Determination of apolipoprotein A and its constitutive A-I and A-II polypeptides by separate electroimmunoassays.

Abstract

Electroimmunoassays ("rocket" electrophoresis) are described for human serum apolipoprotein A and its constitutive A-I and A-II polypeptides. Purified lipoprotein A, A-I, and A-II were used to prepare monospecific antisera and to standardize assays. These specific, rapid (5-8 h), precise (the within-and between-assay coefficients of variations are 5 and 7%, respectively), and accurate (by gravimetry) assays are applicable to measurement of these polypeptides in whole serum and in various density classes of lipoproteins. Comparable results are obtained with intact and delipidized lipoproteins. Results correlated well with those obtained by radial immunodiffusion or radioimmunoassay. However, the present procedure is more rapid than the former and simpler than the latter immunoassay. Concentrations of A-I and A-II in the serum of normal men and women were similar (143 +/- 24 and 146 +/- 78 mg/dl, respectively, for A-I and 78 +/- 17 and 83 +/- 25 mg/dl for A-II). Subjects with type lla, llb, and IV hyperlipoproteinemias had similar concentrations of both polypeptides, while patients with type I disease, lecithin:cholesterol acyltransferase deficiency and LP-A deficiency had lowest concentrations of A-I (0.3-30 mg/dl) and A-II (11-20 mg/dl). The molar ratio of A-I/A-II in the serum and high-density lipoproteins was close to unity.

Variations in apolipoproteins B and A, during the course of myocardial infarction

“…On admission, every morning for the first 7 days, on days 14,2 1 and 30 venous blood samples were taken after a fast of at least 8 h. About 20 ml of blood was placed in centrifuge tubes and stabilized with EDTA (1 [13], with 100 pg of protein in each tube. Apolipoproteins A1 and B were determined in whole plasma and in the ultracentrifuged fractions by electroimmunoassay (rocket electrophoresis), adapting the technique of Laurel1 [14] as described by Curry et al [15]. For this purpose, an LKB 2177 water-cooled Multiphor electrophoresis plate was used and on this twenty-one samples can be run simultaneously.…”

Relationships between the metabolism of high‐density and very‐low‐density lipoproteins in man: studies of apolipoprotein kinetics and adipose tissue lipoprotein lipase activity

“…Electroimmunoassay of apo A1 and apo A11 was performed by a modification of the method of Curry et al [30], using 1.5% agarose in sodium barbitone buffer (50 mmol/l; pH 8.6) and 8.6 V/cm for 16 h. Antisera were prepared in rabbits by the method of Albers et al [31]. Standard curves (90-270 ng of apo AI; 20-60 ng of AII) were constructed in each plate, using serial dilutions of a plasma pool the apoprotein content of which had previously been determined by immunodif-after three to five half-lives.…”

Serum lipoprotein and apolipoprotein concentrations and tissue lipoprotein‐lipase activity in overt and subclinical hypothyroidism: the effect of substitution therapy

“…The accuracy and precision of the lipid determinations have been checked at the Center of Disease Control, Atlanta, Georgia, U.S.A. The concentrations in whole serum of apo B and A-I were determined, using the electroimmunoassay, as described by Laurel1 [23], modified essentially as described by Curry [24]. The details of the antibody production, purification and standardization have recently been described [25].…”