Journal: bioRxiv

Article Title: Isolation and characterization of human monoclonal antibodies to pneumococcal capsular polysaccharide 3

doi: 10.1101/2021.08.02.454853

Figure Lengend Snippet: A) HumAbs (native) with their LC swaps were generated and tested by ELISA for binding reactivity to purified PPS3 and B2. Absorbance at 405 is shown on the Y axis for the humAb concentrations shown on the X axis for each humAb. The numerical half-maximal binding titer (EC 50 ) is depicted on the graph. Results are representative of 3 independent experiments (n = 2). ST3 strain B2 was incubated with increasing concentrations of humAbs (C10, C10 LC swap (C10 H C27 L ), C27, C27 LC swap (C27 H C10 L )) or control IgG1 and analyzed by flow cytometry. B) Representative FACS dot plots showing percentage agglutination of the indicated native humAb or LC swap at various concentrations. C) Line graph depicting percentage agglutination on the Y axis for concentrations of indicated humAbs and LC swaps on the X axis. Results are representative of 2 independent experiments (n = 2 per condition). By one-way ANOVA; at 10ug/ml; (C10 vs IgG1, C10 vs C10 LC swap (C10 H C27 L ) C10 vs C27, C10 vs C27 LC swap (C27 H C10 L ) *** P <0.001); at 20ug/ml (C10 vs IgG1, C10 vs C10 LC swap (C10 H C27 L ), C10 vs C27, C10 vs C27 LC swap (C27 H C10 L ) * P <0.05).

Article Snippet: Concentrations of fluorescently conjugated PPS3 (PPS3-PE) (Fina BioSolutions) were incubated with ST3 mouse hybridoma cells ( ) with or without unlabelled PPS3 (25 μg/well).

Techniques: Generated, Enzyme-linked Immunosorbent Assay, Binding Assay, Purification, Incubation, Control, Flow Cytometry, Agglutination

Journal: Immunity, Inflammation and Disease

Article Title: Enhanced IgG 1 ‐mediated antibody response towards thymus‐dependent immunization in CXCR1‐deficient mice

doi: 10.1002/iid3.380

Figure Lengend Snippet: In vivo IgM responses towards the thymus‐independent (TI) antigens TNP‐Ficoll and Pneumovax23 (P23). For analysis of TI immune responses, wild‐type (WT) and CXCR1‐deficient (KO) mice were intraperitoneally immunized with (A) TNP‐Ficoll or (B) P23. Blood sampling was performed before (Day 0) and 7 and 14 days postimmunization to measure NP7 and NP14 (TNP‐Ficoll) or P23, pPS3, 4, 6B and 19F (P23)‐specific antibodies by enzyme‐linked immunosorbent assay (ELISA). Graphs depict the overtime progression of serum IgM levels given as optical density (OD) at 450 nm measurement wavelength. Accessorily, single data points are shown for Day 14. For every specific assay, all probes were analyzed on one identical ELISA plate in duplicates to allow direct comparison of samples. Curves represent n = 5–8 mice per genotype out of two independent experiments and error bars illustrate the mean ± SEM . Significances were determined by two‐way analysis of variance multiple comparisons and a p < .05 was considered statistically significant. § indicates significance of WT curves and # of KO curves ( §,# p < .05, §§,## p < .01, §§§,### p < .001)

Article Snippet: For detection of P23‐ and pPS‐specific antibodies in the sera of mice, high‐binding 96‐well plates were coated over night at 37°C with 1‐µg/ml P23, pPS3, 4, 6B, or 19F (SSI Diagnostica).

Techniques: In Vivo, Sampling, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Isolation and characterization of human monoclonal antibodies to pneumococcal capsular polysaccharide 3

doi: 10.1101/2021.08.02.454853

Figure Lengend Snippet: A) HumAbs (native) with their LC swaps were generated and tested by ELISA for binding reactivity to purified PPS3 and B2. Absorbance at 405 is shown on the Y axis for the humAb concentrations shown on the X axis for each humAb. The numerical half-maximal binding titer (EC 50 ) is depicted on the graph. Results are representative of 3 independent experiments (n = 2). ST3 strain B2 was incubated with increasing concentrations of humAbs (C10, C10 LC swap (C10 H C27 L ), C27, C27 LC swap (C27 H C10 L )) or control IgG1 and analyzed by flow cytometry. B) Representative FACS dot plots showing percentage agglutination of the indicated native humAb or LC swap at various concentrations. C) Line graph depicting percentage agglutination on the Y axis for concentrations of indicated humAbs and LC swaps on the X axis. Results are representative of 2 independent experiments (n = 2 per condition). By one-way ANOVA; at 10ug/ml; (C10 vs IgG1, C10 vs C10 LC swap (C10 H C27 L ) C10 vs C27, C10 vs C27 LC swap (C27 H C10 L ) *** P <0.001); at 20ug/ml (C10 vs IgG1, C10 vs C10 LC swap (C10 H C27 L ), C10 vs C27, C10 vs C27 LC swap (C27 H C10 L ) * P <0.05).

Article Snippet: PBMCS were stained with PPS3-PE and anti-human fluorescently-conjugated: CD19-PE-Cy7, CD27-APC, IgM-FITC, IgG-V421, CD3-V500, CD4-V500, CD8-V500 and CD14-V500 (BD).

Techniques: Generated, Enzyme-linked Immunosorbent Assay, Binding Assay, Purification, Incubation, Flow Cytometry, Agglutination