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Selection of Recombinant Antibodies 245against the desired antigen could be isolated to construct “immune” libraries(14,18). Immune libraries are typically used in medical research to select abundantantibodies against one particular antigen or group of antigens, for example,of infectious pathogens, whereas they are not the source of choice for theisolation of antibodies with other specificities. “Single-pot” or universal librariesare designed to provide antibody fragments binding to every possible antigen.Naive libraries are constructed from rearranged antibody genes from IgM producingB-cells of nonimmunized donors. An example for this library type is the naivehuman Fab library constructed by de Haardt et al. (19). “Semisynthetic” librariesare derived from not rearranged V-genes from pre-B-cells (germline cells) or froma single antibody framework with at least one complementary determining region(CDR) genetically randomized, such as the library described by Pini et al. (20).Acombination of naive and synthetic repertoire was used by Hoet et al. (21). Theycombined LCs from autoimmune patients with a Fd fragment containing syntheticCDR 1 and CDR2 in one human framework and naive CDR3 regions,origined from autoimmune patients. Fully synthetic libraries have a humanframework with randomly integrated CDR cassettes (22,23). All library types—“immune,” “naïve,” and “synthetic” and their intermediates—have been provento be useful sources for the selection of antibodies for diagnostic and therapeuticpurposes. To date, “single-pot” antibody libraries with a theoretical diversity ofup to 10 11 independent clones have been generated (24) to serve as a molecularrepertoire for phage display selection procedures. An overview of antibodylibraries and the comparison of their construction principles is given by Hustand Dübel (25) (Fig. 1).2. Materials2.1. Coating of Microtiter Wells1. Maxisorb microtiter plates oder stripes (Nunc, Wiesbaden, Germany).2. Phosphate-buffered saline (PBS): 8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na 2HPO 4⋅2H 2O,and 0.24 g/L KH 2PO 4, pH 7.4.3. Dimethyl sulfoxide.4. Phosphate-buffered saline Tween (PBST) PBS + 0.1% Tween-20.2.2. Panning1. Milk phosphate-buffered saline Tween (MPBST) 2% skim milk in PBST, prepare fresh.2. Panning block solution: 1% (w/v) skim milk and 1% (w/v) bovine serum albumin(BSA) in PBST, prepare fresh.3. 10 µg/mL Trypsin in PBS.4. E. coli XL1-blue MRF`(Stratagene), genotype: ∆(mcrA)183 ∆(mcrCB-hsdSMRmrr)173endA1 supE44 thi-1 recA1 gyrA96 relA1 lac (F′ proAB LacI q Z∆M15Tn10 [Tet R ]).