Reference (Avian Influenza Virus) AIV antisera used in the study.

1<p>Homologous HI Titre.</p>2<p>Source of challenged and vaccinated antibodies.</p>3<p>ND: HI Titre not detected.</p

Mean comparisons and analysis of variance of recombinant M2e-MBP ELISA results on sera from challenged, negative, and vaccinated tested groups.

1<p>Means that do not share a letter are significantly different.</p>2<p>Highly significant.</p

Comparison of HI titres and M2e-MBP ELISA OD in chicks vaccinated and then challenged by A/Ck/West Java/Pwt-Wij/2006.

<p>Comparison of HI titres and M2e-MBP ELISA OD in chicks vaccinated and then challenged by A/Ck/West Java/Pwt-Wij/2006.</p

SDS-PAGE and Western blot results of M2e-MBP recombinant protein.

<p>A) Purified recombinant M2e-MBP (lane 1) and MBP (lane 2) proteins and pre-stained molecular marker (M) analysed by SDS–PAGE (12.5%) and stained using Coomassie Brilliant Blue. (B & C) Western blot analysis of purified M2e-MBP (1B) and MBP (1C) with reference AIV antisera to: (1) live A/Chicken/Scotland/1959 (H5N1); (2) inactivated H5N1; (3) live A/Ck/Viet Nam/8/2004 (H5N1); (4) live A/Turkey/Wisconsin/1/1966 (H9N2); (5) live A/Ostrich/Denmark/72420/1996 (H5N2); (6)sera from commercial non-vaccinated non-infected; (7) SPF chicken sera.</p

Positive serum samples used in the study^*.

<p>* Birds were immunized once (D10), twice (B47, B38, B1, B2, PL80) or three times (A17) with a commercial H5N1 vaccine, at four weekly intervals. Two weeks after the last immunization, birds were challenged with H5N1 strains ^a A/chicken/West Java/Sbg-29/2007 or ^b A/Chicken/West Java/PWT-WIJ/2006. Two weeks after the challenge, sera were collected for assay in haemagglutination inhibition test and M2e and tM2e ELISA.</p><p>Positive serum samples used in the study^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108420#nt101" target="_blank">*</a>}.</p

Two-graph receiver operating curve (TG-ROC) analysis of tM2e-MBP ELISA OD for field (vaccinated or non-vaccinated) and challenged (positive) sera.

<p>The cut-off value was chosen to be at 95% sensitivity and 95% specificity.</p

ANOVA and mean comparison by Tukey's method of ELISA OD values of positive, SPF and sera from vaccinated chicks.

<p>* From each positive, vaccinated and SPF groups, three samples were tested in triplicate in both tM2e-MBP and M2e-MBP ELISAs.</p><p>ANOVA and mean comparison by Tukey's method of ELISA OD values of positive, SPF and sera from vaccinated chicks.</p

Boxplot of ODs obtained with tM2e-MBP and M2e-MBP with positive, vaccinated and SPF sera showed in <b>table 2</b>.

<p>The asterisks represent small number of the cases which are far from common ranges of plus and minus standard deviation (±SD).</p

SDS-PAGE analysis of purified tM2e-MBP recombinant protein.

<p>Lane M: Molecular weight markers. Lanes 1 to 7: Fractions of purified recombinant tM2e-MBP protein with molecular weight of 52.9 kD (arrow).</p

Reaction of tM2e-MBP and M2e-MBP proteins with positive sera in ELISA.

<p>The positive sera are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108420#pone-0108420-t001" target="_blank">Table 1</a>. Pared T test highlighted the statistically higher reactivity (p = 0.05) of tM2e-MBP compared to M2e-MBP.</p