**4. Protocols**

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

Cloned scFv antibody libraries are stored at −80°C as bacteria glycerol stocks and has to be packed into scFv-phages for selection using target antigen or virus stock (see Protocol C). The following protocol describes the superinfection of a log phase library bacterial culture with helper phage comprising kanamycin resistance (see Section I for the production of helper phages) and PEG purification of produced scFv-phages.

**A1.** Inoculate prewarmed 2xYT-GA to an initial OD600nm of 0.1 with freshly thawed library glycerol stock (~1 ml). Typically, we inoculate 250 ml in a 1 L baffled Erlenmeyer flask for libraries of large library size up to 109 independent bacteria clones. The inoculated volume depends on library diversity and might vary between 50 ml and > 2 l. For libraries cloned from virus-infected donors, an initial library size of 107 and inoculating 100 ml is sufficient (**Note 1**).

**A2.** Grow bacteria at 250 rpm at 37°C until they reach log phase (OD600nm of about 0.5). This typically takes about 2½ h (**Note 2**).

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

**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 bacteria lawn the next day.

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

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

• 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). • Lysozyme solution: 50 mg/ml lysozyme, in ultrapure water, prepared right before use.

• Complete culture medium: e.g., Vero cells, 10% heat-inactivated fetal bovine serum (FBS)

• Carboxymethyl cellulose (CMC) medium: 2/3 volume of sterile complete culture medium

• CMC solution: 20 g/l carboxymethyl cellulose sodium salt dissolved in PBS (added slowly

• Crystal violet solution: 200 mg/l crystal violet dissolved in 1 ml ethanol, filled up to 1 l with

• 5% formaldehyde solution: 135 ml/l formaldehyde stock solution (37%), diluted in PBS,

• 2xYT-agar plates (100 mm): 16 g/l tryptone, 10/l g yeast extract, 5/l g NaCl, and 15/l g agar

• 2xYT medium, helper phage VCSM13 or M13K07, kanamycin stock solution, PEG/NaCl

Cloned scFv antibody libraries are stored at −80°C as bacteria glycerol stocks and has to be packed into scFv-phages for selection using target antigen or virus stock (see Protocol C). The following protocol describes the superinfection of a log phase library bacterial culture with helper phage comprising kanamycin resistance (see Section I for the production of helper

**A1.** Inoculate prewarmed 2xYT-GA to an initial OD600nm of 0.1 with freshly thawed library glycerol stock (~1 ml). Typically, we inoculate 250 ml in a 1 L baffled Erlenmeyer flask for

dissolved in ultrapure water, autoclaved, poured as thin layers, stored at 4°C.

, 300 mM NaCl, 10 mM imidazole, dissolved in ultrapure

PO<sup>4</sup>

(optional: supplemented with penicillin/streptomycin solution).

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

• Sterile cell culture plates (6-well, 12-well, and 48-well): e.g., by Greiner Bio-One.

water, adjusted pH to 8.0, filter-sterilized, stored at 4°C.

and 1/3 volume of CMC solution, stored at 4°C.

• Top-agar: 2xYT supplemented with 7.5 g/l agar, autoclaved.

• TG1 bacteria strain, M9/+Thi minimal plates: see B.

phages) and PEG purification of produced scFv-phages.

under stirring), autoclaved, stored at RT.

ultrapure water, stored at RT.

**I. Production of helper phage**

**A. Preparation of scFv-phages**

stored at RT.

solution: see A.

**4. Protocols**

• NPI-10 buffer: 50 mM NaH<sup>2</sup>

84 Antibody Engineering

• Ni-NTA Spin Columns (Qiagen).

**A7.** On the next day, pellet bacteria (4000 × *g*, 10 min, 4°C) and transfer supernatant containing the antibody phages into fresh 50 ml PP tubes (40 ml per tube).

**A8.** Add 8 ml of prechilled PEG/NaCl to 40 ml supernatant (1/5 volume). Mix well and incubate for at least 1 h on ice (**Note 6**).

**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 remaining solution (**Note 7**).

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

*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, long-term storage of packaged library can be done by adding sterile glycerol (15% final concentration) and freezing at −80°C (**Note 8**).

**C3.** The next day, wash tube three times with PBS by filling the tube with PBS using a wash

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**C4.** Block the remaining binding sites by filling the empty immunotube completely with

**C5.** In parallel, preincubate packaged library (1012 to 1013 cfu) or phages from subsequent rounds (1011 to 1012 cfu) in MPBS (2–4% final milk concentration) for at least 1 h by slow overhead rotation in PP tubes (volume depends on volume used for coating immunotubes). Especially in the first round of selection, use 100-fold excess of scFv-phages compared with

**C6.** Inoculate 20 ml of 2xYT with 200 μl of overnight culture and grow culture until the logphase is reached (OD600 of about 0.5). This will take about 2½ h. Log phase TG1 can be stored on ice until infection with eluted phage. *Optional*: Check for infection of overnight TG1 culture

**C7.** Empty the immunotube, add the preblocked phage solution, seal it with parafilm, and

**C8.** In the last 30 min of incubation, start washing the immunotubes. In the first selection round, wash 10× (5× with PBST and 5× with PBS). In the following rounds, increase stringency of washing by adding more wash cycles (second round: 20× cycles, third round: 30× cycles,

**C9.** Elution of antigen-bound scFv-phages can be done by different methods. We prefer either

*Option 1* (*acid elution*): completely remove remaining buffer, add 1 ml acid elution buffer (pH 2.2) and incubate for about 8 min (longer incubation can destroy the phage). Rotate sealed tubes in an overhead rotator if using >1 ml for coating. Pipette eluted phage to about 100 μl

*Option 2* (*protease elution*): many common phagemid vectors possess a trypsin cleavage site between the scFv and pIII protein (e.g., all phagemids containing the myc or FLAG tag) that can be used for elution. Add 1 ml freshly prepared trypsin-PBS to the tubes and incubate for 10 min standing (1 ml coated tubes) or overhead rotation (>1 ml coated tubes) (**Note 15**).

**C10.** Per selection, inoculate 14 ml of log-phase TG1 culture with the eluted phage solution in fresh 50 ml tubes. Infect at 37°C by 30 min standing and 30 min shaking. *Optional*: fill the empty immunotube with 5 ml of log-phase TG1 and perform infection at 37°C as described

**C11.** To monitor the success of an antibody selection, the eluted phage titer can be determined after each selection round. Therefore, make serial dilutions of the TG1 culture after infection

Incubate plates at 30°C overnight and count colonies to determine the eluted phage titer after each round. Typically for successful enrichment of specific binders, the eluted phage titer

, 10<sup>4</sup>

, and 106

in the first round up to the total number used for bacterial

dilutions onto 2xYT-GA plates).

incubate for 90 min gently shaking followed by 30 min standing at the bench.

bottle and removing the liquid immediately by pouring.

final library size to increase capturing promising binders (**Note 13**).

