Monoclonal phage ELISA

Monoclonal phage ELISA

1. After the final round of screening, add 50 µL of OmniMAX bacteria (OD600 ~0.6) to each well and incubate at room temperature for 20 min with shaking at 600 rpm to allow phage infection.
2. Collect all the bacteria. Take 100 µL bacteria for 10-fold serial dilutions, spread the diluted bacteria on ampicillin LB plates (without helper phage infection), and incubate the plates at 37 °C overnight.
3. The next day, add 100 µL of 2×YT medium supplemented with ampicillin to each well of a 96-well, 2 mL deep-well plate (Corning Axygen, P-2ML-SQ-C). Pick one single colony into each well and culture at 37 °C with shaking at 220 rpm for 8 hours.
4. After the 8-hour culture, transfer 50 µL of culture from each well to a new plate for preservation. Add 5 µL of M13KO7 helper phage (titer > 1 × 10¹¹) to the remaining culture and incubate at 37 °C with shaking at 220 rpm for 40 min for helper phage infection.
5. Add 1 mL of 2×YT medium supplemented with ampicillin and kanamycin to each well and culture at 37 °C with shaking at 220 rpm overnight. In the meantime, coat a MaxiSorp plate with purified antigen-hIgG and mCherry-hIgG proteins (100 ng/well) at 4 °C overnight.
6. After the overnight coating, remove the coating solution from the MaxiSorp plate and wash three times with TBST. Block the plate with freshly prepared 5% skim milk in PBST (MPBST; PBS with 0.05% Tween-20) at room temperature (RT) for 1–2 hours.
7. After the overnight culture, centrifuge the deep-well plate at 3000 rpm for 5 min and transfer 600 µL of the phage-containing supernatant from each well to a new plate.
8. Once blocking is complete, transfer 100 µL of the phage-containing supernatant from each well to the antigen-hIgG and mCherry-hIgG coated wells and incubate at RT for 1 hour.
9. Remove the solution and wash the MaxiSorp plate 10 times with TBST. Detect bound phage by adding 50 µL of anti-M13-HRP antibody (1:1000 in 5% MPBST; Santa Cruz Biotechnology, sc-53004 HRP) to each well and incubating at RT for 1 hour.
10. Remove the solution and wash the plate three times with TBST. Add 100 µL of TMB substrate solution (Thermo Fisher Scientific, 34028) to each well.
11. Before the color saturates, stop the reaction by adding 50 µL of 0.4 M H₂SO₄ per well, and measure the absorbance at 450 nm.