M13 phage

Aβ42 (ab120301), Aβ40 (ab120479), and anti-β amyloid antibody (ab2539) were purchased from Abcam (Cambridge, UK). 1,1,1,3,3,3-Hexafluoro-2-propanol was purchased from Sigma-Aldrich (St. Louis, MO, USA). Protein G–coated plates were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-β-amyloid 1–42 (AB5078P) and anti-β-amyloid 1–40 (AB5074P) antibodies were purchased from Merck (Kenilworth, NJ, USA). M13 phages were purchased from New England Biolabs (Ipswich, MA, USA). The studied Aβ42-binding peptide probe (ABPP) sequences were synthesized by AnyGen (Gwangju, Korea). Streptavidin-HRP was obtained from BD Difco Laboratories (Sparks, NV, USA). 3,3′,5,5′-tetramethylbenzidine (TMB) solution was purchased from R&D Systems (Minneapolis, MN, USA). All chemicals were obtained from commercial sources and were of the highest quality available.

Epitope mapping of both isotypes was performed as we described previously [12 (link)], using linear 12-mer peptide libraries displayed on the protein pIII of M13 phages (New England Biolabs) as recommended by the manufacturer. In brief, magnetic beads were incubated with Fab IgA or IgG on a rotating wheel for 2 h at room temperature and epitope screening was initiated by incubating each Fab IgA or IgG coupled beads with the original 12-mer (10¹³) phage-displayed peptide library containing different phages, overnight at 4°C. After three rounds of selection with increasing stringency, the phages were tittered, and single clones were picked and tested by phage ELISA for specific binding on each FabA or FabG. Positive clones were amplified, and each specific peptide insert was sequenced. Importantly, two rounds of negative selection using beads coated with normal human IgA or IgG (Jackson ImmunoResearch) were introduced between two rounds of positive selections. Phages remaining from the negative selection were amplified in Escherichia coli ER2738, precipitated, and used for second and third rounds of selection similar to the first round, but with increased buffer stringency as described [12 (link)].