MPBS to the brim and incubate for about 2 h at RT.

by plating 100 μl on 2xYT-GA and 2xYT-GK plates.

acid elution (*Option 1*) or protease elution (*Option 2*).

neutralization buffer in a novel PP tube (**Note 14**).

(e.g., by plating 100 μl of undiluted culture, 10<sup>2</sup>

should increase from about 10<sup>4</sup>

infection in subsequent rounds (~1010).

above. After infection, combine both cultures for following steps.

–106

etc.).

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

Prior to selection, titer of produced scFv-phages should be determined. Phage titration can be done by different methods including counting plaque-forming units (pfu) on bacterial lawns or counting the total particle number. More easily, phage titer is determined as colony-forming units (cfu) by easy countable bacteria colonies on selection plates as described below.

**B1.** Streak out TG1 from frozen, uninfected bacteria stock onto minimal M9/+Thiamine plates (**Note 9**).

**B2.** Inoculate 5 ml of 2xYT with grown single colony of TG1 and grow overnight (250 rpm, 37°C).

**B3.** On the next day, inoculate 10 ml of 2xYT with 100 μl of overnight culture and grow culture until the log-phase is reached (OD600 of about 0.5). This will take about 2½ h. *Optional*: Check overnight TG1 cultures for infection by plating 100 μl/plate on 2xYT-GA agar and 2xYT-GK agar. No colonies should grow (**Note 10**).

**B4.** In parallel, prepare serial dilution for phage titration. Expect about 1013 to 1014 cfu/ml out of 1 l culture. To guarantee that all phages can infect bacteria, prepare serial dilutions of phage in PBS by diluting 10 μl into 990 μl (10<sup>2</sup> , 10<sup>4</sup> , and 106 ). Pipette 10 μl of the 106 dilution to 990 μl of the log-phase TG1 culture (108 dilution) and infect bacteria at 37°C (30 min standing, 30 min shaking) (**Note 11**).

**B5.** After infection, prepare up to five 1:10 serial dilutions (100–900 μl) of infected bacteria in 2xYT medium. Plate 100 μl of infected bacteria and dilutions onto 2xYT-GA plates. Additionally, plate 100 μl of TG1 culture as negative controls on 2xTY-GA and 2xYT-GK plates. Incubate plates overnight at 30°C. Alternatively, spot 10 μl/dilution in triplicates onto 2xYT-GA plates and dry before overnight incubation.

**B6.** The next day, count colonies on countable plates and calculate phage titer as colonyforming units (cfu/ml). If colonies are too small to count, increase temperature to 37°C. No colonies should be visible on control plates.

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

The following protocol describes the selection of target-specific scFv-phages using recombinant viral protein or virions immobilized on immunotubes. Usually ≥3 selection rounds are performed to enrich specific binding scFv-phages.

**C1.** For the first round of selection, coat an immunotube with 10–50 μg protein dissolved in 1 ml PBS and incubate the parafilm-sealed tube overnight at 4°C. For subsequent rounds, decrease the protein concentration for more stringent conditions (1–5 μg/ml). If oligopeptides are used for the selection procedure, coat 2–5 μg/ml for all rounds. Alternatively, immunotubes might be coated with a virus stock if the target antigen is unknown (**Note 12**).

**C2.** On the same day, prepare uninfected TG1 overnight culture as described (see B1–B3).

**C3.** The next day, wash tube three times with PBS by filling the tube with PBS using a wash bottle and removing the liquid immediately by pouring.

long-term storage of packaged library can be done by adding sterile glycerol (15% final con-

Prior to selection, titer of produced scFv-phages should be determined. Phage titration can be done by different methods including counting plaque-forming units (pfu) on bacterial lawns or counting the total particle number. More easily, phage titer is determined as colony-forming

**B1.** Streak out TG1 from frozen, uninfected bacteria stock onto minimal M9/+Thiamine plates

**B2.** Inoculate 5 ml of 2xYT with grown single colony of TG1 and grow overnight (250 rpm,

**B3.** On the next day, inoculate 10 ml of 2xYT with 100 μl of overnight culture and grow culture until the log-phase is reached (OD600 of about 0.5). This will take about 2½ h. *Optional*: Check overnight TG1 cultures for infection by plating 100 μl/plate on 2xYT-GA agar and

**B4.** In parallel, prepare serial dilution for phage titration. Expect about 1013 to 1014 cfu/ml out of 1 l culture. To guarantee that all phages can infect bacteria, prepare serial dilutions of

, 10<sup>4</sup>

**B5.** After infection, prepare up to five 1:10 serial dilutions (100–900 μl) of infected bacteria in 2xYT medium. Plate 100 μl of infected bacteria and dilutions onto 2xYT-GA plates. Additionally, plate 100 μl of TG1 culture as negative controls on 2xTY-GA and 2xYT-GK plates. Incubate plates overnight at 30°C. Alternatively, spot 10 μl/dilution in triplicates onto

**B6.** The next day, count colonies on countable plates and calculate phage titer as colonyforming units (cfu/ml). If colonies are too small to count, increase temperature to 37°C. No

The following protocol describes the selection of target-specific scFv-phages using recombinant viral protein or virions immobilized on immunotubes. Usually ≥3 selection rounds are

**C1.** For the first round of selection, coat an immunotube with 10–50 μg protein dissolved in 1 ml PBS and incubate the parafilm-sealed tube overnight at 4°C. For subsequent rounds, decrease the protein concentration for more stringent conditions (1–5 μg/ml). If oligopeptides are used for the selection procedure, coat 2–5 μg/ml for all rounds. Alternatively, immuno-

tubes might be coated with a virus stock if the target antigen is unknown (**Note 12**).

**C2.** On the same day, prepare uninfected TG1 overnight culture as described (see B1–B3).

, and 106

). Pipette 10 μl of the 106

dilution) and infect bacteria at 37°C (30 min standing,

dilution to

units (cfu) by easy countable bacteria colonies on selection plates as described below.

centration) and freezing at −80°C (**Note 8**).

2xYT-GK agar. No colonies should grow (**Note 10**).

2xYT-GA plates and dry before overnight incubation.

colonies should be visible on control plates.

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

performed to enrich specific binding scFv-phages.

phage in PBS by diluting 10 μl into 990 μl (10<sup>2</sup>

990 μl of the log-phase TG1 culture (108

30 min shaking) (**Note 11**).

**B. Determination of phage titer**

(**Note 9**).

86 Antibody Engineering

37°C).

**C4.** Block the remaining binding sites by filling the empty immunotube completely with MPBS to the brim and incubate for about 2 h at RT.

**C5.** In parallel, preincubate packaged library (1012 to 1013 cfu) or phages from subsequent rounds (1011 to 1012 cfu) in MPBS (2–4% final milk concentration) for at least 1 h by slow overhead rotation in PP tubes (volume depends on volume used for coating immunotubes). Especially in the first round of selection, use 100-fold excess of scFv-phages compared with final library size to increase capturing promising binders (**Note 13**).

