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Title Page
Abstract
Contents
I. Introduction 8
II. Materials and Methods 17
1. Bio-panning of phage-displayed human synthetic Fab library 17
2. Indirect and quantitative ELISAs of phage-displayed Fabs selected after panning 18
3. Screening for CRM197-specific Fab clones(이미지참조) 18
4. Conversion of Fabs into IgG, expression, and purification of antibodies 19
5. Quantitative and indirect ELISAs s of purified IgG 20
6. Western blot analysis 20
7. Production of mouse anti-CRM197 polyclonal antibody(이미지참조) 21
8. Sandwich ELISA 21
9. Affinity determination of human mAb using Octet Red 22
10. Cleavage of CRM197 by trypsin treatment(이미지참조) 22
III. Results 23
1. Bio-panning of phage-displayed human synthetic Fab library against CRM197(이미지참조) 23
2. Selection of anti-CRM197 phage Fabs(이미지참조) 27
3. Conversion of Fabs into IgG1 format and characterization of anti-CRM197 antibodies(이미지참조) 29
4. Development of sandwich ELISA for the detection of CRM197(이미지참조) 33
5. Affinity determination of 3F9 antibody 38
6. Domain mapping of anti-CRM197 mAb(이미지참조) 38
IV. Discussion 41
V. References 43
Abstract(Korean) 45
Figure 1. Structure of diphtheria toxin showing the catalytic, transmembrane and... 9
Figure 2. Schematic representation of the endosomal pathway of cellular entry of diphtheria... 10
Figure 3. CRM197 as carrier protein for Vaccine. A polysaccharide is chemically...(이미지참조) 11
Figure 4. Schematic representation of an IgG. An antibody consists of two heavy... 14
Figure 5. Schematic representat ion of the Fab displayed phage. 15
Figure 6. Schematic representation of panning procedures. 16
Figure 7. 10% SDS-PAGE of purified CRM197.(이미지참조) 24
Figure 8. Titres of enriched phages following each round of panning. 25
Figure 9. Polyclonal phage ELISA. Each well was coated BSA, CRM197, or anti-...(이미지참조) 26
Figure 10. Ranking of positive clones of antigen binding activities to CRM197 by...(이미지참조) 28
Figure 11. (A) Purification of IgG 1E1, 1F3, 2A9, and 3F9. (B) 10% SDS-PAGE... 30
Figure 12. Antigen biding activities of purified four antibodies by indirect ELISA. 31
Figure 13. Western blot analysis of purified 1F3, 2A9, 1E1, and 3F9 antibodies under... 32
Figure 14. Indirect ELISA of mouse sera contained anti CRM197 polyclonal antibody....(이미지참조) 34
Figure 15. Schematic representation of sandwich ELISA using mAb and mouse... 35
Figure 16. Antigen biding activities of purified antibodies (1E1, 1F3, 2A9, and 3F9)... 36
Figure 17. Analysis of antigen binding activities of purified 3F9 to CRM197 diluted...(이미지참조) 37
Figure 18. (A) Affinity determination of 3F9 for CRM197 using Octet Red. CRM197...(이미지참조) 39
Figure 19. Domain mapping of 3F9 (A) 10% SDS-PAGE under reducing conditions... 40
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