Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

NOTE: Filter sterile tips must be used throughout when dealing with phage to avoid contamination to pipette gun and surrounding area. Aseptic area or hood must be used when handling with bacteria and phage experiments. Phage experiment area must be cleaned up using 2% sodium dodecyl sulfate (SDS) followed by 70% ethanol to avoid phage contamination. For making serial dilutions in this protocol, new tips should be used for each dilution.

1. E. Coli SS320 Electro-competent Cell Preparation

1. Pre-warm LB/tet agar plate (prepared and stored at 4 °C for less than 1-week old) at 37 °C incubator for 1 h. Use a sterile inoculation loop or sterile tip to streak out a glycerol stock of E. coli SS320 cells onto the pre-warmed LB/tet agar plate in a zig-zag direction gently along the surface of the plate. Incubate the plate at 37 °C incubator overnight for around 12-15 h.
2. The following day, use a sterile inoculation loop or sterile tip to pick a well-separated single colony along the zig-zag line and inoculate into 10 mL of 2YT/tet medium in a 50-mL round bottom tube by dipping the loop into the medium and stirring briefly.
3. Incubate at 37 °C with shaking at 200 rpm for around 3-4 h until OD₆₀₀ is reached at around 0.4-0.8 (log phase growth). Monitor OD₆₀₀ at 2 h. During this period, pre-warm 5 LB/tet agar plates (prepared and stored at 4 °C for less than 1-week old) in a 37 °C incubator for at least 1 h.
4. Add 20 μL M13KO7 helper phage from lab stock with titer around 1 x 10¹³ colony forming unit (cfu)/mL into 180 μL of sterile 1X PBS in autoclaved 1.5-mL tubes to prepare 10 ten-fold serial dilutions.
5. Aliquot 4 mL autoclaved and liquid 2YT top agar into 5 X 14 mL round bottom tubes, label each tube with one dilution from the fifth to the ninth of the ten-fold dilution, respectively, and incubate in a 65 °C incubator to maintain the 2YT top agar in the liquid state.
6. Mix 500 μL of log phase E. coli SS320 cells into the M13KO7 dilution tube (choose the fifth ten-fold dilution to the ninth ten-fold dilution) and incubate in a 37 °C incubator for 5-10 min.
7. During the incubation of step 1.6, transfer 2YT top agar from step 1.5 to room temperature (RT) to cool down for around 5 min to about 42 °C. Use inner wrist to test temperature of 2YT top agar; it should remain as liquid. Transfer each dilution mixture from step 1.5 to each corresponding 2YT top agar tube. Tighten the lid of each tube and mix by turning upside down several times gently and briefly to avoid bubble generation.
8. Carefully pour each mixture along the edge of a pre-warm LB/tet agar plate (from step 1.3) and slant the plate slightly to fully and evenly fill the plate with the mixture while avoiding the introduction of bubbles. Keep the plates at RT for around 5-10 min to solidify the top agar within each plate and incubate the 2YT top agar plates at 37 °C overnight for around 15-18 h.
9. Choose the dilution plate after overnight growth from step 1.8 with around 100-200 average-sized, single, well-separated plaques for plaque inoculation. Use one hand to hold the agar plate against a light source, and the other hand to hold a pipette gun loaded with a long sterile pipette tip to vertically stab into the top agar, and collect a single and well-separated plaque.
   1. Slant the pipette tip slightly to separate top agar with plaque. Pipette up and down several times to dislodge the agar with plaque into a 14-mL round bottom culture tube pre-loaded with 1 mL of 2YT/kan/tet medium (see Table 1).
   2. Repeat this procedure to pick 3-5 plaques in total. The purpose of picking several plaques is to ensure successful inoculation, as a plaque can be faint and relatively small when compared with a bacteria colony. Grow the plaques for 8 h at 37 °C with shaking at 200 rpm.
10. Transfer a tube of growing culture into 50 mL of 2YT/kan/tet medium in a 250-mL baffled flask. Grow at 37 °C with shaking at 200 rpm overnight for around 12 h.
11. Inoculate three 2-L baffled flasks containing 900 mL of superbroth/tet/kan medium each with 5 mL of the overnight culture. Incubate at 37 °C with shaking at 200 rpm for 6-7 h to OD₆₀₀ around 0.6-0.8.
12. Chill the three flasks of bacterial culture in an ice bath for 5 min with gentle constant swirling by hand. The following steps should be done in a cold room, on ice, with prechilled solutions and equipment.
13. Use absorbance tissue towel to dry the outer glass of each flask; use one hand to slant the 1-L autoclaved centrifuge bottle on bench and the other hand to pour the medium gently from each flask into each centrifuge bottle.
14. Spin at 5,000 × g and 4 °C for 10 min to pellet the bacteria.
