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Phage Display Methods and Protocols Edited by Michael Hust 2018

Construction of Naive and Immune Human Fab Phage-Display Library Noorsharmimi Omar and Theam Soon Lim

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Naive libraries are unique as the antibody gene repertoire used is generally from healthy individuals. In this way, the antibody repertoire would not have been exposed to any disease-specific antiges resulting in a naive unbiased repertoire. Naive libraries are generally generated through the isolation of IgM repertoire instead of other isotypes.

2 Materials 2.1 Isolation of B-Cells presented on phage surfaces due to the size tolerance of the phage packaging mechanism. The Fab is structurally formed by the heavy chain (HC) and light chain (LC) variable regions and constant regions juxtaposed to each other, whereby the HC and LC are connected by inter- chain disulphide bridges [23]. The size of Fab antibodies is double the size of scFv making it challenging for phage presentation. Nevertheless, the size of the Fab is still under the display tolerance of the phage particle to be presented at the pIII position. Although the generation of the Fab phage-display library is challenging in terms of cloning and phage packaging, Fab is still a preferred antibody format together with scFv for recombinant antibody production. There are several different approaches employed for the clon- ing of the genetic diversity of the antibody repertoires. After the isolation of the mRNA from B-lymphocytes, cDNA templates are prepared by RT-PCR. There are several different approaches used for antibody library cloning like PCR assembly [17], intermediate TOPO cloning [18], and isothermal rolling circle amplification [24]. However, the method commonly used is the two-step clon- ing. The two-step cloning strategy is typically done by the insertion of the light chain into the phagemid for an intermediate mini light chain library followed by the cloning of the heavy chain repertoire to yield the full antibody repertoire [25, 26]. The main challenge when developing such libraries is the diversity and size of the library at the final point. Therefore, great care is taken into consideration during the downstream cloning process to ensure the antibody repertoire is maintained as diverse as possible to reduce sequence redundancy and cloning bias. Here, we describe the application of the two-step cloning strategy for the generation of human Fab antibody phage-display libraries. The choice of the library being a naive or immune library would mainly depend on the isotype of the heavy chain repertoire that will ultimately be used for library cloning. The construction of a large diverse antibody library would require balancing the diver- sity with the limitations of E. coli transformation efficiencies. The protocol described here is optimized for Fab library cloning to generate a diverse library for phage display. 1. Ficoll-PaqueTM PLUS solution (GE Healthcare, Life Sciences). 2. 1  Phosphate-Buffered Saline (PBS), pH 7.4: Add 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4.2H2O, 0.24 g KH2PO4 in 1 L dH2O, autoclave and store at room temperature. 3. QIAamp RNA Blood Mini Kit (Qiagen, Germany). 2.2 First-Strand cDNA Synthesis 2.3 Amplification of HC and LC Fab Gene Repertoire 1. 300–500 ng RNA sample used as template. 2. SuperscriptTM II Reverse Trancriptase (InvitrogenTM, USA). 3. Oligo dT 12–18, 500 μg/mL (InvitrogenTM, USA). 4. Random hexamers, 3 μg/mL (InvitrogenTM, USA). 5. 10 mM dNTP mixture (InvitrogenTM, USA). 1. Vent® DNA polymerase (NEB® Inc.,USA) and Pfu DNA poly- merase (Thermo Fisher) together with theirs’ corresponding buffer. 2. Forward and Reverse Forward and Reverse primers for first amplification of HC and LC (see Table 1) primers for second amplification of HC and LC with restriction endonuclease sites (see Table 2). 3. 10 mM dNTP mixture (Thermo Scientific). 4. Agarose powder (InvitrogenTM,USA). 5. 10  TBE buffer pH 8.0: Add 108 g Tris base, 55 g Boric acid and 7.4 g EDTA in 1 L dH2O, autoclave and kept in room temperature. 6. QIAquick Gel extraction kit (Qiagen, Germany). 1. NcoI and corresponding buffer (NEB® Inc., USA). 2. MluI and corresponding buffer (NEB® Inc., USA). 3. 100  BSA (NEB® Inc., USA). 4. Antarctic phosphatase and corresponding buffer (NEB® Inc., USA). 5. T4 DNA Ligase and corresponding buffer (NEB® Inc., USA). 6. 3 M sodium acetate pH 5.2. 7. ElectroMAX Escherichia coli, E. coli DH10β cells (Invitro- genTM, USA)   :F mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 endA1araD139Δ(ara, leu)7697 galU galK λ  rpsL nupG. 8. MicropulserTM electroporator (BIO-Rad, USA). 9. 50 mg/mL ampicillin stock solution: Add 0.5 g ampicillin in 10 mL of 50% (v/v) ethanol, filter-sterilize and store at  20  C. 10. 2YT media pH 7.2: Add 16 g tryptone, 10 g yeast extract and 5 g NaCl in 1 L dH2O, autoclave and store at room temperature. 11. 2YT media containing 100 μg/mL ampicillin and 2% glucose.

