Rhmpl

ELISA plates were coated with rhMPL (R&D Systems) or bovine serum albumin (BSA; negative control; BD Biosciences, Franklin Lakes, NJ, US) at 4 °C overnight. Each well was washed with PBS and blocked with MPBS (5% skim milk in PBS) at 37 °C for 1 h. Next, the phages were added, and incubated at 37 °C for 2 h. After washing five times with PBS, the bound phages were detected using anti-M13 phage-HRP-conjugated antibody (1:2000; Sino Biological Inc, Chesterbrook, PA, US). Absorbance was measured at 450 nm using a VersaMax™ Microplate Reader (Molecular Devices, San Jose, CA, US). The 2R13 was detected using anti-human IgG Fc-HRP-conjugated antibody (1:2000; Abcam, Cambridge, MA, US).

The naïve human combinatorial antibody phage libraries (diversity ≈ 10⁹) were obtained from Scripps Research (La Jolla, CA, US). The overall antibody panning was done as described in a previous study [17 (link)]. Briefly, Dynabeads™ M-270 Epoxy beads (Invitrogen, San Diego, CA, US) were coupled with recombinant human MPL (rhMPL, R&D Systems, Minneapolis, MN, US) at 37 °C overnight with end-over-end rotation. The beads were blocked using MPBST (2% skim milk and 0.05% Tween 20 in phosphate-buffered saline [PBS]) at room temperature for 1 h. After adding the antibody phage libraries, the blocked beads-libraries mixture was incubated at room temperature with end-over-end rotation for 1 h. Subsequently, the supernatant was removed, and the beads were rinsed three times with PBST (0.05% Tween 20 in PBS). The bound phages were eluted using 0.2 M glycine–HCl (pH 2.2) and subsequently neutralized with 2 M Tris–HCl (pH 8.0).

The binding kinetics of 2R13 were measured using the iMSPR mini-instrument (icluebio, Seongnam, Republic of Korea). rhMPL (R&D Systems) or recombinant mouse MPL (rmMPL, R&D Systems) was immobilized on the research-grade carboxylic (COOH) sensor chip (icluebio) by using 10 mM sodium acetate buffer (pH 4.0) at approximately 500 response units (RUs). The chip was blocked with 1 M ethanolamine (pH 8.0) and two-fold serial dilutions (256–16 nM) of 2R13 were individually injected. Data were analyzed using the iMSPR analysis software (TraceDrawer; icluebio).