Fig. 4. The cDNA library screening process. cDNA libraries contain millions of different bacteriophage particles. To find the one bacteriophage out of the millions that contain the cDNA of interest requires an efficient and rapid technique to simultaneously analyze (nondestructively) all of these different cloned molecules. The procedure begins with the library plated on petri dishes. Typically, up to 500,000 phages might be grown on each 10-cm plate. A nitrocellulose filter is placed on top of the plate. This adsorbs some of the material on the surface of the dish, including bacteria, phage, and debris from lysed bacteria. This adsorbed material also includes some of the fusion protein expressed by bacterial cells before lysis kills an infected cell. This fusion protein leaks out of the bacterial cell, and some of the fusion protein will absorb onto the nitrocellulose filter where the plaque is located. For orientation purposes, marks are made in the filter and the petri dish with a syringe needle. These are needed to align the processed filter and the master plate to pick out the phage plaque with the positive signal on the filter. To detect the positive plaque, we incubate antibodies raised against the protein for which we want the cDNA clone.181,182 These antibodies bind to the fusion protein containing antigenic determinants common to it and the protein. Excess antibodies are rinsed off, leaving only the antibodies bound to the specific fusion protein. Again, these are located on the filter positioned directly over the spot where the plaque producing the fusion protein is on the plate. Second antibodies (anti-antibodies) conjugated with an enzyme such as alkaline phosphatase are incubated with the filter. After rinsing off excess antibodies, a substrate, which produces a colored product, is added, and a colored spot develops on the filter corresponding to the plaque producing the fusion protein. We align the filter and the plate, and with a glass pipet excise the plaque(s) corresponding to the signal. The phage from this plug of agar are replated, and the procedure is repeated until the phage are 100% positive for the antibody reaction, indicating that the bacteriophage is pure. Each of the several methods described previously has been used successfully, but the most effective has been the first, the use of antibodies and an expression cDNA library.