12. 2YT agar plate containing100 μg/mL ampicillin and 2% glucose. 13. Sterile Petri dish, 25 cm (Brandon). 14. Sterile Bio-assay dish, 25 cm   25 cm (Nunc). 15. Plasmid purification Maxi-prep kit (Qiagen, Germany). 16. 80% Glycerol: Add 80 mL glycerol in 20 mL of dH2O, auto- clave and store at room temperature. 17. pLABEL-Fab phagemid vector. 1. SalI HF and corresponding buffer (NEB® Inc., USA). 2. NheI HF corresponding buffer. (NEB® Inc., USA). 3. E. coli XL1 Blue cells (Agilent Technologies, USA): recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F ́ proAB lacIq ZΔM15 Tn10 (Tetr). 4. Other materials are the same as first step cloning. Oligonucleotide primers used:  1. LMB3Fw:50 –CAGGAAACAGCTATGAC–30.   2. pIIIRv:50 –TTAGATCGTTACGCTAAC-30. 1. 2YT media pH 7.2: Add 16 g Tryptone, 10 g Yeast extract and 5 g NaCl, autoclave and store at room temperature. 2. 2YT media containing 100 μg/mL ampicillin and 2% glucose. 3. 2YT media containing 100 μg/mL ampicillin and 60 μg/mL kanamycin. 4. M13 K07 helper phage (NEB® Inc.,USA). 5. 20% Polyethylene glycol 6000/2.5 M NaCl (PEG/NaCl) solu- tion: Add 200 g PEG and 146 g NaCl in 1 L of dH2O, autoclave and store at room temperature. 6. 1 M Isopropyl-β-D-thiogalactopyranoside, IPTG dioxane free: Add 2.38 g IPTG in 10 mL dH2O and store in 1 mL aliquots. 7. 1  PBS buffer.

