Quantification of Bacterial Lipopolysaccharides by the Purpald Assay: Measuring Formaldehyde Generated from 2-keto-3-deoxyoctonate and Heptose at the Inner Core by Periodate Oxidation

Lee, Che-Hung; Tsai, Chao-Ming

doi:10.1006/abio.1998.2961

Cited by 161 publications

(124 citation statements)

References 25 publications

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“…Bacterial LPS concentrations were determined by measuring the presence of KDO according to a previously described colorimetric assay (43,44). KDO forms part of the oligosaccharide core of the bacterial LPS molecule, and E. coli LPS contains two reactive KDO moieties per molecule (45). OMV samples (45 μL) in PBS were combined with 0.2 N H 2 SO 4 (5 μL) and heated at 100°C for 20 min.…”

Section: Methodsmentioning

confidence: 99%

Delivery of foreign antigens by engineered outer membrane vesicle vaccines

Chen

Osterrieder

Metzger

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

256

247

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As new disease threats arise and existing pathogens grow resistant to conventional interventions, attention increasingly focuses on the development of vaccines to induce protective immune responses. Given their admirable safety records, protein subunit vaccines are attractive for widespread immunization, but their disadvantages include poor immunogenicity and expensive manufacture. We show here that engineered Escherichia coli outer membrane vesicles (OMVs) are an easily purified vaccine-delivery system capable of greatly enhancing the immunogenicity of a low-immunogenicity protein antigen without added adjuvants. Using green-fluorescent protein (GFP) as the model subunit antigen, genetic fusion of GFP with the bacterial hemolysin ClyA resulted in a chimeric protein that elicited strong anti-GFP antibody titers in immunized mice, whereas immunization with GFP alone did not elicit such titers. Harnessing the specific secretion of ClyA to OMVs, the ClyA-GFP fusion was found localized in OMVs, resulting in engineered recombinant OMVs. The anti-GFP humoral response in mice immunized with the engineered OMV formulations was indistinguishable from the response to the purified ClyA-GFP fusion protein alone and equal to purified proteins absorbed to aluminum hydroxide, a standard adjuvant. In a major improvement over current practice, engineered OMVs containing ClyA-GFP were easily isolated by ultracentrifugation, effectively eliminating the need for laborious antigen purification from cell-culture expression systems. With the diverse collection of heterologous proteins that can be functionally localized with OMVs when fused with ClyA, this work signals the possibility of OMVs as a robust and tunable technology platform for a new generation of prophylactic and therapeutic vaccines.protein subunit | adjuvant

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Section: Methodsmentioning

confidence: 99%

Delivery of foreign antigens by engineered outer membrane vesicle vaccines

Chen

Osterrieder

Metzger

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

256

247

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“…Briefly, short carbohydrate chain-containing LPS was isolated from E. coli and alkaline-hydrolyzed for removal of lipid A moieties, followed by ethanol precipitation. The resulting modified LPS was quantitated using the 2-keto-3-deoxyoctonate assay (26), and the reduced size of the modified LPS was checked on a silver-stained SDSpolyacrylamide gel.…”

Section: Methodsmentioning

confidence: 99%

Bacterial DNA Containing Methylated CpG Motifs Retains Immunostimulatory Activity in Synergy with Modified Lipopolysaccharides

Cho

Ahn²,

Song

et al. 2007

Microbiology and Immunology

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We previously described the immunostimulatory activity of CIA07, a combination of bacterial DNA fragments and modified LPS, and demonstrated that CIA07 has antitumor activity in a mouse bladder cancer model. In this study, we investigated whether methylation of the CpG motifs on the bacterial DNA fragments affects the immunostimulatory potential of CIA07. E. coli DNA fragments were methylated with CpG methylase, and then combined with modified LPS for experiments. Our results revealed that methylated CIA07 (mCIA07) and unmethylated CIA07 were equally active in inducing cytokine secretion from human whole blood cells. In addition, both methylated DNA fragments and mCIA07 retained the ability to activate expression and nuclear translocation of NF‐κB in RAW 264.7 cells. Finally, methylated DNA fragments and mCIA07 exhibited an antitumor activity comparable to those of their unmethylated counterparts in our mouse bladder cancer model. These data demonstrate that CpG methylation of E. coli DNA does not abrogate the immunostimulatory activity of DNA fragments or CIA07, suggesting that the synergistic activity by bacterial DNA in combination with LPS may be independent of the methylation status of CpG motifs.

