Monoclonal IgE antibodies against birch pollen allergens: Novel tools for biological characterization and standardization of allergens

Kaul, Susanne; Scheurer, Stephan; Danz, Nicole; Schicktanz, S.; Vieths, Stefan; Hoffmann, Andreas

doi:10.1067/mai.2003.1510

Cited by 10 publications

(10 citation statements)

References 29 publications

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“…In SDS-PAGE gels, rBet v 1 forms bands at 17, 34, and 68 kDa, identified as Bet v 1 monomers, dimers, and tetramers by murine mAb as well as by human IgE Abs in our study and by others (10,(55)(56)(57).…”

Section: Discussionmentioning

confidence: 86%

Dimerization of the Major Birch Pollen Allergen Bet v 1 Is Important for its In Vivo IgE-Cross-Linking Potential in Mice

Schöll¹,

Kalkura

Shedziankova

et al. 2005

The Journal of Immunology

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In type I allergy, the cross-linking of membrane IgE on B lymphocytes and of cytophilic IgE on effector cells by their respective allergens are key events. For cross-linking two IgE molecules, allergens need at least two epitopes. On large molecules, these could be different epitopes in a multivalent, or identical epitopes in a symmetrical, fashion. However, the availability of epitopes may be limited on small allergens such as Bet v 1, the major birch pollen allergen. The present work analyzes whether dimerization is required for the cross-linking capacity of this allergen. In immunoblots, murine monoclonal and polyclonal human Bet v 1-specific Abs detected, besides a Bet v 1 monomer of 17 kDa, a dimer of 34 kDa. In dynamic light scattering, Bet v 1 appeared as dimers and even multimers, but a single condition could be defined where it behaved exclusively monomerically. Small-angle x-ray scattering of the monomeric and dimeric samples resulted in diagrams agreeing with the calculated models. Circular dichroism measurements indicated that the structure of Bet v 1 was preserved under monomeric conditions. Skin tests in Bet v 1-allergic mice were positive with Bet v 1 dimer, but remained negative using the monomer. Furthermore, in contrast to dimeric Bet v 1, the monomer was less capable of activating murine memory B cells for IgE production in vivo. Our data indicate that the presentation of two identical epitopes by dimerized allergens is a precondition for cross-linking of IgE on mast cells and B lymphocytes.

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Section: Discussionmentioning

confidence: 86%

Dimerization of the Major Birch Pollen Allergen Bet v 1 Is Important for its In Vivo IgE-Cross-Linking Potential in Mice

Schöll¹,

Kalkura

Shedziankova

et al. 2005

The Journal of Immunology

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“…4). excluded as well [29], since no novel peaks were induced in CIEF when Bet v 1a was injected alone with its concentration varied over a range of 0.9-14.9 mmol/L [38].…”

Section: Migg Clonementioning

confidence: 99%

“…The application of recombinant Ags and their immunochemical standardization by (preferably) monoclonal (mAb) or at least oligoclonal Abs of known specificity will overcome this dilemma [6,29]. Both, recombinant Ags and mAbs are theoretically available in inexhaustible supply [6,14,34].…”

Section: Introductionmentioning

confidence: 98%

“…In vivo at least two epitopes of a single Ag have to be recognized simultaneously in order to crosslink membrane-bound IgEs and evoke allergic symptoms [17,60]. Consequently, the current approach with one mAb addresses the proof of single epitope integrity but not the allergenicity and biological activity of the tested product [29]. The concurrent application of several mAbs is a mid-term scope to allow for a simultaneous integrity testing of multiple epitopes.…”

Section: Introductionmentioning

confidence: 99%

“…Immunochemical characterization of Ag products utilizes the same techniques, but employs Ab mixtures with high specificity for the recombinant target Ag. Thereto comprehensive pools of sera from atopic patients are employed to prove integrity of conformational IgE epitopes [29]. However, once exhausted they have to be replaced by novel serum blends.…”

Section: Introductionmentioning

confidence: 99%

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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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Generation mechanism of novel, huge protein bodies containing wild type or hypoallergenic derivatives of birch pollen allergen Bet v 1 in rice endosperm

et al. 2014

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Tree pollen chimera 7 (TPC7), a hypoallergenic Bet v 1 tolerogen against birch pollen allergy, induces the formation of novel, huge protein bodies (referred to as TPC7 bodies) in rice endosperm, and is accumulated in high level. In the present study, we found that native Bet v 1 and TPC9, analog proteins of TPC7, were also deposited into novel protein bodies in rice endosperm. However, the novel protein bodies in Bet v 1 and TPC9 rice were much smaller and less abundant than those in TPC7 rice, reflected in lower amounts of accumulation of Bet v 1 and TPC9 than that of TPC7. A domain swapping experiment between TPC7 and Bet v 1 revealed that the latter half of TPC7 is important for the formation of the TPC7 body. We found that chaperons and folding enzymes such as BiP and protein disulfide isomerase were localized within the TPC7 body. TPC7 protein was extracted from TPC7 seeds as large aggregates with molecular masses greater than 669 kDa, or approximately 75 kDa under native or semi-native conditions. These TPC7 aggregates are thought to be responsible for the induction of TPC7 body formation. TPC7 accumulated to a maximum level of 550 μg/seed, which amounts to 23% of total seed protein, while Bet v 1 and TPC9 accumulated much lower levels. The TPC7 body represents a promising reservoir, which may serve as a fusion partner for high-level production and sequestering storage of recombinant proteins.

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Monoclonal IgE antibodies against birch pollen allergens: Novel tools for biological characterization and standardization of allergens

Cited by 10 publications

References 29 publications

Dimerization of the Major Birch Pollen Allergen Bet v 1 Is Important for its In Vivo IgE-Cross-Linking Potential in Mice

Dimerization of the Major Birch Pollen Allergen Bet v 1 Is Important for its In Vivo IgE-Cross-Linking Potential in Mice

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

Generation mechanism of novel, huge protein bodies containing wild type or hypoallergenic derivatives of birch pollen allergen Bet v 1 in rice endosperm

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