@phdthesis{oai:sucra.repo.nii.ac.jp:00019183,
 author = {RANWADANA, MUDIYANSELA GEDARA CHATHUNI SAMANTHIKA KUMARI JAYATHILAKE},
 month = {},
 note = {viii, 92 p., Gluten intolerance, or adverse intestinal reactions to gluten, is a fairly common problem among certain groups of people. Celiac disease is the most severe form of gluten intolerance, which can lead to permanent damage in the digestive system. Since lifelong avoidance of gluten is the only available treatment, development of reliable techniques to identify gluten contamination in food is important. Gliadin, a component of gluten, is known to play a major role in gluten toxicity. The cDNA display method is used in this study which is a promising in vitro display technique, which uniquely converts an unstable mRNA-protein fusion molecule to a stable mRNA/cDNA-protein fusion molecule using a well-designed puromycin linker. This study was aimed to select specific single-domain antibodies against toxic gliadin from an alpaca-derived naïve VHH library using cDNA display method and to apply newly developed cDNA display mediated immuno-PCR (cD-iPCR) method in determining the affinity of selected VHHs against gliadin and finally to compare the results of cD-iPCR with other affinity assays (Pull-down assay and ELISA). Three candidate VHHs were successfully selected and the affinities of the VHHs were observed by pulldown assay and indirect ELISA method. Those affinity results were in line with the novel cD-iPCR assay results indicating the accurate applicability of the method. In addition, cD-iPCR method was successfully applied to detect gliadin in actual food samples. VHH1 and VHH2 were the best binders toward gliadin compared with VHH3 in all assays performed, including the cD-iPCR assay. We believe this work demonstrates the potential application of the cDNA display method in selecting binders against toxic and heterogeneous targets such as gliadin with an immunization-free preparation manner., Chapter one: Introduction and Literature review………………………………1
1.1 Introduction…………………………………………………………………………………1
1.2 Gliadin Intolerance and celiac disease ……………………………………………………6
1.2.1 Wheat Storage Protein…………………………………………………………………6
1.2.2 Gliadins…………………………………………………………………………………7
1.2.3 Gluten Intolerance………………………………………………………………………8
1.2.4 Autoimmune enteropathy of celiac disease…………………………………………10
1.3. Camelid single domain antibodies (VHH)………………………………………………12
1.4 Principle of evolutionary molecular engineering…………………………………………15
1.4.1 Evolutionary molecular engineering …………………………………………15
1.4.2 In vivo selection of polypeptides by cell-dependent…………………………………17
1.4.3 In vitro selection of polypeptides by cell-indipendant…………………………………18
1.4.4 cDNA Display………………………………………………………………………20
1.4.5 cDNA display mediated immune-PCR (cD-iPCR)…………………………………22
1.5 Research Objectives………………………………………………………………………25
Chapter 2: In vitro selection of anti-gliadin single-domain antibodies from a naïve library using cDNA display method……………………………26
2.1 Introduction………………………………………………………………………………26
2.2. Chemicals and instruments………………………………………………………………27
2.3. Methodology……………………………………………………………………………28
2.3.1 Preparation of gliadin immobilized Beads……………………………………………28
2.3.2 Construction of VHH-coding DNA library…………………………………………28
2.3.3 Construction of protein-coding DNA full construct………………………… ………29
2.3.4 Synthesis of VHH cDNA display molecules…………………………………………30
2.3.5 Confirmation of cDNA display formation of VHH by SDS-PAGE…………………32
2.3.6 In vitro affinity selection……………………………………………………………33
2.3.7 Next Generation Sequence analysis…………………………………………………35
2.3.8 Pull-down assay with cell-free translation for VHH candidates……………………36
2.3.9 Expression of VHH proteins…………………………………………………………37
2.3.10 ELISA for candidate VHHs…………………………………………………………38
2.4 Results and Discussion……………………………………………………………………38
2.4.1 Target Immobilization………………………………………………………………38
2.4.2 Confirmation of VHH cDNA display molecule formation on SDS-PAGE……………39
2.4.3 In vitro affinity selection of VHH against gliadin……………………………………42
2.4.4 Pull-down assay for candidate VHHs………………………………………………44
2.4.5 Indirect ELISA for candidate VHHs…………………………………………………47
2.5 Conclusions………………………………………………………………………………49
Chapter 3: Detection of gliadin using cDNA display mediated immune-PCR (cD-iPCR)…………………………………………………………………50
3.1 Introduction………………………………………………………………………………50
3.2 Chemicals and instruments………………………………………………………………51
3.3 Methodology………………………………………………………………………………51
3.3.1 Preparation of Immunoglobulin G (IgG) immobilized Beads………………………51
3.3.2 Preparation of Green Fluorescent protein (GFP) immobilized Beads…………………51
3.3.3 Determination of sensitivity using cD-iPCR…………………………………………52
3.3.4 Determination of the specificity using cD-iPCR………………………………………52
3.3.5 Determination of gliadin with the presence of food components (cD-iPCR)…………53
3.3.6 Extraction of gliadin from commercial food samples for cD-iPCR…………………54
3.3.7 Detection of gliadin in commercial food samples using cD-iPCR……………………55
3.4 Results and discussion……………………………………………………………………55
3.4.1 Detection sensitivity using cD-iPCR………………………………………………55
3.4.2 Specificity confirmation using cD-iPCR……………………………………………57
3.4.3 Detection of gliadin in food samples using cD-iPCR…………………………………58
3.4.4 Extraction of gliadin from commercial food samples…………………………………59
3.4.5 Detection of gliadin in commercial food samples using cD-iPCR……………………60
3.5 Conclusions………………………………………………………………………………61
Chapter 4: Overall discussion and conclusion………………………………………62
4.1 Discussion……………………………………………………………………………………62
4.2 Conclusions………………………………………………………………………………67
Acknowledgment…………………………………………………………………………68
References………………………………………………………………………………………69
Annex I (Materials, compositions and sequences)………………………………………………83, 指導教員 : 根本直人, text, application/pdf},
 school = {埼玉大学},
 title = {In vitro selection of anti-gliadin single-domain antibodies from a naïve library and its application for cDNA-display mediated immuno-PCR},
 year = {2020},
 yomi = {ランワダナ, ムディヤンセラ ゲダラ チャトゥニ サマンティカ クマリ ジャヤティラケ}
}