Mantle cell lymphoma (MCL) is one of several sub-types of B-cell lymphomas. The malignancy is very aggressive and average survival time is short. The hallmark of MCL is over expression of cyclin D1, however about 15% of all MCL cases do not display this over expression and are easily misdiagnosed. Recently the transcription factor Sox11 has been shown to be specifically over expressed in the nucleus of MCL-tumour cells, and polyclonal rabbit anti-Sox11 antibodies have been used to successfully identify MCL in both cyclin D1 positive and negative cases. However, human recombinant MCL-specific antibodies as have several advantages over these polyclonal rabbit antibodies; they can easily be produced in large quantities in vitro, their specificity is constant from batch to batch and they can possibly be used for therapeutic purposes. Because of this, it is desirable to produce human recombinant antibodies against proteins over expressed in MCL. In this study human recombinant IgGs have been produced towards two pro-teins over expressed in MCL, Sox11 and KIAA0882. This was done by cloning of single chain variable fragments (scFvs), previously selected from a large scFv library through phage display selection against Sox11- and KIAA0882-protein epitope signature tag (PrEST), into vectors containing human IgG constant regions followed by expression of human IgG antibodies in human embryonic kidney (HEK) 293 cells. One IgG clone for each antigen was shown to be functional and specific…
Contents
1. Introduction
1.1 Recombinant antibodies
1.2 Mantle cell lymphoma (MCL)
1.3 Sox11
1.4 Protein epitope signature tags (PrESTs) and PrEST-specific polyclonal rabbit antibodies
1.5 Antibody structure and Single Chain Variable Fragments (scFv)
1.6 Aim of this thesis
2. Material and methods
2.1 Plasmids and vectors
2.3 Enzyme-linked immunosorbent assay (ELISA) protocol
2.4 Production and evaluation of scFv peptides
2.4.1 Production and purification of scFv peptides
2.4.2 Binding ability of purified scFvs
2.5 Production and evaluation of PrESTs
2.5.1 Production and purification of PrESTs
2.5.2 Functional analysis of purified PrESTs
2.6 Production and purification of human recombinant antibodies
2.6.1 Cloning of scFv into human IgG1-format
2.6.2 Transfection and expression of human IgGs in HEK 293 cells
2.6.3 Purification of produced IgGs
2.6.4 Quantification of IgG production in HEK293 cells
2.7 Evaluation of produced human recombinant IgG antibodies
2.7.1 Quantification of purified IgGs
2.7.2 Functional study of purified IgGs
2.7.3 Epitope mapping of purified IgGs by competitive ELISA
2.7.4 Detection of Sox11 in cell lysate by Western blot
2.7.5 Affinity of purified IgGs measured by Biacore
3. Results
3.1 Functional analysis of produced scFvs and PrESTs
3.2 Sequence analysis
3.3 IgG production
3.4 Evaluation of produced IgGs
3.4.1 Quantification of purified IgGs by ELISA and NanoDrop™
3.4.2 Evaluation of binding abilities of purified IgGs using ELISA
3.4.4 Detection of Sox11 in cell lysate by Western blot
3.4.5 Affinity of produced IgGs vs. original scFvs
4. Discussion
4.1 IgG production
4.2 Epitope mapping
4.3 Detection of Sox11 in Western blot
4.4 Affinity of produced IgGs
5. Conclusions and future work
6. Acknowledgements
7. References
Author: Gärdefors, Katarina
Source: Linköping University
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