**C6.** Inoculate 20 ml of 2xYT with 200 μl of overnight culture and grow culture until the logphase is reached (OD600 of about 0.5). This will take about 2½ h. Log phase TG1 can be stored on ice until infection with eluted phage. *Optional*: Check for infection of overnight TG1 culture by plating 100 μl on 2xYT-GA and 2xYT-GK plates.

**C7.** Empty the immunotube, add the preblocked phage solution, seal it with parafilm, and incubate for 90 min gently shaking followed by 30 min standing at the bench.

**C8.** In the last 30 min of incubation, start washing the immunotubes. In the first selection round, wash 10× (5× with PBST and 5× with PBS). In the following rounds, increase stringency of washing by adding more wash cycles (second round: 20× cycles, third round: 30× cycles, etc.).

**C9.** Elution of antigen-bound scFv-phages can be done by different methods. We prefer either acid elution (*Option 1*) or protease elution (*Option 2*).

*Option 1* (*acid elution*): completely remove remaining buffer, add 1 ml acid elution buffer (pH 2.2) and incubate for about 8 min (longer incubation can destroy the phage). Rotate sealed tubes in an overhead rotator if using >1 ml for coating. Pipette eluted phage to about 100 μl neutralization buffer in a novel PP tube (**Note 14**).

*Option 2* (*protease elution*): many common phagemid vectors possess a trypsin cleavage site between the scFv and pIII protein (e.g., all phagemids containing the myc or FLAG tag) that can be used for elution. Add 1 ml freshly prepared trypsin-PBS to the tubes and incubate for 10 min standing (1 ml coated tubes) or overhead rotation (>1 ml coated tubes) (**Note 15**).

**C10.** Per selection, inoculate 14 ml of log-phase TG1 culture with the eluted phage solution in fresh 50 ml tubes. Infect at 37°C by 30 min standing and 30 min shaking. *Optional*: fill the empty immunotube with 5 ml of log-phase TG1 and perform infection at 37°C as described above. After infection, combine both cultures for following steps.

**C11.** To monitor the success of an antibody selection, the eluted phage titer can be determined after each selection round. Therefore, make serial dilutions of the TG1 culture after infection (e.g., by plating 100 μl of undiluted culture, 10<sup>2</sup> , 10<sup>4</sup> , and 106 dilutions onto 2xYT-GA plates). Incubate plates at 30°C overnight and count colonies to determine the eluted phage titer after each round. Typically for successful enrichment of specific binders, the eluted phage titer should increase from about 10<sup>4</sup> –106 in the first round up to the total number used for bacterial infection in subsequent rounds (~1010).

**C12.** Centrifuge the remaining TG1 culture (3500 × *g*, 10 min, RT), resuspend pellets in about 1 ml 2xYT, and plate onto either one large square plate (245 × 245 mm) or 3 round 15 cm plates containing 2xYT-GA agar.

**E-I. Monoclonal phage (mpELISA) screening**

at −20°C.

37°C.

log-phase (37°C, 200 rpm).

phase bacteria containing 5 × 107

plates and incubate overnight at 4°C.

30 min standing and 30 min shaking at 37°C.

lets in 180 μl/well of induction medium 2xYT-AKI. No glucose!

**E12.** The next day, block ELISA plates with MPBS for 2 h at RT. **E13.** Remove blocking solution and add 50 μl/well 4% MPBS.

transferred to a new PP microplate and stored at 4°C.

**E16.** Wash plate 3× with PBST and 3× with PBS.

bate for 1 h at RT.

**E1.** Plate TG1 glycerol stocks from target-enriched selection rounds as confirmed in ppELISA (see Protocol D) on 2xYT-GA selection plates to obtain single colonies. Alternatively, use the plates that have been prepared for the determination of the phage titer (see Protocol B).

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**E3.** Pick (single!) TG1 colonies from selection plates that are positively enriched rounds using sterile toothpicks or pipette tips and inoculate one well/colony. Keep wells H6 and H12 free

**E4.** Incubate plate at 37°C overnight while shaking. This will be your master plate (**Note 20**). **E5.** On the next day, transfer an aliquot of the culture to a new plate containing 100 μl/well 2xYT-GA. Pipette either about 5 μl/well from the master plate using a multichannel pipette to the new induction plate or use a 96-well induction device with sterile metal pins for induction. **E6.** Add 50 μl/well 2xYT containing 50% glycerol to the masterplate and freeze sealed plate

**E7.** Incubate induction plate for about 2½ h in the phage orbital shaker until bacteria reach

**E8.** Infect bacteria 1:20 with helper phage, i.e., 10 μl/well of a 1011 cfu/ml dilution (100 μl log-

**E9.** Centrifuge plate (10 min, 3000 × *g*, 4°C), discard supernatant, and resuspend bacteria pel-

**E10.** Seal plate with a breathable membrane and incubate the induction plate overnight at

**E11.** Coat two ELISA Maxisorb™ plates per induction plate by pipetting 100 μl/well of antigen/virus stock to the first half (columns 1–6) and control protein to the second-half (columns 7–12) of the plates. Use 2–10 μg/ml antigen/control protein diluted in PBS for coating. Seal

**E14.** Centrifuge the induction plate for 10 min at 3000 × *g* and 4°C. The supernatant can be

**E15.** Pipette 50 μl/well of the supernatant from the induction plate to one antigen-coated column and one control protein-coated column (e.g., column 1 of the induction plate to columns

**E17.** Add 100 μl/well of HRP-conjugated anti-M13 antibody diluted 1:5000 in MPBS and incu-

1 and 7 of the ELISA plate, column 2 to columns 2 and 8) and incubate for 1 h at RT.

bacteria and should be infected by 109

phages). Infect for

**E2.** Fill a sterile 96-well round-bottom plate with 100 μl/well of 2xYT-GA.

as blank. Seal plate with breathable membrane (**Note 19**).

**C13.** Grow plates overnight at 30°C and harvest bacteria by adding sterile-filtered 15% glycerol in 2xYT (about 10 ml totally). Harvest cells with flamed glass spreader. Mix very well and freeze bacteria glycerol stock at −80°C.

**C14.** Perform subsequent selection rounds. Resolve 100–400 μl of the glycerol stock from the previous round in 100 ml 2xYT-GA to start OD600nm of 0.1 and perform selection following the Protocols A, B, and C (**Note 16**).

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

After performing repeated rounds of selection, the scFv-phage preparations from the different rounds should be analyzed by ppELISA to identify if target-specific scFv-phage were successfully enriched.

**D1.** Coat a 96-well ELISA plate with the target antigen (or virus stock: usually 1 × 105 −1 × 107 pfu/ml) and control proteins at 2–10 μg/ml (100 μl/well) in PBS, seal plate with parafilm, and incubate overnight at 4°C. In total, coat two wells of target and control proteins plus two wells as blank for each selection round (R0, R1, R2, R3, etc.).

**D2.** The next day, remove coating solution and block entire plate with MPBS (400 μl/well) for 2 h at RT.

**D3.** Dilute phages from the different selection rounds to 1012 cfu/ml in MPBS, pipette 100 μl/ well, and incubate for 1 h at RT.

