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Title: A study of anti-DNA autoantibodies in systemic lupus erythematosus
Author: Wall, Gerard
ISNI:       0000 0001 3556 981X
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1993
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Two anti-DNA autoantibody-secreting hybridomas, derived from murine models of the disease systemic lupus erythematosus (SLE), were provided. MAb-32, an IgG1, kappa, bound both single- (ss) and double-stranded (ds) DNA while mAb F-423, an IgG3, kappa, bound only ssDNA. F-423 also reacted with RNA and poly (dA) and its binding to ssDNA was found to be greatly amplified by histones. The VH genes of both antibodies and the VK gene of mAb F-423 were isolated by PCR, cloned, and sequenced. The two VH genes were also cloned into the pSW1.Fab expression vector and bacterial clones which showed DNA-binding were isolated. Continued induction of these clones led to the loss of their DNA-binding activity, due to a recombination event within the expression vector leading to the loss of its heavy chain-encoding genes. This event, seen only in anti-DNA clones, was proposed to be due to toxicity of the anti-DNA Fab fragments, leading to selection of recombinant bacteria. A number of experiments were carried out to improve the stability of the anti-DNA vector in E.coli. E.coli XL1-Blue was found to be preferable to E.coli TG1 as host strain both in terms of its lower frequency of recombination and tighter control of expression, while maximal production of Fab fragments was shown to be achieved after only 1-3 hours of induction. Furthermore, antigen binding of antibody fragments produced at 25[Special character omitted]C was found to be considerably higher than that of fragments produced at 37[Special character omitted]C. Mixing of CDRs from the two anti-DNA autoantibodies was carried out by CDR grafting.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Autoimmune disease Medicine Biochemistry Molecular biology Cytology Genetics