Molecular characterisation of the capsid proteins from enteric calciviruses
Molecular characterisation of the capsid proteins from enteric calciviruses
The purpose of this research was therefore to produce monoclonal antibodies against recombinant Southampton virus-like particles (rSV VLPs) and Lordsdale virus-like particles (rLV VLPs) expressed in insect cells. These VLPs can be purified by CsCl density gradient centrifugation and used as antigen.
Twenty-one hybridoma secreting antibodies against rSV, rLV at both antigens were obtained from three separate fusions. Ascitic fluid was prepared from four hybridomas exhibiting various antigen specificities. The monoclonal antibody designated 222 was incorporated into an Enzyme Linked Immunosorbent Assay (ELISA) for the detection of genogroup II NLVs in clinical samples.
To characterise the monoclonal antibody binding sites, full-length and truncated forms of LV and SV ORF-2 were expressed in an in vitro coupled transcription/translation system. However, only three monoclonal antibodies immunoprecipitated full-length LV capsid protein (83, 222 and 26). When the LV capsid protein was expressed as N- and C- terminal fragments no immunoprecipitation by these monoclonal antibodies was observed. It is likely that these monoclonal antibodies bind conformational epitopes which are lost when the capsid protein is expressed as two fragments.
This project also aimed to produce a construct containing the ORF-2, ORF-3 and 3'UTR of a new Genotype I NLV. The subgenomic regions of the new genogroup I Bab/96/UK NLV was assessed in pSP73 and sub-cloned into pBlueBacI for subsequent expression in insect cells. The Bab/96/UK capsid protein was expressed as stable VLPs which were observed by EM. This has increased the repertoire of VLPs available for the characterisation of these viruses. Polyclonal sera was raised against Bab/96/UK VLPs and antigenicity of Bab/96/UK was assessed. Bab/96/UK demonstrated reactivity with both genogroup I and genogroup II antisera. Bab/96/UK will therefore be invaluable for studying the relationship between calicivirus phylogeny and antigenicity.
University of Southampton
1999
Shipway, Sarah Louise
(1999)
Molecular characterisation of the capsid proteins from enteric calciviruses.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The purpose of this research was therefore to produce monoclonal antibodies against recombinant Southampton virus-like particles (rSV VLPs) and Lordsdale virus-like particles (rLV VLPs) expressed in insect cells. These VLPs can be purified by CsCl density gradient centrifugation and used as antigen.
Twenty-one hybridoma secreting antibodies against rSV, rLV at both antigens were obtained from three separate fusions. Ascitic fluid was prepared from four hybridomas exhibiting various antigen specificities. The monoclonal antibody designated 222 was incorporated into an Enzyme Linked Immunosorbent Assay (ELISA) for the detection of genogroup II NLVs in clinical samples.
To characterise the monoclonal antibody binding sites, full-length and truncated forms of LV and SV ORF-2 were expressed in an in vitro coupled transcription/translation system. However, only three monoclonal antibodies immunoprecipitated full-length LV capsid protein (83, 222 and 26). When the LV capsid protein was expressed as N- and C- terminal fragments no immunoprecipitation by these monoclonal antibodies was observed. It is likely that these monoclonal antibodies bind conformational epitopes which are lost when the capsid protein is expressed as two fragments.
This project also aimed to produce a construct containing the ORF-2, ORF-3 and 3'UTR of a new Genotype I NLV. The subgenomic regions of the new genogroup I Bab/96/UK NLV was assessed in pSP73 and sub-cloned into pBlueBacI for subsequent expression in insect cells. The Bab/96/UK capsid protein was expressed as stable VLPs which were observed by EM. This has increased the repertoire of VLPs available for the characterisation of these viruses. Polyclonal sera was raised against Bab/96/UK VLPs and antigenicity of Bab/96/UK was assessed. Bab/96/UK demonstrated reactivity with both genogroup I and genogroup II antisera. Bab/96/UK will therefore be invaluable for studying the relationship between calicivirus phylogeny and antigenicity.
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Published date: 1999
Identifiers
Local EPrints ID: 464013
URI: http://eprints.soton.ac.uk/id/eprint/464013
PURE UUID: 9078870b-17f6-4aad-bf2f-bcf58e243ac6
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Date deposited: 04 Jul 2022 21:00
Last modified: 04 Jul 2022 21:00
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Author:
Sarah Louise Shipway
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