**3. Materials**

#### **A. Preparation of scFv-phages**


#### **B. Determination of the phage titer**

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].

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

• 2xTY medium: 16 g/l tryptone, 10 g/l yeast extract, 5 g/l NaCl, dissolved in ultrapure water,

• 2xYT-GA: 900 ml 2xYT medium, supplemented with 100 ml glucose stock solution and

• Kanamycin stock solution (2000×): 100 mg/ml kanamycin sulfate dissolved in ultrapure

• Ampicillin stock solution (1000×): 100 mg/ml ampicillin sodium salt dissolved in ultrapure

• 20% (w/v) glucose stock solution (10×): 200 g/l d-glucose dissolved in ultrapure water, fil-

• Helper phage VCSM13 (Agilent Technologies) or M13K07 (e.g., NEB Biolabs), Kanamycin-

• 2xYT-GK 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 500 μl kanamycin stock solution right before pour-

• 1 M IPTG stock solution (20,000×): 2.38 g isopropyl β-d-1-thiogalactopyranoside dissolved

• Optional for oligomeric display: hyperphage M13 K07ΔpIII (Progen Biotechnik).

in 10 ml ultrapure water, filter-sterilized, stored at −20°C.

bovine serum albumin.

**A. Preparation of scFv-phages**

autoclaved, and stored at room temperature (RT).

1 ml ampicillin stock solution right before use.

water, filter-sterilized, stored at −20°C.

water, filter-sterilized, stored at −20°C.

ter-sterilized, stored at 4°C.

resistant.

ing, stored at 4°C.

**3. Materials**

80 Antibody Engineering


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


• Phage neutralization buffer: 1 M Tris-HCl dissolved in ultrapure water, adjusted to pH 9.5, filter-sterilized, stored at 4°C.

• Potassium phosphate buffer: 0.17 M KH<sup>2</sup>

phate buffer, filter-sterilized, stored at 4°C.

search): 1:10,000 diluted in MPBS.

water, autoclaved, stored at RT.

• 2xYT-glycerol: see E-I

• PBS: see C.

• HB2151 bacterial strain: see E-II.

• M9/+Thi minimal plates, MgSO<sup>4</sup>

diameter) by EMD Millipore.

quick Gel Extraction Kit (optional): all from Qiagen.

• 96-well PCR plate and sealing (e.g., BRAND).

sterilized, stored at 4°C.

in MPBS.

PO<sup>4</sup>

• Buffered 2xYT (pH 7.0): 900 ml 2xYT medium supplemented with 100 ml potassium phos-

• Induction medium 2xYT-SAI: buffered 2xYT supplemented with 50 ml sucrose stock solution, 1 ml ampicillin stock solution, and 50 μl IPTG stock solution (IPTG concentration

• Primary scFv-tag-specific monoclonal antibody (e.g., anti-myc, anti-His): 5 μg/ml diluted

• Secondary anti-primary polyclonal antibody HRP conjugate (e.g., Jackson ImmunoRe-

• LB high salt medium: 10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl, dissolved in ultrapure

• Taq PCR Core Kit, QIAquick PCR Purification Kit, QIAprep Spin Miniprep Kit, and QIA-

• Colony PCR/sequencing primers flanking the scFv insert (most pHEN phagemids):

P1 (LMB3long, forward) 5′-CAGGAAACAGCTATGACCATGATTAC-3′; and P2 (fdseqlong, reverse) 5′-GACGTTAGTAAATGAATTTTCTGTATGAGG-3′.

• Sterile 96-well PP round-bottom microplates (e.g., Greiner Bio-One).

**G. Small-scale expression of soluble scFvs for functional characterization**

• 2xYT-GA, 2xYT-GK agar plates (100 mm), IPTG stock solution: see A.

ultrapure water, adjusted to pH 8.0, stored at 4°C.

stock solution: see B.

• Induction medium 2xYT-GA with 0.1% glucose: 995 ml 2xYT medium, supplemented with

• Periplasmic preparation buffer: 200 g/l sucrose, 30 mM Tris-HCl, 1 mM EDTA, diluted in

• Low protein-binding sterile syringe filters (0.22 μm): e.g., Millex-GV Filter (PVDF, 4 mm

5 ml glucose stock solution and 1 ml ampicillin stock solution right before use.

depending on phagemid vector used), prepared right before use.

**F. Identification of complete scFv fragments by colony PCR and sequencing**

, 0.72 M K<sup>2</sup>

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

HPO<sup>4</sup>

, adjusted to pH 7.0, filter-

83

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

• Trypsin-PBS solution: 10 μg/ml trypsin (e.g., from porcine pancreas by Sigma Aldrich) dissolved in PBS, pH 7.4, prepared right before use.

#### **D. Polyclonal phage ELISA (ppELISA)**


#### **E. Screening for monoclonal binders**

#### *(I) Monoclonal phage (mpELISA) screening:*


#### *(II) Soluble scFv screening:*


#### **F. Identification of complete scFv fragments by colony PCR and sequencing**


P2 (fdseqlong, reverse) 5′-GACGTTAGTAAATGAATTTTCTGTATGAGG-3′.


• Phage neutralization buffer: 1 M Tris-HCl dissolved in ultrapure water, adjusted to pH 9.5,

• Trypsin-PBS solution: 10 μg/ml trypsin (e.g., from porcine pancreas by Sigma Aldrich) dis-

• Murine anti-M13 monoclonal antibody HRP-conjugate (GE Healthcare), diluted 1:5000 in

• 2xYT-glycerol: 2xYT dissolved in 50% glycerol and 50% ultrapure water, autoclaved, stored

• 2xYT-GA medium, ampicillin stock solutions, glucose stock solution, kanamycin stock solution, IPTG stock solution, helper phage VCSM13 or M13K07, and induction medium

• Maxisorb™ ELISA plates, anti-M13 monoclonal HRP-conjugate, TMB substrate, and stop

• Sterile 96-well PP round-bottom microplates, breathable sealing membrane, microplate

• 1 M sucrose stock solution: 342.3 g/l dissolved in ultrapure water, filter-sterilized, stored

): concentrated sulfuric acid dissolved 1:9 in ultrapure water.