15. Following centrifugation, gently decant the supernatant into a 5-L autoclaved waste beaker.
16. Fill each bottle with 100 mL of sterile-filtered 1.0 mM HEPES, pH 7.4, and add an autoclaved magnetic stir bar to each bottle to aid in pellet resuspension at 200 rpm. Swirl and dislodge the entire pellet from the bottle wall every several minutes during the magnetic stirring. Once the pellet is dissolved, fill each bottle with an additional 400 mL of sterile-filtered 1.0 mM HEPES, pH 7.4.
17. Centrifuge at 5,000 × g and 4 °C for 10 min. Decant the supernatant, retaining the stir bar in the bottle.
18. Repeat steps 1.16 to 1.17 once.
19. Resuspend each pellet in 500 mL of sterile-filtered, 10% ultrapure glycerol with the aid of the stir bars. Swirl and dislodge the entire pellet from the bottle wall every several minutes during the magnetic stirring.
20. Centrifuge and decant as in step 1.17. Repeat step 1.19 once. Use long-arm autoclaved forceps to remove the stir bar.
21. Centrifuge at 5,000 × g and 4 °C for 15 min. Decant the supernatant and remove any remaining traces of supernatant from each centrifuge bottle with a pipette.
22. Add 3.0 mL of 10% ultrapure glycerol to one bottle and gently resuspend the pellet by pipetting. Transfer the suspension to the next bottle and repeat until all of the pellets are resuspended and combined in one bottle. Approximately 6 mL of highly concentrated cells are obtained with a titer of around 3 x 10¹¹ cfu/mL.
23. Pre-chill 1.5-mL autoclaved microcentrifuge tubes and one 96-well tube storage box without dividers at -80 °C for at least 1 h before this step. Transfer the pre-chilled microcentrifuge tubes to the cold room on ice before pipetting aliquots of the cell pellet suspension. Use a pipette to aliquot 350 μL of cell suspension into each 1.5 mL microcentrifuge tube.
24. Transfer the aliquots into a foam box container filled with liquid nitrogen for flash freezing (3-5 min).
    1. Transport the foam box with liquid nitrogen to a -80 °C freezer (see step 1.23).
    2. Remove the 1.5-mL microcentrifuge tube storage box from the -80 °C freezer (see step 1.23) and put on ice.
    3. Quickly use a metal mesh to sieve the aliquots from liquid nitrogen and place them in the tube storage box. Store at -80 °C.
       CAUTION: Liquid nitrogen can cause burns and care must be taken for safety protection.Use a Fab backbone phagemid to check the efficiency of the prepared electro-competent cells (the Fab backbone phagemid stock should be in ultrapure (MilliQ) water at 400 ng/µL). Thaw 1 μg of the Fab backbone phagemid in 10 µL ultrapure water and a 350 μL aliquot of electro-competent SS320 on ice, and prechill a 0.2-cm gap electroporation cuvette on ice.
25. Add the 350 μL electro-competent SS320 cells to the 10 µL DNA after thawing and mix by pipetting several times while keeping the mixtures on ice. Avoid introducing bubbles.
26. Pre-warm 15 mL of SOC medium in a 50-mL tube in a 37 °C water bath for at least 30 min before electroporation. Transfer the 360 μL mixture to the cuvette and perform electroporation following the manufacturer's instructions.
27. Immediately rescue the cells after electroporation by adding 1 mL of pre-warmed SOC medium (from step 1.27) and pipetting. Transfer the medium from the cuvette to a 125-mL flask pre-loaded with 5 mL of pre-warmed SOC.
28. Rinse the cuvette twice, each time with 1 mL of pre-warmed SOC medium, and transfer the medium to the 125-mL flask (see step 1.27). Add pre-warmed SOC medium to a final volume of 10 mL to the 125-mL flask.
29. Incubate the 10-mL cell culture for 30 min at 37 °C with shaking at 200 rpm.
30. Make serial dilutions to determine the transfection efficiency and M13KO7 pre-infection rate.
    1. Use a multi-channel pipette to add 180 μL of 2YT media to each well of a single column of a 96-microwell plate.
    2. Make 8 tenfold serial dilutions: transfer 20 μL of culture from step 1.29 to the first well of the plate, mix by pipetting, and transfer 20 μL of the mixture to the next well. Repeat this step to the end of the serial dilution.
    3. Plate 10 μL of each serial dilution on the LB/carb, LB/kan, and LB/tet plates in duplicate.
    4. Incubate overnight at 37 °C.
    5. Efficiency calculation formula: Assume that M is the average colony number counted from the diluted fold 10^N (N is from 1-8). The E. coli SS320 electro-competent cell transfection efficiency of the Fab backbone phagemid from LB/carb plate is equal to M X 10^N+3 cfu/µg. The M13KO7 pre-infection rate is estimated from the ratio of the colonies in LB/kan and LB/tet. The percentage of E. coli SS320 competent cells transfected with the Fab backbone phagemid is estimated from the ratio of colonies in LB/carb and LB/kan.

