Ph d 12 phage display peptide library kit

Ph.D.™-12 Phage Display Peptide Library Kit (New England Biolabs, Ipswich, MA, USA) was used to map epitopes on CD24 [42 (link)]. cG7 was coated in the plate for 12 h at 4°C and incubated with 1.5×10¹⁰ phages for 2 h at 37°C. After washing thrice with PBS, the bound phages were eluted and amplified in E. coli ER2738 for the next cycles. Four cycles later, high affinity individual clones were selected by ELISA and sequenced. Based on the sequencing results, we analyzed the cG7 epitope. The ZDOCK program (version 3.0.2) was used to dock cG7 and epitope residues on CD24 using a structure downloaded from the Protein Model Portal (PMP) [43 (link), 44 (link)]. The superior modeled complex structure was analyzed by MOE and the residues that played an important role in the molecular interactions were identified.

The fibrillized d- or l-enantiomeric PHF6 peptide, prepared as described above, was immobilized on polystyrene 96-well microtiter plates (Thermo-Fischer Scientific, Waltham, USA). The assay plate to be coated was shaken for 1 hour at room temperature with gentle agitation. Until the selection started, the coated assay plate was stored at 4°C. Phage display was performed using the Ph.D.-12 Phage Display Peptide Library Kit (New England Biolabs, Ypswich, USA) according to the instructions of the manufacturer.

The PhD-12 Phage Display Peptide Library Kit (New England Biolabs) was used to identify mimotope peptide sequence(s), as previously reported in detail (33 (link)). For each mAb at least 12 phage clones were sequenced. The obtained 12-mer peptide sequences of each screened mAbs were first ranked and then the unique sequence(s) or motif(s) were determined. Unique sequence(s) of each antibody were used to determine epitope-level redundancy based on the frequencies of common mimotope peptide(s) of compared mAb pairs according to the following formula: $\mathrm{R}\mathrm{e}\mathrm{d}XY=\left[\left(Xn1\ast Ym1\right)\ast 100\right]+...+\left[\left(Xni\ast Ymi\right)\ast 100\right]$ where
Xn = the frequency of the nth peptide of mAb X (number of unique mimotope peptides n divided by the number of all peptides of mAb X)
Ym = frequency of the mth peptide of mAb Y (number of unique mimotope peptides m divided by the number of all peptides of mAb Y)
Xn1 = the frequency of the first common mimotope peptide of mAb X between mAbs X & Y
Ym1 = the frequency of the first common mimotope peptide of mAb Y between mAbs X & Y
Xni = the frequency of the ith common mimotope peptide of mAb X between mAbs X & Y, and
Ymi = the frequency of the ith common mimotope peptide of mAb Y between mAbs X & Y.
A list of the unique peptides of the QP library mAbs can be found in supplemental Table S1. A database containing all peptide data shown in supplemental Fig. S2. can be found there.

Anti-PPRV-H C77 monoclonal antibody (mAb) derived from a hybridoma line was purified from cell culture supernatant using the Melon Gel Monoclonal IgG Purification Kit (Pierce). Purified C77 was dialysed against 0.1 M NaHCO₃ (pH 8.6) and its concentration measured using a BCA Protein Assay Kit (Pierce) [15] (link). The Ph.D.-12™ Phage Display Peptide Library Kit (New England BioLabs) was used to screen for C77 binding epitopes as described previously [15] (link). Data obtained from several rounds of panning were analysed using the Vector NTi software package (Invitrogen) and amino acid sequences were mapped to the native PPRV Nigeria75/1 H protein.

Ph.D.™-12 Phage Display Peptide Library Kit (New England Biolabs) was used for biopanning. This library contained 2 × 10¹¹ unique 12-mer peptides linked to the N-terminus of a phage coat protein by a 4 amino acid spacer (GGGS). The library was diluted in TBS (50 mM pH 7.5 Tris–HCl, 150 mM NaCl). Microtubes coated with 100 µg/mL purified proteins in 0.1 M pH 8.6 NaHCO₃ were used for the selection of phages. BORIS-del N_1-258 was used in the first step of elutriation for 60 min at room temperature (RT) to remove non-specific phage clones. The pre-cleared phage library was used for further elutriations on BORIS-N_1-258-coated microtubes for 60 min at room temperature and eluted with a general buffer (0.2 M pH 2.2 Glycine–HCl, 1 mg/mL BSA). The eluate enriched in BORIS-N_1-258 binding phage clones was neutralized with 1 M pH 9.1 Tris–HCl. By two additional rounds of selection, phage clones enriched in BORIS-N_1-258 binding clones were prepared.

The purified ORF2 mAbs were applied as targets and subjected to biopanning using a Ph.D.-12 Phage Display Peptide Library Kit (New England Biolabs, MA, USA). The procedure was performed according to the manufacturer’s instructions and a previous study [13 (link)]. The ratio in each round was calculated to analyze the enrichment efficiency using the titer of the amplified phages in the input buffer (input value) and in the elution buffer (output value).
Individual phage clones were selected from the final round and amplified in E. coli ER2738 (New England Biolabs, USA), followed by precipitation according to the manufacturer’s protocols. Genes encoding the exogenous peptides of M13 were amplified by polymerase chain reaction (PCR) using the forward primer 5'-TCACCTCGAAAGCAAGCTGA and reverse primer 5'-CCCTCATAGTTAGCGTAACG. The PCR parameters were: 95°C for 5 min, 30 cycles of 95°C for 30 s, 56.5°C for 30 s, 72°C for 30 s, with a final extension at 72°C for 5 min.
The binding activities of phage clones displaying different high frequency peptide sequences, at concentrations of 10¹⁰–10⁴ plaque-forming units (pfu) diluted in 0.1 M NaHCO₃ (pH 8.6), to ORF2 mAbs were detected by ELISA, according to the instruction of Ph.D.-12 Phage Display Peptide Library Kit.

Before biopanning on cancer cells, the phage peptide library (Ph.D.™-12 Phage Display Peptide Library Kit, New England BioLabs, USA) was depleted on a primary culture of untransformed human breast BN-2 cells. To do this, BN-2 cells were incubated with the phage peptide library (0.3 × 10¹¹ PFU/mL) at 37°C for 1 h. The depleted library contained in the supernatant was amplified according to the manufacturer’s protocol and used in biopanning.
Biopanning of the phage peptide library on MDA-MB-231 cancer cells was performed as described previously with some modifications [10 (link)].