Phage ELISA Binding Assay with Direct Target Coating

• It is useful to include a phage ELISA in any panning experiment since artifacts of the panning process cannot always be anticipated or prevented.
• The following ELISA protocol is sufficient for rapidly determining whether a selected phage clone binds the target, without the need for an antibody specific for the target.
• In this procedure a microtiter plate is coated with the target at high density, and each purified phage clone is applied to the plate at various dilutions. Bound phage is then detected with an anti-M13 antibody (Thermo Fisher Scientific, PA1-26758 or MA1-34468).
• The amount of target coated on the plate is not quantifiable but is present at sufficiently high density to allow multivalent binding to the phage.
• This method will not determine whether the selected phage binds with high or low affinity. The method is useful for qualitative determination of relative binding affinities for a number of selected clones in parallel and will distinguish true target binding from binding to the plastic support.
  
  

1. Prepare 50 μl of ~10^13-14 pfu/ml stocks of phage to assay as described in the protocol Plaque Amplification for ELISA Samples.
   
   
2. Coat one row of ELISA plate wells for each clone or pool to be characterized with 100–200 μl of 100 μg/ml of target in 0.1 M NaHCO ₃, pH 8.6. Incubate overnight at 4°C in an air-tight humidified box (e.g., a sealable plastic box lined with damp paper towels).
   
   
3. Shake out excess target solution and slap plate face-down onto a paper towel. Fill each well completely with blocking buffer. Additionally, one row of uncoated wells per clone to be characterized should also be blocked to test for binding of each selected sequence to BSA-coated plastic (this test for background signal is extremely important, especially if panning was carried out on a polystyrene surface). A second, fully uncoated microtiter plate should be blocked for use in serial dilutions of phage (Step 5) before addition to the target-coated plate. Dilutions are done in a separate blocked plate to ensure that phage are not absorbed onto the target while performing dilutions, which would result in a sudden “falling-off” of signal as the phage is diluted. Incubate the plates filled with blocking buffer for 1–2 hours at 4°C.
   
   
4. Shake out the blocking buffer and wash each plate 6 times with TBST, slapping the plate face-down onto a clean section of paper towel each time. The percentage of Tween should be the same as the concentration used in the panning wash steps.
   
   
5. In the separate blocked plate, carry out four-fold serial dilutions of the phage in 200 μl of TBST per well, starting with 10¹² virions in the first well of a row and ending with 2 x 10⁵ virions in the 12^th well.
   
   
6. Using a multichannel pipettor, transfer 100 μl from each row of diluted phage to a row of target-coated wells, and transfer 100 μl to a row without target. Incubate at room temperature for 1–2 hours with agitation.
   
   
7. Wash plate 6 times with TBST as in Step 4.
   
   
8. Follow manufacturer instructions for antibody application and development.