Usefulness of ELISA Methods for Assessing LPS Interactions with Proteins and Peptides

Abstract

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, can trigger severe inflammatory responses during bacterial infections, possibly leading to septic shock. One approach to combatting endotoxic shock is to neutralize the most conserved part and major mediator of LPS activity (lipid A) with LPS-binding proteins or peptides. Although several available assays evaluate the biological activity of these molecules on LPS (e.g. inhibition of LPS-induced TNF-α production in macrophages), the development of simple and cost-effective methods that would enable preliminary screening of large numbers of potential candidate molecules is of great interest. Moreover, it would be also desirable that such methods could provide information about the possible biological relevance of the interactions between proteins and LPS, which may enhance or neutralize LPS-induced inflammatory responses. In this study, we designed and evaluated different types of ELISA that could be used to study possible interactions between LPS and any protein or peptide. We also analysed the usefulness and limitations of the different ELISAs. Specifically, we tested the capacity of several proteins and peptides to bind FITC-labeled LPSs from Escherichia coli serotypes O111:B4 and O55:B5 in an indirect ELISA and in two competitive ELISAs including casein hydrolysate (hCAS) and biotinylated polymyxin B (captured by deglycosylated avidin; PMX) as LPS-binding agents in the solid phase. We also examined the influence of pH, detergents and different blocking agents on LPS binding. Our results showed that the competitive hCAS-ELISA performed under mildly acidic conditions can be used as a general method for studying LPS interactions, while the more restrictive PMX-ELISA may help to identify proteins/peptides that are likely to have neutralizing properties in vitro or in vivo.

Delineating distinct heme-scavenging and -binding functions of domains in MF6p/helminth defense molecule (HDM) proteins from parasitic flatworms

“…In accordance with previous findings (6), our data confirm that the entire Fasciola MF6p/FhHDM-1 protein and its C-terminal region are able to bind LPS and, moreover, to compete with PMX for LPS binding. The latter aspect is of major importance because it demonstrates that the binding is specific and because recent reports indicate that proteins and peptides able to interact with the PMX-binding site on LPS are potential can-didates for neutralizing LPS toxicity (23). In agreement with this hypothesis, MF6p/FhHDM-1 and its C-terminal region were reported to be able to significantly inhibit the LPS-induced production of TNF by murine macrophages (6,10).…”

“…laps that of heme. For this purpose, we used a recently developed competitive ELISA (23), which uses biotinylated PMX immobilized on deglycosylated avidin to capture LPS. For the study, we compared the LPS-binding ability of PMX (positive control for inhibition) with that of the Fasciola sMF6p/ FhHDM-1 protein (either native or synthetic), the N-terminal region (sFhMF6a), the C-terminal region (sFhMF6c), two shorter fragments derived from the sFhMF6c region (sFhMF6c1 and sFhMF6c2), and the C-terminal regions of the three MF6p/FhHDM-1 orthologs from C. sinensis, O. viverrini, and P. westermani.…”

Inhibition of Neuroinflammation by AIBP: Spinal Effects upon Facilitated Pain States

“…LPS binding to MD2 and AIBP was assessed in plate-based assay as described (Martínez-Sernández et al, 2016). …”

Clonorchis sinensis MF6p/HDM (CsMF6p/HDM) induces pro-inflammatory immune response in RAW 264.7 macrophage cells via NF-κB-dependent MAPK pathways

“…The structural integrity of the C-terminal region in the MF6p/HDM has been considered essential for interaction with LPS [9]; however, this interaction may not be universal across similar peptides derived from MF6p/HDM orthologs in other trematodes. Otherwise, this phenomenon might be attributed to differences in the physicochemical properties of CsMF6p/ HDM and FhMF6p/HDM, such as isoelectric point [9], hydrophobicity [37] and LPS serotypes (O127:B8 for CsMF6p/HDM; LPS O111:B4 and O55:B5 for FhMF6p/ HDM) [38]. To understand the different patterns of CsMF6p/HDM and FhMF6p/HDM in LPS binding and neutralization of LPS-induced inflammatory responses, the docking poses of LPS with CsMF6p/HDM and FhMF6p/HDM were compared.…”