Sheep anti human c3c fitc

Antibody-mediated C3b/iC3b deposition on the surface of whole GBS bacteria was measured on the surface of formaldehyde-fixed GBS using a flow cytometric assay performed in 96-well microtitration plates. Briefly, 35 μL serotype Ia, Ib, II, III and V GBS bacteria at 5.14 × 10⁷ CFU/mL in blocking buffer (1% BSA in PBS) were added to 10 μL IgG-depleted human complement [22] (link) and 5 μL of each test serum. Plates were incubated for 7.5 min at 37 °C with shaking (900 rpm) then pelleted. Supernatant was removed and the pellet washed once with 200 μL blocking buffer. Samples were resuspended in 200 μL blocking buffer containing 1:500 sheep anti-human C3c FITC (Abcam) and incubated as for the surface binding assay.

Antibody-mediated C3b/iC3b deposition onto the surface of formaldehyde-fixed GBS was measured using a flow cytometric assay performed in 96-well microtitre plates [14] (link). Briefly, 35 μL serotype Ia, II, III or V GBS bacteria at 5 · 14 × 10⁷ CFU/mL in blocking buffer (1% BSA in PBS) were added to 10 μL IgG-depleted human plasma as the complement source [15] (link) and 5 μL of each test serum. Plates were incubated for 7.5 min at 37 °C with shaking (900 rpm), and the bacteria pelleted by centrifugation at 3000g for 5 min. Supernatant was removed and the bacteria washed once with 200 μL blocking buffer. Bacteria were resuspended in 200 μL blocking buffer containing 1:500 sheep anti-human C3c FITC (Abcam) and incubated for 20 min before washing and analysis by flow cytometry.