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 sepa rately 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, a nd a comigration of fluorescence and optical density signals using gel filt ration. This method, in combination with the use of specific antibodies ind ucing additional shifts, demonstrated that in serum high-density lipoprotei ns (HDL), albumin and low-density lipoproteins (LDL) were able to monomeriz e LPS. An ELISA on collected fractions of the gel filtration revealed bindi ng of the recently identified LPS-binding protein, serum amyloid P componen t (SAP), to the high molecular weight LPS aggregate. In serum, binding of s oluble CD14 (sCD14) and LPS-binding protein (LBP) to LPS could not be detec ted. 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 tes ted, only SAP clearly bound to the LPS-coated sensor chip. These results sh ow that gel filtration of FITC-LPS is a quick and reliable method to study the binding characteristics of LPS-binding components. (C) 2000 Elsevier Sc ience B.V. All rights reserved.