2. Preparing Uracil-containing ssDNA (dU-ssDNA) from the Phagemid Template

NOTE: A previously reported Fab backbone phagemid was used as the template for dU-ssDNA preparation¹⁵. The architecture of the Fab backbone phagemid is shown in Figure 2. A plasmid spin kit (QIAprep Spin M13) is used for extraction of dU-ssDNA with slight modifications.

1. Pre-warm a LB/cmp plate (prepared and stored at 4 °C for less than 1-week old) in a 37 °C incubator for 1 h. Streak out a glycerol stock of E. coli CJ236 cells (or another dut−/ung− strain) with sterile loops or tips onto the pre-warmed LB/cmp plate. Incubate the plate at 37 °C overnight for around 12-15 h.
2. Pick a single colony with a sterile tip and inoculate into 2 mL of 2YT/cmp medium in a 14-mL polypropylene round-bottom tube.
3. Incubate the medium at 37 °C with shaking at 200 rpm for 3-4 h until OD₆₀₀ is reached at around 0.4-0.8 (log phase growth).
4. Add 5-10 μL phage of Fab backbone template into the culture, and incubate at 37 °C with shaking at 200 rpm for 30 min to allow phage infection.
5. After incubation, use a sterile tip to streak out 10 μL of culture onto a pre-warmed LB/carb plate at 37 °C. Incubate at 37 °C overnight for around 12-15 h.
6. Pick a single colony of the E. coli CJ236 containing Fab backbone phagemid to inoculate a starter culture of 3 mL 2YT/carb/cmp in a 14-mL polypropylene round-bottom tube, and grow at 37 °C with shaking at 200 rpm overnight for around 12 h.
7. Inoculate the 0.3 mL starter culture into 30 mL 2YT/carb/cmp in a 250-mL baffled bottle.
8. Incubate the cell culture at 37 °C with shaking at 200 rpm for 3-4 h until OD₆₀₀ is reached at around 0.4-0.8 (log phase growth).
9. Add the M13KO7 (lab stock, titer of approximately 1 x 10¹³ cfu/mL) to the culture from step 2.8 with a multiplicity of infection (MOI) of approximately 10 and the final titer of M13KO7 is approximately 1 x 10¹⁰ cfu/mL.
10. Incubate at 200 rpm and 37 °C for 1 h.
11. Pellet the culture by centrifugation in a 50-mL round-bottom tube at 5,000 × g and 25 °C for 20 min.
12. Decant the supernatant and resuspend the pellet with 30 mL of fresh 2YT/carb/kan/uridine. Transfer the resuspension into a new 250-mL baffled bottle.
13. Incubate at 200 rpm and 25 °C for 22-24 h.
14. Transfer the culture from step 2.13 into a 50-mL round-bottom tube and centrifuge the culture at 12,000 × g for 20 min to separate the phage supernatant from bacterial cell pellet. Transfer the phage supernatant into a new 50-mL round-bottom tube and add 1/5 the final volume of PEG/NaCl solution to precipitate the phage. Mix well and incubate on ice for 30 min to precipitate the phage particles.
15. Centrifuge at 12,000 × g and 4 °C for 30 min. Decant the supernatant and centrifuge at 4,000 × g and 4 °C for 2 min. Aspirate the remaining supernatant.
16. Resuspend the phage pellet in 2 mL of sterile-filtered 1X PBS and transfer to 1.5-mL microcentrifuge tubes. Centrifuge at 12,000 × g for 5 min in a benchtop microcentrifuge to remove any remaining bacterial debris and transfer the phage supernatant to new 1.5-mL microcentrifuge tubes and store at 4 °C.
