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The Development and Application of a Novel Assay for Apolipoprotein B-48.

Peel, Andrew. (1993) The Development and Application of a Novel Assay for Apolipoprotein B-48. Doctoral thesis, University of Surrey (United Kingdom)..

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Abstract

The lipid composition of chylomicrons is directly affected by diet and the remnants of these particles may contribute to atherogenesis. Apolipoprotein B-48 is uniquely associated with chylomicrons of dietary origin, whereas apolipoprotein B-100 is associated with lipoproteins of hepatic origin. Apolipoprotein B-48 is identical to the N-terminal of apolipoprotein B-100 and this has previously prevented production of specific antibodies to apolipoprotein B-48. Antibodies were directed to the charged carboxyl C-terminus of apolipoprotein B-48 because it had been identified as a potential discriminatory region between the two apolipoproteins. This was achieved by conjugating heptapeptides, which corresponded to the C-terminal of apolipoprotein B-48, to carrier proteins to enhance their immunogenicity. A screening enzyme-linked immunosorbent assay was developed to monitor the antibody response to immunisation. The specificity of the resultant antisera was tested by immunostaining of chylomicron-enriched serum and lymph samples following separation of the proteins by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Subsequently, measurement of apolipoprotein B-48 by three different enzyme-linked immunosorbent assay formats and a slot blot was investigated. These were unsuccessful and so a semi-quantitative assay based on sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by immunoblotting and detection by enhanced-chemiluminescence and densitometric scanning was developed. Levels of apolipoprotein B-48 detected by this method correlated (r=0.68) with those measured on a gel which was stained with Coomassie Blue for protein. This assay was tested using chylomicron-enriched plasma samples obtained at regular intervals from eleven healthy subjects following two test meals which differed in their fatty acid composition. Apolipoprotein B-48 levels showed similar patterns to triacylglycerol in response to the two test meals. Differences in the pattern and nature of response were found in comparison of these two parameters with retinyl palmitate. Retinyl palmitate loading is currently the most widely used method of monitoring chylomicron metabolism and hence the production of an antibody and development of an assay for apolipoprotein B-48 are major contributions to the study of postprandial lipid metabolism.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors : Peel, Andrew.
Date : 1993
Additional Information : Thesis (Ph.D.)--University of Surrey (United Kingdom), 1993.
Depositing User : EPrints Services
Date Deposited : 06 May 2020 14:23
Last Modified : 06 May 2020 14:34
URI: http://epubs.surrey.ac.uk/id/eprint/856245

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