Purification of murine immunoglobulin E (IgE) by thiophilic interaction chromatography (TIC)

Vukovic, Natasa; Harraou, Salim; Duijnhoven, Sander M.J. van; Zaiss, Dietmar M.; Elsas, Andrea van

doi:10.1016/j.jim.2020.112914

Cited by 3 publications

(4 citation statements)

References 10 publications

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“…We successfully purified the IgCw-gek protein on a laboratory scale by affinity chromatography on KappaXP-agarose and IgG-C H 1-agarose; however, other purification methods will be cheaper and more effective if the IgCw-gek protein is to be purified on a large scale. Such methods include mixed-mode chromatography with MEP HyperCel (Pall Corporation), which has been used successfully to purify human IgE (20); thiophilic interaction chromatography, The Journal of Immunology which has been used for mouse IgE purification and is capable of purifying any IgE, irrespective of species (21); and conventional protein purification methods, such as gel filtration, ion-exchange chromatography, or a combination of these processes. In addition, understanding the stability of the IgCw-gek protein would be very helpful when working with IgCw-gek as an alternative to human IgE.…”

Section: Discussionmentioning

confidence: 99%

A Recombinant Ig Fragment (IgCw-γεκ) Comprising the Cγ1-Cε2–4 and Cκ Domains Is an Alternative Reagent to Human IgE

Kim

Lee

Choi

et al. 2022

The Journal of Immunology

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Human IgE is useful for immunological assays, such as sensitization of FcεRI-positive cells and IgE measurement. In this study, we report the development of a recombinant Ig fragment, designated IgCw-γεκ, as an alternative reagent to human IgE. IgCw-γεκ (∼130 kDa) comprises two hybrid constant H chain regions (Cγ1-Cε2–4, each ∼53 kDa) and two constant κ L chains (Cκ, each ∼12 kDa) and lacks a V domain. The presence of Cγ1 instead of Cε1 within the H chain increased the production yield and facilitated assembly of the H and L chains. IgCw-γεκ was produced in cultured human embryonic kidney 293F cells, with a yield of ∼27 mg/l. IgCw-γεκ bound to human FcεRIαRs expressed on the surface of rat basophilic leukemia-2H3 cells. A β-hexosaminidase release assay revealed that the biological activity of IgCw-γεκ was comparable with that of IgE. The IgE concentration measured using IgCw-γεκ as a standard was similar to that measured using IgE as a standard. These results suggest that the IgCw-γεκ molecule retains the basic characteristics of IgE, but does not cross-react with Ags, making it an alternative to the IgE isotype references used in a variety of immunological assays.

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Section: Discussionmentioning

confidence: 99%

A Recombinant Ig Fragment (IgCw-γεκ) Comprising the Cγ1-Cε2–4 and Cκ Domains Is an Alternative Reagent to Human IgE

Kim

Lee

Choi

et al. 2022

The Journal of Immunology

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“…Amino acid sequences of all anti-Thy1.1 antibodies are provided in Supplementary Table S1 . The design and production of murine anti-Thy1.1 IgG1 and IgE have been done as described before ( 10 ). In short, the starting point was OX7 hybridoma (anti-Thy1.1 IgG1) which was sequenced to obtain heavy (HC) and light chain (LC) variable domain sequences (VH, VL).…”

Section: Methodsmentioning

confidence: 99%

“…Anti-Thy1.1 IgG2a and anti-Thy1.1 IgG2a-LALA-PG were produced in ExpiCHO-S cells and FreeStyle293 cells, respectively, as described before ( 10 ). Purification was done with MabSelect SuRe LX resin.…”

Section: Methodsmentioning

confidence: 99%

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Mouse IgG2a Isotype Therapeutic Antibodies Elicit Superior Tumor Growth Control Compared with mIgG1 or mIgE

Vukovic

Segués

Huang

et al. 2023

Cancer Research Communications

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In the last decades, antibody based tumor therapy has fundamentally improved the efficacy of treatment for cancer patients. Currently, almost all tumor-antigen targeting antibodies approved for clinical application are of IgG1 Fc-isotype. Similarly, the mouse homolog mIgG2a is the most commonly used in tumor mouse models. However, in mice the efficacy of antibody based tumor therapy is largely restricted to a prophylactic application. Direct isotype comparison studies in mice in a therapeutic setting are scarce. In this study, we assessed the efficacy of mouse tumor-targeting antibodies of different isotypes in a therapeutic setting using a highly systematic approach. To this end, we engineered and expressed antibodies of the same specificity but different isotypes, targeting the artificial tumor antigen CD90.1 / Thy1.1 expressed by B16 melanoma cells. Our experiments revealed that in a therapeutic setting mIgG2a was superior to both mIgE and mIgG1 in controlling tumor growth. Furthermore, the observed mIgG2a anti-tumor effect was entirely Fc-mediated as the protection was lost when antibodies with a Fc silenced mIgG2a isotype (LALA-PG mutations) was used. These data confirm mIgG2a superiority in a therapeutic tumour model.

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Recent progress of chromatographic techniques for antibody purification

LIU,

TANG,

XIA

et al. 2024

CJCSP

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Purification of murine immunoglobulin E (IgE) by thiophilic interaction chromatography (TIC)

Cited by 3 publications

References 10 publications

A Recombinant Ig Fragment (IgCw-γεκ) Comprising the Cγ1-Cε2–4 and Cκ Domains Is an Alternative Reagent to Human IgE

A Recombinant Ig Fragment (IgCw-γεκ) Comprising the Cγ1-Cε2–4 and Cκ Domains Is an Alternative Reagent to Human IgE

Mouse IgG2a Isotype Therapeutic Antibodies Elicit Superior Tumor Growth Control Compared with mIgG1 or mIgE

Recent progress of chromatographic techniques for antibody purification

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