Preparation of lipopolysaccharide derived from Pantoea agglomerans labeled with fluorescence as a tracer for kinetics analysis.
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.
Kadowaki
Takeshi
T
Department of Faculty of Medicine, Kagawa University, Miki-cho, Kida-gun, Kagawa-ken, 761-0793, Japan.
Inagawa
Hiroyuki
H
Kohchi
Chie
C
Hirashima
Mitsuomi
M
Soma
Gen-Ichiro
G
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
D008070:Lipopolysaccharides; C524847:lipopolysaccharide IP-PA1, Pantoea agglomerans