The binding phage were eluted by treatment with 100 uL of 100 mM glycine HCl pH 2. 0 for 10 min, and the solution was neutralized www.selleckchem.com/products/VX-770.html by addition of 50 uL of 2 M Tris, pH 8. 0. The neutralized phage solution was then added to 5 mL of log phase XL1 Blue E. coli in 2��YT broth supplemented with tetracycline. After 1 hr, 50 ug mL carbencillin along with helper phage were added and the culture was grown at 37 C for 1 hr. Subsequently, 25 mL of 2��YT containing 50 ug mL carbenicillin and 25 ug mL kanamycin were added and the culture was grown at 30 C for 18 hrs. The cells were removed by centrifugation, then the phage was isolated as above and used immediately for subsequent rounds of infection. Se lection progress was monitored by 1 large scale sequencing of the phage populations and 2 output phage titers from wells containing the target to wells containing a BSA control.

Individual clones were grown small scale for high throughput phage ELISA analysis in deep 96 well plates. Cultures of 1 mL LB broth containing carbencillin were inoculated with colonies corresponding to selectants, helper phage were added and the culture grown at 30 C for 18 hrs. The cells were removed by centrifugation and the supernatant applied directly to ELISA plate wells in which the antigen or control pro tein had been immobilized. Phage solutions were allowed to bind for 15 mins, the wells washed with PBS T, and then the bound phage detected with the anti M13 HRP conjugate as above. For specificity profile analysis, LF and KLH were purchased from Sigma Aldrich.

Single point competitive ELISAs were similar except that the phage solutions were preincubated with 40 nM 5 Helix for 30 min before addition to wells containing the immobilized 5 Helix. Both specificity pro file analysis and single point competition analysis were spotchecked for reproducibility and, in general, gave consistent results among independent experiments. Competitive phage ELISAs were performed essentially as described. Expression of scFv proteins and monoclonal ELISAs Phagemid vectors were converted to expression vectors by replacement of the hinge, GCN4 and pIII CT seg ment downstream of the scFv segment with a hexahistidine tag. The scFv proteins were expressed in the periplasm of E. coli BL21. Cultures were grown in low phosphate media at 30 C for 14 16 hrs and the cells harvested by centrifugation. Cell lysis was AV-951 achieved by treatment with Bug Buster. The lysate was clarified by ultracentrifugation and puri fied by nickel affinity chromatography. Purified scFv pro teins were dialyzed into PBS then used immediately for analysis or flash frozen and stored at 80 C. Analysis by ELISA was similar to phage ELISA except that an anti FLAG HRP conjugate was used to detect the scFv protein.