Nunc immuno microwell 96 well solid plates

Cellular fibronectin, from human foreskin fibroblasts (cat. no. F2518), Monoclonal anti-fibronectin cellular antibody produced in mouse clone 3E2 (cat. no. F6140), Anti-Fibronectin antibody produced in rabbit (F3648), TLR4 inhibitor TAK-242 (cat. no. 614316), Nunc-Immuno MicroWell 96 well solid plates (cat. no. M9410) were procured from Sigma-Aldrich, MO, USA). Fetal bovine serum and D-Glucose (cat. no. 50-99-7) were procured from Gibco, Applied biosystems, Bangalore, India. Streptozotocin (STZ) extra pure (cat. no. 14653) and Nicotinamide extra pure (cat. no. 72860) were procured from Sisco Research Laboratories Pvt. Ltd. Delhi, India. TMB Substrate Solution (cat. no. N301) from Thermo Scientific Waltham, MA, USA). Goat anti-rabbit IgG H&L (cat. no. ab6721) and goat anti-mouse IgG H&L (cat. no. ab6789) from Abcam, MA, USA. Insulin (Huminsulin R 100 IU Injection) from Eli Lily and Company (India) Pvt. Ltd. Glucometer (On Call Plus G113-214) and blood glucose test strips (On Call Plus, G133-119) were procured from ACON Biotech (Hangzhou) Co. Ltd., Hangzhou, China.

A 59-peptide array covering the amino acid sequence of the N protein as 17 or 13 mer with 10-amino-acid overlaps was obtained from BEI Resources (catalog no. NR-52404). One milligram of lyophilized peptide was resuspended in 1 mL phosphate-buffered saline (PBS) and diluted to 5 μg/mL in PBS, and 100 μL was added to each well of a 96-well microtiter plate (Nunc-Immuno MicroWell 96-well solid plates; Fisher Scientific). Plates were stored at 4°C for 24 to 48 h and washed three times with 250 μL PBS plus 0.05% Tween 20 (PBS-T). Plates were blocked with 200 μL PBS-T plus 2% bovine serum albumin for 4 h. Blocking buffer was removed and replaced with 100 μL patient sera diluted 1:1,000 in blocking buffer. Plates were incubated for 1 h at room temperature and then washed three times with PBS-T. One hundred microliters of 1 μg/mL horseradish peroxidase (HRP)-conjugated anti-human IgG in blocking buffer was added to wells and incubated for 1 h at room temperature with shaking. The plate was washed three times with 250 μL PBS-T. We added 100 μL 3,3′,5,5′-tetramethylbenzidene (TMB) substrate to wells for 1 min, and then reactions were stopped with 100 μL stop solution. Absorbance was read at 450 nm on a Molecular Devices SpectraMax M3 spectrophotometer using SoftMax Pro v6.5.1 software.

A 59-peptide array covering the amino acid sequence of the N protein as 17 or 13 mer with 10-amino-acid overlaps was obtained from BEI Resources (catalog no. NR-52404). One milligram of lyophilized peptide was resuspended in 1 mL phosphate-buffered saline (PBS) and diluted to 5 μg/mL in PBS, and 100 μL was added to each well of a 96-well microtiter plate (Nunc-Immuno MicroWell 96-well solid plates; Fisher Scientific). Plates were stored at 4°C for 24 to 48 h and washed three times with 250 μL PBS plus 0.05% Tween 20 (PBS-T). Plates were blocked with 200 μL PBS-T plus 2% bovine serum albumin for 4 h. Blocking buffer was removed and replaced with 100 μL patient sera diluted 1:1,000 in blocking buffer. Plates were incubated for 1 h at room temperature and then washed three times with PBS-T. One hundred microliters of 1 μg/mL horseradish peroxidase (HRP)-conjugated anti-human IgG in blocking buffer was added to wells and incubated for 1 h at room temperature with shaking. The plate was washed three times with 250 μL PBS-T. We added 100 μL 3,3′,5,5′-tetramethylbenzidene (TMB) substrate to wells for 1 min, and then reactions were stopped with 100 μL stop solution. Absorbance was read at 450 nm on a Molecular Devices SpectraMax M3 spectrophotometer using SoftMax Pro v6.5.1 software.