TLC-based Detection of Glycolipid Antibodies

Polyclonal chicken IgY anti-Gb3Cer and anti-Gb4Cer antibodies with previously described specificities [42 (link),74 (link),75 (link)] and a monoclonal rat IgM anti-Forssman GSL antibody (clone IIC2), produced by Bethke and co-workers [41 (link)] and described as a highly specific tool for the detection of Forssman GSL [25 (link),38 (link),76 (link)], were employed for TLC overlay assays. Stx2e-containing supernatant of STEC strain S123G of serotype O139:K82 was used as previously described [25 (link)]. Mouse anti-Stx2 antibody (clone VT 135/6-B9, 2.75 mg/mL) was purchased from SIFIN GmbH (Berlin, Germany). Secondary alkaline phosphatase (AP)-conjugated affinity-purified polyclonal rabbit anti-chicken IgY (code 303-055-033), goat anti-rat IgG + IgM (code 112-055-044) and goat anti-mouse IgG (code 115-055-003) antibodies were from Dianova (Hamburg, Germany).

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Steil D., Bonse R., Meisen I., Pohlentz G., Vallejo G., Karch H, & Müthing J. (2016). A Topographical Atlas of Shiga Toxin 2e Receptor Distribution in the Tissues of Weaned Piglets. Toxins, 8(12), 357.

Publication 2016

goat anti-mouse IgG

Alkaline phosphatase Anti antibody Anti igg Anti igm Antibodies Antibodies specificities Assays Chicken Clone Gb3cer Gb4cer Goat Monoclonal antibody Mouse Rabbit Stec Strain Stx2 Workers

Corresponding Organization : University of Münster

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Variable analysis

independent variables

Polyclonal chicken IgY anti-Gb3Cer and anti-Gb4Cer antibodies
Monoclonal rat IgM anti-Forssman GSL antibody (clone IIC2)
Stx2e-containing supernatant of STEC strain S123G of serotype O139:K82

dependent variables

Results of TLC overlay assays

control variables

Previously described specificities of the polyclonal chicken IgY anti-Gb3Cer and anti-Gb4Cer antibodies
Highly specific tool for the detection of Forssman GSL described for the monoclonal rat IgM anti-Forssman GSL antibody (clone IIC2)
Mouse anti-Stx2 antibody (clone VT 135/6-B9, 2.75 mg/mL) purchased from SIFIN GmbH (Berlin, Germany)
Secondary alkaline phosphatase (AP)-conjugated affinity-purified polyclonal rabbit anti-chicken IgY, goat anti-rat IgG + IgM, and goat anti-mouse IgG antibodies from Dianova (Hamburg, Germany)

controls

Positive control: Previously described specificities of the polyclonal chicken IgY anti-Gb3Cer and anti-Gb4Cer antibodies
Positive control: Highly specific tool for the detection of Forssman GSL described for the monoclonal rat IgM anti-Forssman GSL antibody (clone IIC2)
Positive control: Mouse anti-Stx2 antibody (clone VT 135/6-B9, 2.75 mg/mL) purchased from SIFIN GmbH (Berlin, Germany)
Negative control: Not explicitly mentioned

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