Preparation of lipopolysaccharide derived from Pantoea agglomerans labeled with fluorescence as a tracer for kinetics...

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).

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.

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.

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.