Purification of soluble CD14 fusion proteins and use in an electrochemiluminescent assay for lipopolysaccharide binding.
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.
Burkhardt
M
M
Biosynexus Incorporated, 9119 Gaither Rd, Gaithersburg, MD, USA. martyburkhardt@biosynexus.com
LopezAcosta
A
A
Reiter
K
K
Lopez
V
V
Lees
A
A
D000818:Animals; D019149:Bioreactors; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004563:Electrochemistry / Q000379:methods; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000302:isolation & purification* / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D012995:Solubility
D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D011993:Recombinant Fusion Proteins