1. Corning® Costar® 96 Microtiter strip well, flat-bottom plate (Sigma Aldrich, Inc). 2. Carbonate bi-carbonate coating buffer, pH 9.6: Add 3.03 g Na2CO3, 6.0 g NaHCO3 in 1 L dH2O, autoclave and keep in 4  C. 3. 0.5% PBS-T: Add 5 mL Tween 20 into 1 L PBS. 4. 2% (w/v) PTM blocking buffer: Add 2 g skimmed milk in 100 mL 0.1% PBS-T (Need to be prepared freshly). 1. 2YT media containing 100 μg/mL ampicillin and 60 μg/mL kanamycin. 2. 2YT media containing 100 μg/mL ampicillin and 2% glucose). 3. 20% Polyethylene glycol 6000/2.5 M NaCl (PEG/NaCl) solu- tion: Add 200 g PEG and 146 g NaCl in 1 L of dH2O, autoclave and store at room temperature. 4. 1  PBS (for phage library titration). 1. 0.5% PBS-T: Add 5 mL Tween-20 into 1 L PBS. 2. 10 μg/mL Trypsin: Add 10 μg Trypsin in 1 mL dH2O. 3. E. coli XL1 Blue F0 (Agilent Technologies, USA): recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F ́ proAB lacIq ZΔM15 Tn10 (Tetr). 4. 2YT media, pH 7.2: Add 16 g Tryptone, 10 g Yeast extract and 5 g NaCl in 1 L dH2O, autoclave and store at room temperature. 5. 2YT media containing kanamycin 60 μg/mL. 6. 2YT media containing100 μg/mL ampicillin and 2% glucose). 7. Sterile 10 cm disposable Petri dishes. 8. 1 M Isopropyl-β-D-thiogalactopyranoside, IPTG dioxane free: Add 2.38 g IPTG in 10 mL dH2O and store in 1 mL aliquots. 1. 2YT amp/glu agar plates: Add 31 g premixed 2 YT and 15 g agar in 1 L dH2O, autoclave, add 2% glucose and 100 μg/mL ampicillin. 2. 2YT kan agar plates: Add 31 g premixed 2 YT and 15 g agar in 1 L dH2O, autoclave, add 2% glucose and 100 μg/mL kanamycin. 1. 2% (w/v) PTM blocking buffer: Add 2 g skimmed milk in 100 mL 0.1% PBS-T (Need to be prepared freshly). 2. 0.5% PBS-T: Add 5 mL Tween 20 into 1 L PBS. 3. Anti-M13 Horseradish peroxidase: Prepare 1:5000 dilution in 2% PTM. 4. 2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) sub- strate, ABTS developing solution: add 10 mg tablet ABTS in 5 mL of 50 mM citric acid, 5 mL of 50 mM trisodium citrate and 10 μL H2O2. Store in the dark. 5. 2.0 M Sulphuric Acid, H2SO4. (to Stop the ABTS Reaction).

 3 Methods 3.1 Blood Collection

Blood sample can be collected from healthy or disease related donors for human naive and immune library generation respectively (see Note 1 (a, b)). 1. Extract B-cells from 10 mL whole blood using Ficoll-PaqueTM Plus (see Note 2). 2. The isolated lymphocytes were diluted with 1  PBS, pH 7.4 and centrifuged at 12,000   g, 15 min, 18  C. 3. Remove the plasma cells using a clean filtered pippete tip. 4. Gently transfer the lymphocytes layer into a new 50 mL conical tube and fill with 1  PBS until 50 mL. 5. Centrifuge the mixture at 12,000   g for 10 min at 18  C.

3.2 Total RNA Preparation

1. Extract total RNA from the B-cells using a total RNA extrac- tion kit based on the manufacturer’s protocols (see Note 3). 2. Keep total RNA at  80  C until use.

3.3 cDNA Synthesis 1. Synthesize the cDNA using SuperscriptTM II Reverse Transcriptase according to the manufacturer’s procedure. 2. A reaction mix will contain 500 ng RNA of each donor, 1 μL of oligo dT 12–18 (500 μg/mL), 0.1 μL random hexamer (3 μg/ mL), and 1 μL of 10 mM dNTP and filled up to a final volume of 12 μL with dH2O. 3. Denature the RNA at 65  C for 5 min and immediately chill down on ice. 4. Add 4 μL 5  first-strand buffer (250 mM Tris-HCl pH 8.3, 375 mM KCl and 5 mM MgCl2), 2 μL of 0.1 M DTT and 40 units of RNAseOUTTM into the first reaction mixture. 5. Incubate the mixture at 42  C for 2 min. 6. Add 200 units of the SuperscriptTM II Reverse Transcriptase and mix it gently. 7. Next, incubate the mixture at 42  C for 50 min and heat inactivate it at 70  C for 15 min. 8. Add 2 units of RNAse H and incubate at 37  C for 20 min. 9. Store the cDNA at  20  C until use.

