Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.296008
Title: Expression of humanised antibodies in mammalian and insect cells
Author: Bentley, Kathryn Jane
ISNI:       0000 0001 3460 5639
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1995
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Abstract:
Two expression systems for humanised antibody production were studied: stably transfected mammalian cells and recombinant baculovirus-infected insect cells, in each case attempts being made to improve recombinant antibody yield. In this study, "in vitro amplification", involving the amplification of antibody heavy and light chain genes prior to their transfection into cells, was used in an attempt to isolate stable mammalian cell transfectants secreting antibody at an elevated level. Two forms of amplified DNA were constructed: in one, multiple copies of the heavy and light chain expression cassettes were cloned into separate cosmid vectors; in the other, a vector containing both the heavy and light chain cassette was linearised and self-ligated to form multimers. The success of the procedure was different for the two forms: co-transfection with the cosmids led to the isolation of clones secreting antibody at an increased level, whilst transfection with the multimerised double construct did not. In the bucolovirus/insect cell expression system, foreign gene expression is generally controlled by the very late polyhedrin promoter. A different baculoviral promoter, Immediate Early I, active early in infection, was used in this study, to determine whether a higher level of functional antibody was obtained with its use. This was found not to be the case, with higher yields of equally functional antibody being achieved with the polyhedrin promoter. It is demonstrated that insect cell-derived antibody has similar antigen-binding and antibody dependent cellular cytotoxicity activity, but reduced complement dependent cellular cytotoxicity activity, compared to mammalian-cell derived antibody.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.296008  DOI: Not available
Keywords: Genetics Molecular biology Cytology Genetics Biochemistry
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