The Tomlinson I+J human single fold synthetic naïve phage display single-chain antibody fragment (scFv) libraries constructed in pIT2 (HIS MYC tag) vector with the size of 1.4 × 108 for each, and DAb human VH domain library constructed in PR2 (MYC VSV tag) with the size of 3× 109, helper phage KM13, E. coli strains TG1 and HB2151 for selection of specific antibody clones and production of phage and soluble single-chain Fvs antibodies, respectively, were purchased from GeneService (Cambridge, UK). C. perfringens epsilon toxoid was purchased from National Institute for Biological Standards and Control (NIBSC), UK.
Biopanning of phage display libraries
The purity of the epsilon toxoid was initially confirmed by SDS-PAGE. The libraries and KM13 helper phage stocks were amplified to have enough quantities for use in several rounds of selection. Biopanning was performed using Tomlinson I+J and DAb libraries in parallel to ensure the researchers select the most epsilon toxoid binding clones. For selection, MaxiSorp® immunotubes (Nunc, Denmark) were coated with 5 ml pure C. perfringens epsilon toxoid (100 μg/ml) in carbonate buffer (pH 9.4) and incubated overnight at 4°C, washed three times with PBS and blocked with 4% MPBS (4 % Dry skimmed milk in PBS) buffer overnight at 4°C. The next day, 5×1012 purified phages from each library in 5 ml MPBS buffer were added to each immunotube and incubated for one hour with gentle agitation at room temperature. The unbound phages were removed by washing 10 times with PBS containing 0.1% Tween 20 and twice with PBS buffer. To recover bound phages, 5 ml trypsin solution (1 mg/10 ml) was added to each immunotube and incubated for one hour at room temperature with gentle agitation. Then, eluted phages (5 ml) were used to infect exponentially growing E. coli TG1 (30 ml), incubated at 37°C for one hour in a water bath and plated onto TYE agar plates (100 μg/ml ampicillin, 4% glucose) and incubated at 30°C overnight. The following day, cells were scraped from agar plates and diluted into 500 ml of 2xTY medium (100 μg/ml ampicillin, 4% glucose) and infected with KM13 phage. Then, grown phages were precipitated and purified by cold PEG/NaCl solution (20% Polyethylene glycol 6000, 2.5 M NaCl). The purified phages were used in the next round of selection and repeated binding, elution and infection steps for 2 times.
Screening of clones by polyclonal phage ELISA
The screening was done with the entire population of phages eluted (fused to the antibody fragments) after each round of selection. ELISA plates (Nunc, Denmark) were coated with epsilon toxoid (100 μg/ml) overnight at 4°C. The next day, plates were washed and blocked by 4% MPBS buffer per well. After washing, 10 μl of PEG precipitated phages recovered from each round of selection were diluted in 100 μl MPBS buffer and added to each well. One hour later, plates were washed and the binding of phages were detected using a monoclonal anti-c-Myc antibody (Biolegend) and anti-mouse HRP conjugate (Sigma Aldrich, USA) and detected with TMB substrate (Biobasic, Canada). Sulfuric acid solution 1 M was used to stop the reaction and enzymatic activity of HRP–antibody conjugate and the absorbance was read at 450 nm and 620 nm with a microplate reader.
Screening of clones by monoclonal phage ELISA
For monoclonal phage ELISA, individual colonies were picked after the second and third rounds of selection and grown into 2xTY medium containing 100 μg/ml ampicillin and 4% glucose in a 96-well plate and incubated at 37°C, 250 rpm for 12 hours. Then, the overnight culture for each clone diluted 100-fold into 200 μl 2xTY medium (100 μg/ml ampicillin and 0.1% glucose) and incubated at 37°C, 250 rpm until OD600=0.4. The culture was infected with 4×108 KM13 helper phages for 30 min at 37°C, the bacteria pelleted by centrifugation and resuspended in 150 μl of 2×YT containing ampicillin (100 μg/ml) and kanamycin (50 μg/ml) before growth overnight at 25°C, 250 rpm for 16 hours. Then, the plate was centrifuged and 100 μl of culture supernatant of each well was used for ELISA plates that pre-coated with epsilon toxoid (100 μg/ml) and blocked with 4% MPBS buffer. Phage binding was detected with a monoclonal anti-c-Myc antibody (Biolegend) and anti-mouse HRP conjugate (Sigma).
