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ELISA for antibodies to lipid A, lipopolysaccharides and other hydrophobic antigens

ELISA-Methode zur Bestimmung von Anti-Lipoid-A-Antikörpern, Lipopolysacchariden und anderen hydrophoben Antigenen

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Summary

A simple, sensitive and rapid ELISA method for the quantification and characterization of antibodies to lipid A has been developed, which can also be applied to other hydrophobic antigens. For coating, antigens were applied to the wells of ELISA plates as solutions in a mixture of chloroform and ethanol 1:9 (v/v), and the solvent evaporated in a stream of warm air. Under these conditions a high coating efficiency was achieved, which made the assay highly sensitive. The use of the above organic solvent considerably reduced the nonspecific adsorption of immunoglobulins to the solid phase, making the usual blocking of unspecific binding sites with BSA or gelatine unnecessary. For screening of lipid A antibodies in the sera of immunized animals, coating with 0.2 µg of the corresponding antigen per well was found to be suitable. For optimal measurement of antibodies in pre-immune sera, sera of healthy human donors, and of monoclonal antibodies, higher amounts of antigen (1–2 µg/well) had to be used. The coating method described here proved excellent also for other antigens directly soluble in organic solvents, such as Re-lipopolysaccharide (LPS) or gangliosides. In addition, the method was successfully applied to less hydrophobic antigens, such as LPS of the classes Ra to Rd and S forms, and lipoteichoic acid. These could be brought into solution in chloroform/ethanol by diluting their aqueous solutions with a large volume of the organic mixture.

Zusammenfassung

Eine einfache, empfindliche und schnelle ELISA-Methode zur Bestimmung und Charakterisierung von Anti-Lipoid-A-Antikörpern wurde ausgearbeitet. Zum Beschichten der ELISA-Platten wird eine Lösung des Antigens in Chloroform/Äthanol (1:9 [v/v]) in die Löcher eingetragen und das Lösungsmittel dann mit einem Strom warmer Luft verdampft. Damit wird ein hoher Beschichtungsgrad erreicht, wodurch die Bestimmung sehr empfindlich wird. Gleichzeitig wird die unspezifische Adsorptionskapazität der Platten für Immunglobuline erniedrigt, so daß sich eine Blockierung unspezifischer Bindungsstellen erübrigt, wie sie üblicherweise mit BSA oder Gelatine durchgeführt wird. Zum Auffinden von Lipoid-A-Antikörpern sind 0,2 µg des entsprechenden Antigens pro Plattenloch bei Immunseren, und 1–2 µg bei Nicht-Immunseren und monoklonalen Antikörpern optimal. Die hier beschriebene Beschichtungsmethode kann generell bei Antigenen angewandt werden, die in organischen Lösungen direkt lösbar sind, wie zum Beispiel bei Re-LPS und Gangliosiden. Des weiteren ist diese Methode für Antigene geeignet, die zwar primär in Chloroform/Äthanol unlöslich sind, die aber nach Lösen in einem geeigneten wäßrigen Medium in die Chloroform/Äthanol-Mischung überführt werden können. Dies wird an mehreren Beispielen gezeigt (LPS der Klassen Ra-Rd und S-formen, sowie Lipoteichonsäure).

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Authors and Affiliations

  1. Max-Planck-Institut für Immunbiologie, Stübeweg 51, D-7800, Freiburg, FR Germany

    Marina A. Freudenberg, A. Fomsgaard,  I. Mitov &  C. Galanos

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  1. Marina A. Freudenberg
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  2. A. Fomsgaard
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  3. I. Mitov
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  4. C. Galanos
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Freudenberg, M.A., Fomsgaard, A., Mitov, I. et al. ELISA for antibodies to lipid A, lipopolysaccharides and other hydrophobic antigens. Infection 17, 322–328 (1989). https://doi.org/10.1007/BF01650719

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