Generation and epitope analysis of human monoclonal antibody isotypes with specificity for the timothy grass major allergen Phl p 5a

Hecker, Julia; Diethers, Andrea; Etzold, Stefanie; Seismann, Henning; Michel, Yvonne; Plum, Melanie; Bredehorst, Reinhard; Blank, Simon; Braren, Ingke; Spillner, Edzard

doi:10.1016/j.molimm.2011.03.005

Cited by 18 publications

(18 citation statements)

References 50 publications

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“…Free light or heavy chain was not detected, suggesting that the antibody chains are assembled into whole antibody molecules. The molecular sizes corresponding to the heavy and light chains suggest the secreted antibodies are properly folded and glycosylated2930. Similar results were obtained when assessing the IgG 2 , IgG 3 , IgA 1 and IgA 2 isotypes under reducing conditions, with protein bands corresponding to the heavy chains (50–60 kDa) and light chains at 25 kDa (Figure 5).…”

Section: Resultssupporting

confidence: 74%

“…Although these facilitate expression of a large number of mAbs in a short time, in our experience, the transient co-transfection of separate vectors doubles the DNA preparation work, requires maxi or mega plasmid purifications and large amount of transfection reagent for the production of milligrams of antibody, which is a costly, labor- and time-intensive scale-up process. This limitation has been addressed by the generation of vectors hosting a dual antibody expression cassette with a selection marker293031. However, all these antibody cloning systems rely on restriction enzyme- and ligation-dependent cloning methods, which adds an additional cost, prevents high-throughput analysis of antibody candidates, and may sometimes be impractical due to the lack of compatible restriction sites.…”

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confidence: 99%

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A tool kit for rapid cloning and expression of recombinant antibodies

Dodev

Karagiannis

Gilbert

et al. 2014

Sci Rep

104

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Over the last four decades, molecular cloning has evolved tremendously. Efficient products allowing assembly of multiple DNA fragments have become available. However, cost-effective tools for engineering antibodies of different specificities, isotypes and species are still needed for many research and clinical applications in academia. Here, we report a method for one-step assembly of antibody heavy- and light-chain DNAs into a single mammalian expression vector, starting from DNAs encoding the desired variable and constant regions, which allows antibodies of different isotypes and specificity to be rapidly generated. As a proof of principle we have cloned, expressed and characterized functional recombinant tumor-associated antigen-specific chimeric IgE/κ and IgG1/κ, as well as recombinant grass pollen allergen Phl p 7 specific fully human IgE/λ and IgG4/λ antibodies. This method utilizing the antibody expression vectors, available at Addgene, has many applications, including the potential to support simultaneous processing of antibody panels, to facilitate mechanistic studies of antigen-antibody interactions and to conduct early evaluations of antibody functions.

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Section: Resultssupporting

confidence: 74%

mentioning

confidence: 99%

A tool kit for rapid cloning and expression of recombinant antibodies

Dodev

Karagiannis

Gilbert

et al. 2014

Sci Rep

104

View full text Add to dashboard Cite

show abstract

“…Human/mouse chimaeric IgEs were also generated by hybridoma technology from a knock‐in mouse strain , and more efficient cloning strategies using mammalian expression vectors are available . In future fully human IgE antibodies could be generated from synthetic human antibody repertoire libraries , or cloned directly from the B cells of patients .…”

Section: Translational Strategies To Target Cancermentioning

confidence: 99%

AllergoOncology – the impact of allergy in oncology: EAACI position paper

Jensen‐Jarolim

Bax

Bianchini

et al. 2017

Allergy

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Th2 immunity and allergic immune surveillance play critical roles in host responses to pathogens, parasites and allergens. Numerous studies have reported significant links between Th2 responses and cancer, including insights into the functions of IgE antibodies and associated effector cells in both anti-tumour immune surveillance and therapy. The interdisciplinary field of AllergoOncology was given Task Force status by the European Academy of Allergy and Clinical Immunology in 2014. Affiliated expert groups focus on the interface between allergic responses and cancer, applied to immune surveillance, immunomodulation and the functions of IgE-mediated immune responses against cancer, to derive novel insights into more effective treatments. Co-incident with rapid expansion in clinical application of cancer immunotherapies, here we review the current state-of-the-art and future translational opportunities, as well as challenges in this relatively new field. Recent developments include improved understanding of Th2 antibodies, intra-tumoural innate allergy effector cells and mediators, IgE-mediated tumour antigen cross-presentation by dendritic cells, as well as immunotherapeutic strategies such as vaccines and recombinant antibodies, and finally, the management of allergy in daily clinical oncology. Shedding light on the cross-talk between allergic response and cancer is paving the way for new avenues of treatment.

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“…Since IgE‐producing B‐cells in blood are scarce, the first studies with IgE monoclonal antibodies were performed with murine allergen‐specific antibodies, originally IgG but subsequently heterologously expressed as IgE antibodies . In addition, human monoclonal IgE antibodies were obtained by construction of phage display ScFv hybrid libraries with allergic donor‐derived epsilon heavy chain and synthetic variable regions for the light chain . However, these approaches did not address the whole allergen‐specific antibody repertoire of one subject.…”

Section: Different Epitope Mapping Approaches May Increase the Contrimentioning

confidence: 99%

“…34 In addition, human monoclonal IgE antibodies were obtained by construction of phage display ScFv hybrid libraries with allergic donor-derived epsilon heavy chain and synthetic variable regions for the light chain. 114,115 However, these approaches did not address the whole allergen- 77 Another aspect to take into account is the expression of the low-affinity FcεRII (CD23) on non-allergen specific translational B-cells in the blood stream. 123,124 The cells can bind allergen-specific IgE antibodies leading to the selection of B-cells irrelevant for allergy.…”

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confidence: 99%

Can alternative epitope mapping approaches increase the impact of B‐cell epitopes in food allergy diagnostics?

Ehlers

Blankestijn

Knulst

et al. 2018

Clin Experimental Allergy

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Summary In vitro allergy diagnostics are currently based on the detection of specific IgE binding on intact allergens or a mixture thereof. This approach has drawbacks as it may yield false‐negative and/or false‐positive results. Thus, we reviewed the impact of known B‐cell epitopes of food allergens to predict transience or persistence, tolerance or allergy and the severity of an allergic reaction and to examine new epitope mapping strategies meant to improve serum‐based allergy diagnostics. Recent epitope mapping approaches have been worthwhile in epitope identification and may increase the specificity of allergy diagnostics by using epitopes predominately recognized by allergic patients in some cases. However, these approaches did not lead to discrimination between clinically relevant and irrelevant epitopes so far, since the polyclonal serum IgE‐binding epitope spectrum seems to be too individual, independent of the disease status of the patients. New epitope mapping strategies are necessary to overcome these obstacles. The use of patient‐derived monoclonal antibodies instead of patient sera for functional characterization of clinically relevant and irrelevant epitope combinations, distinguished by their ability to induce degranulation, might be a promising approach to gain more insight into the allergic reaction and to improve serum‐based allergy diagnostics.

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Generation and epitope analysis of human monoclonal antibody isotypes with specificity for the timothy grass major allergen Phl p 5a

Cited by 18 publications

References 50 publications

A tool kit for rapid cloning and expression of recombinant antibodies

A tool kit for rapid cloning and expression of recombinant antibodies

AllergoOncology – the impact of allergy in oncology: EAACI position paper

Can alternative epitope mapping approaches increase the impact of B‐cell epitopes in food allergy diagnostics?

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