17. Check the uracil incorporation efficiency in E. coli CJ236 through side by side comparison with E. coli SS320.
    1. Add 180 μL of 2YT media to each well of a single row of a 96-well plate.
    2. Make ten 10-fold serial dilutions: transfer 20 μL of the phage supernatant to the first well of the plate, mix by pipetting, and transfer 20 μL of the mixture to the next well. Repeat this step to the end of the serial dilution.
    3. Add 10 μL of phage from last eight serial dilutions to infect 90 μL of E. coli CJ236 and E. coli SS320 in log phase (OD₆₀₀ = 0.4-0.8). Incubate at 37 °C for 30 min with gentle shaking.
    4. Plate 10 μL from the serial dilution of each infection in E. coli CJ236 and E. coli SS320 on 2YT/Carb plates in duplicate.
    5. Incubate overnight at 37 °C.
    6. Titer calculation formula: Assume that M is the average colony number counted from the diluted fold 10^N (N is from 1-10). The titer from E. coli CJ236 or E. coli SS320 is equal to M X 10^N+2 cfu/mL. Estimate the efficiency of uracil incorporation from the titer ratio of E. coli CJ236 and E. coli SS320.
18. Add 1/100 volume of the phage precipitation buffer MP into the phage supernatant in 1.5-mL microcentrifuge tubes, and turn upside down gently to mix for several times. Incubate at RT for at least 2 min. Phage particles are precipitated from the culture medium, and thus, a cloudy solution should be visible at this point.
19. Apply the sample from step 2.18 to a plasmid spin column (e.g., QIAprep) in a 1.5-mL microcentrifuge tube. The binding capacity of one spin column for ssDNA can reach at least 10 µg. Centrifuge at 6,000 × g and 25 °C for 30 s in a benchtop microcentrifuge. Discard the flow-through which is in the 1.5-mL microcentrifuge tube. Phage particles remain bound to the column matrix at this stage.
20. Add 0.7 mL of the UT-MLB phage lysis and binding buffer (see Discussion) to the column and incubate at RT for at least 1 min.  Centrifuge at 6,000 x g and 25 °C for 30 s and discard the flow-through.
21. Add another 0.7 mL of the UT-MLB buffer and incubate at RT for at least 1 min.
22. Centrifuge at 6,000 × g for 30 s. Discard the flow-through. At this stage, the phage coat protein is separated from the dU-ssDNA, which remains bound to the column matrix.
23. Add 0.7 mL of wash buffer PE containing ethanol following the manufacturer's instructions. Centrifuge at 6,000 × g for 30 s and discard the flow-through.
24. Repeat step 2.23, then centrifuge once more at 6,000 × g for 30 s to remove residual buffer PE.
25. Transfer the column to a new 1.5-mL microcentrifuge tube and add 100 μL of elution buffer EB (10 mM Tris·CL, pH 8.5) to the center of the column membrane.
26. Incubate at RT for 10 min and centrifuge at 6,000 × g for 1 min to elute the dU-ssDNA. Approximately, 1.5-2.5 μg dU-ssDNA/mL culture can be obtained.
27. Analyze the eluted DNA by electrophoresing 1 μL on a 1% agarose TAE gel. The DNA should appear as a predominantly single band with no smearing.
28. Determine the DNA concentration by measuring absorbance on a nanodrop spectrophotometer at 260 nm (A₂₆₀ = 1.0 for 33 ng/μL of ssDNA). Typical dU-ssDNA concentrations are within 200-500 ng/μL.

