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250 Hust et al.3.4. Titering1. Inoculate 5 mL 2X TY-T in a 100-mL Erlenmeyer flask with XL1-blue MRF′ andgrow overnight at 37°C and 250 rpm.2. Inoculate 50 mL 2X TY-T with 500 µL overnight culture and grow at 250 rpm at37°C up to OD 600approx 0.5 nm (see Note 6).3. Make serial dilutions of the phage solution in PBS. The number of eluted phagesdepends on several parameters (e.g., antigen, library, panning round, washingstringency, and so on). In case of a successful panning, the phage titer usually is10 3 –10 5 phage per well in the first round increasing to 10 6 –10 9 phage per well inthe succeeding rounds. The phage preparation after reamplification of the elutedphage have a titer of about 10 12 –10 13 phage/mL.4. Infect 50 µL bacteria with 10 µL phage dilution and incubate for 30 min at 37°C(see Note 7).5. Titrations can be done in two different ways:a. Plate the 60 µL infected bacteria on 2X TY-GA agar plates (9-cm Petri dishes).b. Pipet 10 µL (better as triplicate) on 2X TY-GA agar plates. Here, about 20titering spots can be placed on one 9-cm Petri dish.6. Incubate the plates overnight at 37°C.7. Count the colonies and calculate the colony-forming units titer, according tothe dilution.3.5. ELISA of a Polyclonal Antibody Phage Suspension1. To investigate the enrichment of antigen-specific antibody phage after a panninground, prepare microtiter plates with 100–1000 ng antigen per well for each panninground (for method see Subheading 3.1.). As a control, prepare wells with100–1000 ng BSA in 150 µL PBS overnight at 4°C (see Note 8).2. Wash the coated microtiter plate wells three times with PBST (washing proceduresee Subheading 3.2. and Note 3).3. Block the antigen-coated wells with MPBST for 2 h at RT. The wells must becompletely filled.4. Wash the coated microtiter plate wells three times with PBST (washing proceduresee Subheading 3.2. and Note 3).5. Resuspend 10 10 antibody phage from each panning round in 150 µL 2%MPST andincubate them for 1.5 h on the antigen and the BSA control, respectively.6. Wash the microtiter plate wells three times with PBST (washing procedure seeSubheading 3.2. and Note 3).7. Incubate each well with 100 µL HRP-conjugated anti-M13 antibody 1:5000diluted in MPST for 1.5 h.8. Wash the microtiter plate wells three times with PBST (washing procedure seeSubheading 3.2 and Note 3).9. Shortly before use, mix 19 parts TMB solution A and one part TMB solution B.Add 100 µL of the prepared TMB solution to each well and incubate for 1–15 min.