96 well polystyrene microtiter plates

Target proteins (folate receptor-α, programmed death 1 and programmed death-ligand 1) were dissolved in carbonate buffer pH 9.6 and coated on polystyrene 96-well microtiter plates (Corning, Inc., NY, USA) overnight at 4 °C at a concentration of 50–150 μg/mL. The next day, each well was blocked with 250 μL 3% (w/v) non-fat milk (NFM) (Sangon Biotech Co., Ltd., Shanghai) for 2 hours at 37 °C. After washing 5 times with TBS containing 0.05% (v/v) Tween 20 (TBST), 100 μL Ph.D.-12 peptide library (New England BioLabs, Inc., USA) aliquot containing 10¹¹ phages was added to each well and the plate was incubated for 1 hour at 37 °C. After washing 10 times with TBST and 3 times with TBS to remove the unbounded phages, the target-bound phages were eluted with 150 μL 100 mM Glycine-HCl (pH 2.2) and neutralized immediately with 9 μL 2 M Tris-HCl (pH 9.1). 5 μL of eluate was used for titering and the rest was used to infect E. coli ER2738 host strain (New England BioLabs, Inc., USA) for amplification.

Minimal inhibitory concentration (MIC) was performed using colistin sulfate and polymyxin B by employing broth microdilution in Cation-Adjusted Mueller–Hinton Broth (CAMHB), following the guidelines set by the Clinical and Laboratory Standards Institute (CLSI) (Weinstein, 2018 ). Each A. baumannii strain was diluted with LB broth to get 0.1 OD₆₀₀ from an overnight culture, growth to 0.4 OD₆₀₀ at 37 °C, 220 rpm till mid-log phase. The bacterial concentration was adjusted to 1 × 10⁷ CFU/mL with CAMHB. Colistin sulfate (0.5, 1, 2, 4, 6, 8, 10, 12, 16, 32, 64, 128, 256, 512 μg/mL) and polymyxin B (1, 2, 3, 4, 5, 6, 7, 8 μg/mL) was prepared at various concentrations in CAMHB. A mixture of 50 μl of A. baumannii and 50 μl of the polymyxin solutions was added to the wells of polystyrene 96-well microtiter plates (Corning, New York, NY, United States). The microtiter plates were then incubated overnight at 37 °C, and the OD₆₀₀ of each well was measured. The MIC for each tested strain was determined as the lowest concentration of colistin sulfate or polymyxin B that completely inhibited the growth of the bacteria.

The optimal dilution of antibodies was determined by ELISA according to Voller et al. [22 (link)]. Briefly, polystyrene 96-well microtiter plates (Corning, USA) were coated with 5 μg/mL snake venom in a coating buffer (0.1 M carbonate bicarbonate, pH 9.6) for 12 h at 4 °C. The wells were washed six times with washing buffer (PBS, pH 7.4, containing 0.05 % Tween-20). The wells were blocked for 2 h at 37 °C with a blocking buffer (3 % BSA in washing buffer). The wells were washed again three times with washing buffer. Serial dilutions of IgY samples in blocking buffer were prepared and 100 μL of each diluted IgY sample was added into individual coated wells and the plates were incubated at 37 °C for 1.5 h. The wells were washed five times with the same washing buffer.
The plates were incubated with peroxidase conjugated rabbit anti-chicken IgY (1:5000) for 45 min at 37 °C. After the plates were washed five times, 100 μL of substrate buffer (0.1 M citric acid, plus 0.2 M sodium diphosphate, 5.0 mL H₂O, 5.0 mg OPD, 5 mL of H₂O₂) were added and incubated at room temperature in the dark for 20 min. The reaction was stopped by addition of 50 μL of 2 N sulfuric acid. Absorbance was read at 490 nm on an ELISA plate reader (Molecular Devices Corporation, USA). IgY samples from the eggs collected before immunization were used as negative control. Wells free of venom were used as blanks.