**5. Notes**

**H7.** Add 500 μl/well of the scFv-virus mixtures to each well and tilt plates as described above.

**H8.** Incubate plates at 37°C for about 1 h (time can vary between 30 and 90 min depending on

**H10.** Add 3 ml/well CMC medium and incubate plates in the humidified incubator (37°C, 5%

**H11.** Fixate the cells with formaldehyde solution (3 ml/well for 5 min) and stain with crystal

% of neutralization = 100 − [(no. of plaques: virus + antibody)/no. of plaques: virus only) × 100]. When analyzing serial dilutions of scFvs, percentage of neutralization can be plotted against

After functional characterization, produced scFvs should be characterized for specificity and affinity in binding to recombinant antigen and, more importantly, to intact virions. The specificity of scFvs to recombinant proteins can be easily analyzed by ELISA using recombinant virus antigens (or virus lysates). While scFv affinities has to be determined by kinetic measurements (e.g., by surface plasmon resonance or biolayer interferometry), estimation of apparent scFv affinities can be quickly performed by ELISA. Therefore, recombinant antigen is coated on ELISA plates and 1:2 serial dilutions of purified scFvs (e.g., 1 μM start concentration) are incubated in triplicates followed by detection via tagspecific IgGs and anti-IgG HRP conjugate. The half-maximal effective concentration (EC50) of saturated binding corresponds to the KD value and can be used for affinity ranking of scFvs. However, immobilization of antigens/virions on plastic can alter conformation of coated proteins leading to antibodies recognizing epitopes that are not found on intact virions. Alternatively, binding affinity of antibody fragments can also be estimated by flow cytometry using antigen overexpressing cell lines [30], e.g., by using Vero cell being

In conclusion, broadly neutralizing human monoclonal antibodies represent an excellent opportunity for the prevention and therapy of viral infections and are a potent tool to identify neutralizing epitopes on viral proteins for vaccine approaches. Phage display technology became a potent tool to isolate human neutralizing antibodies and should be considered as a

Larger preparation of helper phage (VCSM13 or M13K07) being used for superinfection to prepare scFv-phages for selection (Protocol A) and screening (Protocol E-I) can be obtained following the protocol below. Hyperphage cannot be produced without recombinant *E*. *coli*

Include virus only to every plate. ScFvs should be tested in at least duplicates.

the cell/virus). During incubation, carefully tilt all the plates 10–15 min.

**H12.** Wash wells once with PBS and twice with ultrapure water (2 ml/well). **H13.** Count plaques and calculate percentage of neutralization as follows:

**H9.** Remove inoculum and wash cells with 3 ml/well PBS.

scFvs concentration to determine the PRNT50 values.

CO<sup>2</sup>

94 Antibody Engineering

) for three days.

infected with HSV [18, 28].

**I. Production of helper phage**

strain and must be purchased.

validated technique for future approaches.

violet solution (800 μl/well) for 2 min.

**Note 1:** To minimize loss of diversity, very large libraries (>109 independent clones) should always be stored as sublibraries that can be separately packed and combined prior to selection. Antibody selection should be only performed with freshly packaged (sub-)libraries that have been kept at 4°C for short as possible. Due to loss of diversity, we do not recommend selection with frozen phage preparations or phage antibodies that have been packaged from secondary library stocks. Importantly, minimize freeze and thaw steps of your primary library and keep it frozen at −80°C until needed.

**Note 2:** Correct growing temperature is crucial for phage display. Too low a temperature (<34°C) might result in ineffective formation of pili that are necessary for successful infection by phage.

**Note 3:** Oligomeric display of scFvs in the first round of selection by infection with hyperphage can greatly improve selection efficacy and can reduce loss of interesting binders during the initial selection step improving the average display from 0.01 up to 5 antibody fragments per phage. Please note that hyperphage infection results in scFv-phages without wild-type pIII proteins that are necessary for successful infection. To restore the wild-type infectivity, hyperphage-packed libraries should be eluted by a protease cutting between the antibody fragment and the pIII protein (e.g., trypsin works well for pSEX and most pHEN derivatives). To check for suitable proteases for your phagemid, you might analyze your vector on PeptideCutter (ExPASy database) and perform Western blot analysis of digested antibody phages using an anti-pIII antibody for detection (see Note 8).

**Note 11:** One ml of log phase TG1 (OD600nm of 0.5) corresponds to about 5 × 108

plates or be captured in solution using streptavidin dynabeads [31].

5 × 107

binders.

est infectivity of eluted phages.

phages for 1 ml log-phase TG1.

Always infect with at least 1:10 phage-to-cell ratio for titration, i.e., do not use more than

Detailed Protocols for the Selection of Antiviral Human Antibodies from Combinatorial Immune...

**Note 12:** If enough antigen is available, increase volume of coating solution up to 5 ml, especially in the first round of selection. Only use highly pure (>90%), freshly prepared protein from sources you can trust. Do not use proteins that have been stored at 4°C for prolonged periods. If using oligopeptides or virions for selection, immobilization condition should be optimized due to lower coating efficacies and/or reduced accessibility of epitopes. Test different buffer/pH/additives for immobilization on MaxiSorp™ plates (e.g., 50 mM carbonate buffer pH 9.6) using tag-specific antibody enzyme conjugates for detection in ELISA. Alternatively, antigens might be biotinylated and immobilized on streptavidin-coated tubes/

**Note 13:** If using proteins with large tags/fusions (e.g., Fc region, GST tag) for antibody selection, supplement the preincubation solution with respective proteins to reduce enrichment of binders against those parts. If using biotinylated oligopeptides immobilized on streptavidincoated immunotubes, preincubate phages with streptavidin to deplete streptavidin-specific

**Note 14:** To check pH of final solution, perform microtitration using pH indicator strips to

**Note 15:** Always elute hyperphage-packaged libraries by protease elution to guarantee high-

**Note 16:** We usually perform three rounds of selection, but this might be increased up to five or more. However, performing additional rounds might lead to loss of diversity and enrich-

**Note 17:** Washing can be done by hand using a small plastic box filled with buffer. Remove all liquid by inverting the plates and hitting it onto paper towels. Change buffer and towels frequently. For better reproducibility and when screening larger amounts of plates, wash plates

**Note 18:** Successful enrichment of specific binding scFv-phages should be seen as increasing of target-specific signals compared to control antigens. For a typical result see [18], Figure 4A. **Note 19:** Try to pick single colonies. Sometimes picking of double colonies or a spillover from well to well might occur. Secondary screen of positive well (hit-picking) can be performed. If performing high-throughput screening on a regular basis, automation of the picking process

**Note 20:** Most universal orbital shakers for bacterial culture possess an orbit diameter of about one inch that works very well for Erlenmeyer flasks (25 ml up to 2 l) and shaking at about 200 to 250 rpm. However, growing bacterial culture in microtiter plates can be more challenging due to evaporation, contamination, and oxygen transfer. To prevent contamination, shaking

determine volume of neutralization buffer required to get a final pH of 7.2–7.4.

ment of binders having growth advantages that might occur (e.g., truncated scFvs).

using a microplate washer (e.g., BioTek Instruments, Tecan).

by colony pickers (e.g., Molecular Devices) might be considered.

bacteria.

97

http://dx.doi.org/10.5772/intechopen.70139

**Note 4:** Expression of scFv-pIII fusion proteins with *lac* promoter-based phagemid vectors can be performed by using glucose-free media without or a low concentration of IPTG (about 5 μM to 500 μM final concentration depending on used phagemid). However, strong induction of the *lac* promotor by too high concentration of IPTG might reduce the expression of complete scFv-pIII fusion proteins. Induction conditions should be optimized and antibody presentation on phages can be analyzed by SDS PAGE and Western blot using primary antipIII detection system (see Note 8).

**Note 5:** Reducing temperature to 30°C or lower guarantees better expression and folding of complete antibody fusions that otherwise might be overgrown by incomplete fusions. Moreover, lower temperature helps to reduce degradation/cleavage of antibody fragments on the phage surface.

**Note 6:** Longer incubation on ice or 4°C might result in better precipitation of antibody phage and higher yields.

**Note 7:** To save time, we perform phage pelletation in Beckman centrifuges, equipped with fixed angel rotors such as JA 16.250 that allows higher speed centrifugation in 250 ml reusable or 50 ml disposable PP tubes if using adapters. If not available, centrifugation with swinging buckets and lower speed can be performed, but centrifugation time should be increased accordingly.

**Note 8:** Helper phage/hyperphage packaged libraries can be analyzed by SDS-PAGE and Western blot. Run reduced phage samples on 10% SDS PAGE and incubate membrane with primary anti-pIII monoclonal antibody and secondary anti-mouse polyclonal serum HRP conjugate, using ECL substrate for detection. Although wild-type pIII has a calculated molecular weight of about 45 kDa, it runs at about 60 kDa in SDS-PAGE. Accordingly, complete scFv-pIII constructs can be detected at about 90 kDa.

