D25 competitive ELISA

DS-Cav1 was coated on microtiter plates (Greiner Bio-One) overnight at 4°C and then blocked with PBS–0.05% Tween–5% nonfat dry milk for 1 hour at 37°C. Sera were serially diluted twofold (initial dilution, 1:5) and incubated for 1.5 hours at 37°C. After PBS–0.05% Tween washes, competitive biotinylated D25 Ab was added and incubated for 30 min at 37°C. Plates were washed and an anti-biotin HRP conjugate (Invitrogen) was added for 1.5 hours at 37°C. After washing, the plates were developed using 3,3,5,5-tetramethylbenzidine (tebu-bio) and stopped with 1 M HCl (Prolabo). ODs were read at 450 to 650 nm. After blank subtraction, the percentage of inhibition was calculated as follows: [1 − (sera OD)/(baseline OD)] × 100. The dilution needed to achieve 50% of inhibition was obtained by a four-parameter logistic regression.