3.4 V-Gene (Heavy and Light Chain) Amplification by PCR

1. Different reverse primers are used for the different libraries. The naive library uses the IgM-derived heavy chain V-genes reverse primer and 19 different VH forward primers are used for full Fab amplification. The immune library requires the IgG-derived heavy chain V-genes reverse primer is use instead with the same VH forward primers. (All the primers are shown in Table 1) (see Note 4). 2. For light chain amplification, the kappa (κ) and lambda (λ) reverse primers are used with six different λ forward primers and 4 different κ primers. (All the primers are shown in Table 1). 3. A total of 200 ng cDNA is used as a template for V-gene amplification. 4. In a total of 20 μL, add 200 μM dNTPs, 0.2 μM forward and reverse primers, 2 μL of 10  Pfu buffer (MgSO4), and lastly 0.2 μL of 2.5 U/μL Pfu DNA Polymerase. Add dH2O until 20 μL. The PCR program is as follows: 95  C for 2 min, annealing at 55  C or 62  C for 1 min 30 sec, elongation at 72  C for 2 min and a final elongation at 72  C for 5 min. Perform the PCR with 30 cycles of amplification (see Note 5). 5. Purify the amplified V-gene (VH—750 bp, VL kappa/lambda  750 bp) products separated on the 1% agarose gel using the QiaQuick Gel extraction kit according to the manufacturer’s protocol. 6. Determine concentrations of each donor sample and store the products at  20  C until used.

3.5 Restriction Sites Amplification of the V-Gene by PCR 1. A total of 20 μg of first amplification of V-genes products is used as DNA template for the amplification of the restriction site. Perform the PCR individually. 2. In a total of 20 μL, add 10 μM of dNTPs, 0.2 μM forward and reverse primers, 2 μL of 10  Thermo pol® buffer, and lastly 0.2 μL of 2000 U/mL Vent DNA polymerase. Add dH2O until 20 μL. The PCR program is as follows: 95  C for 1 min 30 s, annealing at 55  C or 62  C for 30 s, elongation at 72  C for 2 min, and a final elongation at 72  C for 5 min. Perform the PCR for 20 cycles of amplification. 3. Pool all VH and VL (κ and λ) into individual and subfamilies separately.

3.6 Two-Step Cloning

The cloning of the V-genes was carried out with a two-step cloning strategy. The first step is to generate a mini library containing the Fab VH repertoire in pLABEL-Fab using DH10β E. coli cells. The second cloning step is to introduce the VL (κ and λ) repertoire to the VH-mini library in XL1 Blue E. coli cells.

3.6.1 VH Cloning (First Step)

1. Digest 10 μg of pLABEL-Fab and 2 μg VH pools with 50 U and 10 U of NcoI and MluI (NEB® Inc., USA) respectively at 37  C for 16 hours (h). All the subfamilies are pooled according to five subsequently smaller groups of five donors. Heat inacti- vate the digestion mixture at 65  C for 10 min (see Note 6). 2. Add 1 U of alkaline phosphatase and incubate at 37  C for 1 h. 3. Purify the treated products using QIAquick PCR Purification Kit according to the manufacturer’s protocol. 4. Ligate 500 ng of digested pLABEL-Fab and 250 ng digested VH pools using 1 U of T4 DNA Ligase (see Notes 7 and 8). 5. Incubate the ligation mixture for 16 h at 8  C. 6. Heat inactivate the mixture at 65  C for 10 min. 7. Precipitate the ligated DNA using 1 μL of 3 M sodium acetate and 100 μL of 100% ethanol, incubate in  80  C for 1 h and centrifuge for 10 min at 12,000   g at 4  C (see Note 9). 8. Wash the DNA pellet with 70% Ethanol. 9. Resuspend the DNA pellet with 4 μL dH2O. 10. Mix 2 μL of DNA with 50 μL of thawed DH10β E. coli cells on ice for 2 min. 11. Transfer the 52 μL mixture to a prechilled 0.1 cm eletropora- tion cuvette. 12. Electroporate the mixture using the bacteria program using an electroporator (see Note 8). 13. Add 1 mL of pre-warmed 2YT media into the mixture and transfer the suspension to a 1.5 mL microcentrifuge tube. Shake the culture for 1 h at 37  C and 700 rpm. 14. Plate out the transformations on 30 bioassay dishes (25 cm   25 cm) of 2YT agar containing 100 μg/mL ampicil- lin and 2% glucose. 15. Incubate the plates at 37  C for 16 h. 16. Scrape the colonies with 300 mL 2YT containing 100 μg/mL ampicillin and 2% glucose. 17. Centrifuge the cells at 8000   g for 5 min and resuspend the cell pellet with 50 mL 2YT containing 100 μg/mL ampicillin and 2% glucose. 18. A glycerol stock of the library is prepared (see Subheading 3.7). 19. Estimate the library diversity by titrating the library stock (see Subheading 3.8). 20. Perform a colony PCR to confirm cloning of the heavy chain repertoire insert (see Subheading 3.9) and calculate the library size (see Notes 8 and 10).

