We describe a method for routine immunoturbidimetry of apolipoproteins (apo) A-I, A-II, and B in both normo- and hyperlipemic sera. A special antiserum reagent, consisting of a highly concentrated mixture of nonionic and anionic detergents (final concentration in the assay, 36 g/L), rapidly removes intrinsic turbidities of even strongly lipemic sera without interfering with the antigen-antibody precipitation reaction. The method has good precision, and obviates the need for special sample pretreatment, extended incubation periods, and measurment of sample blanks. A comparison with established immunoephelometric assays generally showed close agreement for analytical recoveries of the three apolipoproteins. However, in samples containing greater than or equal to 18 g of triglycerides per liter, the nephelometric assays yielded about two- to threefold higher values for apo A-II and B than did the turbidimetric procedure. To elucidate this discrepancy, we used the turbidimetric methods to assay sera with and without enzymatic lipolytic pretreatment. Even for samples with triglyceride concentrations up to 60 g/L, complete enzymatic lipolysis (as evidenced by thin-layer chromatography) did not significantly alter the recoveries of apo A-II and B from those obtained with the untreated specimens. Thus the immunoturbidimetric methods yield reliable results for apo A-I, A-II, and B, not only in normo- but also in hyperlipemic sera.
The plasma levels of total and high-density lipoprotein cholesterol and of the major apolipoproteins (apo B and apo A-I) were studied in 30 newborns, on cord blood and after 7 and 30 days of life. The evolution of these parameters during the first month of life shows that newborns have low LDL cholesterol and apo B levels at birth, which increase drastically during the first week and remain constant between 7 and 30 days. The HDL cholesterol and apo A-I levels are proportionally high at birth and keep increasing slowly up to 30 days. During this period, the cholesterol/apoprotein ratio remains constant in the LDL and HDL class. These data suggest that lipid and apoprotein levels at 7 days are more representative than cord-blood levels and more meaningful for a screening of congenital hypercholesterolemia. The cholesterol/apo B and apo B/apo A-I ratios, which are considered to be better predictive factors for atherosclerosis, should be included as screening parameters.
A specific and sensitive "sandwich"-type enzyme-linked immunosorbent assay (ELISA) has been developed for quantifying human apo A-IV. Using apo A-IV immunosorbent columns, we isolated monospecific anti-apo A-IV antibodies for coating the ELISA plates and for preparing peroxidase-antibody conjugate. The assay can detect as little as 0.20 ng of apo A-IV, with mean intra- and interassay CVs of 3.6% and 8.2%, respectively. The apoA-IV concentrations in normolipemic and hyperlipemic plasma were unaffected by either delipidation or treatment with detergents or urea. To validate the ELISA assay we compared it with an immunoelectrophoretic technique. ApoA-IV concentrations in plasma from normo- and dyslipemic subjects compared well by the two assays (r = 0.89). The mean apo A-IV concentration, measured by ELISA in plasma from 50 normolipemic subjects, was 143 (SD 52) mg/L; values for dyslipemic subjects were not significantly different. We also used this new assay to monitor apo A-IV profiles of normolipemic and hypertriglyceridemic plasma after chromatographic fractionation.
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