9, 429C434 [PMC free article] [PubMed] [Google Scholar] 39. of an N-terminal biotin, a SGSG-linker, followed by the C-terminal located selection motif (Innovagen, Lund, Sweden). Selection of CIMS antibodies Human recombinant scFv antibodies were selected from the phage display library, n-CoDeR (29). Three consecutive rounds of selection were performed, using biotinylated peptide motifs as antigens. In selection round one, about 1013 colony-forming units of phage were mixed with 50 nm antigen in a total volume of NU6300 3 ml. The selection buffer was phosphate-buffered saline (PBS) made up of 3% (w/v) bovine serum albumin (BSA), 0.05% (v/v) Tween-20, and 0.02% (w/v) sodium azide. The antigen and phage mixture was incubated for 16 h at room temperature. Biotinylated peptides were captured on 108 streptavidin-conjugated magnetic beads (Dynabeads M-280, Dynal, Oslo, Norway) during a 30 min incubation. NU6300 Before use, Dynabeads were blocked with 5% (w/v) BSA in selection buffer. Following peptide capture, beads were washed a total of nine times, using a Magnetic Particle Concentrator (Dynal, Oslo, Norway), three times with selection buffer, three times with PBS made up of 0.05% (v/v) Tween-20 and three times with PBS. Captured phages were then eluted by addition of 400 l of a 1 mg/ml trypsin solution for 30 min, after which trypsin was inactivated by addition of 40 l of a 2 mg/ml aprotinin solution. All incubations were performed with gentle end-over-end rotation. Log phase was infected with the eluted phage pool and a new, amplified phage pool was produced essentially as described by Engberg (30), using strain HB101F (constructed from HB101, Invitrogen, Carlsbad, CA) and 20-fold excess of helper phage R408 (Stratagene, La Jolla, CA). In selection round two, about 1011 colony-forming units of amplified phage were mixed with 20 nm antigen in a total volume of 1 ml and 3 107 streptavidin-conjugated magnetic beads were used to capture biotinylated peptide motifs. Bound phage were eluted by addition of 400 l of 10 mm glycin-HCl, pH 2.2 for 30 min. A few l of 1 1 m Tris-HCl, pH 9.0, was then added to neutralize the acid. The eluted phage pool was not amplified, but used directly in the third selection round. Thus, in selection round three, peptides were preloaded on avidin-coated wells of a microtiter plate, with 8 wells each coated with 0.5 g avidin and loaded with 10 pmol peptide. Wells were then blocked with 5% (w/v) BSA in selection buffer. About 106 eluted phages from round two were diluted to 800 l in selection buffer and then added to peptide-loaded wells, 100 l per well. The plate was incubated for 16 h at room temperature with gentle agitation. Wells were NU6300 washed three times with selection buffer, three times with PBS made up of 0.05% (v/v) Tween-20, and three times with PBS. Captured phages were CEACAM5 eluted using trypsin, 100 l per well, as described above. To counteract selection of irrelevant (nonspecific) phages, each selection round was stringently preceded by a preselection, designed to eliminate phage clones of certain antigen specificities. The starting phage stocks of selection rounds one and two were preselected against irrelevant biotinylated peptide motifs followed by capture on streptavidin-conjugated magnetic beads. The phage stock used in round three was preselected against avidin coated on a microtiter plate. Enrichment of irrelevant phages was also counteracted by addition of irrelevant nonbiotinylated peptide motifs.
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