3. Kunkel's Method Based Oligonucleotide-directed Mutagenesis

Notes: It is advisable to conduct small-scale reactions prior to full scale reactions to ensure the quality of the mutagenic oligonucleotide and reaction components. A cartoon of Kunkel's method based oligonucleotide-directed mutagenesis is shown in Figure 3. Various amino acid diversities are introduced into CDRH1, CDRH2, CDRH3, and CDRL3 regions with IMGT numbering nomenclature¹⁶ (Table 2). The theoretical amino acid diversity of each CDR, total theoretical amino acid diversity, and oligonucleotide sequences are listed in Table 2.

1. Oligonucleotide phosphorylation with T4 polynucleotide kinase
   1. Combine 0.6 μg of mutagenic oligonucleotides, designed to mutate a single CDR, with 2 μL 10X TM buffer, 2 μL 10 mM ATP, and 1 μL 100 mM DTT. Add ultrapure H₂O to a total volume of 18 μL in a 1.5-mL tube. For the library construction, 4 separate and parallel phosphorylation reactions are set up corresponding to CDRH1, CDRH2, CDRH3, and CDRL3, respectively.
   2. Add 20 U (2 uL) of T4 polynucleotide kinase to each tube and incubate for 1 h at 37 °C (Reaction 1 in Table 3). Use immediately for annealing.
2. Annealing of the oligonucleotides to the template
   1. To 20 μg of dU-ssDNA template, add 25 μL of 10X TM buffer, 20 μL of each phosphorylated oligonucleotide solution, and ultrapure H₂O to a final volume of 250 μL in a 0.5-mL tube. Mix well and transfer into 0.20 mL PCR tubes (50 μL each) as Reaction 2 in Table 3. These DNA quantities provide a molar ratio of 3:1 between oligonucleotide and template, assuming that the length ratio of oligonucleotide and template is 1:100.
   2. Incubate the reaction in a PCR machine at 90 °C for 3 min, 50 °C for 3 min, and 20 °C for 5 min.
3. Enzymatic synthesis of CCC-dsDNA
   1. To the 250 μL annealed oligonucleotide/template mixture, add 10 μL of 10 mM ATP, 10 μL of 25 mM dNTP mix, 15 μL of 100 mM DTT, 30 Weiss units T4 DNA ligase, and 30 U T7 DNA polymerase as Reaction 3 in Table 3.
   2. Incubate the reaction in the 1.5-mL tube at 20 °C overnight.
   3. Wash and concentrate the synthesized CCC-dsDNA in a 0.5-mL centrifugal filter device with a 30 kDa pore size membrane at RT.
      1. Transfer the overnight reaction mixture to the filter device and add ultrapure H₂O to 400 µL final volume. Spin at 14,000 × g for 10 min; the volume is less than 50 μL.
      2. Discard the flow through, add 400 µL of ultrapure H₂O into the filter, and spin at 14,000 × g for 10 min.
      3. Repeat step 3.3.3.2 once more.
      4. Place the filter upside down in a clean microcentrifuge tube to recover the CCC-dsDNA. Spin at 1,000 × g for 2 min; the recovered volume is generally around 20-40 μL. The recovered CCC-dsDNA can be used immediately for electroporation of E. coli or frozen at -20 °C for later use. Normally 20-40 μg CCC-DNA can be obtained.
   4. Electrophorese 1.0 µL of the eluted reaction product alongside the dU-ssDNA template to visualize the outcome of the reaction.

