Lipopolysaccharide (LPS) labeled with Alexa 488 hydrazide as a novel probe for LPS binding studies.
BACKGROUND
Lipopolysaccharide (LPS) comprises the outer cell wall of all gram-negative bacteria. It consists of an oligosaccharide core and lipid A. All LPS-induced biological responses are lipid A-dependent. Once released, LPS triggers a host systemic inflammatory response that leads to septic shock. Binding studies have helped to reveal some of the molecular interactions behind septic shock. Such studies have employed methods of labeling bacterial LPS with either radiochemicals or fluorescent dyes. Poor labeling of the LPS has resulted in the use of high concentrations of LPS in order to detect its binding.


METHODS
In this study, we have devised a new methodology for labeling LPS, using hydrazide and galactose oxidase in order to oxidize galactose residues to aldehyde groups in the oligosaccharide core of the LPS.


RESULTS
We have managed to generate a conjugate that is highly fluorescent (LPS-to-Alexa 488 labeling ratio of 1:5) and biologically active.


CONCLUSIONS
For the first time, this probe has enabled us to detect LPS binding even at pg/ml concentrations. Using this methodology, any Alexa-hydrazide dye can be conjugated to LPS, providing us with novel probes for imaging studies.
Triantafilou
K
K
Department of Biological Sciences, University of Essex, Colchester, United Kingdom.
Triantafilou
M
M
Fernandez
N
N
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005434:Flow Cytometry / Q000379:methods; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D005692:Galactose Oxidase / Q000378:metabolism; D006834:Hydrazines / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D018950:Lipopolysaccharide Receptors / Q000276:immunology / Q000378:metabolism; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D012475:Salmonella; D012680:Sensitivity and Specificity; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
C417561:Alexa 488 hydrazide; D005456:Fluorescent Dyes; D006834:Hydrazines; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D005692:Galactose Oxidase; D016650:Fluorescein-5-isothiocyanate