Immunoreactive proteins to the pooled sera of individuals from the schistosomiasis endemic area.

<p>All <i>Smp</i> IDs can be found in schistodb.net. AW-TOT: adult worm total protein extract, AW-TEG: adult worm tegumental protein extract.</p>1<p>: actin was co-extracted from the same spot with reticulocabin, except in pH 5–8 IPG strip;</p>2<p>: annexin was co-extracted from the same spot with one of major egg antigen;</p>3<p>: the same protein corresponding to different spots and protein sequences;</p>4<p>: short-chain dehydrogenase was co-extracted from the same spot with four and A half lim domains;</p>5<p>: gene ID corresponding to Smp_018890 and Smp_187370;</p>6<p>: troponin I from <i>S. japonicum</i>;</p><p>*: proteins selected to <i>in vitro</i> recombinant protein expression.</p

Immunoreactive spots to the pooled sera of <i>Schistosoma mansoni</i> infected, non-infected from endemic area and non-infected from non-endemic area volunteers identified in adult worm total and tegumental protein extracts.

<p>AW-TOT: adult worm total protein extract, AW-TEG: adult worm tegumental protein extract. INF: 2D-WB using pooled serum of <i>S. mansoni</i> infected individuals, NE: 2D-WB using pooled serum of non-infected individuals from endemic area and NI: 2D-WB using pooled serum of non-infected individuals from non-endemic area. (-): spots not immunoreactive in the corresponding protein extracts. The numbers (0, 1, 2 and 3) indicate how many experiments each spot was detected in the triplicate of the corresponding 2D-WB. In bold: spots immunoreactive exclusively in 2D-WB using INF serum pool. Highlighted line: spot immunoreactive exclusively to NE serum pool. Spot numbers are related to the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002745#pntd-0002745-g004" target="_blank">Figure 4</a>.</p

2D-PAGE and 2D-WB of <i>Schistosoma mansoni</i> adult worm protein extracts using different IPG strip pH ranges.

<p>A) 2D-PAGE of adult worm total (AW-TOT) and tegumental (AW-TEG) protein extracts using 7 cm, pH 3–10, 3–10NL and 5–8 IPG strips and SDS-PAGE 12%, stained by Colloidal Coomassie Blue G-250. B) Corresponding 2D-WB using pool of <i>S. mansoni</i> infected individuals serum and anti-human Ig's polyvalent antibody HRPO conjugated. The dashed circle indicates a region of strongly stained protein spots in 2D-PAGE that are weakly or not immunoreactive in 2D-WB and the circle indicates a region of protein spots barely visible in 2D-PAGE and highly immunoreactive. C) Schematic representation of immunoreactive spots excised from the corresponding 2D-PAGE to MS/MS identification. Gray circles represent immunogenic spots not identified and black circles represent immunogenic spots identified by MS/MS. All the identified proteins are listed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002745#pntd-0002745-t002" target="_blank">Table 2</a>. The figure shows one representative experiment of three replicates.</p

Indication on the 2D-PAGE of the immunoreactive spots identified in 2D-WB experiments.

<p>Adult worm total (AW-TOT) and tegumental (AW-TEG) protein extracts were separated by 2-DE using 7 cm pH 3–10 IPG strips followed by SDS-PAGE 12%, and stained by Colloidal Coomassie Blue G-250. Protein spots immunoreactive to INF, NE and/or NI pooled sera on the 2D-WB were extracted from the corresponding 2D-PAGE to mass spectrometry identification. The identified protein spots are numbered according to the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002745#pntd-0002745-t003" target="_blank">Table 3</a>. The figure shows one representative experiment of three replicates.</p

<i>In vitro</i> expression and western blotting analysis of recombinant <i>Schistosoma mansoni</i> major egg antigen and hemoglobinase proteins.