**Note 9.** TG1 is an amber suppressor *E*. *coli* strain most widely used for antibody phage display. Growing TG1 on proline-deficient M9 minimal plates guarantees maintenance of the F' episome important for production of pili necessary for phage infection. For growing of TG1, M9 plates must be additionally supplemented with thiamine due to a chromosomal mutation in the thiamine biosynthesis.

**Note 10:** OD600 of culture is critical. Do not overgrow or grow below 34°C. Bacteria can be kept on ice for a while (30 min up to a few hours), but cells might start losing pili after longer incubations.

**Note 11:** One ml of log phase TG1 (OD600nm of 0.5) corresponds to about 5 × 108 bacteria. Always infect with at least 1:10 phage-to-cell ratio for titration, i.e., do not use more than 5 × 107 phages for 1 ml log-phase TG1.

per phage. Please note that hyperphage infection results in scFv-phages without wild-type pIII proteins that are necessary for successful infection. To restore the wild-type infectivity, hyperphage-packed libraries should be eluted by a protease cutting between the antibody fragment and the pIII protein (e.g., trypsin works well for pSEX and most pHEN derivatives). To check for suitable proteases for your phagemid, you might analyze your vector on PeptideCutter (ExPASy database) and perform Western blot analysis of digested antibody

**Note 4:** Expression of scFv-pIII fusion proteins with *lac* promoter-based phagemid vectors can be performed by using glucose-free media without or a low concentration of IPTG (about 5 μM to 500 μM final concentration depending on used phagemid). However, strong induction of the *lac* promotor by too high concentration of IPTG might reduce the expression of complete scFv-pIII fusion proteins. Induction conditions should be optimized and antibody presentation on phages can be analyzed by SDS PAGE and Western blot using primary anti-

**Note 5:** Reducing temperature to 30°C or lower guarantees better expression and folding of complete antibody fusions that otherwise might be overgrown by incomplete fusions. Moreover, lower temperature helps to reduce degradation/cleavage of antibody fragments

**Note 6:** Longer incubation on ice or 4°C might result in better precipitation of antibody phage

**Note 7:** To save time, we perform phage pelletation in Beckman centrifuges, equipped with fixed angel rotors such as JA 16.250 that allows higher speed centrifugation in 250 ml reusable or 50 ml disposable PP tubes if using adapters. If not available, centrifugation with swinging buckets and lower speed can be performed, but centrifugation time should be increased

**Note 8:** Helper phage/hyperphage packaged libraries can be analyzed by SDS-PAGE and Western blot. Run reduced phage samples on 10% SDS PAGE and incubate membrane with primary anti-pIII monoclonal antibody and secondary anti-mouse polyclonal serum HRP conjugate, using ECL substrate for detection. Although wild-type pIII has a calculated molecular weight of about 45 kDa, it runs at about 60 kDa in SDS-PAGE. Accordingly, complete

**Note 9.** TG1 is an amber suppressor *E*. *coli* strain most widely used for antibody phage display. Growing TG1 on proline-deficient M9 minimal plates guarantees maintenance of the F' episome important for production of pili necessary for phage infection. For growing of TG1, M9 plates must be additionally supplemented with thiamine due to a chromosomal mutation

**Note 10:** OD600 of culture is critical. Do not overgrow or grow below 34°C. Bacteria can be kept on ice for a while (30 min up to a few hours), but cells might start losing pili after longer

phages using an anti-pIII antibody for detection (see Note 8).

scFv-pIII constructs can be detected at about 90 kDa.

in the thiamine biosynthesis.

pIII detection system (see Note 8).

on the phage surface.

and higher yields.

96 Antibody Engineering

accordingly.

incubations.

**Note 12:** If enough antigen is available, increase volume of coating solution up to 5 ml, especially in the first round of selection. Only use highly pure (>90%), freshly prepared protein from sources you can trust. Do not use proteins that have been stored at 4°C for prolonged periods. If using oligopeptides or virions for selection, immobilization condition should be optimized due to lower coating efficacies and/or reduced accessibility of epitopes. Test different buffer/pH/additives for immobilization on MaxiSorp™ plates (e.g., 50 mM carbonate buffer pH 9.6) using tag-specific antibody enzyme conjugates for detection in ELISA. Alternatively, antigens might be biotinylated and immobilized on streptavidin-coated tubes/ plates or be captured in solution using streptavidin dynabeads [31].

