Validation of capillary zone electrophoresis and capillary isoelectric focusing separations optimized for the characterization of two recombinant products of the birch pollen allergen Bet v 1a

Kronsteiner, Barbara; Dullnig, Verena; Stutz, Hanno

doi:10.1002/elps.200700892

Cited by 9 publications

(10 citation statements)

References 68 publications

(94 reference statements)

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“…Separations with various volatile and non‐volatile BGEs between pH 3.0 and 3.30 in SMIL (3) capillaries with HDMB/DS/HDMB coating provided selectivities only slightly higher than previously achieved in bare fs or TEPA‐coated capillaries (data not shown) 11, 12. Alternatively, volatile 75 and 100 mmol/L NH 4 HCO 3 buffers, pH 6.50, were selected since this pH domain has previously provided best selectivity in dynamically coated capillaries 11–13. Compared with the acidic pH region, selectivity improved in SMIL capillaries but fluctuations in EOF and t m with up to 2.40 and 2.20% (CV, n =6) occurred, respectively (data not shown).…”

Section: Resultsmentioning

confidence: 92%

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Glucosylceramide Mimics: Highly Potent GCase Inhibitors and Selective Pharmacological Chaperones for Mutations Associated with Types 1 and 2 Gaucher Disease

et al. 2013

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A series of iminoxylitol derivatives carrying a C-linked di-O-acyl or di-O-alkyl glyceryl substituent were prepared and characterized. All of these compounds, which were designed as glucosylceramide (GlcCer) mimics, were nanomolar inhibitors of lysosomal β-glucosidase (glucocerebrosidase, GCase). Two of these pseudoglycolipids were further evaluated for their ability to enhance the activity of mutant GCase in human Gaucher cells. Although the di-O-hexyl ether was surprisingly devoid of chaperoning activity on both N370S and L444P GCases, the di-O-decanoyl ester was a potent chaperone of the L444P hydrolase, capable of increasing the residual activity of the enzyme by a factor of two at a very low concentration (50 nM); such a significant effect on the L444P mutation in human fibroblasts has not yet been observed. In heat-stress studies, the diether was found to be much more effective in stabilizing the wild-type enzyme than the diester. Four representative pseudoglycolipids were also assayed as inhibitors of GlcCer synthase, because such compounds could find use in the substrate reduction therapy approach to treat lysosomal storage diseases, but these compounds revealed only moderate activity. As efficient pharmacological chaperones, new structures such as the di-C10 -ester constitute leads for the development of therapeutic agents for types 2 and 3 Gaucher disease, the most severe neuronopathic forms of this lysosomal disease.

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Section: Resultsmentioning

confidence: 92%

“…Recently, various CE modes, e.g. CZE, CIEF and affinity CIEF, have been applied for the characterization of recombinant allergens 11–14. Although CE provides an outstanding selectivity, it becomes merely gradually implemented within the arsenal of analytical methods applied by the pharmaceutical industry 15.…”

Section: Introductionmentioning

confidence: 99%

Glucosylceramide Mimics: Highly Potent GCase Inhibitors and Selective Pharmacological Chaperones for Mutations Associated with Types 1 and 2 Gaucher Disease

et al. 2013

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“…For MS/MS identification of a protein spot from a 2D gel a relatively high amount of protein is needed ( (Bakker et al, 2006), LC-ESI-MS (Liquid Chromatography Electron Spray Ionisation Mass Spectrometry) or by lectin binding assay (Kronsteiner et al, 2008). It allows the identification of amino acid sequence of peptides using computational methods to analyse the MS fragmentation patterns.…”

Section: Allergen Detection and Identification Using Mass Spectrometrmentioning

confidence: 99%

“…Kronsteiner et al (2008) used capillary zone electrophoresis to characterise two recombinant products of the birch pollen allergen Bet v1a. CE offers attractive features for the analysis of proteins, as the analysis times can be relatively short and only minute amounts of sample are needed.…”

Section: Allergen Detection and Identification Using Mass Spectrometrmentioning

confidence: 99%

Scientific Opinion on the assessment of allergenicity of GM plants and microorganisms and derived food and feed

