Fig. 7.

Neutralizing antibodies to BoNT/A1 were quantitated using the RSC serum neutralization assay. Primary rat spinal cord cells were exposed to 0.5 units (0.5 pM) of BoNT/A1 preincubated with the indicated mouse serum. Cell lysates were analyzed by Western blotting using an anti-SNAP-25 antibody that recognizes both BoNT/A1-cleaved (c) and uncleaved (u) SNAP-25. Uncleaved SNAP-25 is indicative of toxin neutralization. BALB/c mice were immunized with 7.4 pmol of HcmR4 or Hc and serum collected on day 14. (A) Sera were analyzed for neutralizing antibodies. As a control, sera from nonimmunized BALB/c mice (nI) were also analyzed. Data presented are representative of three assays performed. (B) The level of toxin neutralization was determined by quantitative analysis of SNAP-25 cleavage by Western blot densitometry and expressed as percent protection. Shown are the means ± SD for three independent analyses for each serum sample analyzed and for all volumes analyzed. (C) Protection is plotted against serum volume to reveal the extent of protection provided by immunization with HcmR4 or Hc. Results show means ± SEM for each volume of serum analyzed. *, P < 0.05.