**Note 13:** If using proteins with large tags/fusions (e.g., Fc region, GST tag) for antibody selection, supplement the preincubation solution with respective proteins to reduce enrichment of binders against those parts. If using biotinylated oligopeptides immobilized on streptavidincoated immunotubes, preincubate phages with streptavidin to deplete streptavidin-specific binders.

**Note 14:** To check pH of final solution, perform microtitration using pH indicator strips to determine volume of neutralization buffer required to get a final pH of 7.2–7.4.

**Note 15:** Always elute hyperphage-packaged libraries by protease elution to guarantee highest infectivity of eluted phages.

**Note 16:** We usually perform three rounds of selection, but this might be increased up to five or more. However, performing additional rounds might lead to loss of diversity and enrichment of binders having growth advantages that might occur (e.g., truncated scFvs).

**Note 17:** Washing can be done by hand using a small plastic box filled with buffer. Remove all liquid by inverting the plates and hitting it onto paper towels. Change buffer and towels frequently. For better reproducibility and when screening larger amounts of plates, wash plates using a microplate washer (e.g., BioTek Instruments, Tecan).

**Note 18:** Successful enrichment of specific binding scFv-phages should be seen as increasing of target-specific signals compared to control antigens. For a typical result see [18], Figure 4A.

**Note 19:** Try to pick single colonies. Sometimes picking of double colonies or a spillover from well to well might occur. Secondary screen of positive well (hit-picking) can be performed. If performing high-throughput screening on a regular basis, automation of the picking process by colony pickers (e.g., Molecular Devices) might be considered.

**Note 20:** Most universal orbital shakers for bacterial culture possess an orbit diameter of about one inch that works very well for Erlenmeyer flasks (25 ml up to 2 l) and shaking at about 200 to 250 rpm. However, growing bacterial culture in microtiter plates can be more challenging due to evaporation, contamination, and oxygen transfer. To prevent contamination, shaking at a lower speed (about 180 rpm) can be performed although oxygen supply is reduced as well. Alternatively, PP deep well plates can be used for screening. To prevent evaporation, plates can be tape-fixed in an additional plastic bag or use PBS-filled plates at the bottom and top when using microplate holders. The highest yield of soluble scFvs can be obtained with special temperature-controlled shakers with a small orbital diameter (0.12 inch) and shaking at high speed (1000 rpm).

formed. PRNT assay can be adapted to plates with higher well number (e.g., 12–24 well/ plates) by reducing pfu and volumes used. Presented protocol was optimized for PRNT with HSV and Vero cells. Cell line and optimal conditions are different for each virus and should be optimized for best results (e.g., scFv-virus incubation time, absorption time on cells, percentage of CMC for overlay, working virus dilution and volume, incubation time after infection). **Note 29:** Since performing of the PRNT is laborious and time-consuming, using a single concentration of scFvs for neutralization usually allows a good head-to-head comparison to identify best neutralizing scFvs. In a second step, most promising scFvs can be cloned into other (bivalent) Ab formats (e.g., scFv-Fc) and tested in more detail by analyzing PRNT50 (or more

Detailed Protocols for the Selection of Antiviral Human Antibodies from Combinatorial Immune...

http://dx.doi.org/10.5772/intechopen.70139

99

**Note 30:** The temperature of melted top agar should be exactly at 42°C before casting. Higher temperatures might kill *E*. *coli* bacteria, while at lower temperatures, the top agar might solid-

**Note 31:** Although helper phage supernatant can be directly used after heat treatment, we recommend purifying and concentrating by one or two steps of PEG/NaCl precipitation.

**Note 32:** Using 0.22 μm filter for phage filtration is not recommended due to loss of filamentous phages. If clogging of 0.45 μm filter occurs, centrifuge phage preparation at high g-force

bacteria for infection. Avoid additional freeze-and-thaw cycles of helper phage preparations. Titer of frozen helper phage might drop over time during storage and titration should be repeated after several months. Sterile glycerol up to a final concentration of 50% prior to snap freezing can be added to prolong storage time. Review Ref. [33] for more information about

We thank Buck Rogers and Cedric Mpoy for discussions and corrections on the manuscript.

\*

1 Department of Radiation Oncology, Washington University School of Medicine, St. Louis,

2 Institute for Virology, University Hospital Essen, University of Duisburg-Essen, Essen,

and Adalbert Krawczyk<sup>2</sup>

\*Address all correspondence to: adalbert.krawczyk@uni-due.de

phage dilution guarantees an at least tenfold excess of helper phage to

precisely even PRNT70 or PRNT90).

at 4°C prior to filtration.

correct storage of phages.

