Sed after the treatment, whereas anti-GM1 monoclonal antibody reactivity was increased.

Sed after the treatment, whereas anti-GM1 monoclonal antibody reactivity was increased. This indicates that the neuraminidase-treatment transformed GD1a-like LOS into GM1-like LOS by removing the terminal neuraminic acid. (D) Absorption studies using antibodies against cM1/D1a with various antigens. Anti-cM1/D1a IgG antibodies from the serum of a patient with GBS (S382) were absorbed by the intact GC016 LOS (black bar; GM1/GD1a mimics) and a mixture of GM1 and GD1a gangliosides (gray bar), but not by neuraminidase-treated GC016 LOS (GM1 mimic as shown in A) and NCTC11168 LOS (GM1/GM2 mimics). doi:10.1371/journal.pone.0124004.ggenome strain NCTC11168 bears GM1-like and GM2-like LOSs, but no GD1a-like LOS [13]. The mice were immunized intraperitoneally 5 times at 2-week intervals with 1 mg (dry weight) of heat-killed lysate of C. jejuni [14]. This research was approved by the Animal Care and Use Committee, Dokkyo Medical University, Japan (approval no. 00?2). The mice were treated according to the Guidelines for the Care and Use of Laboratory Animals, Dokkyo Medical University, Japan.Enzyme-linked immunosorbent assayIgG antibodies to individual gangliosides (GM1, GM1b, GM2, GD1a, GalNAc-GD1a, GD1b, GD2, GT1a, GT1b or GQ1b; 10 pmol/well) were measured in sera (starting at 1:500 dilution) from the patients and mice using peroxidase-conjugated anti-human or anti-mouse IgG antibodies [15]. IgG antibodies to ganglioside complex GM1/GD1a (cM1/D1a) were tested with a mixture of GM1 and GD1a (each 5 pmol/well) as antigen. Anti-cM1/D1a antibodies were judged positive when the PXD101 site optical density of the antibodies was 0.5 greater than the sum of optical densities of antibodies to individual GM1 and GD1a [16]. IgG antibodies to other ganglioside complexes were measured as well. Frequency differences between the groups were compared by means of Fisher’s exact test using SPSS 12.0J software (SPSS Inc., Chicago, IL). A difference was considered significant when the two-sided p value was less than 0.05. Absorption studies were performed as described elsewhere [17]. Absorption rates ( ) were calculatedPLOS ONE | DOI:10.1371/journal.pone.0124004 April 13,3/Campylobacter LOS Complex in GBSfrom [1-(optical densities in wells with serum with absorption-treatment) / (optical densities in reference wells with serum without absorption-treatment)] x 100. Crude LOS fractions were prepared from C. jejuni strains, and the presence of GM1-like, GD1a-like, GM1/GD1a-like or GM1b-like LOS was determined using monoclonal antibodies [anti-GM1 (GB2) and anti-GD1a (GB1)], cM1/D1a serum (S382) or GM1b-specific serum (S8056) from patients with GBS as described elsewhere [18]. GD1a-like LOS was treated overnight with the neuraminidase from Arthrobacter ureafaciens (Nakalai Tesque, Inc., Kyoto, Japan) at a concentration of 0.3 mU/ml acetate buffer (pH 5.0) at 37 . This treatment in the absence of surfactant is expected to SP600125 web cleave only the terminal sialic acid in the ganglioside.Thin-layer chromatography with immunostainingBovine brain ganglioside mixture, authentic GM1b and a mixture of GM1 and GD1a were spotted on a thin-layer chromatography plate and developed with chloroform/methanol/12 mM magnesium chloride in water (5:4:1, by volume) [19]. After the development, a mixture of GM1 and GD1a was spotted on a different lane. Each plate was overlaid with serum (1:50 dilution,) from Patient 4 in Table 1, followed by peroxidase-conjugated anti-human IgG antibodies.Results Structural c.Sed after the treatment, whereas anti-GM1 monoclonal antibody reactivity was increased. This indicates that the neuraminidase-treatment transformed GD1a-like LOS into GM1-like LOS by removing the terminal neuraminic acid. (D) Absorption studies using antibodies against cM1/D1a with various antigens. Anti-cM1/D1a IgG antibodies from the serum of a patient with GBS (S382) were absorbed by the intact GC016 LOS (black bar; GM1/GD1a mimics) and a mixture of GM1 and GD1a gangliosides (gray bar), but not by neuraminidase-treated GC016 LOS (GM1 mimic as shown in A) and NCTC11168 LOS (GM1/GM2 mimics). doi:10.1371/journal.pone.0124004.ggenome strain NCTC11168 bears GM1-like and GM2-like LOSs, but no GD1a-like LOS [13]. The mice were immunized intraperitoneally 5 times at 2-week intervals with 1 mg (dry weight) of heat-killed lysate of C. jejuni [14]. This research was approved by the Animal Care and Use Committee, Dokkyo Medical University, Japan (approval no. 00?2). The mice were treated according to the Guidelines for the Care and Use of Laboratory Animals, Dokkyo Medical University, Japan.Enzyme-linked immunosorbent assayIgG antibodies to individual gangliosides (GM1, GM1b, GM2, GD1a, GalNAc-GD1a, GD1b, GD2, GT1a, GT1b or GQ1b; 10 pmol/well) were measured in sera (starting at 1:500 dilution) from the patients and mice using peroxidase-conjugated anti-human or anti-mouse IgG antibodies [15]. IgG antibodies to ganglioside complex GM1/GD1a (cM1/D1a) were tested with a mixture of GM1 and GD1a (each 5 pmol/well) as antigen. Anti-cM1/D1a antibodies were judged positive when the optical density of the antibodies was 0.5 greater than the sum of optical densities of antibodies to individual GM1 and GD1a [16]. IgG antibodies to other ganglioside complexes were measured as well. Frequency differences between the groups were compared by means of Fisher’s exact test using SPSS 12.0J software (SPSS Inc., Chicago, IL). A difference was considered significant when the two-sided p value was less than 0.05. Absorption studies were performed as described elsewhere [17]. Absorption rates ( ) were calculatedPLOS ONE | DOI:10.1371/journal.pone.0124004 April 13,3/Campylobacter LOS Complex in GBSfrom [1-(optical densities in wells with serum with absorption-treatment) / (optical densities in reference wells with serum without absorption-treatment)] x 100. Crude LOS fractions were prepared from C. jejuni strains, and the presence of GM1-like, GD1a-like, GM1/GD1a-like or GM1b-like LOS was determined using monoclonal antibodies [anti-GM1 (GB2) and anti-GD1a (GB1)], cM1/D1a serum (S382) or GM1b-specific serum (S8056) from patients with GBS as described elsewhere [18]. GD1a-like LOS was treated overnight with the neuraminidase from Arthrobacter ureafaciens (Nakalai Tesque, Inc., Kyoto, Japan) at a concentration of 0.3 mU/ml acetate buffer (pH 5.0) at 37 . This treatment in the absence of surfactant is expected to cleave only the terminal sialic acid in the ganglioside.Thin-layer chromatography with immunostainingBovine brain ganglioside mixture, authentic GM1b and a mixture of GM1 and GD1a were spotted on a thin-layer chromatography plate and developed with chloroform/methanol/12 mM magnesium chloride in water (5:4:1, by volume) [19]. After the development, a mixture of GM1 and GD1a was spotted on a different lane. Each plate was overlaid with serum (1:50 dilution,) from Patient 4 in Table 1, followed by peroxidase-conjugated anti-human IgG antibodies.Results Structural c.