**D4.** Wash plate 3× with PBST and 3× with PBS. **Note 17**

**D5.** Add 100 μl/well of HRP-conjugated anti-M13 antibody diluted 1:5000 in MPBS and incubate for 1 h at RT.

**D6.** Wash plate 3× with PBST and 3× with PBS.

**D7.** Add 100 μl/well of TMB substrate solution and incubate until blue color has developed (up to 30 min). Stop reaction by adding 50 μl/well of stop solution. Read absorbance at 450 nm in a microplate reader (**Note 18**).

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

After successful antibody selection (see Protocols A–D), you will end up with an enriched pool of target-specific scFv-phages that must be screened for single binding antibodies ("monoclonals"). Screening can be done differently, using scFv-phages, scFv-pIII fusion proteins, or soluble scFv fragments. For beginners, we recommend screening as monoclonal phages by mpELISA (see Protocol E-I) since scFv-phages can be easily produced and detected by anti-phage HRP conjugates. Sometimes, screening as soluble fragments (see Protocol E-II) is preferred since screening as scFv-phages might result in false positive binders; meaning binding is dependent on the entire antibody-pIII fusions/phage. However, soluble screening requires switching the bacteria strain to a nonamber suppressor strain (e.g., HB2151).

## **E-I. Monoclonal phage (mpELISA) screening**

**C12.** Centrifuge the remaining TG1 culture (3500 × *g*, 10 min, RT), resuspend pellets in about 1 ml 2xYT, and plate onto either one large square plate (245 × 245 mm) or 3 round 15 cm plates

**C13.** Grow plates overnight at 30°C and harvest bacteria by adding sterile-filtered 15% glycerol in 2xYT (about 10 ml totally). Harvest cells with flamed glass spreader. Mix very well and

**C14.** Perform subsequent selection rounds. Resolve 100–400 μl of the glycerol stock from the previous round in 100 ml 2xYT-GA to start OD600nm of 0.1 and perform selection following the

After performing repeated rounds of selection, the scFv-phage preparations from the different rounds should be analyzed by ppELISA to identify if target-specific scFv-phage were

pfu/ml) and control proteins at 2–10 μg/ml (100 μl/well) in PBS, seal plate with parafilm, and incubate overnight at 4°C. In total, coat two wells of target and control proteins plus two wells

**D2.** The next day, remove coating solution and block entire plate with MPBS (400 μl/well) for

**D3.** Dilute phages from the different selection rounds to 1012 cfu/ml in MPBS, pipette 100 μl/

**D5.** Add 100 μl/well of HRP-conjugated anti-M13 antibody diluted 1:5000 in MPBS and incu-

**D7.** Add 100 μl/well of TMB substrate solution and incubate until blue color has developed (up to 30 min). Stop reaction by adding 50 μl/well of stop solution. Read absorbance at 450 nm

After successful antibody selection (see Protocols A–D), you will end up with an enriched pool of target-specific scFv-phages that must be screened for single binding antibodies ("monoclonals"). Screening can be done differently, using scFv-phages, scFv-pIII fusion proteins, or soluble scFv fragments. For beginners, we recommend screening as monoclonal phages by mpELISA (see Protocol E-I) since scFv-phages can be easily produced and detected by anti-phage HRP conjugates. Sometimes, screening as soluble fragments (see Protocol E-II) is preferred since screening as scFv-phages might result in false positive binders; meaning binding is dependent on the entire antibody-pIII fusions/phage. However, soluble screening requires switching the bacteria strain to a nonamber suppressor strain

−1 × 107

**D1.** Coat a 96-well ELISA plate with the target antigen (or virus stock: usually 1 × 105

containing 2xYT-GA agar.

88 Antibody Engineering

freeze bacteria glycerol stock at −80°C.

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

as blank for each selection round (R0, R1, R2, R3, etc.).

**D4.** Wash plate 3× with PBST and 3× with PBS. **Note 17**

**D6.** Wash plate 3× with PBST and 3× with PBS.

Protocols A, B, and C (**Note 16**).

well, and incubate for 1 h at RT.

in a microplate reader (**Note 18**).

**E. Screening for monoclonal binders**

successfully enriched.

2 h at RT.

bate for 1 h at RT.

(e.g., HB2151).

**E1.** Plate TG1 glycerol stocks from target-enriched selection rounds as confirmed in ppELISA (see Protocol D) on 2xYT-GA selection plates to obtain single colonies. Alternatively, use the plates that have been prepared for the determination of the phage titer (see Protocol B).

**E2.** Fill a sterile 96-well round-bottom plate with 100 μl/well of 2xYT-GA.

**E3.** Pick (single!) TG1 colonies from selection plates that are positively enriched rounds using sterile toothpicks or pipette tips and inoculate one well/colony. Keep wells H6 and H12 free as blank. Seal plate with breathable membrane (**Note 19**).

**E4.** Incubate plate at 37°C overnight while shaking. This will be your master plate (**Note 20**).

**E5.** On the next day, transfer an aliquot of the culture to a new plate containing 100 μl/well 2xYT-GA. Pipette either about 5 μl/well from the master plate using a multichannel pipette to the new induction plate or use a 96-well induction device with sterile metal pins for induction.

**E6.** Add 50 μl/well 2xYT containing 50% glycerol to the masterplate and freeze sealed plate at −20°C.

**E7.** Incubate induction plate for about 2½ h in the phage orbital shaker until bacteria reach log-phase (37°C, 200 rpm).

**E8.** Infect bacteria 1:20 with helper phage, i.e., 10 μl/well of a 1011 cfu/ml dilution (100 μl logphase bacteria containing 5 × 107 bacteria and should be infected by 109 phages). Infect for 30 min standing and 30 min shaking at 37°C.

**E9.** Centrifuge plate (10 min, 3000 × *g*, 4°C), discard supernatant, and resuspend bacteria pellets in 180 μl/well of induction medium 2xYT-AKI. No glucose!

**E10.** Seal plate with a breathable membrane and incubate the induction plate overnight at 37°C.

**E11.** Coat two ELISA Maxisorb™ plates per induction plate by pipetting 100 μl/well of antigen/virus stock to the first half (columns 1–6) and control protein to the second-half (columns 7–12) of the plates. Use 2–10 μg/ml antigen/control protein diluted in PBS for coating. Seal plates and incubate overnight at 4°C.

**E12.** The next day, block ELISA plates with MPBS for 2 h at RT.

**E13.** Remove blocking solution and add 50 μl/well 4% MPBS.

**E14.** Centrifuge the induction plate for 10 min at 3000 × *g* and 4°C. The supernatant can be transferred to a new PP microplate and stored at 4°C.

**E15.** Pipette 50 μl/well of the supernatant from the induction plate to one antigen-coated column and one control protein-coated column (e.g., column 1 of the induction plate to columns 1 and 7 of the ELISA plate, column 2 to columns 2 and 8) and incubate for 1 h at RT.

