a. Gently agitate the filters for 1.5-2 hours in the AP- or HRP-conjugated antibody solution at room temperature.
b. Wash the filters as described in Step 14.
c. Prepare the chemiluminescent substrates according to the manufacturer's instructions.
d. Incubate the washed filters in the chemiluminescent substrates for 1-5 minutes (again, consult the manufacturer's recommendations for optimal exposure times).
e. Drain the excess solution from the filters, and immediately wrap the filters in Saran Wrap. Do not allow the filters to dry out.
f. Establish an autoradiogram. Typically, the initial exposure is 1 minute. This interval provides enough information to establish the proper exposure time.
16. Identify the locations of positive plaques or, if made, compare the duplicate filters, searching for coincident signals. For screens involving radiolabeled or chemiluminescent probes, compare the resulting autoradiograms with the agar plates on a light box. For screens involving chromogenic reagents that leave a visible positive residue on the filter, carry out the following steps:
a. Lay a sheet of Saran Wrap or Mylar film over the filters.
b. On the surface of the Saran Wrap, mark the locations of the holes in the filters and the locations of antigen-positive clones with different colored waterproof markers. Label the Saran Wrap to identify the plates from which the filters were derived.
c. Place the sheet of Saran Wrap on a light box, and align the plates containing the original bacteriophage A, plaques on top of it.
d. Identify the area containing the positive plaque, and remove a plug of agar from this area using the large end of a Pasteur pipette. Transfer the plug to 1 ml of SM containing 2 drops of chloroform.
e. Keep the sheet of Saran Wrap, which provides a permanent record of the locations of the positive plaques. The colored spots on the original filters fade quite rapidly.
17. Allow the bacteriophage particles to elute from the agar plug for several hours at 4°C. Measure the titer of the bacteriophages in the eluate, and then replate them so as to obtain approx. 3000 plaques per 90mm plate. Rescreen the plaques as described above (from Step 4 onward), and repeat the process of screening and plating until a homogeneous population of immunopositive recombinant bacteriophages is obtained.
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