ELISA was conducted as previously described [23 (link)] with some
modifications. His-BL0675A was added to 96-well microtiter plates coated with PCMs and incubated at room
temperature for 1 hr. After washing three times with HBS-EP buffer (pH 7.4), an anti-His-tag antibody
(1:3,000; Roche) was added and the plates were incubated at room temperature for 40 min. After washing,
alkaline phosphatase-conjugated anti-mouse IgG (1:4,500; Dako, Glostrup, Denmark) was added for 40 min at room
temperature. Antibody-specific signals were detected with BluePhos MicroWell Substrate Kit (KPL, Gaithersburg,
MD, USA) by measuring the absorbance at 620 nm.
modifications. His-BL0675A was added to 96-well microtiter plates coated with PCMs and incubated at room
temperature for 1 hr. After washing three times with HBS-EP buffer (pH 7.4), an anti-His-tag antibody
(1:3,000; Roche) was added and the plates were incubated at room temperature for 40 min. After washing,
alkaline phosphatase-conjugated anti-mouse IgG (1:4,500; Dako, Glostrup, Denmark) was added for 40 min at room
temperature. Antibody-specific signals were detected with BluePhos MicroWell Substrate Kit (KPL, Gaithersburg,
MD, USA) by measuring the absorbance at 620 nm.
