Analytical determination of ribulose-1, 5-bisphosphate carboxylase/oxygenase using monoclonal antibodies

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dc.contributor.authorMdluli, Khisimuzien_US
dc.date.accessioned2024-08-14T22:51:27Z
dc.date.available2024-08-14T22:51:27Z
dc.date.copyright1986en_US
dc.date.issued1986
dc.degree.departmentDepartment of Biology
dc.degree.levelMaster of Science M.Sc.en
dc.description.abstractMonoclonal antibodies produced against a partially purified ribulose-1, 5-bisphospha te carboxylase/oxygenase (RuBPC/O-ase) from Skeletonema costatum were used to study the enzyme. To establish that the antibodies were monospecific for the enzyme in a crude plant extract, a sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was conducted on the crude plant extract and the separated proteins were electrotransferred onto nitrocellulose paper (Western blot) where they were available for immunodetection. It was found that all the protein bands seen on the gel were successfully transfered onto the nitrocellulose paper, but the only band that immunostained was the one corresponding to the large subunit (LS) of RuBPC/O-ase. It was concluded that the antibody was monospecific for the LS of RuBPC/O-ase. An attempt was made to immunopurify RuBPC/O-ase from a crude extract using a column of CN-Br activated sepharose 4B onto which the monoclonal antibodies were immobilized. This was not successful due to the fact that the antibodies had too high an affinity for the enzyme. This conclusion was reached after repeated failure to elute the enzyme off the column. Its presence on the column was shown by boiling the sepharose beads in SDS sample buffer and preforming an SDS-PAGE and a Western blot on the soluble phase which contained the denatured protein. After failing to immunopurify, another method of obtaining a pure enzyme from a crude extract was used. This involved ammonium sulphate precipitation at 50% followed by sucrose density gradient ultracentrifugation. The resulting enzyme was electrophoretically pure when visualized by staining with Coomassie brilliant blue. An immunoquantitation assay which involved a modified Enzyme Linked Immuno Sorbent Assay (ELISA), was worked out using pure spinach RuBPC/O-ase (Sigma). The ascites solution was titrated to determine an appropriate antibody dilution (limiting dilution) to be used in the assay. A standard curve was successfully constructed showing a linear relationship between the logarithm of the enzyme concentration and the light absorbance at 490nm by the reaction solution. The successful construction of a standard curve indicated that unknown enzyme concentrations in crude plant extracts can be determined by extrapolating from the absorbance on the standard curve if the extracts are reacted with the antibody on the ELISA. Since the antibody had been found to be monospecific for the LS, it was assumed that it would readily cross-react with enzymes from other photosynthetic species. This would enable unlimited use of the quantitative assay. When cross-reactivity was assayed for on the Western blot, it was non-existent. The antibody stained only the enzyme from Skeletonema costatum, the antigen donor. When a dot immunobinding assay of crude extracts was conducted, extensive cross-reactivity was found, but this method could not identify the cross-reacting molecule as being the RuBPC/O-ase. To resolve this problem of identity without using the Western Blot, an SDS-denatured pea (Pisum sativum) extract was separated by gel permeation chromatography using a TSK 3000 column. The RuBPC/O-ase fraction (as indicated by SDS-PAGE) was examined for antibody reactivity using a dot immunobinding assay. The pea enzyme cross-reacted with the antibody. Cross-reactivity had already been shown on the ELISA with pure spinach RuBPC/O-ase, which had also been used for assaying for positivity when cloning the hybridoma. It was concluded that the antibody was cross-reacting between species and failure to detect cross-reactivity on the Western Blot was a consequence of physical artifacts during the transfer. Monoclonal antibodies against RuBPC/O-ase, having been shown to be monospecific for the LS of RuBPC/O-ase, are hereby presented as a convenient tool for qualitative and quantitative determination of RuBPC/O-ase in soluble plant extracts.
dc.format.extent141 pages
dc.identifier.urihttps://hdl.handle.net/1828/18988
dc.rightsAvailable to the World Wide Weben_US
dc.titleAnalytical determination of ribulose-1, 5-bisphosphate carboxylase/oxygenase using monoclonal antibodiesen_US
dc.typeThesisen_US

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