**E16.** Wash plate 3× with PBST and 3× with PBS.

**E17.** Add 100 μl/well of HRP-conjugated anti-M13 antibody diluted 1:5000 in MPBS and incubate for 1 h at RT.

**E18.** Wash plate 3× with PBST and 3× with PBS.

**E19.** Add 100 μl/well of TMB substrate solution and incubate until blue color has developed (up to 30 min). Stop reaction by adding 50 μl/well of stop solution. Read absorbance at 450 nm in a microplate reader (**Note 21**).

electroporation in HB2151, or direct expression of soluble scFv (Protocol G) that can be used

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**F1.** Streak out the masterplate glycerol stocks from wells that have been identified as antigenspecific binders by screening (see Protocol E) on 2xYT-GA plates and grow overnight at 30°C.

**F3.** Prepare PCR master mix using Taq PCR Core Kit according to manufacturer's recommendations preparing 50 μl/reaction and using scFv insert flanking primers, e.g., P1 (LMB3long)

**F5.** Use sterile pipette tips to pick single colonies and inoculate one well of the PCR plate and

**F6.** Run PCR according to manufacturer's recommendations using an annealing temperature

**F7.** Analyze 25 μl/sample on a 1% agarose gel containing ethidium bromide. Complete scFv

**F8.** Purify remaining 25 μl PCR product of full-length scFvs by QIAquick PCR Purification Kit according to manufacturer's recommendations by eluting DNA in ultrapure water

**F9.** Sequence inserts using a primer designed to either bind downstream or upstream of the forward (P1) or reverse primer (P2) used for colony PCR. Although colony PCR primer (or shorter versions thereof) might be used as well, sequencing primer binding within the ampli-

**F10.** Use 100 μl of infected medium from positive binders for inoculation of 6 ml LB high-salt medium supplemented with 100 μg/ml ampicillin and grow culture overnight at 37°C.

**F11.** The next day, use 1 ml of grown culture to prepare bacterial stock by adding 0.5 ml 50% 2xYT/glycerol and freeze at −20°C. Use remaining culture to prepare plasmid DNA using QIAprep Spin Miniprep Kit according to manufacturer's recommendations by eluting DNA

**F12.** Analyze obtained scFv sequences for sequence inaccuracies using molecular cloning software (e.g., SnapGene). VH and VL genes can be analyzed by the Fab Analysis online tool by aligning the scFv sequences to the VBASE2 database [26] to identify closest antibody germline

**Preliminary notes prior to antibody fragment production and functional characterization** Antigen/virus-specific scFvs needs to be further characterized for virus neutralization. This step is challenging since the neutralization capacity of antibodies often depends on the antibody valency as observed for HSV, varicella-zoster virus, HIV, and rabies [27, 28]. In these

inserts run at about 1 kb when using pHEN phagemids and primer P1 and P2.

for subsequent functional analysis (see Protocol H).

and P2 (fdseqlong) if using pHEN derivatives.

fied scFv insert might increase sequence quality.

sequences as well as to identify the CDR regions of the scFvs.

in ultrapure water (**Note 24**).

of 53°C and 30 cycles.

(**Note 23**).

**F2.** Fill sterile 96-well plate with 100 μl/well LB medium.

**F4.** Distribute 50 μl/well of PCR master mix into a 96-well PCR plate.

afterward the corresponding well of the medium-filled masterplate.

#### **E-II. Soluble scFv screening**

**E20.** Grow bacteria strain HB2151 on M9/+Thi minimal plates and prepare log phase-HB2151 culture (OD600nm of 0.5) as described before (see B1–B3).

**E21.** Take 10 μl of eluted phages from the different positively enriched selection rounds and infect 1 ml of log-phase HB2151 at 37°C (30 min standing, 30 min shaking).

**E22.** Prepare different dilutions of the phage-infected HB2151 culture in 2xYT (e.g., 10<sup>2</sup> , 10<sup>4</sup> , 106 ), plate 100 μl of the dilutions on 2xYT-GA plates, and grow overnight at 30°C. Alternatively, spot 10 μl/dilution on 2xYT-GA plates in triplicates.

**E23.** The next day, prepare a masterplate as described above (see E2–E4) by picking single HB2151 colonies.

**E24.** Inoculate an induction plate (see E5) and freeze the masterplate after adding 50% 2xYT/ glycerol (see E6).

**E25.** Grow induction plate for around 3 h until OD600nm of about 1 is reached.

**E26.** Centrifuge the induction plate (10 min, 3000 × *g*, 4°C) and completely remove the supernatant by carefully pipetting without disturbing the bacteria pellet.

**E27.** Resuspend the bacteria pellets in 180 μl/well of buffered 2xYT-SAI medium. No glucose! Seal the plate with breathable membrane and incubate the induction plate overnight at 30°C while shaking (**Note 22**).

**E28.** Perform ELISA as described in E11 to E19. However, soluble scFvs must be detected by a tag-specific antibody (e.g., anti-myc and anti-His) followed by washing and incubation with anti-primary HRP conjugated antibody. Alternatively, when using scFv libraries entirely based on the kappa light chain, detection can be performed with protein A-HRP or protein L-HRP using 3% BSA for blocking (lower backgrounds).

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

After screening for monoclonal binders (scFv-phages or soluble scFv fragments), hits should be analyzed for the presence of the full-length scFv insert (VH and VL) prior to sequencing. Selection of bacterial clones with incomplete inserts is a common issue observed with antibody phage display. Bacteria encoding for incomplete fragments with lower affinities often show growth advantages compared with those encoding for full-length scFvs and might be predominantly enriched during selection. To retrieve complete scFvs, we recommend analysis of colonies after screening by colony PCR as described below prior to sequencing. The following protocol allows PCR amplification of scFv inserts for size analysis and fast sequencing. Moreover, prepared plasmid DNA and glycerol stocks are useful for sequencing, cloning, electroporation in HB2151, or direct expression of soluble scFv (Protocol G) that can be used for subsequent functional analysis (see Protocol H).

**F1.** Streak out the masterplate glycerol stocks from wells that have been identified as antigenspecific binders by screening (see Protocol E) on 2xYT-GA plates and grow overnight at 30°C.

**F2.** Fill sterile 96-well plate with 100 μl/well LB medium.

**E18.** Wash plate 3× with PBST and 3× with PBS.

culture (OD600nm of 0.5) as described before (see B1–B3).

spot 10 μl/dilution on 2xYT-GA plates in triplicates.

in a microplate reader (**Note 21**).

**E-II. Soluble scFv screening**

90 Antibody Engineering

106

HB2151 colonies.

glycerol (see E6).

while shaking (**Note 22**).

