSLN (M5 scFv) CAR ldiotype Antibody, Alexa Fluor 647 into the bottom of the tube.
2. Add dead cell staining solution and additional fluorochrome conjugated antibodies into the bottom of the tube.
3. Pipette 100 µL of well-mixed, anticoagulated whole blood into the bottom of the tube. Mix gently and thoroughly.
Note Avoid smearing sample down the side of the tube. If the sample remains on the side of the tube, it will not be stained with the reagents.
4. Incubate for 25 minutes in the dark at room temperature (18-25°C).
5. Pipette Red Blood Cell Lysis Solution to the tube. Mix gently and thoroughly. Incubate for 15 minutes in the dark at room temperature (18-25°C).
6. Add 500 μL FACS buffer to the tube. Mix well and centrifuge at 300g for 5 minutes at room temperature (18-25°C). Aspirate supernatant
completely.
7. Repeat step 6 twice.
8. Add a suitable amount of FACS buffer to resuspend cell and analysis by flow cytometry.
(Optional) For Cell Sample
1. Harvest the cells and wash the cells twice by FACS buffer.
2. Count the cells number and the viability.
3. Resuspend the cell suspension to a concentration up to 1×106 nucleated cells per 100 μL of buffer.
4. Add 1 μL Anti-MSLN (M5 scFv) CAR ldiotype Antibody, Alexa Fluor 647, dead cell staining solution and additional fluorochrome. Mix gently and
thoroughly.
5. Incubate for 25 minutes in the dark at room temperature (18-25°C).
6. Add 500 μL FACS buffer to the tube. Mix well and centrifuge at 300 g for 5 minutes at room temperature (18-25°C). Aspirate supernatant
completely.
7. Repeat step 6 twice.
8. Add a suitable amount of FACS buffer to resuspend cell and analysis by flow cytometry.
Tabd 1 Content
