Detection of Lipopolysaccharide(LPS)‐Binding Membrane Proteins by Immuno‐Coprecipitation with LPS and Anti‐LPS Antibodies

In this study we describe a general method for the detection and characterization of endotoxin‐(lipopolysaccharide, LPS)‐binding membrane proteins. In the past, experimental procedures to detect LPS‐binding sites on cells were generally performed with chemically modified LPS derivates. Since any modification of a ligand may lead to a modification of its binding characteristics, the results of those studies are controversial. In our assay, cell membrane preparations are treated with free lipid A, the endotoxic center of LPS, in the presence of normal human serum. After binding of lipid A, membrane proteins are solubilized by mild detergent treatment without disruption of the lipid A‐protein complexes. Addition of anti‐(lipid A) mAbs and subsequent adding of protein A agarose lead to the precipitation of complexes of lipid A and its binding proteins. By SDS/PAGE and western blot, these precipitates can be screened for the presence of LPS/lipid A‐binding proteins. We describe the use of this method for the immuno‐coprecipitation of lipid A (or LPS) with an 80‐kDa LPS‐binding membrane protein (LMP80), which we have previously identified on several human cells. In addition, CD14, the well‐known functional LPS receptor on monocytes and macrophages, can be detected. By means of this immuno‐coprecipitation approach we could demonstrate binding of either purified LPS preparations or synthetic lipid A to these LPS/lipid A‐binding membrane proteins at physiological pH under conditions in which the proteins are in their natural membranous environment.