**E19.** Add 100 μl/well of TMB substrate solution and incubate until blue color has developed (up to 30 min). Stop reaction by adding 50 μl/well of stop solution. Read absorbance at 450 nm

**E20.** Grow bacteria strain HB2151 on M9/+Thi minimal plates and prepare log phase-HB2151

**E21.** Take 10 μl of eluted phages from the different positively enriched selection rounds and

), plate 100 μl of the dilutions on 2xYT-GA plates, and grow overnight at 30°C. Alternatively,

**E23.** The next day, prepare a masterplate as described above (see E2–E4) by picking single

**E24.** Inoculate an induction plate (see E5) and freeze the masterplate after adding 50% 2xYT/

**E26.** Centrifuge the induction plate (10 min, 3000 × *g*, 4°C) and completely remove the super-

**E27.** Resuspend the bacteria pellets in 180 μl/well of buffered 2xYT-SAI medium. No glucose! Seal the plate with breathable membrane and incubate the induction plate overnight at 30°C

**E28.** Perform ELISA as described in E11 to E19. However, soluble scFvs must be detected by a tag-specific antibody (e.g., anti-myc and anti-His) followed by washing and incubation with anti-primary HRP conjugated antibody. Alternatively, when using scFv libraries entirely based on the kappa light chain, detection can be performed with protein A-HRP or protein

After screening for monoclonal binders (scFv-phages or soluble scFv fragments), hits should be analyzed for the presence of the full-length scFv insert (VH and VL) prior to sequencing. Selection of bacterial clones with incomplete inserts is a common issue observed with antibody phage display. Bacteria encoding for incomplete fragments with lower affinities often show growth advantages compared with those encoding for full-length scFvs and might be predominantly enriched during selection. To retrieve complete scFvs, we recommend analysis of colonies after screening by colony PCR as described below prior to sequencing. The following protocol allows PCR amplification of scFv inserts for size analysis and fast sequencing. Moreover, prepared plasmid DNA and glycerol stocks are useful for sequencing, cloning,

, 10<sup>4</sup> ,

**E22.** Prepare different dilutions of the phage-infected HB2151 culture in 2xYT (e.g., 10<sup>2</sup>

infect 1 ml of log-phase HB2151 at 37°C (30 min standing, 30 min shaking).

**E25.** Grow induction plate for around 3 h until OD600nm of about 1 is reached.

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

natant by carefully pipetting without disturbing the bacteria pellet.

L-HRP using 3% BSA for blocking (lower backgrounds).

**F3.** Prepare PCR master mix using Taq PCR Core Kit according to manufacturer's recommendations preparing 50 μl/reaction and using scFv insert flanking primers, e.g., P1 (LMB3long) and P2 (fdseqlong) if using pHEN derivatives.

**F4.** Distribute 50 μl/well of PCR master mix into a 96-well PCR plate.

**F5.** Use sterile pipette tips to pick single colonies and inoculate one well of the PCR plate and afterward the corresponding well of the medium-filled masterplate.

**F6.** Run PCR according to manufacturer's recommendations using an annealing temperature of 53°C and 30 cycles.

**F7.** Analyze 25 μl/sample on a 1% agarose gel containing ethidium bromide. Complete scFv inserts run at about 1 kb when using pHEN phagemids and primer P1 and P2.

**F8.** Purify remaining 25 μl PCR product of full-length scFvs by QIAquick PCR Purification Kit according to manufacturer's recommendations by eluting DNA in ultrapure water (**Note 23**).

**F9.** Sequence inserts using a primer designed to either bind downstream or upstream of the forward (P1) or reverse primer (P2) used for colony PCR. Although colony PCR primer (or shorter versions thereof) might be used as well, sequencing primer binding within the amplified scFv insert might increase sequence quality.

**F10.** Use 100 μl of infected medium from positive binders for inoculation of 6 ml LB high-salt medium supplemented with 100 μg/ml ampicillin and grow culture overnight at 37°C.

**F11.** The next day, use 1 ml of grown culture to prepare bacterial stock by adding 0.5 ml 50% 2xYT/glycerol and freeze at −20°C. Use remaining culture to prepare plasmid DNA using QIAprep Spin Miniprep Kit according to manufacturer's recommendations by eluting DNA in ultrapure water (**Note 24**).

**F12.** Analyze obtained scFv sequences for sequence inaccuracies using molecular cloning software (e.g., SnapGene). VH and VL genes can be analyzed by the Fab Analysis online tool by aligning the scFv sequences to the VBASE2 database [26] to identify closest antibody germline sequences as well as to identify the CDR regions of the scFvs.

#### **Preliminary notes prior to antibody fragment production and functional characterization**

Antigen/virus-specific scFvs needs to be further characterized for virus neutralization. This step is challenging since the neutralization capacity of antibodies often depends on the antibody valency as observed for HSV, varicella-zoster virus, HIV, and rabies [27, 28]. In these cases, bivalent antibodies (IgG) or antibody fragments (F(ab)<sup>2</sup> ) neutralized the virus with an exceptional efficiency, while monovalent Fab or scFv fragments did not or only when used at very high concentrations (e.g., 300 times higher than IgG). Although the production of soluble scFvs from phagemid libraries for functional characterization is a simple procedure (see Protocol G), it allows only the production of monovalent fragments that might be insufficient for virus neutralization in some cases. Therefore, the cloning of the VH and VL genes of virusspecific scFvs into other bivalent antibody formats (e.g., scFv-Fc, diabody, minibody) using a high-throughput, recombinant protein expression systems should be considered to increase the likelihood of isolating potent antiviral antibodies (e.g., transient HEK 293 expression platform from National Research Council Canada). The screening of virus-specific antibodies for virus neutralization can be performed either with bivalent or monovalent antibodies. The choice of the method (e.g., reporter virus and cell line) strongly depends on the virus itself. You need the suitable cell culture system to test neutralizing effects of antibodies on the viral replication. In this chapter, we demonstrate the identification of an HSV-neutralizing scFvs as an example for the isolation of neutralizing antibodies from phage display libraries. As mentioned above, this procedure can be modified for the screening of bivalent antibodies and for other viruses.

**G8.** Dialyze scFvs using D-Tube™ Dialyzer (MWCO 12–14 kDa) overnight at 4°C against

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

**G9.** Purify scFvs by IMAC employing Ni-NTA spin columns following the manufacturer's

**G10.** Analyze samples by reduced SDS-PAGE and Coomassie stain (and/or Western blot) to

**G11.** Dialyze scFvs against PBS buffer, filter-sterilize using low-protein-binding filter (0.22 μm) and measure protein concentration (e.g., by Nanodrop using calculated extinction coefficient

**G12.** Store scFvs at 4°C and proceed as soon as possible to characterization. ScFv stability can differ dramatically depending on the sequence from few days to several years. Do not freeze scFvs since many tend to aggregate after thawing. Oligomeric state of scFv can be analyzed

In the case of screening antiviral antibodies, scFvs can be screened in neutralization and/or protection assays that detect antiviral activities of antibodies *in vitro* or *in vivo* [29]. As the "gold standard" assay for screening and neutralizing capacity of antibodies, plaque reduction neutralization test (PRNT) can be performed where the PRNT50 value is used to describe the neutralization activity. The protocol below describes a PRNT-based assay to screen for best neutralizing

**H1.** Cultivate cell line suitable to form plaques when infected with virus of interest (e.g., Vero cells for HSV, dengue virus) according to supply recommendations. Most virus sustainable cell lines are recommended to be propagated and stored in low passage levels to guarantee

**H3.** To screen for the best neutralizing antibody, dilute all scFvs to a constant concentration (e.g., 4 μM) in appropriate culture medium supplemented with penicillin/streptomycin solution. For some viruses, reduction of the FBS concentration is recommended. If the PRNT50 should be determined, prepare twofold serial dilution of scFvs (at least five steps) that is the amount of antibody required to neutralize 50% of the infectious virus particles (**Note 29**).

