Protein g coated plates

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.

Isotype-specific detection of immunoglobulins (Igs) was carried out according to the protocol by Germundson and Nagamoto-Combs [49 (link)] with modifications. Eight-well RIA strips (Corning, Inc., Corning, NY, USA) were coated with 2 μg/mL BLG in a sodium carbonate/bicarbonate buffer (pH 9.5) overnight at 4 °C. The wells were washed and blocked in the PBS containing 0.5% bovine serum albumin (BSA). The serum samples were diluted to 1:40 and incubated with protein G-coated plates (Thermo Fisher, Waltham, MA, USA) for 1 h at 37 °C to adsorb total IgG, and the resulting supernatant was subsequently added to each well. Allergen-specific IgE was detected using secondary anti-mouse IgE and avidin HRP (eBioscience, San Diego, CA, USA) [49 (link)]. The substrate reaction was terminated with 2N sulfuric acid, and the plates were immediately read at 450 nm with a reference wavelength of 550 nm on an ELx800 Universal Microplate Reader (BioTek Instruments, Winooski, VT, USA).