Quantitative Determination of Globotriaosylceramide by Immunodetection of Glycolipid-Bound Recombinant Verotoxin B Subunit

Zeidner, Ken M.; Desnick, Robert J.; Ioannou, Yiannis A.

doi:10.1006/abio.1998.2966

Cited by 43 publications

(46 citation statements)

References 58 publications

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“…In the 4 patients, GlcCer concentrations decreased during both ERT and SRT. In the 2 Fabry patients, concentrations of CTH decreased upon therapy, as has been reported (13,30,31 ). These data illustrate the suitability of the new method for the follow-up of patients with Gaucher and Fabry disease during therapy.…”

Section: Discussionsupporting

confidence: 78%

HPLC for Simultaneous Quantification of Total Ceramide, Glucosylceramide, and Ceramide Trihexoside Concentrations in Plasma

Groener¹,

Poorthuis²,

Kuiper³

et al. 2007

Clinical Chemistry

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Background: Simple, reproducible assays are needed for the quantification of sphingolipids, ceramide (Cer), and sphingoid bases. We developed an HPLC method for simultaneous quantification of total plasma concentrations of Cer, glucosylceramide (GlcCer), and ceramide trihexoside (CTH). Methods: After addition of sphinganine as internal calibrator, we extracted lipids from 50 L plasma. We deacylated Cer and glycosphingolipids by use of microwave-assisted hydrolysis in methanolic NaOH, followed by derivatization of the liberated amino-group with o-phthaldialdehyde. We separated the derivatized sphingoid bases and lysoglycosphingolipids by HPLC on a C18 reversed-phase column with a methanol/water mobile phase (88:12, vol/vol) and quantified them by use of a fluorescence detector at ex 340 nm and em 435 nm. Results: Optimal conditions in the Solids/Moisture System SAM-155 microwave oven (CEM Corp.) for the complete deacylation of Cer and neutral glycosphingolipids without decomposition were 60 min at 85% power, fan setting 7. Intra-and interassay CVs were <4% and <14%, respectively, and recovery rates were 87%-113%. The limit of quantification was 2 pmol (0.1 pmol on column), and the method was linear over the interval of 2-200 L plasma. In samples from 40 healthy individuals, mean (SD) concentrations were 9.0 (2.3) mol/L for Cer, 6.3 (1.9) mol/L for GlcCer, and 1.7 (0.5)

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Section: Discussionsupporting

confidence: 78%

HPLC for Simultaneous Quantification of Total Ceramide, Glucosylceramide, and Ceramide Trihexoside Concentrations in Plasma

Groener¹,

Poorthuis²,

Kuiper³

et al. 2007

Clinical Chemistry

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“…The strategy of this assay method may be applicable for other binding assays, such as Cholera toxin B-subunit for ganglioside GM1. 25) Although the ELISA method using verotoxin B subunit for the determination of Gb3 content was already established, 14) the assay procedure in our method (only two-step binding procedure) was simpler than the ELISA method (three steps). We believe minimum steps in a procedure have benefits not only for the quick assay but also for the specific assay.…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, the simple assay method has been reported as an enzyme-linked immunosorbent assay (ELISA) method using verotoxin B subunit. 14) This method has many benefits including no special equipment is required, many samples can be assayed at once, and a specific Gb3 assay can be accomplished because of the high affinity of the verotoxin B subunit toward Gb3.…”

mentioning

confidence: 99%

Histidine-Tagged Shiga Toxin B Subunit Binding Assay: Simple and Specific Determination of Gb3 Content in Mammalian Cells

Shin

Nishikawa

Maruyama

et al. 2006

CHEMICAL & PHARMACEUTICAL BULLETIN

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The recombinant 1B-His, in which 6 histidine residues were added at the carboxy termini of the B subunits, was prepared as described previously.15) The BL21DE cells expressing 1B-His were cultured in 300 ml of LB broth (Difco laboratories, Detroit, MI, U.S.A.) supplemented with 50 mg/ml kanamycin (Nacalai Tesque, Inc., Kyoto, Japan) at 37°C for 2 h. The cells were subsequently treated with 1.0 mmol/l isopropyl b-D(Ϫ)thiogalactopyranoside (Wako Pure Chemical Industries, Ltd., Osaka, Japan) at 37°C for 4 h. Cells were pooled by centrifugation at 6000 rpm for 15 min at 4°C. The 1B-His was extracted from cell pellets with the Bugbuster protein extraction reagent (Novagen, Madison, WI, U.S.A.) and purified by using the His-bind purification kit (Novagen) according to manufacturer's recommendations. Purified 1B-His fractions were applied to an NAP10 column (Amersham Biosciences, Uppsala, Sweden) equilibrated with phosphate-buffered saline (PBS). The purified 1B-His was revealed as a single band on SDS-PAGE (data not shown) and its aliquots were stored at Ϫ20°C.Materials HisProbe-HRP was purchased from Pierce Biotechnology, A two-step binding assay for globotriaosylceramide (Gb3) content was developed by histidine-tagging strategy, which is a well-established method for the purification of recombinant proteins. The complete binding of the recombinant His-tagged Shiga toxin 1B subunit (1B-His) (1 m mg/ml) to the standard Gb3 adsorbed on a multi-well H type plate was observed within 30 min at 37°C; and its binding could be visualized by the following applications of HisProbe-HRP (8 m mg/ml) and tetramethylbenzidine (TMB) peroxidase substrate. The 1B-His binding assay was linear over the range of 1 to 100 ng of Gb3 per well. The binding of 1B-His was specific to Gb3 separated from HeLa cells, and no major cross-reactivity of other glycolipids in Folch's lower fractions extracted from HeLa cells was detected. The glycolipids in Folch's lower fractions from HeLa cells, human fibroblasts and mouse heart were suitable for this assay, but the further purification was needed for glycolipids from human plasma, thus sample preparation is critical factor for the reliable determination of Gb3 content. The 1B-His binding to Gb3 was inhibited by the addition of galactose, but not mannose. This 1B-His binding assay will be useful not only for the determination of Gb3 content, but also for screening for the compounds which inhibit the toxin-binding to Gb3. The strategy of our present method may be applicable for other binding assay, such as Cholera toxin B-subunit for ganglioside GM1.

