Abstract
Lipopolysaccharide is the major constituent of the outer membrane of gram-negative bacteria and, once released from the bacterial surface into the bloodstream, is a potent activator of the host immune system, which can lead to septic shock. LPS has a hydrophilic region consisting of a repeating oligosaccharide that is strain-specific (O-antigen) and a core polysaccharide, which is covalently linked to a hydrophobic lipid moiety (lipid A). Lipid A is the most conserved part and is responsible for the toxicity of LPS. Therefore, finding molecules able to bind to this region and neutralize LPS toxicity is of relevant interest as it may provide new therapies to prevent septic shock (Chen et al., 2006). Several proteins and peptides were reported to bind LPS and alter its toxicity towards reduction and even enhancement (Brandenburg et al., 1998), such as serum albumin (Ohno and Morrison, 1989), lipopolysaccharide binding protein (LBP) (de Haas et al., 1999), casein (López-Expósito et al., 2008), lysozyme, the antibiotic polymyxin B and antimicrobial peptides (Chen et al., 2006). Although some of these proteins are neutral and even anionic/acidic (pI<7) (Jang et al., 2009), due to the amphipathic structure of LPS and the presence of negatively charged phosphate groups on the lipid A, the most important factors that are considered for optimal binding to LPS are a cationic/basic (pI>7) and amphipathic nature (Chen et al., 2006). Here we describe a competitive ELISA that can be used to identify proteins or peptides that bind LPS, as a first approach before analyzing the possible activity in vitro and in vivo. In this ELISA, serial dilutions of the protein or peptide to be tested are preincubated with a fixed concentration of fluorescein isothiocyanate (FITC)-labeled LPS from Escherichia coli serotype O111:B4 and then added to wells of a microtitre plate which are blocked with a casein hydrolysate that binds LPS (Martínez-Sernández et al., 2014). Binding of the protein to LPS displaces LPS from binding to the casein, which is revealed using a horseradish peroxidase (HRP)-labeled anti-FITC polyclonal conjugate. This method allows simultaneous analysis of several proteins or peptides in a short period of time and no recognizing molecules (e.g., antibodies) to a specific protein or peptide are needed.
Keywords: LPS, Lipopolysaccharide, ELISA, Casein, LPS-binding
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Example of inhibition curve obtained with several molecules using the reported method to analyze LPS binding. LPS-FITC (0.25 µg/well) was incubated with four-fold dilutions of polymyxin B (circles), LBP human recombinant (squares), bovine serum albumin (BSA) (inverted triangles) and myoglobin from equine skeletal muscle (triangles). It is noteworthy that an excess of protein/peptide might have a “zone effect” in the assay, as occurs with polymyxin B.
Notes
Recipes
Acknowledgments
This work was supported by grants AGL2011-30563-C03 (Ministerio de Ciencia e Innovación, Spain) and CN 2012/155 (Xunta de Galicia, Spain). Victoria Martínez-Sernández holds a predoctoral fellowship (Programa de Formación del Profesorado Universitario) from the Spanish Ministerio de Educación, Cultura y Deporte. This protocol was modified from Martínez-Sernández et al. (2014).
References
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