Expression and purification of scFv-D1.3-GFP11 and GFP1-10 pEP-D1.3-GFP11 and pEP-D1.3-Ecoil DNA were transformed into BL21 (DE3) (Novagen). Bacteria were grown in 50 ml of auto- induction media [28] at 25uC overnight to produce the recombinant protein. Bacteria were harvested and resuspended in 10 ml PBS. Lysis was performed by means of a pressure press (EmulsiFlex–C5, Avestin Inc.). The bacterial debris was pelleted by centrifugation, and the protein purified from the supernatant using the C-terminal His-tag and Ni-NTA agarose resin (Qiagen Inc.) according to the manufacturer’s instructions. Purified protein was dialyzed against PBS and stored at 220uC. Crude protein extract or bacterial culture supernatant was used. After expression in 1 ml of selective auto-induction media, cells were harvested, the supernatant was recovered, and the pellet resus- pended in 1/10 volume of Pop culture (Novagen) solution. Lysis was performed for 25 minutes at room temperature (RT), with shaking, and the suspension used after centrifugation without further protein purification. GFP 1-10 was prepared as previously described [19,29,30]; 75 mg of inclusion body GFP1-10 was unfolded in 1 ml 9 M urea, and refolded in 20 ml TNG buffer (100 mM Tris HCl pH 7.5, 150 mM NaCl, 10% v/v glycerol) prior to use. Quanti- fication was carried out by comparing the intensity of purified scFv- D1.3-GFP11 to known concentrations of BSA standards on poly- acrylamide gel stained with gel code (Pierce Inc.). Complementation assay A 96-well microplate was first blocked with a solution of 0.5% bovine serum albumin (BSA) in PBS for 30 minutes. Different concentrations of purified scFv-D1.3-GFP11 were prepared (75, 150, 300 pmol) in a final volume of 100 ml. A large molar excess (800 pmol) of GFP 1-10 in 180 ml was added and complementa- tion carried out. Crude Pop-culture E.coli lysate was also tested using two fold serial dilutions in a final volume of 50 ml, incubated with a similar large molar excess of GFP1-10. Fluorescence (lexc = 488 nm/lem = 530 nm) was monitored using a Tecan Infinite M200 reader measuring at 3 minutes intervals for 20 hours for the purified protein and 10 hours for the crude extracts at room temperature (RT). Calibration curve To correlate the fluorescent signal with the D1.3-GFP11 protein quantity present in the crude bacteria extract, two calibration curves were generated by measuring the fluorescence after split- GFP complementation of a control antigen, sulfite-reductase (SR- GFP11), which we have used extensively [29] and of purified scFv- D1.3-GFP11, both of known concentration (range 1.5–160 pmol) and molecular weight. Forty ml of purified protein solutions or total cell extract were mixed with 190 ml of 800 pmol GFP 1-10 protein solution. The samples were incubated over night (ON) at 4uC. Fluorescence was measured using the Beckman Coulter DTX-880 Multimode Detector. The quantity of scFv-D1.3-GFP11 in the crude bacteria extract was calculated using the equations in Canbantou et al [31]. Enzyme linked immunosorbant assay (ELISA) 100 ml of neutravidin solution (10 mg/ml concentration) were added to each well of an ELISA plate (Nunc 442404) and incubated at 37uC for 1 hour in order to coat the plate. The wells were washed twice with PBSLT (PBS, Tween 20 0.05%), then blocked with 200 ml of 1% BSA in PBS for 1 hour at RT. The blocking solution was washed away with PBSLT. 100 ml of Split GFP Labeling of scFvs biotinylated antigen (10 mg/ml) were added to each well and incubated for 30 minutes at RT. After extended washing with PBSLT, 100 ml of different concentrations of the scFv-D1.3- GFP11 or D1.3-Ecoil purified protein (range 0.6 – 300 nM) were added and incubated for 1 hour at room temperature. Unbound proteins were removed by washing three times with PBST (PBS, Tween 20 0.5%) and three times with PBSLT. 100 ml of anti-SV5 AP (1mg/ml) solution was added and incubated for 1 hour at RT. Washing steps were repeated (3xPBST, 3xPBSLT), and alkaline phosphatase activity detected using PNPP solution (Sigma) with absorbance read at 405 nm. Equilibrium binding titration curve for the complemented D1.3-GFP11 to determine the affinity constant Kd of scFv-D1.3- GFP11 was obtained by nonlinear regression using PRISM software (version 5.0 Graph Pad). Fluorescence linked immunosorbant assay (FLISA) Neutravidin coating and antigen binding were performed following the ELISA protocol, except that black (Nunc 43711) ELISA plates were used as previously described [23]. After antigen coating, 50 ml of different concentrations of the scFv-D1.3-GFP11 purified protein (ranging from 0.6 to 300 nM),, were added and incubated for 1 hour at RT. Unbound proteins were removed by washing three times with PBST and three times PBSLT. 90 ml of 800 pmol GFP1-10 were added to each well and fluorescence was read after 4 hours at RT. Equilibrium binding titration curve to determine the affinity constant Kd of scFv-D1.3-GFP11 was obtained by using nonlinear regression running PRISM software (version 5.0 Graph Pad). Yeast display The scFv-D1.3-GFP11 construct was cloned into the yeast display vector pDNL6, to create pDNL6-GFP11, and transformed into S. cerevisiae EBY100 according to the Yeast transformation kit manual (Sigma). A single clone was then grown in SD-CAA selective media at 30uC. Cells were diluted to 0.5 OD600 in induction media SG/R + CAA and incubated at 20uC with Figure 2. Comparison of antigen binding activity assessed by ELISA. The antigen binding activity was analyzed by ELISA using different concentrations of scFv-D1.3 and scFv-D1.3-GFP11 scFvs (ranging from 0.6 to 300 nM). The scFv-D1.3-GFP11 construct showed good binding activity for lysozyme (lys) even after incubation with the GFP1-10 complementing protein, indicating the absence of steric hindrance between the restored GFP protein and the anti-SV5 antibody used for detection. Myoglobin (myo) was used as negative control. doi:10.1371/journal.pone.0025727.g002 PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e25727