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Generation of the IgCw-γ1ε2-4/κ composed of the constant domain Cγ1 and Cε2-4
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 권명희 | - |
| dc.contributor.author | 이정현 | - |
| dc.date.accessioned | 2022-11-29T02:32:30Z | - |
| dc.date.available | 2022-11-29T02:32:30Z | - |
| dc.date.issued | 2021-02 | - |
| dc.identifier.other | 30507 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/20033 | - |
| dc.description | 학위논문(석사)--아주대학교 일반대학원 :의생명과학과,2021. 2 | - |
| dc.description.tableofcontents | Ⅰ. INTRODUCTION 1 Ⅱ. MATERIALS AND METHODS 6 A. Construction of plasmid vectors 6 B. Purification of proteins 16 C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining 16 D. Immunoblotting 17 E. Size-exclusion chromatography (SEC) 17 F. Cell culture 18 G. Establishment of stable cell line 18 H. Flow cytometry 19 I. Enzyme-Linked Immunosorbent assay (ELISA) 20 J. β-hexosaminidase release assay 21 Ⅲ. RESULT 22 A. Production of IgCw-γε/κ using HEK293F cells 22 B. Binding of IgCw-γε/κ to human FcεRⅠα chain expressed on RBL-2H3-cell surface 30 C. Quantitative determination of IgE concentrations using IgCw-γε/κ 34 D. Release of β-hexosaminidase and inhibition of IgE-FcεRⅠ binding by IgCw-γε/κ 37 Ⅳ. DISCUSSION 40 Ⅴ. CONCLUSION 43 REFERENCES 44 국문요약 51 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | Generation of the IgCw-γ1ε2-4/κ composed of the constant domain Cγ1 and Cε2-4 | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.department | 일반대학원 의생명과학과 | - |
| dc.date.awarded | 2021. 2 | - |
| dc.description.degree | Master | - |
| dc.identifier.localId | 1203156 | - |
| dc.identifier.uci | I804:41038-000000030507 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/common/orgView/000000030507 | - |
| dc.subject.keyword | Cε | - |
| dc.subject.keyword | Immunoglobulin E | - |
| dc.subject.keyword | Reference antibody | - |
| dc.subject.keyword | constant region | - |
| dc.subject.keyword | isotype control | - |
| dc.description.alternativeAbstract | In this study, a novel recombinant antibody fragment IgCw-γ1ε2-4/κ (IgCw-γε/κ) was constructed and evaluated for its potential as an alternative isotype control, which can be used in place of IgE. IgCw-γε/κ (~140 kDa) is composed of two hybrid human constant heavy chains (Cγ1-hinge region-Cε2-4) and two human constant kappa light chains (Cκ). IgCw-γε/κ was expressed as an assembled form of the heavy and light chains, and the production yield of IgCw-γε/κ obtained from a culture of transfected HEK293F cells was 17 mg/L, which is comparable to that of full-size IgE. Binding of IgCw-γε/κ to human Fc epsilon receptor Ⅰ (FcεRⅠ) expressed on RBL-2H3-hFcεRⅠα cells was confirmed using flow cytometry. A standard curve generated using IgCw-γε/κ was used for the measurement of IgE concentrations. Further, the sensitization with IgCw-γε/κ of RBL-2H3-hFcεRⅠα cells and subsequent stimulation with anti-human kappa light chain antibody induced β-hexosaminidase release. IgCw-γε/κ also acted as an FcεRⅠ blocker to suppress IgE-FcεRⅠ interaction. Taken together, these results demonstrate that IgCw-γε/κ can be used as a functional alternative isotype control, instead of full-size IgE, in biomedical research fields. | - |
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- Graduate School of Ajou University > Department of Biomedical Sciences > 3. Theses(Master)
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