Devel. be easily expressed and purified in their native conformation. Some molecules with large extracellular domains may adopt a specific conformation upon interaction with other cell surface proteins, thereby forming complexes that are cumbersome to produce by recombinant expression. Moreover, many standard screening Rabbit Polyclonal to CRP1 practices, such as the adsorption of recombinant proteins on plastic, may significantly alter protein conformations (5). For these reasons, Abs selected on the basis of binding to a recombinant protein may not bind the native conformation of this protein. It is thus of high interest to develop procedures entirely based on the use of intact cells expressing the receptor of choice. However, in this case, an extra step is necessary to enrich for phage-Abs binding to the receptor of interest rather than to other cell surface proteins. Because selection steps are performed at 4 C. Supernatant was the final cell lysate. Total protein concentration (average between 2C5 mg/ml) was determined spectrophotometrically using a protein assay kit (Bio-Rad Laboratories, Hercules, CA). Production and Purification of sdAbs For polyclonal production of soluble sdAbs, 10 l of output from selection round 1 and 2 Diosmin were used to inoculate 200 ml of 2YT/ampicillin (100 g/ml). Cells were grown at 37 C (250 rpm) until the OD600 reached 0.5. SdAb expression was induced by the addition of 0.1 mm IPTG (isopropyl-h-d-thiogalactopyranoside) at 30 C (250 rpm) for 20 h. SdAbs were purified by metal affinity chromatography as described (19). In Vitro Biotinylation The biotinylation of protein was performed using Ez-link micro NMHS-PEO4- biotinylation kit (Perbio science) following the recommendation of the manufacturer. Llama Immunization and Library Construction Three young adult llama (Lama glama) were immunized subcutaneously at days 1, 10, 20, and 30 with breast cancer biopsy lysate (two llamas) or with healthy breast biopsy (one llama). One llama was immunized with HER2-Fc protein and HEK-mGluR4 cells. VHH library constructions were performed as previously described (14, 20). Selection of Phage-sdAbs To produce phage-sdAbs, 10 l of the library was grown in 50 ml of 2YT/ampicillin (100 g/ml)/glucose (2%) at 37 C to an OD600 of 0.5. Then, the culture was infected with KM13 or hyperphage (Progen biotechnik) helper phage at a ratio of 20 phages/cell for 30 min at 37 Diosmin C without shaking. The culture was centrifuged for 10 min at 3000 at 4 C and Diosmin the phage-containing pellet was resuspended with 1 ml of PBS. Different strategies of panning were performed. Some phages were selected using magnetic epoxy beads (Dynabeads, invitrogen) coated with antigen or lysates immobilized on epoxy beads during 48 h at 4 C following recommendations of the manufacturer. Other phages were selected directly on cells (30 106 cells). Beads or cells were washed three times in PBS (using a magnetic particle concentrator for magnetic beads and centrifugation step for cells) and phage-sdAb library (1 ml) and beads or cells were saturated in 2% milk PBS. For selection including a depletion step, phage-sdAb library were incubated with the irrelevant immobilized antigen at room temperature or with 80 106 irrelevant cells at 4 C during 2 h, with rotation. Phage-sdAb libraries (depleted or not) were recovered and incubated with beads with rotation during 2 h at room temperature or at 4 C for cells. For masked selection in the presence of soluble sdAbs, 10 m of purified sdAbs were added during this step. Beads, cells, or plates were washed 10 times with 1 ml 0,1% Tween PBS (without Tween for cells) and two times with PBS. Bound phages were eluted with tryspin solution (Sigma) at 1 mg/ml during 30 min at room temperature with rotation. Eluted phages were incubated without shaking with log-phase TG1 cells and Diosmin plated on 2YT/ampicillin (100 g/ml)/glucose (2%) in 15 cm Petri dishes. Some isolated colonies Diosmin were grown overnight in microtiter plate containing 200 l 2YT/ampicillin (100 g/ml)/glucose (2%) and stored at ?80 C after the addition of 15% glycerol (masterplates). The remaining colonies were harvested from the plates, suspended in 2 ml 2YT/ampicillin (100 g/ml)/glucose.