Monoclonal anti c myc antibody

The screening was done with the entire population of phages eluted (fused to the antibody fragments) after each round of selection. ELISA plates (Nunc, Denmark) were coated with epsilon toxoid (100 µg/ml) overnight at 4 °C. The next day, plates were washed and blocked by 4% MPBS buffer per well. After washing, 10 µl of PEG precipitated phages recovered from each round of selection were diluted in 100 µl MPBS buffer and added to each well. One hour later, plates were washed and the binding of phages were detected using a monoclonal anti-c-Myc antibody (Biolegend) and anti-mouse HRP conjugate (Sigma Aldrich, USA) and detected with TMB substrate (Biobasic, Canada). Sulfuric acid solution 1 M was used to stop the reaction and enzymatic activity of HRP–antibody conjugate and the absorbance was read at 450 nm and 620 nm with a microplate reader.

For monoclonal phage ELISA, individual colonies were picked after the second and third rounds of selection and grown into 2xTY medium containing 100 µg/ml ampicillin and 4% glucose in a 96-well plate and incubated at 37 °C, 250 rpm for 12 hours. Then, the overnight culture for each clone diluted 100-fold into 200 µl 2xTY medium (100 µg/ml ampicillin and 0.1% glucose) and incubated at 37 °C, 250 rpm until OD600 = 0.4. The culture was infected with 4 × 10⁸ KM13 helper phages for 30 min at 37 °C, the bacteria pelleted by centrifugation and resuspended in 150 µl of 2 × YT containing ampicillin (100 µg/ml) and kanamycin (50 µg/ml) before growth overnight at 25 °C, 250 rpm for 16 hours. Then, the plate was centrifuged and 100 µl of culture supernatant of each well was used for ELISA plates that pre-coated with epsilon toxoid (100 µg/ml) and blocked with 4% MPBS buffer. Phage binding was detected with a monoclonal anti-c-Myc antibody (Biolegend) and anti-mouse HRP conjugate (Sigma).

Twenty-four to forty-eight hours after transient transfection with Lipofectamine 2000, HEK293T cells grown on coverslips were fixed with 2% formaldehyde/medium, permeabilized with 0.1% TritonX-100/PBS, incubated with monoclonal anti-c-Myc antibody (BioLegend), polyclonal anti-Calreticulin antibody (Thermo Fisher Scientific) and DAPI (Wako, Osaka, Japan), and detected using anti-mouse Alexa Fluor 488 or anti-rabbit Alexa Fluor 546 antibodies (Thermo Fisher Scientific). A laser confocal microscope (TCSSP5, Leica Microsystems, Wetzlar, Germany) was used to capture images.