Blotto blocking buffer

Patient sera were tested on dried slides as previously described.[27 ] In short, slides were incubated in Blotto blocking-buffer (Thermo Fisher Scientific, MA, USA) for one hour at 37°C in an incubation chamber to reduce non-specific binding of serum. Serum was diluted in eight two-fold dilution steps (1:10 to 1:2560) in blotto supplemented with 0.1% Surfact-Amp (Thermo Fisher Scientific, MA, USA) and incubated for 1 hour at 37°C in a moist chamber. Incubation followed with an Fc-fragment specific IgG or Fc5μ-fragment specific IgM specific conjugate with a Cy5-fluorescent dye (Invitrogen, CA, USA) for one hour at 37°C. For IgM detection, serum was first depleted of IgG antibodies using Gullsorb (Meridian Bioscience, OH, USA) according to the manufacturer’s instructions. Between each incubation step, slides were washed three times with a protein array washing buffer (Thermo Fisher Scientifc, MA, USA). After final wash, slides were scanned with a Tecan scanner (Tecan Trading AG, Männedorf, Switzerland). A median fluorescence signal (measured at 647nm) for each of the triplet spots per antigen was determined by ScanArray Express 4.0.0.0001 supporting program (PerkinElmer, MA, USA) using an adaptive circle (diameter 80–200 μm). The fluorescent signal ranged from 0 to a maximum of 65,536 units. Results were imported in R for analysis.[28 ]

IgG concentrations in animal sera were measured using a total human IgG MSD assay optimized for detection of full-length human IgGs and scFv-Fcs. Briefly, 96-well MSD plates (Meso Scale Diagnostics, Rockville, MD, USA) were coated overnight with 100 ng/well of goat anti-human variable light-chain (V + L) fragment antigen-binding region (Fab) (Jackson ImmunoResearch, West Grove, PA, USA). Plates were washed and blocked with 5% BLOTTO Blocking buffer (ThermoFisher). Samples were serially diluted in PBS and incubated for 1 h at room temperature, followed by a wash step and the addition of 25 ng/well goat anti-human-FC Sulfo-Tag capture antibody (Meso Scale Diagnostics). Plates were incubated again for 1 h at room temperature, after which another wash step was performed, and plates were developed using MSD read buffer T (Meso Scale Diagnostics). IgG concentrations were determined using a protein reference standard (ThermoFisher).

Purified P particles and VLPs were diluted in protein array buffer (Maine Manufacturing) and protease inhibitor (BioVision), with final concentration of 1 mg ml⁻¹ (determined by checkerboard titration, data not shown). Proteins were spotted in triplicate with two 333 pL spots onto 64-pad nitrocellulose-coated slides (Oncyte avid, Grace bio-labs) using a non-contact Piezorray spotter (PerkinElmer) as described previously (Koopmans et al., 2012 (link)). Slides were incubated with Blotto blocking buffer (Thermo Fisher Scientific) to avoid non-specific nitrocellulose binding, and subsequently with serial fourfold diluted human sera starting at a 1 : 40 dilution. Rabbit sera and B-cell supernatants were tested at a single dilution (1 : 20 for rabbit sera, 1 : 8 for B-cell supernatant pools and 1 : 4 for individual cultures). After washing, slides were incubated with goat anti-human IgG or anti-rabbit IgG (Fc-fragment specific), conjugated with Alexa Fluor 647 fluorescent dye (Jackson Immuno Research). Bound dye was quantified using a ScanArray Gx Plus microarray scanner (PerkinElmer).