Gullsorb

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 ]

To distinguish between anti-tTG IgA- and IgG-class antibodies, GullSORB (Meridian Bioscience, Inc.) was used to deplete the samples of IgG, as described [18 (link)]. All samples were studied by tTG-LFRET with and without IgG depletion.