Journal:

Article Title: Killing of Streptococcus pneumoniae by capsular polysaccharide-specific polymeric IgA, complement, and phagocytes

doi:

Figure Lengend Snippet: (a) IgA-mediated killing of type 14 S. pneumoniae by differentiated HL-60 cells and baby rabbit complement. Phagocyte/bacteria ratio was 400:1. Tests were run in duplicate; results are shown as the mean ± SEM of 3 experiments. (b) Killing of type 14 S. pneumoniae by human polymorphonuclear leukocytes (PMN) and differentiated HL-60 cells in the presence of 10% baby rabbit complement and 1.2 mg/mL of total IgA (98% IgA, <1% IgG, <2% IgM). Given in Results are means ± SEM of experiments (PMN, n = 4; HL-60 cells, n = 6) with IgA purified from serum of volunteers before and 1 month after immunization with 23-valent pneumococcal vaccine. Levels of type 14–specific IgA were 678 ng/mL before immunization and 2,445 ng/mL after immunization in these pools of purified IgA. *P < 0.0001 for percent kill before immunization vs. 1 month after immunization. (c) The efficacy of molecular forms of IgA on phagocytic killing of type 14 S. pneumoniae. Serial dilutions of purified mIgA and pIgA were tested in the presence of 10% baby rabbit complement for their ability to mediate complement-dependent killing of the organism. Percent kill is given as a function of the concentration of anti–type 14 IgA in purified mIgA and pIgA. Tests were run in duplicate; results are shown as the mean ± SEM of 2 experiments.

Article Snippet: Purified IgA or control IgG were incubated with shaking for 30 minutes at 25°C with 1,000 CFU of log-phase type 14 S. pneumoniae (American Type Culture Collection 6314, Rockville, Maryland, USA) ( 29 – 31 ).

Techniques: Purification, Concentration Assay

Journal:

Article Title: Killing of Streptococcus pneumoniae by capsular polysaccharide-specific polymeric IgA, complement, and phagocytes

doi:

Figure Lengend Snippet: Effect of receptor-blocking mAbs and isotype control antibodies on killing of type 14 S. pneumoniae with 2 μg/mL specific IgA and 10% baby rabbit complement. Differentiated HL-60 cells were preincubated for 30 minutes at 25°C with blocking antibodies (40 μg/mL) before addition of IgA-opsonized organisms and complement. Results are shown as the mean ± SEM of 2 experiments. P = 0.08 for percent kill with anti-Fcα receptor antibody compared with killing without blocking antibody.

Article Snippet: Purified IgA or control IgG were incubated with shaking for 30 minutes at 25°C with 1,000 CFU of log-phase type 14 S. pneumoniae (American Type Culture Collection 6314, Rockville, Maryland, USA) ( 29 – 31 ).

Techniques: Blocking Assay

Journal:

Article Title: Killing of Streptococcus pneumoniae by capsular polysaccharide-specific polymeric IgA, complement, and phagocytes

doi:

Figure Lengend Snippet: Effect of cation chelators on phagocytic killing of type 14 S. pneumoniae by IgA in the presence of baby rabbit complement (a) and human complement (b). EDTA, which blocks both alternative and classical pathways, and MgEGTA, which blocks only the classical pathway, were added to killing reactions to determine the complement activation pathway involved in IgA-mediated phagocytic killing of pneumococci by differentiated HL-60 cells. Chelators (EDTA and MgEGTA) were added at 10 mM. All tests were run in triplicate; results are shown as the mean ± SEM of 6 experiments. *P < 0.05 vs. killing without EDTA. Results were the same whether individual experiments used IgA that was 98% pure (<2% IgM) or 99.9% pure (<0.1% IgM).

Article Snippet: Purified IgA or control IgG were incubated with shaking for 30 minutes at 25°C with 1,000 CFU of log-phase type 14 S. pneumoniae (American Type Culture Collection 6314, Rockville, Maryland, USA) ( 29 – 31 ).

Techniques: Activation Assay

Journal:

Article Title: Killing of Streptococcus pneumoniae by capsular polysaccharide-specific polymeric IgA, complement, and phagocytes

doi:

Figure Lengend Snippet: Effect of depletion and repletion of selected human complement components in human serum on IgA-mediated killing of type 14 S. pneumoniae by IgA. Organisms were incubated with 1 mg/mL of immune IgA, differentiated HL-60 cells, and a complement source consisting of 10% serum from a hypogammaglobulinemic patient, serum depleted of the classical pathway component C2, serum depleted of the alternative pathway component factor B, or serum depleted for factor B and then repleted with a physiologic concentration of factor B (200 μg/mL). All sera were preadsorbed with type 14 pneumococci before the killing assay to remove specific antibody in the complement source. Results are shown as mean ± SEM of 7 experiments. *P < 0.05 vs. normal complement and factor B–repleted complement.

Article Snippet: Purified IgA or control IgG were incubated with shaking for 30 minutes at 25°C with 1,000 CFU of log-phase type 14 S. pneumoniae (American Type Culture Collection 6314, Rockville, Maryland, USA) ( 29 – 31 ).

Techniques: Incubation, Concentration Assay

Journal:

Article Title: Killing of Streptococcus pneumoniae by capsular polysaccharide-specific polymeric IgA, complement, and phagocytes

doi:

Figure Lengend Snippet: Effect of PMN pretreatment on IgA-mediated killing of type 14 S. pneumoniae. Human PMNs were preincubated with media alone (first 2 columns) or TNF-α (10–8M), C5a (10–9M), or both for 30 minutes before addition of log-phase organisms and purified immune IgA (1 mg/mL total IgA). Baby rabbit complement was added to untreated PMNs with IgA as a positive control (first column); IgA with untreated PMNs and no complement served as a negative control (second column). PMNs preincubated with TNF-α and C5a showed no uptake or killing of the organism in the absence of immune IgA (not shown). Results are shown as mean ± SD for 3 experiments.

Article Snippet: Purified IgA or control IgG were incubated with shaking for 30 minutes at 25°C with 1,000 CFU of log-phase type 14 S. pneumoniae (American Type Culture Collection 6314, Rockville, Maryland, USA) ( 29 – 31 ).

Techniques: Purification, Positive Control, Negative Control