**H4.** Freshly thaw cryostock with a known titer of plaque-forming units (pfu) at RT and dilute

**H5.** Pipette 300 μl of diluted scFvs (or PBS as negative control) to 300 μl of the virus prepara-

**H6.** Carefully aspirate old medium from cell plates and wash with 2 ml PBS. Always pipette

cells/well and grow in 5 ml/well com-

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93

for one to three days

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

scFvs and was optimized for HSV-specific and neutralizing scFvs.

plete culture medium in a humidified cell incubator at 37°C and 5% CO<sup>2</sup>

tion in a sterile plate and incubate the scFv-virus mixtures at 37°C for 1 h.

**H2.** Seed cells in 6-well cell culture plates with 5 × 105

and until a confluency of at least 90% is reached (**Note 28**).

to the wall of the wells to not destroy the cell monolayer.

protocol and loading columns several times with the scFv preparations.

NPI-10 buffer (**Note 27**).

check for purity.

and molecular mass).

by gel filtration chromatography.

susceptibility for plaque formation.

to 600 pfu/ml (same medium as in H3).

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

After the identification of scFvs with a unique sequence, soluble scFv can be produced in small scale to analyze their biological function, e.g., for their neutralizing capacity in case of antiviral antibodies. The protocol below describes the soluble expression of scFvs in the periplasm of nonamber suppressor strain HB2151 that allows the correct formation of disulfide bonds. ScFvs are recovered by periplasmic extraction followed by one-step purification using Immobilized metal ion affinity chromatography (IMAC) for His-tagged scFvs. Please adapt the protocol when using other tags for purification or upscale if higher quantities of scFvs are needed.

**G1.** Streak out HB2151 glycerol stock of unique scFv clones on 2xYT-GA agar plates to obtain single colonies (**Note 25**).

**G2.** Inoculate 5 ml 2xYT-GA with single picked colony and grow overnight at 37°C while shaking.

**G3.** The next day, inoculate 200 ml 2xYT-GA containing reduced concentration of 0.1% glucose with 2 ml of overnight culture and grow culture at 37°C until OD600nm of 1 is reached.

**G4.** Add 0.2 ml IPTG stock solution (i.e., 1 mM final concentration) to the cultures and grow culture overnight at RT (**Note 26**).

**G5.** Harvest bacteria by centrifugation (6000 × *g*, 15 min, 4°C) and resuspend the pellets in 5 ml periplasmic preparation buffer supplemented with 1 ml/l of freshly prepared lysozyme stock solution.

**G6.** Incubate preparation on ice for 30 min and stabilize spheroblasts by adding 50 μl 1 M MgSO<sup>4</sup> (i.e., 10 mM final concentration).

**G7.** Centrifuge preparations (12,000 × *g*, 30 min, 4°C) to clarify periplasmic fractions.

**G8.** Dialyze scFvs using D-Tube™ Dialyzer (MWCO 12–14 kDa) overnight at 4°C against NPI-10 buffer (**Note 27**).

cases, bivalent antibodies (IgG) or antibody fragments (F(ab)<sup>2</sup>

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

After the identification of scFvs with a unique sequence, soluble scFv can be produced in small scale to analyze their biological function, e.g., for their neutralizing capacity in case of antiviral antibodies. The protocol below describes the soluble expression of scFvs in the periplasm of nonamber suppressor strain HB2151 that allows the correct formation of disulfide bonds. ScFvs are recovered by periplasmic extraction followed by one-step purification using Immobilized metal ion affinity chromatography (IMAC) for His-tagged scFvs. Please adapt the protocol when using other tags for purification or upscale if higher quantities of scFvs are needed.

**G1.** Streak out HB2151 glycerol stock of unique scFv clones on 2xYT-GA agar plates to obtain

**G2.** Inoculate 5 ml 2xYT-GA with single picked colony and grow overnight at 37°C while

**G3.** The next day, inoculate 200 ml 2xYT-GA containing reduced concentration of 0.1% glucose with 2 ml of overnight culture and grow culture at 37°C until OD600nm of 1 is reached.

**G4.** Add 0.2 ml IPTG stock solution (i.e., 1 mM final concentration) to the cultures and grow

**G5.** Harvest bacteria by centrifugation (6000 × *g*, 15 min, 4°C) and resuspend the pellets in 5 ml periplasmic preparation buffer supplemented with 1 ml/l of freshly prepared lysozyme stock

**G6.** Incubate preparation on ice for 30 min and stabilize spheroblasts by adding 50 μl 1 M

**G7.** Centrifuge preparations (12,000 × *g*, 30 min, 4°C) to clarify periplasmic fractions.

for other viruses.

92 Antibody Engineering

single colonies (**Note 25**).

culture overnight at RT (**Note 26**).

(i.e., 10 mM final concentration).

shaking.

solution.

MgSO<sup>4</sup>

exceptional efficiency, while monovalent Fab or scFv fragments did not or only when used at very high concentrations (e.g., 300 times higher than IgG). Although the production of soluble scFvs from phagemid libraries for functional characterization is a simple procedure (see Protocol G), it allows only the production of monovalent fragments that might be insufficient for virus neutralization in some cases. Therefore, the cloning of the VH and VL genes of virusspecific scFvs into other bivalent antibody formats (e.g., scFv-Fc, diabody, minibody) using a high-throughput, recombinant protein expression systems should be considered to increase the likelihood of isolating potent antiviral antibodies (e.g., transient HEK 293 expression platform from National Research Council Canada). The screening of virus-specific antibodies for virus neutralization can be performed either with bivalent or monovalent antibodies. The choice of the method (e.g., reporter virus and cell line) strongly depends on the virus itself. You need the suitable cell culture system to test neutralizing effects of antibodies on the viral replication. In this chapter, we demonstrate the identification of an HSV-neutralizing scFvs as an example for the isolation of neutralizing antibodies from phage display libraries. As mentioned above, this procedure can be modified for the screening of bivalent antibodies and

) neutralized the virus with an

**G9.** Purify scFvs by IMAC employing Ni-NTA spin columns following the manufacturer's protocol and loading columns several times with the scFv preparations.

**G10.** Analyze samples by reduced SDS-PAGE and Coomassie stain (and/or Western blot) to check for purity.

**G11.** Dialyze scFvs against PBS buffer, filter-sterilize using low-protein-binding filter (0.22 μm) and measure protein concentration (e.g., by Nanodrop using calculated extinction coefficient and molecular mass).

**G12.** Store scFvs at 4°C and proceed as soon as possible to characterization. ScFv stability can differ dramatically depending on the sequence from few days to several years. Do not freeze scFvs since many tend to aggregate after thawing. Oligomeric state of scFv can be analyzed by gel filtration chromatography.

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

In the case of screening antiviral antibodies, scFvs can be screened in neutralization and/or protection assays that detect antiviral activities of antibodies *in vitro* or *in vivo* [29]. As the "gold standard" assay for screening and neutralizing capacity of antibodies, plaque reduction neutralization test (PRNT) can be performed where the PRNT50 value is used to describe the neutralization activity. The protocol below describes a PRNT-based assay to screen for best neutralizing scFvs and was optimized for HSV-specific and neutralizing scFvs.

**H1.** Cultivate cell line suitable to form plaques when infected with virus of interest (e.g., Vero cells for HSV, dengue virus) according to supply recommendations. Most virus sustainable cell lines are recommended to be propagated and stored in low passage levels to guarantee susceptibility for plaque formation.

**H2.** Seed cells in 6-well cell culture plates with 5 × 105 cells/well and grow in 5 ml/well complete culture medium in a humidified cell incubator at 37°C and 5% CO<sup>2</sup> for one to three days and until a confluency of at least 90% is reached (**Note 28**).

**H3.** To screen for the best neutralizing antibody, dilute all scFvs to a constant concentration (e.g., 4 μM) in appropriate culture medium supplemented with penicillin/streptomycin solution. For some viruses, reduction of the FBS concentration is recommended. If the PRNT50 should be determined, prepare twofold serial dilution of scFvs (at least five steps) that is the amount of antibody required to neutralize 50% of the infectious virus particles (**Note 29**).

**H4.** Freshly thaw cryostock with a known titer of plaque-forming units (pfu) at RT and dilute to 600 pfu/ml (same medium as in H3).

**H5.** Pipette 300 μl of diluted scFvs (or PBS as negative control) to 300 μl of the virus preparation in a sterile plate and incubate the scFv-virus mixtures at 37°C for 1 h.

**H6.** Carefully aspirate old medium from cell plates and wash with 2 ml PBS. Always pipette to the wall of the wells to not destroy the cell monolayer.

**H7.** Add 500 μl/well of the scFv-virus mixtures to each well and tilt plates as described above. Include virus only to every plate. ScFvs should be tested in at least duplicates.

**I1.** Thaw primary phage stock and prepare 10<sup>2</sup>

in a prewarmed water bath.

night at 37°C while shaking.

nitrogen prior to storage at ≤−20°C.

using 1:10 serial phage dilutions from 108

ing plaques on countable plates (**Note 33).**

library and keep it frozen at −80°C until needed.

**Note 1:** To minimize loss of diversity, very large libraries (>109

of 2xYT.

**5. Notes**

by phage.

dilutions (100 μl) in 2xYT medium.

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95

to 1013 for infecting 100 μl log phage TG1 and count-

independent clones) should

**I2.** Mix helper phage dilution with equal volume of log-phase TG1 culture prepared as described above (see B1–B3) to infect bacteria at 37°C (30 min standing and 30 min shaking). **I3.** Melt top agar in a microwave, aliquot in glass tubes while hot, and cool down tubes to 42°C

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

**I4.** Pipette infected bacteria to top agar tubes, mix quickly, and immediately cast top agar onto

**I5.** The next day, pick a single small plaque from grown bacteria lawns and transfer into 3 ml

**I6.** Incubate culture for 3 h at 37°C while shaking and use grown culture to inoculate 500 ml

**I7.** After one further hour of growing, add 200 μl kanamycin stock solution and grow over-

**I8.** The next day, pellet bacteria (4000 × *g*, 10 min, 4°C), transfer 40 ml of supernatant into 50 ml

**I9.** To inactivate the remaining bacteria, either heat helper phage preparation for 15 min at

**I10.** Aliquot the phage preparation into 2 ml PP tubes and snap freeze the tubes in liquid

**I11.** Determine phage titer as plaque-forming unit (pfu/ml) as described above (see I1–I4)

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

**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

**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

prewarmed (37°C) 2xYT plates. Grow plates overnight at 30°C (**Note 30).**

2xYT that was inoculated with 100 μl of TG1 overnight culture right before.

PP tubes and precipitate phage as described in Protocols A8–A10 (**Note 31**).

65°C (recommended) or filter through low protein binding 0.45 μm filter (**Note 32**).

**H8.** Incubate plates at 37°C for about 1 h (time can vary between 30 and 90 min depending on the cell/virus). During incubation, carefully tilt all the plates 10–15 min.

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

**H10.** Add 3 ml/well CMC medium and incubate plates in the humidified incubator (37°C, 5% CO<sup>2</sup> ) for three days.

**H11.** Fixate the cells with formaldehyde solution (3 ml/well for 5 min) and stain with crystal violet solution (800 μl/well) for 2 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:

% 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 scFvs concentration to determine the PRNT50 values.

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 infected with HSV [18, 28].

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 validated technique for future approaches.

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

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* strain and must be purchased.

**I1.** Thaw primary phage stock and prepare 10<sup>2</sup> dilutions (100 μl) in 2xYT medium.

**I2.** Mix helper phage dilution with equal volume of log-phase TG1 culture prepared as described above (see B1–B3) to infect bacteria at 37°C (30 min standing and 30 min shaking).

**I3.** Melt top agar in a microwave, aliquot in glass tubes while hot, and cool down tubes to 42°C in a prewarmed water bath.

**I4.** Pipette infected bacteria to top agar tubes, mix quickly, and immediately cast top agar onto prewarmed (37°C) 2xYT plates. Grow plates overnight at 30°C (**Note 30).**

**I5.** The next day, pick a single small plaque from grown bacteria lawns and transfer into 3 ml 2xYT that was inoculated with 100 μl of TG1 overnight culture right before.

**I6.** Incubate culture for 3 h at 37°C while shaking and use grown culture to inoculate 500 ml of 2xYT.

**I7.** After one further hour of growing, add 200 μl kanamycin stock solution and grow overnight at 37°C while shaking.

**I8.** The next day, pellet bacteria (4000 × *g*, 10 min, 4°C), transfer 40 ml of supernatant into 50 ml PP tubes and precipitate phage as described in Protocols A8–A10 (**Note 31**).

**I9.** To inactivate the remaining bacteria, either heat helper phage preparation for 15 min at 65°C (recommended) or filter through low protein binding 0.45 μm filter (**Note 32**).

**I10.** Aliquot the phage preparation into 2 ml PP tubes and snap freeze the tubes in liquid nitrogen prior to storage at ≤−20°C.

**I11.** Determine phage titer as plaque-forming unit (pfu/ml) as described above (see I1–I4) using 1:10 serial phage dilutions from 108 to 1013 for infecting 100 μl log phage TG1 and counting plaques on countable plates (**Note 33).**
