Figure 7.
Figure 7. Role of specific anti-FbsA antibodies in S agalactiae–induced platelet aggregation. (A) S agalactiae cells (1 × 109) were mixed for 1 hour with 3 mg/mL unfractionated Fbg, Fbg from which IgG had been removed, or with 625 μg/mL affinity-purified antibodies. Bacteria were harvested by centrifugation, washed, and adjusted to 1 × 109 cells/mL. Then, 50 μL of the bacterial suspension was added to 0.4 mL GFPs (4 × 108) and tested for platelet aggregation as reported in Figure 6. To examine the effect of FcγRIIA on platelet aggregation, 0.4 mL GFPs was preincubated for 10 minutes with the anti-FcγRIIA antibody IV.3 (20 μg/mL), mixed with 50 μL of a S agalactiae suspension previously treated with unfractionated Fbg or IgG-free Fbg and then tested for aggregation. (A inset) ELISAs were performed to quantify the level of anti-FbsA antibodies in unfractionated Fbg, IgG-depleted Fbg, or affinity-purified IgG. The assay was performed by incubating FbsA-coated microtiter wells (2 μg/well) with 7.5 μg protein of each fraction followed by the addition of peroxidase-conjugated rabbit antihuman IgG. (B) The reactivity of IgG from human donors to recombinant FbsA. Microtiter wells were coated with recombinant FbsA (1 μg/100 μL) and incubated with each IgG preparation (2 μ/100 μL) for 2 hours at 22°C. Bound antibody was detected with peroxidase-conjugated rabbit antihuman IgG. (C) Cells of S agalactiae (1 × 109) were preincubated for 1 hour with 100 μg/mL IgG from an individual with a high anti-FbsA antibody titer (donor no. 19), washed, and adjusted to 1 × 109 cells/mL. Then, 50 μL of the bacterial suspension was added to 0.4 mL GFPs (4 × 108), previously incubated with or without the anti-FcγRIIA antibody IV.3 according to the conditions described in panel A, and tested for platelet aggregation as reported. The effects of IgG from donor no. 19 depleted of anti-FbsA antibodies and of donor no. 20, who lacks anti-FbsA antibodies on GFP aggregation by S agalactiae, are also reported. All the results are representative of those observed in 3 separate experiments.

Role of specific anti-FbsA antibodies inS agalactiae–induced platelet aggregation. (A) S agalactiae cells (1 × 109) were mixed for 1 hour with 3 mg/mL unfractionated Fbg, Fbg from which IgG had been removed, or with 625 μg/mL affinity-purified antibodies. Bacteria were harvested by centrifugation, washed, and adjusted to 1 × 109 cells/mL. Then, 50 μL of the bacterial suspension was added to 0.4 mL GFPs (4 × 108) and tested for platelet aggregation as reported in Figure 6. To examine the effect of FcγRIIA on platelet aggregation, 0.4 mL GFPs was preincubated for 10 minutes with the anti-FcγRIIA antibody IV.3 (20 μg/mL), mixed with 50 μL of a S agalactiae suspension previously treated with unfractionated Fbg or IgG-free Fbg and then tested for aggregation. (A inset) ELISAs were performed to quantify the level of anti-FbsA antibodies in unfractionated Fbg, IgG-depleted Fbg, or affinity-purified IgG. The assay was performed by incubating FbsA-coated microtiter wells (2 μg/well) with 7.5 μg protein of each fraction followed by the addition of peroxidase-conjugated rabbit antihuman IgG. (B) The reactivity of IgG from human donors to recombinant FbsA. Microtiter wells were coated with recombinant FbsA (1 μg/100 μL) and incubated with each IgG preparation (2 μ/100 μL) for 2 hours at 22°C. Bound antibody was detected with peroxidase-conjugated rabbit antihuman IgG. (C) Cells of S agalactiae (1 × 109) were preincubated for 1 hour with 100 μg/mL IgG from an individual with a high anti-FbsA antibody titer (donor no. 19), washed, and adjusted to 1 × 109 cells/mL. Then, 50 μL of the bacterial suspension was added to 0.4 mL GFPs (4 × 108), previously incubated with or without the anti-FcγRIIA antibody IV.3 according to the conditions described in panel A, and tested for platelet aggregation as reported. The effects of IgG from donor no. 19 depleted of anti-FbsA antibodies and of donor no. 20, who lacks anti-FbsA antibodies on GFP aggregation by S agalactiae, are also reported. All the results are representative of those observed in 3 separate experiments.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal