Purification of soluble CD14 fusion proteins and use in an electrochemiluminescent assay for lipopolysaccharide binding

CD14, a 55 kDa 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 CH2–CH3 portion of the human IgG heavy chain or to a 6× 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.