Summary
A reliable method for determining serum apoprotein levels is an essential condition for investigating the role of apoproteins in atherogenesis. Electroimmunoassay according to Laurell has been studied and applied with some modifications for the determination of the two main apoproteins: Apo A-I and Apo B. Apo B immunoplates containing 0.4% v/v of rabbit anti-Apo B antiserum were processed for 4 h at 10 V/cm. Samples were incubated at 52°C for 3 h, diluted and then 10 μl were seeded in each well. Apo A-I immunoplates (8% v/v of sheep antiserum) were processed for 24h at 2 V/cm. Agarose gel concentration, exsiccation procedure and staining were the same for both apoproteins. The method was standardized employing a secondary standard consisting of a serum pool obtained from normal subjects. Apo A-I and Apo B levels of the pool have been previously determined employing as primary standards the HDL, (1.120–1.230 g/ml) and the LP-B (1.035–1.050 g/ml) fractions, respectively, which were isolated by preparative ultracentrifugation. Preliminary observations from a study on 20 healthy, volunteers with normal lipid levels revealed different apoprotein levels in young men and women and significant differences between postmenopausal women and women in the fertile age.
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Alaupovic P., Curry M., McConathy C., Fesmire J.: Electroimmunoassay of apolipoproteins of human plasma. In:Lippei, K. (Ed.): Report of the high density apoprotein methodology workshop. NIH, San Francisco, 1979; p. 227.
Albers J. J., Wahl P. W., Cabana V. G., Hazzard W. R., Hoover J. J.: Quantitation of apolipoprotein A-I of human plasma high density lipoprotein—Metabolism25, 633, 1976.
Avogaro P., Bittolo Bon G., Cazzolato G., Quinci G. B.: Are apolipoproteins better discriminators than lipids for atherosclerosis?—Lanceti, 901, 1979.
Avogaro P., Bittolo Bon G., Cazzolato G., Quinci G. B., Sanson A., Zagatti G. C., Catturelli G.: Vanations in apolipoproteins B and A-I during the course of myocardial infarction—Europ. J. clin. Invest.8, 121, 1978.
Baggio G., Fellin R., Baiocchi M. R., Martini S., Baldo G., Manzato E., Crepaldi G.: Relationship between triglyceride-rich lipoprotein (chylomicrons and VLDL) and HDL2 and HDL3 in the postprandial phase in humans—Atherosclerosis37, 271, 1980.
Baggio G., Fellin R., March A., Baldo G., Martini S., Crepaldi G., Sparrow J., Gotto A. M. Jr.: Lipoprotein metabolism activation by infusion of plasma and apoprotein C-II synthetic fragment 43–78—Europ. J. clin. Invest.11, 2, 1981.
Barr D. P., Russ E. M., Eder H. A.: Protein-lipid relationship in human plasma. II. In atherosclerosis and related conditions—Amer. J. Med.11, 480, 1951.
Berg K., Børresen A.-L., Dahlen G.: Serum high-density-lipoprotein and atherosclerotic heart disease—Lanceti, 499, 1976.
Curry M. D., Alaupovic P., Suenram C. A.: Determination of apolipoprotein A and its constitutive A-I and A-II polypeptides by separate electroimmunoassays—Clin. Chem.22, 315, 1976.
Enholm C., Garoff H., Simon K., Aro H.: Purification and quantitation of human plasma lipoprotein carrying the Lp(a) antigen—Biochim. biophys. Acta (Amst.)236, 431, 1971.
Fielding C. J., Shore V. G., Fielding P. E.: A protein cofactor of lecithin-cholesterol acyltransferase—Biochem. biophys. Res. Commun.46, 1493, 1972.
Glickman R. M., Green P. H. R., Lees R. S., Tall A. R.: Apoprotein A-I synthesis in normal intestinal mucosa and in Tangier disease—New Engl. J. Med.299, 1424, 1978.
Kane J. P.: A rapid electrophoretic technique for identification of subunit species of apoproteins in serum lipoproteins—Ann. Biochem.53, 350, 1973.
Karlin J. B., Rubenstein A. H.: Serum lipoprotein quantification by immunochemical methods. In:Scanu A. M., Wissler R. W., Getz G. S. (Eds): The biochemistry of atherosclerosis. Marcel Dekker, Inc., New York, 1979; p. 189.
Kostner G. M., Pristautz H., Pilger E.: Quantitation of apolipoproteins in PVD patients and controls: comparison with serum lipids and LP-lipids. In: Lipoproteins and coronary atherosclerosis. Autumn Meeting of EAG, Lugano, 1981; p. 98. (Abstract).
Laurell C.-B.: Electroimmunoassay—Scand. J. clin. Lab. Invest.29 (Suppl. 124), 21, 1972.
Lee D. M., Alaupovic P.: Composition and concentration of apolipoprotein in very-low and low-density lipoproteins of human plasma—Atherosclerosis19, 501, 1974.
Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J.: Protein measurement with the Folin-phenol reagent—J. biol. Chem.193, 265, 1951.
Miller G. J., Miller N. E.: Plasma-high-density-lipoprotein concentration and development of ischaemic heart disease—Lanceti, 16, 1975.
Miller J. P., Mao S. J. T., Patsch J. R., Gotto A. M. Jr.: The measurement of apolipoprotein A-I in human plasma by electroimmunoassay—J. Lipid Res.21, 775, 1980.
Scanu A. M., Edelstein C.: Solubility in aqueous solutions of ethanol of the small molecular peptides of the serum very low density and high density lipoproteins: relevance to the recovery problem during delipidation of serum lipoproteins—Ann. Biochem.44, 576, 1971.
Schaefer E. J., Eisenberg S., Levy R. I.: Lipoprotein apoprotein metabolism—J. Lipid Res.19, 667, 1978.
Schonfeld G., Pfleger B.: The structure of human high density lipoprotein and the levels of apolipoprotein A-I in piasma as determined by radioimmunoassay—J. clin. Invest.54, 236, 1974.
Schonfeld G., Pfleger B., Roy R.: Structure of human high density lipoprotein reassembledin vitro—J. biol. Chem.250, 7943, 1975.
Sniderman A., Shapiro S., Marpole D., Skinner B., Teng B., Kwiterovich P. O. Jr.: Association of coronary atherosclerosis with hyperapobetalipoproteinemia [increased protein but normal cholesterol levels in human plasma low density (β) lipoproteins]—Proc. nat. Acad. Sci. (Wash.)77, 604, 1980.
Zilversmit D. B.: Atherogenesis: a postprandial phenomenon—Circulation60, 473, 1979.
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Baggio, G., Gabelli, C., Baiocchi, M.R. et al. Improvement of electroimmunoassay for apolipoproteins B and A-I. La Ricerca Clin. Lab. 13, 321–330 (1983). https://doi.org/10.1007/BF02905875
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DOI: https://doi.org/10.1007/BF02905875