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“…Sandrine Roy, Dominique P. Germain, Arlette Baillet, Patrice Prognon, Pierre Chaminade La technique ELISA [1] est originale dans la mesure où la détection du Gb 3 n'est pas assurée par la mise en oeuvre d'un anticorps anti-Gb 3 , mais par la sous-unité B de la vérotoxine (VTB) d'Escherichia coli, qui présente une affinité importante pour le glycosphingolipide considéré. Le complexe Gb 3 -VTB est, ensuite, révélé par un anticorps anti-VTB.…”

Section: Quantification Et Spéciation Du Globotriaosylcéramideunclassified

Quantification et spéciation du globotriaosylcéramide

Roy¹,

Germain

Baillet³

et al. 2005

Med Sci (Paris)

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Le globotriaosylcéramide (Gb 3 , GL-3 ou CTH) est un glycosphingolipide ubiquitaire de l'organisme. Il est constitué d'un trihexoside, polaire, et d'un squelette céramide, apolaire. La partie osidique, de structure fixe, implique que le Gb 3 constitue une classe lipidique. Au sein de cette classe, les variations structurales du squelette céramide (longueur des chaînes hydrocarbonées, nombre et positions de doubles liaisons et/ou de groupements hydroxylés) permettent, quant à elles, de définir la notion d'espèces moléculaires, ou isoformes, au sein de la classe lipidique. Au cours de la maladie de Fabry, l'α-galactosidase A, rendue non fonctionnelle suite à une mutation du gène GLA, n'est plus à même d'hydrolyser le Gb 3 , ce qui conduit à son accumulation dans le plasma, les tissus et l'urine. La quantification de ce lipide et l'étude de ses variations structurales impliquent dans un premier temps de l'isoler au sein des échantillons biologiques étudiés. Par la suite, l'étude de ce glycosphingolipide revêtira deux aspects : d'une part, l'analyse de la classe dans son ensemble dans un but diagnostic ou quantitatif, d'autre part, la détermination des différentes structures en présence. Différentes approches ont été proposées dans la litté-rature pour procéder à l'analyse de la classe lipidique. Il s'agit notamment de techniques immunologiques, comme la technique ELISA (enzyme linked immunosorbent assay), ou de chromatographie liquide de phases normales. Si les principes de ces techniques peuvent paraître dissemblables, elles possèdent néanmoins un point commun, dans le sens où elles s'adressent à la partie invariable de la molécule, le trihexoside. REVUES SYNTHÈSE Quantification et spéciation du globotriaosylcéramideSandrine Roy, Dominique P. Germain, Arlette Baillet, Patrice Prognon, Pierre Chaminade La technique ELISA [1] est originale dans la mesure où la détection du Gb 3 n'est pas assurée par la mise en oeuvre d'un anticorps anti-Gb 3 , mais par la sous-unité B de la vérotoxine (VTB) d'Escherichia coli, qui présente une affinité importante pour le glycosphingolipide considéré. Le complexe Gb 3 -VTB est, ensuite, révélé par un anticorps anti-VTB. Si cette technique présente l'avantage d'être automatisable et donc de traiter rapidement un grand nombre d'échantillons, le risque de réactions croisées reste important. La technique de chromatographie liquide repose, quant à elle, sur l'affinité qui se développe entre trois parties : le soluté analysé, la phase stationnaire, sur laquelle le soluté peut s'adsorber, et la phase mobile qui permet l'élution du soluté. En optant pour une phase stationnaire polaire, les interactions phase stationnaire-soluté dépendent de la polarité du soluté, et donc de l'importance de la chaîne osidique. Cela permet de séparer les différents glycosphingolipides physiologiques [2], et notamment le Gb 3 et le galabiosylcéramide (Ga 2 ), second glycosphingolipide neutre s'accumulant au cours de la maladie de Fabry. La Figure 1 illustre le chromatogramme obtenu suite à l'analyse du Gb 3 con...

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Quantitative Determination of Globotriaosylceramide by Immunodetection of Glycolipid-Bound Recombinant Verotoxin B Subunit

Cited by 43 publications

References 58 publications

HPLC for Simultaneous Quantification of Total Ceramide, Glucosylceramide, and Ceramide Trihexoside Concentrations in Plasma

HPLC for Simultaneous Quantification of Total Ceramide, Glucosylceramide, and Ceramide Trihexoside Concentrations in Plasma

Histidine-Tagged Shiga Toxin B Subunit Binding Assay: Simple and Specific Determination of Gb3 Content in Mammalian Cells

Quantification et spéciation du globotriaosylcéramide

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