PMC

IgM CDR3-IMGT sequences. Nucleotide sequence, alignment to germline genes, locations of N addition and amino acid translations are shown for the seven IgM clones. CDR3-IMGT encompasses positions 105–117, and the junction includes positions 104 (second CYS) and 118 (J-TRP for the IGH) according to the IMGT unique numbering [].

Functional activity of monoclonal antibodies (mAbs): in vivo protection. Kaplan–Meyer survival curves following intraperitoneal challenge with 103 colony-forming units/mouse (n = 20 per group) with and without passive immunization with one of four IgM mAbs DM17, Bcl, CbH4, 6b5D7 and phosphate-buffered saline control.

Antibody binding to blood group antigens A and B, Gal 1,3 Gal and endotoxin as assessed by enzyme-linked immunosorbent assay (ELISA) and to cytoplasmic and nuclear antigens as assessed by immunohistochemistry. (a) IgM monoclonal antibody (mAb) binding activity to blood group antigens A and B, the xenotransplantation antigen Galα1-3Gal and a pool of four endotoxins [] as measured by ELISA (OD 405), with doubling dilutions of antibody at standardized total IgM concentration. Titration curves for antigen binding are shown for each of the seven mAbs. (b) Anti-nuclear antibody (ANA) staining profiles for each of the seven IgM mAbs at a concentration of 10 μg/ml and ANA-positive and -negative controls on HEp-2 cells. Detection mAb: fluorescein isothiocyanate-labelled anti-Fcμ. Magnification × 400.

Pneumococcal capsular polysaccharides (PncPS) binding studies and competitive inhibition assays. (a) Titration curves of mean fluorescent intensity (MFI) of IgM binding to seven different PncPS serotypes (4; 6B; 9V; 14; 18C; 19F; 23F) by luminex assay. Data are shown for a representative IgM monoclonal antibody (mAb) (DM17), polyclonal standard serum (89SF) and an IgM negative control at specified total IgM concentrations [μg/ml by IgM capture enzyme-linked immunosorbent assay (ELISA)] for mAbs and at serum dilution for polyclonal serum 89SF*. (b) Hierarchy of PncPS binding of the IgM mAbs: mean PncPS serotype-specific binding activity (MFI) [±75% and ±95% confidence interval (CI)] of the seven mAbs (±75% and ±95% CI) is shown to each of seven different pneumococcal capsular polysaccharide (PncPS) serotypes (4; 6B; 9V; 14; 18C; 19F; 23F) at a fixed concentration of total IgM (1·5 μg/ml) as assessed by luminex assay. (c) Detailed comparison of MFI of IgM binding to seven different pneumococcal capsular polysaccharide (PncPS) serotypes (4; 6B; 9V; 14; 18C; 19F; 23F) by luminex at a fixed total IgM concentration for each IgM mAb (n = 7) and a negative control (M1) (total IgM concentration of 1·5 μg/ml) and immune serum 89SF (serotype-specific IgM concentration of 1·5 μg/ml). (d) Competitive inhibition of IgM binding to PncPS and phosphorylcholine (PC) by ELISA using saturating doses of homologous antigen: mean percentage inhibition of binding to PncPS following preincubation with saturating doses (10 μg/ml and 5 μg/ml, respectively) of homologous PncPS (▪) and cell wall polysaccharide (CWPS) (▴) relative to no preincubation is shown for IgM mAbs, polyclonal immune serum IgM (89SF) and PncPS and PC-specific class-switched mAbs (CbE2 [] and TEPC15, respectively). Mean percentage inhibition of binding to PC (◊); following preincubation with saturating doses of PC relative to no preincubation is also shown for the IgM mAbs, polyclonal serum IgM and the PC-specific class-switched mAb (TEPC 15). All experiments were performed in duplicate a minimum of three times. (e) Titration curve showing mAb binding to PC for each of the seven mAbs as measure by ELISA OD (205 nm) with doubling dilutions of antibody at standardized total IgM concentration (10 μg/ml).