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248 Hust et al.interaction during panning as after elution a single antibody phage can give riseto a bacterial colony by its resistance marker. The selection cycle can berepeated by infection of the phagemid bearing E. coli colonies from the formerpanning round with a helperphage to produce new antibody phage, which canbe used for further panning rounds until a significant enrichment of antigenspecificphage is achieved. The number of antigen-specific antibody phageclones should increase with every panning round. Usually two to six panningrounds are necessary to select specifically binding antibody fragments. Highthroughput methods using microtiter plates and robotics can facilitate andenhance the panning procedure (for review see ref. 30).The first step in the evaluation process of potential binders is mostly done byan enzyme-linked immunosorbent assay (ELISA) with polyclonal phage preparationsfrom each panning round against immobilized, i.e., coated, target antigenand on control protein, for example, BSA. In the next step, antibody clonesof panning rounds showing a significant enrichment of specific antigen bindingin the polyclonal phage ELISA are produced as soluble monoclonal antibodyfragments in microtiter plates followed by an ELISA on coated antigen vs oncontrol protein. The following protocols describe the selection of recombinantantibody fragments from antibody gene libraries by phage display and the initialanalysis of the selected antibody fragments.3.1. Coating of Microtiter Plate Wells1. (a) Protein antigen: for the first panning round, use 2–10 µg protein per panning,for the following rounds use 0.1–1 µg protein for more stringent conditions.Dissolve the antigen in 150 µL PBS, transfer into the microtiter plate well and incubateovernight at 4°C (see Note 1) and (b) oligopeptide antigen: use 100–500 ngoligopeptide for each panning round. Dissolve the oligopeptide in 150 µL 5% (v/v)dimethyl sulfoxide containing PBS, transfer into the microtiter plate well andincubate overnight at 4°C (see Note 2).2. Wash the coated microtiter plate wells three times with PBST using an ELISAwasher (see Note 3).3.2. Panning1. (a) Block the antigen-coated wells with MPBST for 2 h at RT. The wells must becompletely filled and (b) perform this step only in the first panning round. In parallel,block an additional well (without antigen) per panning with MPBST for 1 hat RT for preincubation of the antibody gene library. The wells must be completelyfilled. Wash three times with PBST (see Note 3). Incubate 10 11 –10 12 antibodyphage from the library in 150 µL panning block for 1 h at RT. This step removesunspecific binders from the antibody gene library.2. Wash the blocked antigen-coated wells three times with PBST (see Note 3).Either carry over the preincubated antibody phage library to the blocked wells or