Novel quantitative electrophoretic analysis of endotoxins on microchips

Kilár, Anikó; Farkas, Viktor; Kovács, Krisztina; Kocsis, Béla; Kilár, Ferenc

doi:10.1002/elps.200700684

Cited by 12 publications

(7 citation statements)

References 56 publications

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“…The possibility of using this technique within a short time after the preparation of partially purified LPS from bacterial cultures proves that the method has a significant clinical relevance. The improvement in the LOD value compared to previous assays also confirms that the method provides a basis for the detection of endotoxins in different biological matrices.…”

Section: Discussionsupporting

confidence: 57%

“…The fast analyses of Gram‐negative bacterial endotoxins with ME, using noncovalent binding of a fluorescent dye, have been described earlier . The recent ME method is suitable for the determination of endotoxins labeled with covalent binding of a fluorescent dye.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, we have introduced a ME method combined with LIF detection, to visualize bacterial LPSs after complexation with dodecyl sulphate chains followed by the noncovalent binding with a fluorescent dye . The ME system was capable of resolving the various molecular species providing characteristic electrophoretic profiles for the LPS species with increasing molecular masses, reflecting the size heterogeneity mentioned above.…”

Section: Introductionmentioning

confidence: 99%

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Quantitative microfluidic analysis of S‐ and R‐type endotoxin components with chip capillary electrophoresis

Makszin

Kilár²,

Felső

et al. 2012

Electrophoresis

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A novel, fast, and sensitive ME method was developed to analyze and differentiate the smooth (S) and rough (R) type bacterial endotoxin components labeled covalently with a fluorescent dye. The quantitative analysis of purified lipopolysaccharides, or partially purified samples from whole-cell lysates becomes possible with this method. Two groups with three sub-groups in the first group of S-type lipopolysaccharides can be classified based on the electrophoretic profiles. The LOD of the endotoxins from S- and R-type Gram-negative bacteria was found to be 2.6 ng and 6.9 ng, respectively. This method is capable to replace the commonly used SDS-PAGE combined with silver staining.

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Section: Discussionsupporting

confidence: 57%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Quantitative microfluidic analysis of S‐ and R‐type endotoxin components with chip capillary electrophoresis

Makszin

Kilár²,

Felső

et al. 2012

Electrophoresis

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“…Electrophoresis in microchips was performed in the commercially available Agilent 2100 Bioanalyzer system (Agilent Technologies, Waldbronn, Germany) equipped with a diode laser for fluorescence detection. Two kinds of kits have been developed for protein analysis, which were applied in a slightly modified manner for endotoxin analysis, as well . In the present study, the High Sensitivity Protein 250 Kit (Agilent Technologies) was used for the qualitative and quantitative analysis, as described previously .…”

Section: Methodsmentioning

confidence: 99%

Structural background for serological cross‐reactivity between bacteria of different enterobacterial serotypes

et al. 2015

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The structure of the oligosaccharide repeating units of endotoxins from Gram-negative bacteria is characteristic for the different serogroups and serotypes of bacteria. Detailed examination of the cross-reactions of three enterobacterial serotypes, Proteus morganii O34, Escherichia coli O111, and Salmonella enterica sv. Adelaide O35, was performed using sensitive tests (ELISA, immunoblotting). Fine differences between the endotoxins of the bacteria were detected using silver staining of SDS-PAGE gels and chip-technology for the intact lipopolysaccharides (LPSs). The compositions of the O-specific polysaccharides of LPSs extracted from the bacteria were studied, and it was proven that the three cross-reacting bacteria contain O-antigens built from the same monosaccharides, namely colitoses linked to glucose, galactose, and N-acetyl-galactosamine. The NMR and GC-MS studies revealed that the most probable component for the cross-reaction is the rare sugar, colitose.

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“…Besides, the non-specific methods would also give false positive results due to reactions with other microbial products that trigger the LAL reaction, such as peptidoglycan from gram-positive organisms, release of (1-3)-β-D-glucans 9 , and D-glucose polymers from cell walls 24 . Furthermore, the LAL method only provides the general activity of endotoxins in the sample, instead of the detailed structure or distribution profile 25 . LAL method measures endotoxin concentration indirectly, but endotoxins that possess diverse structures and originate from different bacterial serotypes and their mutants may introduce variations.…”

Section: Introductionmentioning

confidence: 99%

Extraction, separation and characterization of endotoxins in water samples using solid phase extraction and capillary electrophoresis-laser induced fluorescence

Fung

Feng

et al. 2017

Sci Rep

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This study focuses on one of the key environmental threats, endotoxins, also known as lipopolysaccharides (LPS). A capillary electrophoresis method in combination with laser induced fluorescence (LIF) detection was developed for the analysis of endotoxins from 16 different bacterial strains. LPSs were derivatized with the amino-reactive fluorescent dye, fluorescein isothiocyanate (FITC), separated by capillary zone electrophoresis (CZE) under the optimized conditions with the use of 50 mM sodium tetraborate buffer (pH 9.30), and detected by LIF detector. To improve the sensitivity of CZE-LIF detection for the determination of trace amounts of endotoxins and to remove possible interference materials in environmental samples, a solid phase extraction (SPE) pre-concentration technique was applied successfully. The SPE targeted at polysaccharide moieties of LPSs and showed LPS enrichment effects too. CE migration time could also reveal the O-antigen chain lengths of LPSs. This CE method and SPE pretreatment showed linearity at 99.84%, and repeatabilities at 8.44% and 11.0% for endotoxins from E. Coli O55:B5 and E. Coli O26:B6. The limit of detection (LOD) could reach around 5 ng/mL at optimized condition. The method was applied successfully to the determination of LPS levels in tap water and wastewater, and demonstrated sensitive, reproducible and reliable results.

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Novel quantitative electrophoretic analysis of endotoxins on microchips

Cited by 12 publications

References 56 publications

Quantitative microfluidic analysis of S‐ and R‐type endotoxin components with chip capillary electrophoresis

Quantitative microfluidic analysis of S‐ and R‐type endotoxin components with chip capillary electrophoresis

Structural background for serological cross‐reactivity between bacteria of different enterobacterial serotypes

Extraction, separation and characterization of endotoxins in water samples using solid phase extraction and capillary electrophoresis-laser induced fluorescence

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