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LAL test for detection of endotoxin unit (EU) in accordance with LPS concentration. A serial dilution of 3 different LPSs [S. minnesota (•), E. coli (▪), and P. aeruginosa (▴)] was assayed using the kinetic turbidimetric method.
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Figure 4: LAL test for detection of endotoxin unit (EU) in accordance with LPS concentration. A serial dilution of 3 different LPSs [S. minnesota (•), E. coli (▪), and P. aeruginosa (▴)] was assayed using the kinetic turbidimetric method.

Mentions: The detection limit of the endotoxin (the purified LPS that originated from E. coli, S. minnesota and P. aeruginosa) in the macrophage culture system, using fluorescein as a pH-indicator, was compared with the LAL test. The stock solution of each strain was 105 ng LPS/ml of DMEM (pH 7.2 and without phenol red) supplemented with 10% FBS. The detection limit of this study was 1 ng/ml LPS of E. coli, S. minnesota, and P. aeruginosa in the case of both the fluorescein absorption and fluorescence assay (Fig. 5). The absorption and fluorescence intensity of fluorescein in the control media without LPS was 0.44 ± 0.008 and 560 ± 20 (mean ± S.E.M.), respectively. Fig. 3A establishes that the absorption of fluorescein in the cultured media was significantly reduced with the increasing LPS concentration (on the whole scale from 1 to 500 ng/ml) of E. coli, S. minnesota, and P. aeruginosa. At a 1 ng/ml LPS concentration of S. minnesota, the absorption of fluorescein decreased from 0.44 of the non-treated control to 0.385, and at 500 ng/ml, it decreased to 0.2. The fluorescence data also demonstrated the dose (LPS)-dependent decrease of fluorescence intensity of fluorescein in cultured media in all cases of E. coli, S. minnesota, and P. aeruginosa (Fig. 3B). In the case of S. minnesota, the emission of fluorescein was reduced from 560 for the non-treated control to 481 at 1 ng/ml of LPS, and it was reduced to 285 at 500 ng/ml. The results in LAL test also had a similar pattern to those results of our methods (Fig. 4). Among the bacteria species examined, the endotoxin unit (EU, the LAL reactivity of 0.1 ng of Reference Standard Endotoxin) of S. minnesota was detected from 0.49 of the control to 1.85 at 1 ng/ml of LPS and to 6982 at 500 ng/ml, which was more sensitive than that of E. coli and P. aeruginosa.

In Vitro Bioassay of Endotoxin Using Fluorescein as a pH Indicator in a Macrophage Cell Culture System

Lee DH, Sung HJ, Han DW, Lee MS, Ryu GH, Aihara M, Takatori K, Park JC - Yonsei Med. J. (2005)

Bottom Line: After RAW 264.7 macrophage cells were treated with lipopolysaccharide (LPS), the addition of fluorescein to the LPS-treated media reproductively reduced its absorption and emission spectra (it was a dose-dependent reduction).Additionally, the absorption and fluorescence intensity of fluorescein, following treatment of RAW 264.7 cells with a high concentration of Staphylococcus aureus (Gram-positive, lysed bacteria), could not generally be detected by the LAL test, but they were found to be reduced, in a dose-response relationship, with this new system.The macrophage culture system-method might be a good supplement to the LAL assay for detection of LPS, Gram-negative and Gram-positive bacteria.

Affiliation: Department of Medical Engineering, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, Korea.

ABSTRACT
Based on the biological activity of endotoxin, we propose a possible new method for detecting endotoxin using a pH-indication system of macrophage culture media. After RAW 264.7 macrophage cells were treated with lipopolysaccharide (LPS), the addition of fluorescein to the LPS-treated media reproductively reduced its absorption and emission spectra (it was a dose-dependent reduction). The advantages of this LPS-detection method were compared with the Limulus Amebocyte Lysate (LAL) test by using purified bacterial LPS (Salmonella minnessota, Escherichia coli, and Pseudomonas aeruginosa). Additionally, the absorption and fluorescence intensity of fluorescein, following treatment of RAW 264.7 cells with a high concentration of Staphylococcus aureus (Gram-positive, lysed bacteria), could not generally be detected by the LAL test, but they were found to be reduced, in a dose-response relationship, with this new system. The macrophage culture system-method might be a good supplement to the LAL assay for detection of LPS, Gram-negative and Gram-positive bacteria.

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