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. Author manuscript; available in PMC: 2015 May 1.
Published in final edited form as: Eur J Immunol. 2014 Feb 27;44(5):1433–1443. doi: 10.1002/eji.201344266

Figure 4. MSA-specific IgG and PPS14-specific IgG secondary responses to PPS14+[MSA]-beads are induced only by MSA and/or PPS14 attached to either micro- or nanobeads; enhancement of responses by CpG-ODN, but not alum.

Figure 4

Serum titers of PPS14-specific IgG in BALB/c mice (n=5) immunized at day 0 and 14 with (A) ≈2.2×108 latex beads containing 280ng of PPS14 attached to beads coated with MSA (PPS14+[MSA]) or normal mouse sera (PPS14+[NMS]), previously collected from the same mice receiving the beads. (B) 2×108 latex beads coated with MSA ([MSA]-beads), 30 ng of free PPS14, or a mixture of 2×108 [MSA]-beads and 30ng of free PPS14 ([MSA]-beads + free PPS14). (C) ≈2×108 PPS14+[MSA]-beads of a preparation in which the PPS14 attachment to the bead was not stabilized by reduction with cyanoborohydride and contained 209ng of PPS14 attached and 20ng of free PPS14, after >1year of storage. This preparation was used for immunization after washings with PBS that reduced the free PPS14 content to <0.8 ng (not stabilized, washed), or directly without washings (not stabilized, free PPS14), and compared to a preparation in which the Schiff’s bases were reduced with cyanoborohydride (stabilized, washed). (D) 2×108 0.96 µm in diameter, or 1.46 × 1011 0.15µm in diameter, PPS14+[MSA]-beads containing 200 ng of attached PPS14 (E) 3.5×108 0.96µm in diameter, or 4.5×109 0.15 µm in diameter, PPS14+[MSA]-beads containing 28 ng of attached PPS14. #p <0.05 (for C and D, primary [day 14] relative to secondary [day 21] responses); Student's t-test. (F) Anti-MSA IgG in sera of BALB/c mice (n=7) immunized at day 0 and 14 with 2×108 beads coated with 100 ng MSA ([MSA]-beads) or glycine ([Gly]-beads) combined with 25µg CpG-ODN, or 50µg of purified MSA (free MSA) admixed with 25µg CpG-ODN and 13µg of alum. A mixture of CpG-ODN and alum (PBS) was used as control. (G) Serum titers of MSA-specific IgG and PPS14-specific IgG and IgM from BALB/c mice (n=7) immunized at day 0 and 14, with 2×108 PPS14+[MSA]-beads containing 240ng of PPS14 attached to the beads. Beads were admixed for 1 hour at room temperature with 13µg of alum and 25µg CpG-ODN (alum+CpG-ODN), 25µg CpG-ODN alone (CpG-ODN), 13µg alum alone (alum), or in the same final volume of PBS (PBS).*p<0.05 (every group relative to mice immunized with PPS14+[MSA]-beads admixed with alum+CpG-ODN). Latex beads used in all the experiments, except for panel D and E, were 0.96µm in diameter. One of three (panel G), or two (panels A, D and F) independent experiments is shown.