4. Electroporation and Calculation of the Library Size

1. Chill the purified CCC-dsDNA (20 μg in a maximum volume of 50 μL) in a 1.5-mL microcentrifuge tube and a 0.2-cm gap electroporation cuvette on ice.
2. Pre-warm 20 mL of SOC media in a 50-mL polypropylene conical centrifuge tube in 37 °C water bath for at least 30 min.
3. Thaw a 350 μL aliquot of electro-competent E. coli SS320 on ice. Add the cells to the DNA and mix thoroughly by pipetting several times. Avoid introducing bubbles.
4. Transfer the mixture to the cuvette and perform electroporation following the manufacturer's instructions. For example, when the BTX ECM-630 electroporation system is used, the relevant settings are 2.5 kV field strength, 125 Ω resistance, and 50 µF capacitance.
5. Immediately rescue the electroporated cells by adding 1 mL of pre-warmed SOC medium and transferring into 17 mL of SOC medium in a 125-mL flask. Rinse the cuvette twice with 1 mL of SOC medium and transfer to the same flask (final volume is 20 mL).
6. Incubate for 30 min at 37 °C with shaking at 200 rpm.
7. Determine the electroporation efficiency.
   1. Add 180 μL of 2YT media to each well of a single column of a 96-microwell plate.
   2. Make 8 ten-fold serial dilutions: transfer 20 μL of the 20-mL culture to the first well of the plate, mix with pipetting, and transfer 20 μL of the mixture to the next well. Repeat this step to the end of the serial dilution.
   3. Plate 10 μL of each of the serial dilutions on one LB/carb plate in duplicate. Plate 100 µL remaining from each of the serial dilutions onto separate LB/carb plates for colony count cross-check. These plates will also provide single clones for ELISA and sequence analysis (see section 5).
   4. Incubate overnight at 37 °C.
   5. Count the colonies from the 10 μL duplicates on the LB/carb plate. Assume that M is the average colony number counted on 10^N fold LB/carb plate (N is from 1-8). The total library size is equal to 2M X 10^N+3 colonies.
8. Aliquot the culture from step 4.6 equally into two 2-L baffled flasks, each containing 500 mL of 2YT/carb/kan medium for phage library generation.
9. Incubate at 37 °C with shaking at 200 rpm overnight for around 16 h.
10. Transfer the culture to two 1-L autoclaved centrifuge bottles and centrifuge for 30 min at 12,000 × g at 4 °C.
11. Transfer the supernatant to two new 1-L autoclaved centrifuge bottles and add 1/5 the final volume of PEG/NaCl solution to precipitate the phage. Incubate on ice for 30 min.
12. Centrifuge for 30 min at 12,000 × g and 4 °C. Carefully decant the supernatant and avoid disturbing of the phage pellet. Spin for 1 min at 4,000 x g and remove the remaining supernatant with a pipette.
13. Resuspend the phage pellet with 20 mL of sterile-filtered 1X PBS buffer and transfer to a new 50-mL tube.
14. Pellet the insoluble matter by centrifuging for 5 min at 12,000 × g and 4 °C. Transfer the supernatant to a new 50-mL tube.
15. Measure the phage concentration by spectrophotometer (OD₂₆₈ = 1.0 for a solution of 5 X 10¹² phage/mL).
16. Adjust the phage concentration to 5 X 10¹² phage/mL in 1X PBS with 10% ultrapure glycerol.
17. Aliquot 1 mL of phage solution per 1.5-mL microcentrifuge tube. Use the libraries immediately for panning or store at -80 °C.

