title: A bioassay for determination of lipopolysaccharide in environmental samples.
abstract: Although the limulus amebocyte lysate (LAL) assay is widely used to determine the concentration of LPS in biological samples, it is known to be susceptible to interference caused by substances of non-bacterial origin. In particular, polysaccharides such as β-glucans and pectic polysaccharides from fungi or plants, respectively, were shown to give higher LPS readings than were actually present in the sample. Here, we describe an assay for the determination of LPS in biological samples based on the stimulation of TLR4/MD2/CD14 transfected HEK293 cells which dose dependently release IL-8 upon stimulation with increasing concentrations of highly purified Escherichia coli LPS. The resulting standard curve is used to determine the LPS concentration in unknown samples. We show that the outcome of the LPS stimulation is not affected by the presence of β-glucans or other environmental substances found in dust extracts. Although, we present evidence that the LPS concentration measured with the kinetic chromogenic LAL test correlates with data from the TLR4 assay, the LAL test displays higher LPS readings. We conclude that the described TLR4 assay is a reliable alternative to assess the concentration of LPS in environmental samples without being influenced by polysaccharides such as β-glucans and other environmental substances found in dust extracts.
authors.affiliation: Department of Experimental Pneumology, Ruhr-University Bochum, Bochum, Germany. marcus.peters@rub.de
authors.forename: Marcus
authors.initials: M
authors.lastname: Peters
authors.forename: Petra
authors.initials: P
authors.lastname: Fritz
authors.forename: Albrecht
authors.initials: A
authors.lastname: Bufe
mesh_terms: D002478:Cells, Cultured; D004391:Dust / Q000032:analysis*; D004777:Environment; D057809:HEK293 Cells; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D008033:Limulus Test; D018950:Lipopolysaccharide Receptors / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000032:analysis* / Q000494:pharmacology; D051218:Lymphocyte Antigen 96 / Q000235:genetics / Q000378:metabolism; D012015:Reference Standards; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism; D019076:Transgenes / Q000235:genetics
chemical_list: D004391:Dust; D016209:Interleukin-8; C495377:LY96 protein, human; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4