Western blot immunoreactivity of acid-eluted antibodies and control rabbit antisera monospecific for <i>S</i>. <i>mansoni</i> egg antigens; IPSE/alpha-1 and kappa-5 against <i>S</i>. <i>manson</i>i egg antigens.

<p>M = molecular size markers. Primary antibodies: 1 = anti-SmSEA; 2 = anti-SmSEA antibodies eluted from the 43 kDa latex molecule; 3 = rabbit antiserum specific for <i>S</i>. <i>mansoni</i> egg antigen IPSE/alpha-1; 4 = rabbit antiserum specific for <i>S</i>. <i>mansoni</i> egg antigen kappa-5.</p

PAGE and electroblots of allergen extracts probed with rabbit anti-SmSEA antiserum.

<p><b>(a</b>) Coomassie blue-stained SDS-PAGE gel of allergen extracts and <i>S</i>. <i>mansoni</i> SEA (SmSEA). <b>(b)</b> Western immunoblot of allergen extracts and SmSEA electro-transferred to nitrocellulose film from a replicate of the gel in (a) and probed with a rabbit anti-SmSEA antiserum. <b>(c)</b> Western immunoblot replicate of (b) except for treatment of the nitrocellulose film with 10 mM sodium meta-periodate after electro-transfer of allergen/SmSEA molecules and before incubation in primary rabbit anti-SmSEA antiserum. M = molecular size markers; Lanes and (amount of BSA-equivalent protein added to each lane):1 = latex (0.20 mg); 2 = SmSEA (0.010 mg); 3 = banana (0.16 mg); 4 = tomato (0.27 mg); 5 = peanut (0.10 mg); 6 = melon (0.24 mg); 7 = avocado (0.22 mg); 8 = Kiwi fruit (0.19 mg); 9 = chestnut (0.22 mg).</p

Purification of the two <i>S</i>. <i>mansoni</i> cercarial antigens which reacted with the acid-eluted antibody in Fig 7.

<p><b>(a)</b> Coomassie blue-stained SDS-PAGE of SmCTF. Arrows indicate the two protein bands of 20 kDa and 14 kDa, which were excised, eluted from the gel and re-electrophoresed in order to purify for mass spectrometric analysis. M = molecular size markers; 1 = lane loaded with SmCTF. <b>(b)</b> Coomassie blue-stained SDS-PAGE gel. Arrow indicates purified 20 kDa protein from SmCTF in lane 1. <b>(c)</b> Coomassie blue-stained SDS-PAGE gel. Arrow indicates purified 14 kDa protein in lane 1.</p

MASCOT search output of tandem MS data from the purified ~14 kDa gel band from cercariae.

<p>MASCOT search output of tandem MS data from the purified ~14 kDa gel band from cercariae.</p

Reactivity of a 43 kDa latex molecule with rabbit antisera raised against <i>S</i>. <i>mansoni</i> cercarial (SmCH) and egg (SmSEA) antigens.

<p>M = molecular size markers. Lanes 1–3 were probed with 3 different rabbit antisera raised against homogenates of <i>S</i>. <i>mansoni</i> cercariae. Lanes 4–8 were probed with sera from 5 different rabbits immunized with homogenates of <i>S</i>. <i>mansoni</i> eggs. Amount of protein in latex extract across all the 8 lanes = 1.80 mg. The serum used for lane 5 was the same as that used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159542#pone.0159542.g001" target="_blank">Fig 1</a>.</p

Purification of a 43 kDa latex antigen that is cross-reactive with <i>S</i>. <i>mansoni</i> for mass spectrometric analysis.

<p><b>(a)</b> Two lanes to the left: M = molecular size markers; 1 = Western immunoblot reactivity of a rabbit anti-SmSEA against latex extract. Two lanes to the right: Coomassie blue-stained SDS-PAGE gel: 2 = latex extract; M = molecular size markers. 0.02 mg of protein was added to each latex lane. <b>(b)</b> Silver-stained SDS-PAGE gel. M = molecular weight markers; 1 = untreated latex extract; 2 = result of purification of 43 kDa latex molecule by repeated immunoelectrophoreses. <b>(c)</b> Coomassie-stained SDS-PAGE gel. M = molecular size markers; 1 = result of purification of 43 kDa latex molecule by a repeated immunoelectrophoresis. Arrow indicates the stained band that was subjected to mass spectrometric analysis.</p

Reactivity of acid-eluted anti-<i>S</i>. <i>mansoni</i> cercarial antibodies that cross-reacted with the 43 kDa latex molecule.

<p>M = molecular size markers. Antigens in lanes: 1 = latex extract; 2 = SmSEA; 3 = SmCH; 4 = SmCTF (0.025 mg). Immunoblot was probed with rabbit anti-SmCH antibodies purified by acid-elution from 43 kDa latex molecule.</p

Reactivity of acid-eluted anti-SmSEA-derived anti-latex antibodies against extracts of some allergens known to be implicated in the latex-fruit syndrome.

<p><b>(a)</b> Western immunoblot of allergen extracts and SmSEA electrophoreses probed with rabbit anti-SmSEA antibodies that were cross-reactive with a 43 kDa latex molecule. <b>(b)</b> Western immunoblot replicate of (a) except for treatment of the nitrocellulose film with 20 mM sodium meta-periodate after electro-transfer of allergen/SmSEA molecules and before incubation in primary rabbit anti-SmSEA antiserum. M = molecular size markers; 1 = latex; 2 = SmSEA; 3 = banana; 4 = tomato; 5 = peanut; 6 = melon; 7 = avocado. The amount of protein in each lane (BSA-equivalent) is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159542#pone.0159542.g001" target="_blank">Fig 1</a>.</p

MASCOT search output of tandem MS data from the purified ~20 kDa gel band from <i>S</i>. <i>mansoni</i> cercariae.

<p>MASCOT search output of tandem MS data from the purified ~20 kDa gel band from <i>S</i>. <i>mansoni</i> cercariae.</p

Western blot showing reactivity of acid-eluted, latex cross-reactive, rabbit anti-SmSEA antibodies against latex extract and extracts of <i>S</i>. <i>mansoni</i> eggs, cercariae and worms.

<p>M = molecular size markers. Antigens in lanes: 1 = latex extract; 2 = SmSEA; 3 = SmCH (0.025 mg); 4 = SmWH (0.025 mg).</p