Nicotine haptens used in this study.

<p>Nicotine haptens used in this study.</p

Day 160 anti-nicotine Ab titers.

<p>C57BL/6 mice (5/grp) were immunized (d0, d14, d146) with either PBS or 2.5 µg of the indicated conjugated hapten carriers and adjuvants, and serum was assayed by ELISA. Comparisons between groups were conducted by unpaired two-tailed <i>t</i>-test; *p<0.003; **p<0.0001.</p

Anti-nicotine antibody responses in immunized mice.

<p>C57BL/6 mice (5/grp) were injected (d0, d14, d131) with either PBS or 2.5 µg of the indicated conjugate hapten carriers and adjuvants, and sera was assayed for anti-nicotine Ab titers by ELISA. TCCnic-12 contained an average 12 haptens per trimer, whereas KLHnic-22 and KLHnic-100 contained, respectively an average 22 and 100 haptens per monomer protein. Comparisons between groups were conducted by unpaired two-tailed <i>t</i>-test; *p<0.004; **p<0.002.</p

Serum nicotine binding capacities improve when using natural (-) enantiomers of two nicotine haptens.

<p>Day 56 serum was collected and pooled from mice immunized with 5 µg (-) 3’ P8 or (+) 3’ P8 <b>(A)</b> or 5 µg (-) 1’ P9 or (+) 1’ P9 <b>(B).</b> Nicotine binding capacities were determined by equilibrium dialysis.</p

A comparison of the relative size and lysine content of the TCC and four hapten carriers; KLH, Exotoxin A (ExoT A), Tetanus toxoid (Tet T), and the inactivated diphtheria toxin protein CRM197; (A) The number of total amino acids with the number of lysines available for hapten conjugation reported above the bar; (B) The percentage of lysines in each carrier protein.

<p>A comparison of the relative size and lysine content of the TCC and four hapten carriers; KLH, Exotoxin A (ExoT A), Tetanus toxoid (Tet T), and the inactivated diphtheria toxin protein CRM197; (A) The number of total amino acids with the number of lysines available for hapten conjugation reported above the bar; (B) The percentage of lysines in each carrier protein.</p

Nicotine haptens used in this study.

<p>Nicotine haptens used in this study.</p

A bivalent nicotine vaccine stimulates an additive increase in antibody titers and equivalent avidities compared to dose-matched monovalent vaccines.

<p>Serum was collected 56 days after priming from mice immunized with: (1) 10 µg (-) 1’ P9, (2) 10 µg (-) 3’ P9 or (3) 5 µg (-) 1’ P9 + 5 µg (-) 3’ P9. Sera titers <b>(A)</b> and affinities <b>(B)</b> were assayed by ELISA using plates coated with a 50/50 mixture of (-) 1’ BSA and (-) 3’ BSA. Comparisons between groups were conducted by one-way ANOVA. ***p<0.01.</p

Serum nicotine binding capacity was determined by measuring bound and free concentrations of nicotine at equilibrium.

<p>Kd values (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114366#pone-0114366-g008" target="_blank">Fig. 8</a>) were used to calculate total antibody concentrations according to the law of mass action equation: Kd  =  [Nic][IgG]/[Nic-IgG]. Comparisons between groups were conducted by unpaired two-tailed <i>t</i>-test; *p<0.04; **p<0.01; ***p<0001.</p

Antibody specificity.

<p>The specificity of nicotine binding to antisera collected from TCCnic-12 immunized mice was determined by competitive ELISA for nicotine, cotinine and acetylcholine. IC₅₀ values for cotinine were 1000-fold greater than nicotine and could not be calculated for acetylcholine due to a lack of inhibition.</p

Anti-carrier Ab responses in immunized mice.

<p>C57BL/6 mice (5/grp) were injected (d0, d14) with the indicated carriers in the presence of GLA-SE. TCCnic-2, TCCnic-12, and TCCnic-42 contained, respectively, an average 2, 12, and 42 haptens per trimer. Day 28 serum from each group was assayed for Ab that bound the corresponding <i>unconjugated</i> hapten carrier.</p