5. Quality Assessment by Protein A/L Direct Binding ELISA Assay and Sequencing

1. Randomly pick 96 single colonies on LB/carb plate from step 4.7.3 into a 96-deep well culture plate containing 800 μL of 2YT/carb in each well. Incubate for 3-4 h at 37 °C with shaking at 1,000 rpm to OD₆₀₀ = 0.4-0.8.
2. Add 100 μL of M13KO7 (1 X 10¹¹ cfu/mL) to each well of a 96-deep well culture plate with a multichannel pipette. Incubate at 37 °C with shaking at 1,000 rpm for 1 h.
3. Add 100 μL of 2YT containing 10X concentration of kanamycin (500 μg/mL) to each well with a multichannel pipette. Incubate overnight at 37 °C with shaking at 1,000 rpm.
4. Dissolve protein L to 1 µg/mL in 1X PBS. Coat 3/4 of the wells in a 384-well high protein-binding polystyrene plate with 30 µL/well of the protein L solution. Incubate overnight at 4 °C.
5. Discard the overnight protein L coating solution from step 5.4. Add 50 µL of M-PBST (the blocking solution) to all of the wells in the 384-well high protein-binding polystyrene plate with a multichannel pipette. Incubate the plates on a microplate shaker for 1 h at RT.
6. Spin down the overnight culture from step 5.3 in a deep 96-well culture plate at 3,000 x g for 10 min at 4 °C. The phage is in the supernatant.
7. Discard the blocking solution from step 5.5. Add 15 μL of M-PBST and 15 μL of each phage supernatant (step 5.6) to 3 of the protein L coated wells as triplicate, and 1 non-coated well as negative control using a multichannel pipette. Incubate at RT for 1 h with shaking at 200 rpm.
8. Discard the phage solution. Wash the plate 6 times with 80 μL of PBST by an automated plate washer.
9. Add 15 μL of M-PBST and 15 μL of Protein A-HRP (1:1,500 diluted in 1X PBS) to each well with a multichannel pipette, and incubate at RT for 1 h with shaking at approximately 200 rpm.
10. Discard the Protein A-HRP/M-PBST solution. Wash the plate 6 times with 80 μL of PBST.
11. Add TMB substrate (30 μL/well) with a multichannel pipette and incubate for 2-3 min until the color develops. Stop the reaction with 1.0 M H₃PO₄ (30 μL/well).
12. Read the plates spectrophotometrically at 450 nm. Positive clones are defined as those that exhibit an average ratio of OD₄₅₀ absorbance of protein L wells to the M-PBST well greater than 3.0.
13. In a 96-deep well, infect 50 μL of SS320 in 2YT/tet at log phase with 5 μL of the same phage used for the ELISA (step 5.6) for 30 min at RT.
14. Add 950 μL of 2YT/carb into the 96-deep well from step 5.13 and incubate overnight at 37 °C with shaking at 1,000 rpm.
15. Extract the phagemid DNA by mini-prep DNA extraction kit. Use the upstream primers of VL (5'-TCGCTTTGTTTTTATTTTTTAATGTA-3') and VH (5'-GACTACTAATAACATAAAGTCTACGCCG-3') for sequence analysis to estimate the library sequence diversity.