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After washing 3 times with phosphate buffered saline (PBS) plus 0.05% Tween 20 (PBS/Tween-20, pH 7.4), various serum dilutions (in two-fold steps from 1:200 to 1:3,200) in PBS/Tween-20 were then applied to the wells at 4 C overnight and the anti-CII antibodies were allowed to bind to the antigen