Analysis of lipopolysaccharide (LPS)-binding characteristics of serum components using gel filtration of FITC-labeled LPS.
Lipopolysaccharide (LPS)-binding components in serum play an important role in modifying LPS toxicity. We analyzed the binding characteristics of LPS in the presence of serum using gel filtration of FITC-labeled LPS (FITC-LPS) with on line detection of optical density and fluorescence. FITC-LPS separately behaves as an aggregate resulting in a low, dequenched, fluorescence. Binding of single LPS molecules, segregated from the aggregate, to serum components results in an increase in the fluorescence due to dequenching, and a comigration of fluorescence and optical density signals using gel filtration. This method, in combination with the use of specific antibodies inducing additional shifts, demonstrated that in serum high-density lipoproteins (HDL), albumin and low-density lipoproteins (LDL) were able to monomerize LPS. An ELISA on collected fractions of the gel filtration revealed binding of the recently identified LPS-binding protein, serum amyloid P component (SAP), to the high molecular weight LPS aggregate. In serum, binding of soluble CD14 (sCD14) and LPS-binding protein (LBP) to LPS could not be detected. However, this was probably due to an overshadowing effect of albumin, as an extra addition of recombinant sCD14 to serum clearly monomerized FITC-LPS. Biosensor technology revealed that, of all LPS-binding components tested, only SAP clearly bound to the LPS-coated sensor chip. These results show that gel filtration of FITC-LPS is a quick and reliable method to study the binding characteristics of LPS-binding components.
de Haas
C J
CJ
Eijkman Winkler Institute for Medical Microbiology, Department of Inflammation, University Medical Center Utrecht, Utrecht, The Netherlands. c.j.c.dehaas@lab.azu.nl
van Leeuwen
H J
HJ
Verhoef
J
J
van Kessel
K P
KP
van Strijp
J A
JA
D000209:Acute-Phase Proteins; D001798:Blood Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000032:analysis; D002850:Chromatography, Gel / Q000379:methods / Q000592:standards; D016650:Fluorescein-5-isothiocyanate; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000032:analysis; D008070:Lipopolysaccharides / Q000378:metabolism*; D008075:Lipoproteins, HDL / Q000378:metabolism; D008562:Membrane Glycoproteins; D012709:Serum Albumin / Q000378:metabolism; D000683:Serum Amyloid P-Component / Q000378:metabolism; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D001798:Blood Proteins; D002352:Carrier Proteins; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008075:Lipoproteins, HDL; D008562:Membrane Glycoproteins; D012709:Serum Albumin; D000683:Serum Amyloid P-Component; D000099231:Lipopolysaccharide-Binding Protein