3.6.2  VL Cloning (Second Step)

1. Culture a tube of glycerol stock containing the VH-mini library at37 Cfor16hwith800rpmshakingin500mLof2YT media containing 100 μg/mL ampicillin and 2% glucose for plasmid extraction using the maxi-prep kit. 2. Digest the VH-mini library and VL (κ and λ) with SalI HF and NheI HF with its compatible buffer. 3. Perform the digestion and ligation as first step cloning (see Subheading 3.6.1). 4. Electroporate the Fab library in XL1Blue MRF’ E. coli cells for phage packaging purposes. 5. Plate out the culture into 40 bioassay dish plates. 6. Perform the library size estimation as described (see Subhead-ing 3.8, Note 11). 7. Colony PCR is carried out to determine the cloning efficiency of the library (see Subheading 3.9).

3.7 Preparation of Bacteria Library Stock 

1. The collected colonies are then scraped into 300 mL 2YT containing100 μg/mL ampicillin and 2% glucose. 2. Centrifuge the collected 2YT suspension for 10 min at 8000   g. 3. Resuspend the library pellet with 80 mL 2YT containing 100 μg/mL ampicillin and 2% glucose. 4. Add 100% glycerol to the suspension to make 20% library stock to preserve the library. 5. Aliquot the final library suspension into several 2.0 mL cryogenic tubes and keep in  80  C until needed (see Note 12).

3.8 Library Size Estimation 1. Dilute a volume of 10 μL from 80 mL bacteria library suspen- sion with 90 μL 2YT containing 100 μg/mL ampicillin and 2% glucose. 2. Perform a serial dilution by 1:10 ratio up to 10 13. 3. Spot the diluted cultures on the 2YT agar plate containing 100 μg/mL ampicillin and 2% glucose. 4. Incubate all the plates o/n at 37  C. 5. Count the number of colonies observed from the plate for single colony growth. 6. Colony PCR can be performed using randomly picked colonies to confirm Fab inserts (see Subheading 3.9).

3.9 Colony PCR for Fab Insert Determination 1. Pick 20–50 colonies randomly and resuspend in 10 μL dH2O. 2. Perform a PCR for each colony using the LMB3 Fw and pIII Rv primers. 3. The PCR reaction mix contains 2 μL template, 2 μL 10  Dream Taq Buffer, 1 μL of 10 μM forward and reverse primers, 0.4 μL of 10 mM dNTPs and 0.2 μL of Taq polymerase and made up to a final volume of 20 μL with dH2O. 4. The PCR program is as follows; 95  C for 1 min 30 s, annealing at 55  C or 62  C for 30 s, elongation at 72  C for 2 min, and a final elongation at 72  C for 5 min. Perform the PCR for 20 cycles amplification. For all VH and Vκ genes amplification the annealing temperature is 55  C while for the Vλ Vκ genes the annealing temperature is at 62  C. 5. The Fab size of ~1.6 kb should be observed on 0.9% agarose gel electrophoresis (see Notes 8 and 13).

3.10 Sequencing Analysis 1. Pick 20–50 colonies randomly and resuspend in 10 μL dH2O. 2. The quality of the library is further confirmed by DNA sequencing. Send the positive colony PCR clones with the correct size for full Fab insertion for sequencing. 3. DNA sequencing data can be analyzed using IMGT/V-quest software (www.imgt.org/IMGT_vquest/vquest) using the human reference directory [27]. 4. The IMGT/V-quest analysis will provide V-D-J gene segment and CDR analysis of the clones.

