Bovine serum albumin (bsa)

ELISA plates (Nalge Nunc, Rochester, NY) were coated with 500 ng/well of NP25 conjugated with BSA (Biosearch Technologies). To detect IgM, IgG1, IgG3 or IgA, plates were coated with unconjugated goat anti‐mouse IgM (Southern Biotech, , Birmingham, AL), IgG (Southern Biotech), or IgA (BD, Franklin Lakes, NJ), respectively. After incubation overnight (4°C), washing with PBS + 0.05% Tween20 and blocking for 1 h with PBS containing 2% dry milk, 50 μL of culture supernatant was added in a total volume of 150 μL, followed by threefold serial dilutions in blocking buffer and incubated for 2 h at room temperature (RT). Plates were washed six times, and primary antibodies, biotinylated goat anti‐mouse IgM, HRP‐coupled anti‐IgG1, HRP‐coupled anti‐IgG3 (Southern Biotech, Birmingham, AL), or biotinylated goat anti‐mouse IgA (BD Franklin Lakes, NJ), were added in 100 μL PBS/well followed by incubation for 1.5 h, at RT. After six washes, streptavidin‐HRP was added to biotinylated antibodies in 100 μL PBS/well and incubated for 1 h at RT. The assay was developed with TMB substrate (KPL), the reaction was stopped with 1 m H₂SO₄, and the OD was read at 450 nm using an Asys Expert 96 ELISA reader (Biochrom Ltd, Cambridge, UK). For detection of soluble TACI, the mouse TACI/TNFRSF13B DuoSet ELISA kit (R&D Systems) was used, according to the manufacturer's instructions.

ELISA plates (Nunc) were coated with 2 µg/ml β-Galactosidase (Sigma) or 500 ng/well of NP(30) conjugated with BSA (Biosearch Technologies). To detect IgM, IgG, IgG1, IgG2b, IgG2c, IgG3 or IgA plates were coated with unconjugated goat anti-mouse IgM, IgG or IgA (Southern Biotech). After incubation overnight (4°C), washing with PBS + 0.05% Tween20 and blocking for 1 h with PBS containing 2% dry milk (blocking buffer), 5 μl of serum was added in a total volume of 150 μl, followed by 3-fold serial dilutions in blocking buffer and incubated for 2 h at room temperature (RT). Plates were washed six times, and primary antibodies, biotinylated goat anti-mouse IgM, or goat anti-mouse IgG (both from Mabtech AB), biotinylated goat anti-mouse IgA (BD Pharmingen), or HRP-coupled anti-IgG1, anti-IgG2b, anti-IgGc or anti-IgG3 (Southern Biotech) were added in 100 μl PBS/well followed by incubation for 1.5 h, at RT. Streptavidin-HRP was added to biotinylated antibodies in 100 μl PBS/well after washing six times and incubated for 1 h, at RT. The assay was developed with TMB substrate (KPL), the reaction was stopped with 1 M H₂SO₄, and the OD was read at 450 nm using an Asys Expert 96 ELISA reader (Biochrom Ltd.).

ELISA was performed by coating ELISA plates (Nunc) with polysaccharide antigens: 500 ng/well of NP (25) conjugated with BSA (Biosearch Technologies) or the pneumococcal polysaccharide antigens Type 1 161-X™ or Type 3 169-X™ (both ATCC) or 1:100 dilution of Pneumovax (corresponding to 50 ng/well of each antigen contained in the vaccine). To measure total IgM and IgG3 levels, plates were coated with unconjugated anti-IgM or anti-IgG3 (Southern Biotech). Plates were incubated overnight (4°C). Following washing (PBS + 2% Tween20) and blocking for 1 h with PBS containing 2% dry milk, serum was added in threefold serial dilutions in blocking buffer and incubated for 1.5 h at room temperature (RT) before addition of secondary antibody HRP-coupled anti-IgM or IgG3 (Southern Biotech). The assay was developed with TMB substrate (KPL) followed by 1M H₂SO₄ and the OD was read at 450 nm using an Asys Expert 96 ELISA reader (Biochrom).