Immulon 2hb elisa plates

HMGCR antigen (Sigma-H7039) (Sigma-Aldrich) was diluted to 2 μg/mL in 0.01 M phosphate-buffered saline (PBS). HMGCR 0.1 μg was placed in each well of Immulon 2 HB ELISA plates (Thermo Fisher Scientific, Waltham, MA) and incubated overnight at 4°C. After washing 3 times with PBS-0.05% Tween 20, residual nonspecific binding sites in ELISA wells were blocked with 1% normal goat serum in PBS (PBS-NGS) (100 μL per well) for 4 hours at room temperature and washed 3 times with PBS-NGS. Subsequent steps were performed at 4°C. Between steps, washing was performed 5 times using PBS-NGS without detergent. Patient sera were tested in duplicate at 1:3,000 dilution with overnight incubation in ELISA wells at 4°C. Binding of serum IgG to HMGCR was measured using a 4-hour exposure to goat anti-human IgG linked to horseradish peroxidase (Organon Teknika–Cappel, West Chester, PA) in PBS with 1% bovine serum albumin (1:20,000). Color was developed with 100 μL substrate buffer (0.1 M citrate buffer, pH 4.5 with 0.004% H₂O₂ and 0.1% phenylenediamine) for 30 minutes. Optical density was measured at 450 nm. Final anti-HMGCR antibody titers were calculated after subtracting levels of nonspecific serum IgG binding to sulfatide. A normal range of values was determined by analysis of sera from 85 adult patients with other immune or inflammatory neuromuscular disorders.