3.11 Fab Phage Library Packaging 1. To package the Fab library, inoculate a glycerol stock of the gene library stock into 500 mL 2YT containing 100 μg/mL ampicillin and 2% glucose in a 1 L flask. Grow at 37  C, 250 rpm up to an OD600 ~ 0.5. 2. Infect a volume of 250 mL bacteria culture with ~1012 M13KO7 helper phage (see Note 14). 3. Incubate the bacteria culture at 37  C for 30 min static. 4. Transfer the cells into several 50 mL conical tubes and centri- fuge at 2400   g for 30 min at 12  C to remove the excess glucose that suppresses the expression of the HC: pIII protein as well as the free LC protein. 5. Resuspend the pellet with 300 mL 2YT containing 100 μg/mL ampicillin, 60 μg/mL kanamycin, and 100 μM IPTG in a 1 L flask. Grow at 30  C, 200 rpm for o/n (see Note 15). 6. Next day, spin down the bacteria cells at 2400   g for 30 min at 12  C. 7. Transfer the supernatant to a new 50 mL conical tube filling it up to 4/5 volume of the tube and add the remaining 1/5 volume with PEG/NaCl and mix well. 8. Incubate the mixture on ice for 1 h (see Note 16). 9. Centrifuge the mixture at 2400   g for 30 min at 12  C. 10. Gently pour the supernatant away and resuspend the phage pellet in 8 mL 1  PBS and immediately fill with 2 mL PEG/NaCl. 11. Continue to incubate the mixture on ice for 1 h. 12. Centrifuge the mixture at 4800   g for 15 min at 12  C. 13. Again, gently pour the supernatant away and resuspend the phage pellet in 4 mL 1  PBS. 14. Next, centrifuge the mixture at 4800   g for 15 min at 12  C. 15. To remove any remaining bacterial cells from the phage pre-cipiatation, repeat steps 13 and 14 for three times. 16. Finally, resuspend the phage pellet in 2 mL freshly prepared 1  PBS and store at 4  C until use (see Note 17).

3.12 Fab Phage Library Titration 1. Dilute 10 μL of Fab phage library in 90 μL 1  PBS. The serial dilution is performed until a range of 10 14. 2. Infect the Fab phage dilutions with E. coli TG1 or XL1Blue cells at OD600 ~ 0.5. 3. Spot 10 μL of the infected phage on a 2YT agar plate contain- ing 100 μg/mL ampicillin and 2% glucose and on a second 2 YT agar plate containing 60 μg/mL kanamycin (see Note 14). 4. Dry the agar plates under laminar flow. 5. Incubate the agar plates at 37  C for o/n.

3.13 Antigen Analysis

Prior to panning, evaluate the antigens using SDS-PAGE and Western-blot analysis to confirm antigen purity. The purity of the antigen used will ensure a good and specific isolation of Fab anti- body binders.

3.14 Phage Selection (Panning)

1. Coat a microtiter well with a minimum amount of antigen (10 μg) o/n at 4  C using the Carbonate bi-carbonate coating buffer. The wells are then washed 3  with 300 μL PBS-T. Block the antigen-coated wells with 300 μL of 2% PTM for 1 h at RT and wash the wells 3  with 300 μL PBS-T. 2. Concurrently, pre-block 1011 phage particles of the Fab phage library (see Note 18) with 300 μL 2% PTM for 1 h on a microtiter well (see Note 19). 3. Transfer the pre-blocked Fab phage library into the antigen- coated wells and incubate for 2 h at RT with constant shaking at 700 rpm (see Note 20). 4. Wash the wells with PBS-T for 3  in the first round; gradually increase the wash to 5  and 10  for each subsequent panning round. 5. Elute the bound phages with 50 μL of 100 μg/mL Trypsin and incubate at 37  C for 30 min. 6. Remove the eluted phages to a new 1.5 mL microcentrifuge tube and infect with 150 μL XL1 Blue E. coli cells OD600 ~ 0.5 static. 7. Incubate the cells for 30 min at 30  C and continue incubation at 37  C, shake at 700 rpm for 30 min. 8. Take out 10 μL of infected phage to determine the rescued phage by titration (see Subheading 3.12). 9. Add 20 μL of 10  ampicillin and glucose into the remaining mixture. 10. Incubate the mixture at 37  C, 250 rpm o/n. 11. Remove 10 μL of o/n cultured cells and continue culturing in a new cell culture plate containing 190 μL of 2YT ampicillin and 2% glucose for 2 1⁄2 h at 37  C and shake at 700 rpm. The remaining o/n culture is added with 20 μL of glycerol and mixed well for storage at  80  C. 12. Infect the cultured cells with 1011 M13KO7 helper phage. 13. Incubate the cells at 37  C for 30 min static. 14. Transfer the infected cells onto the filter plate and centrifuge the plate at 2400   g for 5 min (see Note 21). 15. Resuspend the cell pellet with 2YT containing 100 μg/mL ampicillin, 60 μg/mL kanamycin, and 100 μM IPTG. 16. Culture the cells at 30  C o/n shaking at 700 rpm. 17. Finally, transfer the packaged Fab phage from round 1 to a new filter plate to separate phage and cells by centrifugation at 2400   g for 5 min. 18. Apply 100 μL of the phage for subsequent round of panning. The remaining phage is kept at 4  C for polyclonal evaluation. The phage titer from each round of panning is determined for the recovery and enrichment analysis (see Note 22). The schematic representations of Fab antibody phage library packaging and Fab phage selection on microtiter well is shown in Fig. 1.