• 96-well microtiter ELISA plate (e.g., Nunc Maxisorp™ by Thermo Fisher Scientific).

• Sterile 96-well polypropylene round-bottom microplates (e.g., Greiner Bio-One).

• Breathable sealing membrane for microplates (e.g., Sigma Aldrich).

filter-sterilized, stored at 4°C.

• PBS and PBST: see C.

• Stop solution (2 M H<sup>2</sup>

MPBS.

82 Antibody Engineering

at RT.

2xYT-AKI: see A. • PBS, PBST: see C.

solution: see D.

at 4°C.

*(II) Soluble scFv screening:*

**D. Polyclonal phage ELISA (ppELISA)**

**E. Screening for monoclonal binders**

*(I) Monoclonal phage (mpELISA) screening:*

• 8-channel pipettes (20 μl and 200 μl).

• HB2151 bacterial strain (Nordic BioSite).

replicator, 8-channel pipettes: see E-I.

• M9/+Thi minimal plates, 2xYT-GA plates: see B.

• 2xYT medium, ampicillin stock solution, glucose stock solution: see A.

solved in PBS, pH 7.4, prepared right before use.

• TMB substrate solution (e.g., Thermo Fisher Scientific).

SO<sup>4</sup>

• 96-pin microplate replicator (Boekel Scientific).

#### **G. Small-scale expression of soluble scFvs for functional characterization**


• Dialysis: D-Tube™ Dialyzer (MWCO 12–14 kDa) by EMD Millipore or dialysis membrane (e.g., Spectra/Por 4 dialysis membrane, MWCO 12–14 kDa, by Spectrum Laboratories).

libraries of large library size up to 109

typically takes about 2½ h (**Note 2**).

bacteria lawn the next day.

free induction medium 2×YT-AKI (**Note 4**).

bate for at least 1 h on ice (**Note 6**).

remaining solution (**Note 7**).

**A6.** Incubate culture overnight shaking at ≤30°C (**Note 5**).

the antibody phages into fresh 50 ml PP tubes (40 ml per tube).

virus-infected donors, an initial library size of 107

independent bacteria clones. The inoculated volume

and inoculating 100 ml is sufficient (**Note 1**).

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

85

depends on library diversity and might vary between 50 ml and > 2 l. For libraries cloned from

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

**A2.** Grow bacteria at 250 rpm at 37°C until they reach log phase (OD600nm of about 0.5). This

**A3.** Infect the log phase bacteria culture by adding freshly thawed helper phage VCSM13 or M13K07 (for preparation of helper phage: see Protocol I) with a multiplicity of infection of 20:1 (phage-to-cell-ratio). Infection is best performed by swirling the flask to distribute phages, followed by 30 min standing and 30 min shaking at 250 rpm and 37°C. Exclusively in the first round of selection, infection of the antibody library can be done by hyperphage M13

**A4.** Superinfection of bacterial culture can be monitored by plating 1 μl of the infected bacteria (diluted in 100 μl 2xYT) onto 2xYT-GK agar plates. Successful infection should result in a

**A5.** To induce expression of scFv-pIII fusion proteins, harvest bacteria by centrifugation (4000 × *g*, 10 min, 4°C) in PP tubes (either 50 or 250 ml) and resuspend bacteria in glucose-

**A7.** On the next day, pellet bacteria (4000 × *g*, 10 min, 4°C) and transfer supernatant containing

**A8.** Add 8 ml of prechilled PEG/NaCl to 40 ml supernatant (1/5 volume). Mix well and incu-

**A9.** Harvest phages by centrifugation (10,000 × *g*, 20 min, 4°C). Make sure to remove PEG/ NaCl completely since remaining PEG leads to losing phages in the next step. Therefore, pour away the PEG solution and remove residuals with gauze or centrifuge again and aspirate

**A10.** Pure phage preparation gives white pellets. Brownish pellets indicate contamination with bacteria debris. One (*Option 1*) or two (*Option 2*) precipitation steps may be performed.

*Option 1*: Resuspend phage pellets in 1 ml of PBS per tube transfer in 2 ml tubes and centrifuge at high speed in a microcentrifuge (3 min, 4°C). Transfer the phage-containing supernatant into a fresh tube and determine the phage titer as colony-forming units (see Protocol B).

*Option 2*: Resuspend the phage pellets in 40 ml of ice cold PBS and pellet the bacteria by centrifugation (4000 × *g*, 10 min, 4°C). Save the supernatant and precipitate a second time (8 ml PEG/NaCl to 40 ml supernatant) for ≥1 h on ice or overnight. Proceed as described in *Option 1*. **A11.** Store the phage at 4°C and proceed as soon as possible with scFv selection. *Optional*: filter supernatant through 0.45 μm filter. Filtered phages may be stored up to 2 weeks. To prevent proteolysis of the antibody fragments, proteolysis inhibitors might be added. Although not recommended for selection since displayed antibody fragment might be denatured,

Especially in the first round of selection, we recommend to precipitate twice.

K07ΔpIII for oligomeric display of scFvs on the phage surface (**Note 3**).


#### **H. Functional antibody characterization by plaque reduction neutralization test**


#### **I. Production of helper phage**

