Abstract
CD14, a 55kDa lipopolysaccharide binding glycoprotein, is a key element in both LPS-mediated activation of cells and endotoxin detoxification. A gene fragment containing residues 1-348 of the human LPS receptor CD14, representing the extracellular form of the molecule, was fused to the CH(2)-CH(3) portion of the human IgG heavy chain or to a 6x His tag and transfected into CHO cells. Stable cell lines of each were grown to produce recombinant protein in unsupplemented serum free media and CD14His was purified by ion-exchange chromatography. After passive immobilization onto a carbon surface both forms of the CD14 fusion proteins bound LPS-biotin in a dose-dependent manner in an electrochemiluminescent assay. Binding was inhibited by the anti-CD14 antibody S39 as well as by unlabeled LPS. This report describes an efficient method of purifying CD14 and a novel assay to detect bioactive lipopolysaccharide.