3.15 Polyclonal Fab Antibody Phage ELISA 1. Coat an appropriate number of microtiter wells with a mini- mum amount of antigen (10 μg) using the carbonate bi-carbonate coating buffer for 1 h at RT and wash 3  with 300 μL PBS-T. Block the antigen-coated wells with 300 μL of 2% PTM for 1 h at RT and wash the wells 3  with 300 μL PBS-T. Concurrently, coat an equal number of empty wells with 300 μL of 2% PTM as negative controls to observe for nonspecific binders. 2. Pre-block the phage obtained from each round of panning with 300 μL of 2% PTM for 1 h at RT to reduce nonspecific binding during antigen recognition. 3. Incubate the pre-cleanup phage of each panning round in the antigen-coated wells as well as the control plates for 2 h at RT and shake at 700 rpm. 4. Wash the wells 3  with 300 μL PBS-T (see Note 23). 5. Add 200 μL of anti-M13-HRP (1: 5000 dilution in 2% PTM) into samples and control wells. 6. Incubate the wells for 1 h at RT and shake 700 rpm. 7. Wash the wells 3  with 300 μL PBS-T. 8. Lastly, develop the wells with 100 μL ABTS solution for 30 min in the dark. 9. Measure the readings at 405 nm using a microtiter plate spectrophotometer for absorbance values of the ABTS color change.

3.16 Polyclonal Fab Antibody Phage ELISA Assesment For a successful panning process, an obvious enrichment is observed as the panning round increases. Choose the best round of panning to isolate monoclonal antibodies with good specificity and diversity as this is critical to isolate different monoclonal anti- bodies with varying affinities. 3.17 Monoconal Fab Antibody Phage ELISA

1. Infect the phage of that particular round from the storage plate with XL1 Blue E. coli cells of OD600 ~ 0.5 or plate out the glycerol stock and continue with monoclonal Fab antibody phage ELISA. 1. For a 96 microtiter plate, pick 47 monoclonal antibodies from the respective round of panning including a positive control of any in house antibody clone as positive control. 2. Inoculate randomly selected phage monoclonal antibodies in 200 μL 2YT containing 100 μg/mL ampicillin and 2% glucose. 3. Grow the clones at 37  C o/n with constant shaking at 700 rpm. 4. Follow steps 11 until 17 of the panning method to package the phage monoclonal Fab antibodies (see Subheading 3.14). 5. For ELISA analysis, follow steps 1 until 9 of the polyclonal phage ELISA (see Subheding 3.15).

6. DNA sequencing of the positive monoclonal Fab antibodies with a satisfactory signal-to-noise ratio is performed with the appropriate primers. The primer sets are scFv Fab (CL1 þ CL2) Rv with LMB3 Fw, scFv Fab Kappa Rv with LMB3 Fw and Human Fab IgM/IgG CH1 Rv with Fab Spacer NheI Fw.