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“…Since the LAL assay is very expensive, the Purpald method (Lee, Tsai, 1999) was used when endotoxin concentration was above 75,000 EU/mL. The determinations of the endotoxin concentrations by the Purpald method is based on the reaction between sodium periodate and the unsubstituted terminal vicinal glycol (UTVG) group, present in endotoxin structure, producing formaldehyde and oxidizing the LPS.…”

Section: Methodsmentioning

confidence: 99%

“…Membrane adsorbers were prepared as previously reported in the literature (Beeskow, Kusharyoto, Anspach, 1995;Castilho, Anspach, Deckwer, 2002;Castilho, Deckwer, Anspach, 2000;Lee, Tsai, 1999). Figure 3 shows the experimental apparatus.…”

Section: Membrane Adsorbermentioning

confidence: 99%

Membrane adsorber for endotoxin removal

Almeida

Fingola

et al. 2016

Braz. J. Pharm. Sci.

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The surface of flat-sheet nylon membranes was modified using bisoxirane as the spacer and polyvinyl alcohol as the coating polymer. The amino acid histidine was explored as a ligand for endotoxins, aiming at its application for endotoxin removal from aqueous solutions. Characterization of the membrane adsorber, analysis of the depyrogenation procedures and the evaluation of endotoxin removal efficiency in static mode are discussed. Ligand density of the membranes was around 7 mg/g dry membrane, allowing removal of up to 65% of the endotoxins. The performance of the membrane adsorber prepared using nylon coated with polyvinyl alcohol and containing histidine as the ligand proved superior to other membrane adsorbers reported in the literature. The lack of endotoxin adsorption on nylon membranes without histidine confirmed that endotoxin removal was due to the presence of the ligand at the membrane surface. Modified membranes were highly stable, exhibiting a lifespan of approximately thirty months.Uniterms: Endotoxins/removal. Membrane adsorber/performance. Histidine. Poly(vinyl alcohol).A superfície de membranas planas de nylon foi modificada utilizando-se bisoxirano como espaçador e poli(álcool vinílico) para recobrimento das membranas. O aminoácido histidina foi utilizado como ligante para endotoxinas, visando à sua aplicação na remoção de endotoxinas a partir de soluções aquosas. São discutidas as etapas de caracterização do adsorvedor com membranas, análise do procedimento de despirogenização e avaliação da eficiência de remoção em modo estático. A densidade de ligantes nas membranas foi em torno de 7 mg/g membrana (massa seca), permitindo uma remoção de endotoxinas de até 65%. O desempenho das membranas preparadas com nylon e recobertas com poli(álcool vinílico) contendo histidina como ligante foi superior ao de outros adsorvedores com membranas descritos na literatura. A ausência de adsorção de endotoxinas em membranas sem histidina confirma que a remoção das endotoxinas deve-se exclusivamente à presença do ligante na superfície da membrana. As membranas modificadas mostraram-se bastante estáveis, exibindo um tempo de vida superior a 30 dias.Unitermos: Endotoxinas/remoção. Membrana adsorvente/desempenho. Histidina. Poli(álcool vinílico).

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Quantification of Bacterial Lipopolysaccharides by the Purpald Assay: Measuring Formaldehyde Generated from 2-keto-3-deoxyoctonate and Heptose at the Inner Core by Periodate Oxidation

Cited by 161 publications

References 25 publications

Delivery of foreign antigens by engineered outer membrane vesicle vaccines

Delivery of foreign antigens by engineered outer membrane vesicle vaccines

Bacterial DNA Containing Methylated CpG Motifs Retains Immunostimulatory Activity in Synergy with Modified Lipopolysaccharides

Membrane adsorber for endotoxin removal

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