Thermo max microtiter plate reader

MaxiSorp 96-well microtiter plates (Nunc, Roskilde, Denmark) were precoated with streptavidin (1 µg/mL) for 2 h at room temperature (RT) followed by coating with biotinylated peptides (1µg/mL) for 2 h at RT. Sera diluted (1:200) in TTN were added to the microtiter plates and incubated for 1 h at RT. Patient sera were analyzed in duplicates. After careful washing with TTN buffer, AP-conjugated goat anti-human IgG diluted in TTN (1 µg/mL) was added to the wells and the plates incubated for 1 h at RT. For antibody quantification, AP activity was determined with pNPP (1 mg/mL) diluted in AP substrate buffer. The absorbance was measured at 405 nm, with background subtraction at 650 nm, using a ThermoMax microtiter plate reader (Molecular Devices, Menlo Park, CA, USA).

CRT (1 µg/mL) diluted in carbonate buffer was added to Polysorp microtiter plates (Thermo–Fisher, Waltman, MA, USA) and incubated overnight (ON) at room temperature (RT). Wells were rinsed with TTN (200 µL/well) and blocked in TTN buffer for 1 h (h) at RT on a shaking table. CRT mAb (1 µg/mL), diluted in TTN buffer, were added to the microtiter wells and incubated for 1 h at RT on a shaking table. Hereafter, the wells were rinsed with TTN (200 µL) for 1 min, and this was repeated 3 times. Next, AP-conjugated goat anti-mouse IgG (1 µg/mL) was added and incubated for 1 h at RT followed by washing as above. Bound Abs were quantified using 1 mg/mL p-NPP in AP substrate buffer, 100 µL per well. The absorbance was measured at 405 nm, with background subtraction at 650 nm on a ThermoMax Microtiter Plate Reader from Molecular Devices (San Jose, CA, USA). All samples were tested in duplicates and corrected for background noise.

Resin-bound GAD/CVB peptides truncated systematically from the N-terminal or C-terminal (see Supplementary Materials) were added to a 96-well multiscreen filter plate (Millipore, Copenhagen, Denmark) and rinsed with TTN buffer. All incubations with antibodies diluted in TTN (1:10 for cell culture supernatants, 1:1000 for purified antibodies) were carried out for 1 h at RT followed by three washes in TTN buffer. Resin beads were washed with TTN buffer using a multiscreen vacuum manifold. AP-conjugated goat anti-mouse IgG was used as primary antibody. Bound antibodies were quantified using pNPP (1 mg/mL) diluted in AP substrate buffer. Finally, the buffer was transferred to a Maxisorp microtiter plate (Nunc, Roskilde, Denmark), and the absorbance was measured at 405 nm, with background subtraction at 650 nm, on a Thermo max microtiter plate reader (Molecular Devices, San Jose, CA, USA).