Screening of clones by scFv ELISA
Antibodies fused to the pIII coat protein of phage were converted to soluble scFv proteins by induction of bacteria with IPTG. scFv ELISAs were done similar to monoclonal phage ELISA. Soluble scFvs were produced by induction of culture of individual bacterial colonies at absorbance of about 0.9 at OD of 600 nm with 1 mM of IPTG and growth for 16 hour at 25°C, 250 rpm in 2×YT. For ELISA, the culture supernatants that contained soluble scFvs were transferred to the antigen-coated wells of ELISA plates. Bound scFvs were detected using 1:5000 monoclonal anti-Polyhistidine HRP conjugate (Sigma Aldrich, USA).
Expression and purification of scFvs
The best clone of scFv ELISA from Tomlinson I+J libraries (Clone No. G2) was tested to confirm the presence of full-length insert by double digestion.
To improve the expression of anti-ETX scFv, the gene of selected clone (G2) was subcloned between the NcoI and NotI restriction sites of the pET26b (+) vector. The pET26b (+) vector contains the kanamycin resistance marker and carries an N-terminal pelB signal sequence for potential periplasmic localization in addition to an optional C-terminal His Tag sequence. CaCl2-treated E. coli BL21 (DE3) was used as the host strain and transformation was performed using the heat shock method. G2 scFv antibody was expressed using 0.01 mM IPTG for 24 hour at 25°C. To extract the antibody, one gram of pelleted cell was resuspended in 5 ml ice-cold protein extraction buffer (50 mM Tris-HCl, 1 mM EDTA, 0.1% Triton X100 and 300 µg⁄ml lysozyme, pH 7.8) and was incubated for 30 min on ice. Then, the sample was sonicated at 24-25% amplitude for 6 sec ON and 6 sec OFF cycle on ice and total sonication time was 10 min Following centrifugation at 10,000 g for 10 minutes at 4°C supernatant containing total protein was mixed with 500 mM NaCl and 20 mM imidazole to purify the scFv protein by Ni-NTA affinity chromatography. Samples were loaded at a speed of 1 ml ⁄min onto the affinity column that had previously been equilibrated with cold binding buffer (50 mM Tris–HCl, 500 mM NaCl and 20 mM imidazole, pH 7.4). Unrelated bacterial proteins were eluted with binding buffer containing 50 mM imidazole before scFv recovery with binding buffer containing 400 mM imidazole. The purified protein fractions containing anti-ETX scFv were analyzed by SDS-PAGE and western blotting.
Development a sandwich ELISA for quantitation of ETX
The sandwich ELISA was designed using B1 (the best clone isolated from DAb library) phage antibody as a capture antibody and soluble G2 scFv (the best clone isolated from Tomlinson I+J libraries) as a detection antibody. The optimal concentrations of capture (100 μg/ml to 0.005 μg/ml) and detection (100 μg/ml to 0.005 μg/ml) antibody were determined by checkerboard titration. To obtain a standard curve of the sandwich ELISA, microtiter plates were coated with 100 μl (25 μg/ml) capture phage VH antibody (B1) for overnight at 4°C. After washing and blocking with 3% BSA/PBS (3% BSA in PBS), the 100 μl 10-fold serial dilutions of pure C. perfringens epsilon toxoid antigen from 5 to 50,000 ng/ml were added into the wells and incubated at 37°C for 1 hour. The wells were washed five times with PBST (0.1% Tween-20 in PBS) buffer and once with PBS buffer and then 100 μl (50 μg/ml) G2 scFv antibody was added into the wells and incubated at 37°C for 1 hour. Subsequently, 1:5000 dilution of HRP-conjugated monoclonal anti-Polyhistidine antibody as the conjugate antibody was added into all wells after washing. Subsequent addition of TMB substrate, the OD value was measured at 450 nm. The standard curve of the sandwich ELISA, were calculated using GraphPad Prism 8 software and 4- parameter logistic curve (4pl) to fit the standard curve. The limits of detection (LOD), limits of quantification (LOQ) were calculated based on the standard deviation of the blank (n=6) as follows :
LOD=Mean (Blank) +3.3× Stdev blank (one sided 95% × 2)
LOQ = Mean blank + 10 × Stdev blank (one sided 95% × 6)
The designed ETX sandwich ELISA test was used to determine the concentration of ETX in an inactivated enterotoxemia commercial vaccine (RVSRI).