**Acknowledgements**

**Author details**

Philipp Diebolder1

USA

Germany

**Note 33:** Using 108

ify too fast before pouring nice layers on plates.

**Note 21:** Usually, antigen-specific binding is defined as signals at least five times higher than the background. For a typical result, see [18], Figure 4B and C.

**Note 22:** The 2% glucose guarantees repression of antibody expression within the first 3 h of growing. To induce antibody expression, medium must be replaced by induction medium without glucose and optimized concentration of IPTG. Many scFvs show very low yield in the supernatant after expression in *E*. *coli* and periplasmic extraction is highly recommend prior to ELISA, e.g., by resuspending bacteria pellets in 180 μl/well periplasmic preparation buffer (see Protocol G) for 30 min on ice. However, as shown by Hust et al. [32], buffered 2xYT-SAI and growing cultures overnight at 30°C can improve production in *E*. *coli* for some scFvs even without performing periplasmic extraction.

**Note 23.** Adjusting PCR conditions to obtain single scFvs bands on agarose gel is important for successful sequencing of PCR fragments, e.g., by increasing annealing temperature. Alternatively, scFv bands can be recovered by QIAquick Gel Extraction Kit or sequencing can be done using plasmid DNA (see F10).

**Note 24:** Plasmid DNA can be used for electroporation into self-made electrocompetent HB2151 if scFv-phage screening with TG1 bacteria was performed beforehand. If already in HB2151 after soluble screening, the glycerol stock can be used to obtain single colonies on 2xYT-GA plates for small-scale production of soluble scFvs (see Protocol G). Alternatively, plasmid DNA can be used for subcloning into bacterial expression vectors without pIII gene.

**Note 25.** For time reasons, we recommend soluble expression of scFvs in nonamber suppressor strain such as HB2151 when using phagemid with the amber stop codon between the scFv and the pIII protein. Otherwise, scFvs can also be subcloned into expression vector without the pIII gene. If clones are still in TG1 after phage screening, phagemid DNA can be transformed into self-made competent HB2151. Use standard protocol for generation of chemically or electrocompetent HB2151. Alternatively use commercially available competent nonamber suppressor strains for soluble production (e.g., SS320 by Lucigen, or Express *Iq* by NEB).

**Note 26.** Reducing the temperature for soluble scFv expression is important for proper folding and stability of produced scFvs.

**Note 27:** As a cheaper option for dialysis in small scale, we recommend using 2 ml PP tubes without lids, filled with scFv preparations, and sealed with square cut dialysis membrane fixed with rubber band and parafilm. If using other tags for purification, dialyze in recommended buffer prior purification.

**Note 28:** Determine best number/growing conditions for your cell line. Gently tilt plate about five times horizontally after seeding to guarantee that uniform monolayers of cells are formed. PRNT assay can be adapted to plates with higher well number (e.g., 12–24 well/ plates) by reducing pfu and volumes used. Presented protocol was optimized for PRNT with HSV and Vero cells. Cell line and optimal conditions are different for each virus and should be optimized for best results (e.g., scFv-virus incubation time, absorption time on cells, percentage of CMC for overlay, working virus dilution and volume, incubation time after infection).

**Note 29:** Since performing of the PRNT is laborious and time-consuming, using a single concentration of scFvs for neutralization usually allows a good head-to-head comparison to identify best neutralizing scFvs. In a second step, most promising scFvs can be cloned into other (bivalent) Ab formats (e.g., scFv-Fc) and tested in more detail by analyzing PRNT50 (or more precisely even PRNT70 or PRNT90).

**Note 30:** The temperature of melted top agar should be exactly at 42°C before casting. Higher temperatures might kill *E*. *coli* bacteria, while at lower temperatures, the top agar might solidify too fast before pouring nice layers on plates.

**Note 31:** Although helper phage supernatant can be directly used after heat treatment, we recommend purifying and concentrating by one or two steps of PEG/NaCl precipitation.

**Note 32:** Using 0.22 μm filter for phage filtration is not recommended due to loss of filamentous phages. If clogging of 0.45 μm filter occurs, centrifuge phage preparation at high g-force at 4°C prior to filtration.

**Note 33:** Using 108 phage dilution guarantees an at least tenfold excess of helper phage to bacteria for infection. Avoid additional freeze-and-thaw cycles of helper phage preparations. Titer of frozen helper phage might drop over time during storage and titration should be repeated after several months. Sterile glycerol up to a final concentration of 50% prior to snap freezing can be added to prolong storage time. Review Ref. [33] for more information about correct storage of phages.
