Hrp conjugated donkey anti goat igg antibody

Cell-based enzyme-linked immunosorbent assay (ELISA) was used to evaluate the factor H deposition at the bacterial surface of different mutants obtained in this study, as previously described with some modifications [10 (link)]. Briefly, washed harvested bacteria were adjusted to an OD₆₀₀ of 0.2 in 50 mM carbonate buffer (pH 9.6). One-hundred microliters per well were added to flat-bottom 96-well microplates (Nunc-immuno^® Polysorp; Nalge Nunc International, Rochester, NY, USA) and incubated for 2 h at room temperature. Bacterial suspension was removed and bound bacteria were fixed with glutaraldehyde (0.05%) for 45 min. Wells were then washed three times with PBS-Tween 0.05% (PBS-T) and blocked with PBS-T supplemented with 2% fat-free milk for 1 hour. After washing, fixed bacteria were then incubated in presence of 100 µL of human factor H (10 µg/mL in PBS) for 2 hours at 37 °C. Plates were then washed three times to remove unbound factor H. The deposition of factor H at the bacterial surface was detected by goat antisera against human factor H (Quidel) and HRP-conjugated donkey anti-goat IgG antibody (Jackson ImmunoResearch, West Grove, PA, USA). The OD₄₅₀ was recorded with a microplate reader after adding horseradish peroxydase substrate. Each assay was repeated in duplicates in four independent experiments.