<p>A) <i>S. mansoni</i> major egg antigen (MjE) and hemoglobinase precursor (Hem) proteins, lanes 1 and 2 respectively, were expressed using TNT SP6 High-Yield Wheat Germ Protein Expression System and the FluoroTect Green_Lys labeled proteins were analyzed in SDS-PAGE 4–20%. The gel image was obtained with a laser-based fluorescent gel scanner. The negative control using no DNA template was indicated in lane 3 and using no DNA template and no FluoroTect Green_Lys, in lane 4. Lane 5 contains the Precision Plus Protein Kaleidoscope molecular weight marker (BioRad). B) Western blotting analysis of the <i>in vitro</i> recombinant proteins probed with INF (2, 3, 4), NE (5, 6, 7) and NI (8, 9, 10) serum pools. The TNT wheat germ extract using MjE flexi-vector as DNA template was loaded in lanes 2, 5 and 8, and using Hem flexi-vector in lanes 3, 6 and 9. TNT wheat germ extract with no DNA template was loaded in lanes 4, 7 and 10. Rabbit anti-human IgG and anti-rabbit IgG HRP conjugated were used as secondary and tertiary antibodies. The membranes were developed using ECL Plus-Western Blotting Detection System and the proteins were visualized by chemiluminescence detection using a Fujifilm LAS-4000 Imaging System. Pre-stained Precision Plus Protein Kaleidoscope molecular weight marker was loaded in lane 1.</p

2D-WB of <i>Schistosoma mansoni</i> adult worm total and tegumental protein extracts using pooled sera of infected, non-infected from endemic area and non-infected from non-endemic area individuals.

<p>Adult worm total (AW-TOT) and tegumental (AW-TEG) protein extracts were separated by 2-DE using 7 cm pH 3–10 IPG strips and SDS-PAGE 12%. The proteins were blotted onto PVDF membranes and probed with <i>S. mansoni</i> infected (INF), non-infected from endemic area (NE) and non-infected from non-endemic area (NI) serum pools, following an additional incubation with anti-human Ig's polyvalent antibody HRPO conjugated. Circle, arrows and arrowhead indicate immunogenic protein spots which reacted exclusively with INF serum pool, while dotted circle indicates immunogenic spot which reacted exclusively with NE pooled sera. The figure shows one representative experiment of three replicates.</p

Selected volunteers from the endemic area of schistosomiasis, Virgem das Graças, Brazil.

<p>Volunteers ID: identity number of the selected volunteers. Localization: sites of volunteers in the village (cVDG: central village of Virgem das Graças; Suss: Suçuarana; Card1/2/3: Cardoso 1, 2 or 3). Gender: M-Male and F-Female. Age: age of volunteers at the serum collection in 2001. EPG: Eggs Per Gram of faeces, from 3 samples, 2 slides each, by Kato-Katz method. TBM: Total Body Minutes, time in minutes of body exposure to water. (-): missing data.</p

Immunoreactive proteins to the pooled sera of individuals from the schistosomiasis endemic area.

<p>All <i>Smp</i> IDs can be found in schistodb.net. AW-TOT: adult worm total protein extract, AW-TEG: adult worm tegumental protein extract.</p>1<p>: actin was co-extracted from the same spot with reticulocabin, except in pH 5–8 IPG strip;</p>2<p>: annexin was co-extracted from the same spot with one of major egg antigen;</p>3<p>: the same protein corresponding to different spots and protein sequences;</p>4<p>: short-chain dehydrogenase was co-extracted from the same spot with four and A half lim domains;</p>5<p>: gene ID corresponding to Smp_018890 and Smp_187370;</p>6<p>: troponin I from <i>S. japonicum</i>;</p><p>*: proteins selected to <i>in vitro</i> recombinant protein expression.</p

Heat map profile of immunoreactive <i>Schistosoma mansoni</i> adult worm proteins to specific pooled sera.

<p>S. <i>mansoni</i> adult worm total (A) and tegumental (B) protein extracts were analyzed by 2D-WB and the proteins identified by mass spectrometry were clustered according to the reactivity pattern against to <i>S. mansoni</i> infected (INF), non-infected from endemic area (NE) and non-infected from non-endemic area (NI) serum pools, in triplicate assays. Those proteins which reacted to the sera in all three assays were assigned by red color, whereas those with no detected reactivity, by black color. Proteins which were not identified in one of the protein extracts were shown by gray color. The immunogenic proteins which reacted exclusively to the INF serum pool are indicated by (*) and protein which reacted exclusively to the NE serum pool, by (#).</p

Venn diagram showing the number of unique and shared proteins identified between and among 2D-WB assays.

<p>The number of immunoreactive proteins identified using AW-TOT protein extract and pH 3–10 IPG strip are represented in the yellow circle, using pH 3–10NL IPG strip in red and using pH 5–8 IPG strip in green. Spots using AW-TEG protein extract and pH 3–10 IPG strip are represented in the blue circle. Common spots identified between and among the assays are represented overlapped by the circles.</p