**2. Preliminary notes**

for displaying antibody fragments on the phage surface have been developed over time using different vectors and phage coat proteins for display. The most common type (3 + 3 system) is based on phagemid vectors where the antibody gene fragments are cloned as fusions with the pIII phage gene. Cloning of the antibody gene repertoires can be done by using different strategies in one, two, or three independent steps where the variable light and heavy chain genes are PCR-amplified and randomly combined into reliable phagemid vectors. In the-one step cloning strategy, the VH and VL genes are separately amplified with an overlapping, additional linker sequence and combined by assembly PCR [19]. In the two-step cloning strategy, mostly the VL gene repertoire is cloned first into the phagemid followed by insertion of

activity in functional assays like the plaque reduction neutralization test (PRNT) (see Protocol H).

78 Antibody Engineering

**Figure 2.** Workflow of the selection and screening procedure. For the selection of antigen-specific scFv-phages, logphase library cultures are packed by superinfection with helper phages (for the preparation of helper phages, see Protocol I) that provide all proteins necessary for phage propagation (see Protocol A). After IPTG induction, expressed scFv-pIII fusions are inserted within the produced phages leading to the presentation of scFv antibody fragments on the phage surface. After determination of phage titer (see Protocol B), specific binding scFv-phages are enriched over several selection rounds by stringent washing and elution on recombinant antigen/virions that have been immobilized onto immunotubes (see Protocol C). Successful enrichment of specific binding scFv-phages can be analyzed by polyclonal phage ELISA (ppELISA) (see Protocol D) prior to screening of monoclonal antibodies as scFv-phages by monoclonal phage ELISA (mpELISA) (see Protocol E-I) or as soluble scFvs (see Protocol E-II). After the identification of bacterial colonies encoding for full-length scFvs by colony PCR and sequencing (see Protocol F), soluble scFvs can be produced in the periplasm of bacteria (see Protocol G) or variable antibody genes can be cloned into mammalian expression vectors to produce Fab or various bivalent antibody fragments. Finally, antibody fragments can be analyzed for their neutralizing

Before starting antibody phage display, please be aware that phages are highly stable and decontamination of workspace and consumables is hard to achieve. It is best to do phage work in a special lab keeping equipment/material separated from the common bacterial workspace, especially when antibody library construction is performed. If not possible, phage work should be carried out in at least a separate workspace including a separate hood, shaker, and centrifuge. Inactivation of phage solutions can be done by incubation with diluted bleach (caution, always wear personal protection during handling) and/or sterilizing workspaces with UV light. For decontamination of tubes and Erlenmeyer flasks, bleach can be added to water-filled tubes and incubated overnight before washing, rinsing, and autoclaving. In common, single-use material is preferred for phage work. Collect phage-contaminated solutions in glass flasks and inactivate by adding bleach before dumping. Only use polypropylene (PP) tubes since phages might stick to other kinds of plastics. To prevent contamination to pipettes, always use barrier tips.

Presented protocols are intended for the selection and screening of antibody libraries based on the scFv antibody format being cloned in phagemid vectors as pIII fusion with an intrinsic amber stop codon and under the *lac* promoter (inducible by IPTG, repressible by glucose). Many current antibody phage display libraries are constructed in phagemid vectors with listed features (e.g., most derivatives of pHEN, pComb3X, pHAL, and pCANTAB), although other selection relevant features such as signal peptides, molecular tags, etc., might differ. Please check features of your antibody library used prior to selection and screening and change protocols accordingly if necessary. If using libraries based on phagemid vectors with *lac* promoter, **always add ≥2% glucose** to the media to repress scFv-pIII protein expression as long as antibody phages are not produced. Lower amounts of glucose results in background expression of pIII fusions and clones with growth advantages (e.g., truncated scFvs) might overgrow leading to a reduced library diversity. More detailed information about antibody phage display [24] and commonly used phagemid vectors can be found elsewhere [25].

• Induction medium 2xYT-AKI: 2xYT supplemented with 1 ml ampicillin stock solution, 500 μl kanamycin stock solution, and 50 μl IPTG stock solution (IPTG concentration depending on

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

• PEG/NaCl solution: 200 g/l polyethylene glycol 6000, 146.1 g/l NaCl, dissolved in ultrapure

• Optional for Western blot analysis: primary murine anti-pIII monoclonal IgG (MoBiTec, diluted 1:1000 milk in phosphate-buffered saline (MPBS)) and secondary goat anti-mouse

• M9 minimal stock solution (5×): 56.4 g/l M9 minimal salt (Sigma Aldrich) dissolved in ul-

• Thiamine stock solution: 50 mg thiamine hydrochloride dissolved in 50 ml ultrapure water,

• M9/+Thi minimal plates (100 mm): 15 g agar dissolved in 780 ml ultrapure water, autoclaved and supplemented with 200 ml M9 minimal salt stock solution, 20 ml glucose stock

• 2xYT-GA agar plates (100 mm): 16 g tryptone, 10 g yeast extract, 5 g NaCl, and 15 g agar dissolved in 900 ml ultrapure water, autoclaved, cooled down to 50°C, and supplemented with 100 ml glucose stock solution and 1 ml ampicillin stock solution right before pouring,

NaCl, dissolved in ultrapure water, adjusted to pH 7.4, autoclaved, stored at RT.

• MPBS: 2% nonfat dry milk in phosphate-buffered saline, prepared right before use.

• 2xYT-GA agar plates (100 mm round, 150 mm round, or 245 mm square): see B.

• Phage elution buffer: 0.1 M glycine-HCl, 0.5 M NaCl, dissolved in ultrapure water, adjusted

solution, and 1 ml thiamine stock solution.

HPO<sup>4</sup>

, 0.24 g/l KH<sup>2</sup>

PO<sup>4</sup>

, 0.2 g/l KCl, 8.0 g/l

dissolved in 50 ml ultrapure water, filter-sterilized,

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81

• Polypropylene (PP) centrifugation tubes: 2 and 50 ml (single-use), 250 ml (reusable).

HRP-conjugated antibody (Jackson ImmunoResearch, diluted 1:10,000 in MPBS).

phagemid vector used), no glucose (!), prepared right before use.

water, autoclaved, stored at 4°C.

**B. Determination of the phage titer**

• TG1 bacteria strain (e.g., Lucigen).

filter-sterilized, stored at −20°C.

solution (see A), 1 ml MgSO<sup>4</sup>

**C. Selection of antigen-specific scFv-phages**

• TG1 bacteria strain (e.g., Lucigen).

to pH 2.2, filter-sterilized, stored at 4°C.

• Phosphate-buffered saline (PBS): 1.42 g/l Na<sup>2</sup>

• 5 ml Nunc MaxiSorp™ immunotubes (Thermo Fisher Scientific).

• PBST: phosphate-buffered saline supplemented with 0.1% Tween20.

• 1 M MgSO<sup>4</sup>

stored at 4°C.

stored at 4°C.

trapure water, autoclaved, stored at 4°C.

stock solution: 6.02 g MgSO<sup>4</sup>

Independent of the source of your antibody library, always try to package antibody libraries from primary bacteria stocks, never from secondary stocks or using phage-packaged libraries to infect bacteria. Only correctly stored (−80°C) primary glycerol bacteria stocks guarantee highest initial antibody diversity. For novel libraries or if not familiar with antibody phage display, perform test selection and subsequent screening using not relevant proteins such as bovine serum albumin.