4 Notes

1. (a) Donors with family backgrounds of severe illnesses, suffering from autoimmune disorder, had fever, on any medi- cation including antibiotics and immunosuppressors within a month are not suitable for naive library sampling. (b) Donor history including treatment and state of illness should be recorded for immune library sampling. The type of treatment regimen is important to standardize the library gen- eration process to ensure the donors are capable of eliciting an immune response toward the disease. Donors that have under- gone immunosuppresors are not suitable as the immune response is ineffective. Conditions may vary depending on the mechanism of disease. 2. The blood layer must not mix with the Ficoll-PaqueTM PLUS as it leads to aggregation of erhythrocytes resulting in lower yields. 3. The total RNA obtained can be analyzed by Agilent Bioanaly- zer for RNA integrity and total concentration. 4. The amplification of the VH Fab repertoire for naive library generation will use the IgM CH1 Rv-specific primer, whereas the immune library will use the IgG CH1 Rv-specific primer. This is to amplify isotype specific repertoire of the heavy chain. A band size of ~750 bp Fab fragment is expected; however, the presence of other bands is expected due to the rearranged V-genes. The excision of the targeted band size must be carried out with care. 5. The V-gene repertoire from each donor is amplified indepen- dently to avoid bias and loss of repertoire by sample pooling. This is to ensure higher repertoire diversity. 6. Repeat digestion until the amount of DNA required is suffi- cient for the corresponding cloning experiment. A sequential digestion may be required if the reaction buffer compatibility affects the digestion efficiency. 7. Multiple ligation reactions may be required to obtain sufficient amount of DNA material for a highly diverse antibody. 8. The library size for the heavy chain repertoire should be as high as possible as the variable heavy chain is the predominant region for antigen binding. The working range should be within107–109 or higher for naive libraries and 105–107 for immune libraries. The highest possible diversity should be achieved for a good quality library. 9. Alternatively, liquid nitrogen may be used for snap freeze to allow a rapid purification process. After snap freezing, centri- fuge for 10 min at 12,000   g at 4  C. 10. The expected band size of the VH repertoire cloning is approx- imately 800 bp. 11. The targeted range of the final library size should be 109–1012 or higher for naive libraries and 106–108 for immune libraries. These are acceptable working sizes although larger library sizes are preferred. 12. The bacteria library stocks are normally prepared to allow a starting OD value of OD600 ~ 0.1. A tube of immune or naive Fab bacteria library stock is enough to be cultured for phage Fab library preparation. 13. The cloning efficiency may vary between users. A cloning efficiency of more than 80% inserts of VH-mini library and full Fab library is preferred. 14. A normal ratio of helper phage to be used when packaging is to ensure 102 more helper phage from the library size. This is to ensure at least 100 copies of each clone are represented in the packaging process. 15. The addition of 100 μM IPTG during Fab phage packaging is to aid the packaging process by inducing the expression of the HC: pIII fused protein and LC monomers during phage library packaging. This will increase the natural formation of Fab antibody molecules on the phage particle. 16. For a higher phage library packaging efficiency, the ice-cold incubation can be pro-longed to 2 h to increase the amount of phage particles to be precipitated. 17. Avoid storing the phage preparation for more than 2–4 weeks at 4  C. 18. For better panning results, fresh phage preparations are used for each panning round to avoid loss from proteolysis of the displayed antibodies through time. 19. To check the quality of the packaged Fab phage library, the colony growth on ampicillin agar plate should be at least two-fold higher in comparison with the kanamycin plate. This is to make sure less helper phage is being produced concur- rently with the phage library. In addition, the colony growth can be used to quantify the phage library size prior for panning. 20. As a guide, the amount of phage titer to be used for the first panning round should be twofolds higher than the library size. 21. Occasionally, clogging may occur during cell separation using the filter plates. If clogging occurs, remove the remaining cells to another well and proceed. 22. The phage recovery (Input/Output) can be calculated from the titer of the input and output phage used for each panning round. 23. Care must be taken to ensure no bubbles are present during washing steps by agitation. Tap the microtiter wells on a pile of clean dry paper towel to remove any remaining PBS-T in the microtiter wells.
[[Category:Antibodies]]

Human Naive Library Construction - Revision history

Paul.xu at 23:38, 6 May 2018