The binding clones from the soluble phage ELISA were selected for soluble scFv expression as described earlier (43). The pAK100 vector carrying scFv DNA and pAK400 vector were digested with SfiI restriction enzyme (New England Biolabs). The digested scFv DNA fragments and pAK400 vector were gel purified using gel extraction kit (Qiagen). The scFv DNA was ligated into pAK400 vector at a 3:1 M ratio using T4 DNA ligase (New England Biolabs) in a reaction volume of 20 µl followed by transformation into HB2151 cells using calcium chloride method. The HB2151 cells carrying scFv cloned pAK400 plasmid were grown and induced by 1 mM IPTG for 4 at 24°C (48). Next, scFv was purified from the periplasmic extract by Ni-NTA (Qiagen) affinity chromatography as described earlier (44). The protein was eluted with 300 mM imidazole followed by extensive dialysis against ice cold 1× PBS (pH 7.4), and protein samples were concentrated using ultrafiltration columns (Ambion) over a 10 kDa cut off and filtered by 0.2 µm sterile syringe filter. The purity of the purified proteins was assessed by SDS-PAGE and Western blot. Briefly, resolving the scFv on 12% SDS-PAGE followed by Western blotting, the scFv was detected using anti-His-tag antibody raised in mouse (Sigma) at a 1:1,000 dilution in 1× PBS with 0.05% Tween 20. Secondary anti-mouse HRP (1:3,000 dilutions) antibody was added and incubated at RT for 2 h, and color was developed using 3,3′-diaminobenzidine tetrahydrochloride (DAB) as substrate. Proteins with >95% purity were stored at −80°C immediately after snap freezing in liquid nitrogen.