2010

EFSA Journal

411

231

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The weight-of-evidence, case-by-case approach is considered the most appropriate way of assessing the allergenicity of genetically modified (GM) food and feed. This scientific opinion discusses various aspects to increase the strength and accuracy of this approach, including the latest developments pertaining to clinical aspects of allergic reactions, structural aspects of GM food and feed and in silico approaches, as well as IgE binding studies and cell-based methods, profiling techniques and animal models. In this context, conclusions and recommendations are provided to update and complement current risk assessment strategies for the allergenicity assessment of newly expressed protein(s) and whole GM food and feed. In summary, it is recommended that with regard to the search for sequence homology and structural similarities, the local alignment method with a known allergen with a threshold of 35% sequence identity over a window of at least 80 amino acids is considered a minimal requirement. When IgE binding tests are considered necessary, e.g. when there is sequence homology and/or structure similarity with known allergens, the use of individual sera from allergic individuals rather than pooled sera is recommended. In addition to the pepsin resistance test, it is recommended that the resistance to digestion of the newly expressed proteins is evaluated using other in vitro digestibility tests mimicking physiological conditions of humans. Finally, when the recipient of the introduced gene is allergenic, in order to compare the allergenicty of the whole GM plant with that of its appropriate comparator(s), it is recommended that relevant characterised endogenous allergens are included in the comparative compositional analysis of the GM plant and its appropriate comparator(s). Proposals for the use of additional testing that may improve the weight-of-evidence approach and 1 On request of EFSA, Question No EFSA-Q-2005-125, adopted on 30 June 2010. 2 Panel members:

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“…CE has the capability to cover major physico-chemical aspects of Ag products as demonstrated recently [36][37][38]. Moreover, CE has been employed in the quality control and characterization of mAbs [39][40][41][42][43].…”

Section: Introductionmentioning

confidence: 99%

Confirmation of immuno‐reactivity of the recombinant major birch pollen allergen Bet v 1a by affinity‐CIEF

et al. 2009

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Affinity-CIEF has been applied to characterize a recombinant product of the major birch pollen allergen Betula verrucosa isoform 1a (Bet v 1a) immuno-chemically. For this purpose mAbs of the IgG-type have been produced in-lab from two murine hybridoma lines, specified as clones 2 and 5.1. Both IgG clones were characterized by SDS-PAGE, MALDI-TOF-MS and CIEF. The purified IgG solutions had to be dialysed against 10 mmol/L phosphate (pH 7.4) to prevent IgG precipitation and to ensure appropriate CIEF separation. Both tested monoclonal IgGs (mIgGs) comprised four constituents covering pI ranges of 6.98-7.09 and 6.78-7.03 for clones 2 and 5.1 with major peaks at pI 7.09 and 7.03, respectively. When increasing amounts of Bet v 1a (pI 4.95) were incubated with 2.0 mumol/L mIgG, novel peaks were progressively induced in a pI range slightly more acidic than the focusing region of mIgGs. These peaks grew on the expense of original mIgG peaks. All pI values were calculated using two pI marker compounds with a repeatability of better than 0.03 units. New peaks represent complexes between Bet v 1a and mIgG either of 1:1 or of 2:1 binding stoichiometry. At a molar ratio of 2:1, saturation of both IgG paratopes with allergen (Ag) molecules was achieved as indicated by unbound Bet v 1a. The current CIEF approach addresses the proof of single epitope integrity in the course of immuno-chemical characterization of Bet v 1a. Contrary to traditional immunoassays, affinity CIEF allows for a distinction and relative quantification of mAbs, Ag-antibody complexes and Ag variants coexisting in one sample.

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Validation of capillary zone electrophoresis and capillary isoelectric focusing separations optimized for the characterization of two recombinant products of the birch pollen allergen Bet v 1a

Cited by 9 publications

References 68 publications

Glucosylceramide Mimics: Highly Potent GCase Inhibitors and Selective Pharmacological Chaperones for Mutations Associated with Types 1 and 2 Gaucher Disease

Glucosylceramide Mimics: Highly Potent GCase Inhibitors and Selective Pharmacological Chaperones for Mutations Associated with Types 1 and 2 Gaucher Disease

Scientific Opinion on the assessment of allergenicity of GM plants and microorganisms and derived food and feed

Confirmation of immuno‐reactivity of the recombinant major birch pollen allergen Bet v 1a by affinity‐CIEF

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