Anti mouse cd19 antibody

To detect sCD19‐SA, reduced or unreduced samples were loaded and electrophoresed on an 8% sodium dodecyl sulfate‐polyacrylamide gel and subsequently electrotransferred onto a polyvinylidene difluoride membrane (Bio‐Rad). The blotted membrane was blocked with 5% skim milk in Tris‐buffered saline with 0.5% Tween‐20 (TBST) at 37°C for 1 h and subsequently incubated with anti‐mouse CD19 antibody (Santa Cruz Biotechnology) at 1: 2500 dilution in blocking buffer at 37°C for 1 h. The membrane was washed three times with TBST and then incubated in anti‐mouse IgG‐conjugated horseradish peroxidase at 1:5000 for another hour at 37°C. After extensive washing, the DAB reaction was visualized using a DAB display liquid kit (Boshide). Primary antibodies for CD3ζ (BD Biosciences) and anti‐rabbit HRP‐conjugated IgG (Santa Cruz Biotechnology) were used to detect anti‐CD19 CAR.

To identify positive cell clones secreting sCD19‐SA protein, cell culture supernatants were collected for dot blotting. When the cells were confluent in 96‐well plates, 5 μl of culture supernatant was collected from each well and dotted on the nitrocellulose filter membrane. After drying, the membrane was placed in the prepared 5% milk blocking solution at 37°C for 1 h and subsequently incubated with anti‐mouse CD19 antibody (Santa Cruz Biotechnology; 1: 2500 dilution in blocking buffer) and horseradish peroxidase‐conjugated anti‐mouse IgG (1: 5000) for 1 h at 37°C. Finally, a 3,3′‐diaminobenzidine (DAB) reaction was performed using a DAB display liquid kit (Boshide Corporation) after extensive washing.