title: Preparation of lipopolysaccharide derived from Pantoea agglomerans labeled with fluorescence as a tracer for kinetics analysis.
abstract: BACKGROUND
  Intradermal and/or oral administration of lipopolysaccharides derived from Pantoea agglomerans (IP-PA1) have shown multiple positive effects such as phylactic, anti-allergic and anti-tumor effects. It has been reported that the effects of IP-PA1 were derived from the induction of activated macrophages. However, it has not been actually clarified whether or not the orally administered IP-PA1 absorbed in the intestine reached and activated tissue macrophages. The aim of this study was to prepare and evaluate IP-PA1 labeled with fluorescence as a tracer, which could be used for IP-PA1 functional studies (administration, distribution, metabolism and excretion).
  
  
  METHODS
  IP-PA1 was labeled with fluorescein isothiocyanate (FITC). IP-PA1 was converted to the monomeric form using triethylamine solvent. Borate buffer (pH 10.5) containing FITC was added to the IP-PA1 solution, and then a sodium deoxycholate solution was added and the mixture incubated for 18 h. The conjugate in the supernatant was dialysed against phosphate-buffered saline and then the purified FITC-IP-PA1 was analyzed on thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) gel filtration. The biological activity of FITC-IP-PA1 was tested with a Limulus assay using a commercial endospecy kit and with a nitric oxide (NO) assay in the RAW 264.7 cell line using Griess reagent. The binding of FITC-IP-PA1 on RAW 264.7 cells was measured by flow cytometry.
  
  
  RESULTS
  FITC-IP-PA1 and free FITC molecules had different Rf values on TLC. The peak of FITC-IP-PA1 and unlabeled IP-PA1 had the same retention time by HPLC. The Limulus activity of FITC-IP-PA1 was 101% compared with unlabeled IP-PA1 (±18.4%). NO production by FITC-IP-PA1 induced dose dependency. In the flow cytometric analysis, RAW 264.7 cells exhibited high fluorescence intensity (99.4%) when cells were incubated with FITC-IP-PA1 (1 μg/ml). The binding of FITC-IP-PA1 to RAW 264.7 cells was inhibited by adding unlabeled IP-PA1.
  
  
  CONCLUSIONS
  These results demonstrate that both FITC-IP-PA1 and unlabeled IP-PA1 are biologically active. The described FITC-IP-PA1 could be utilized in a variety of IP-PA1 functional studies such as biochemistry, immunohistochemistry, and molecular cell biology.
authors.affiliation: Department of Faculty of Medicine, Kagawa University, Miki-cho, Kida-gun, Kagawa-ken, 761-0793, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kadowaki
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Inagawa
authors.forename: Chie
authors.initials: C
authors.lastname: Kohchi
authors.forename: Mitsuomi
authors.initials: M
authors.lastname: Hirashima
authors.forename: Gen-Ichiro
authors.initials: G
authors.lastname: Soma
mesh_terms: D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D005434:Flow Cytometry; D005453:Fluorescence; D007700:Kinetics; D008070:Lipopolysaccharides / Q000008:administration & dosage / Q000378:metabolism*; D051379:Mice
chemical_list: D008070:Lipopolysaccharides; C524847:lipopolysaccharide IP-PA1, Pantoea agglomerans