Immunonephelometry of apolipoprotein A-II (apo A-II) in serum requires that factors responsible for nonspecific increases of light scattering be eliminated. Complete delipidation is not necessary for immunonephelometry of apo A-II; in fact, light scattering of immunocomplexes from delipidated HDL is greater than from isolated apo A-II. Therefore, using the isolated peptide as a standard does not suffice for measurements in serum. The concentration of protein influences the development of light scattering and must be considered in standardizing this measurement.

The intensity of scattered light varies with the size of the scattering particles. Studying the quantitative immunonephelometric determination of apolipoprotein A-1 (apo A-1), we observed that the different particle sizes of lipoproteins must be taken into account in immunonephelometry if apo A-1 is a constituent of the lipoprotein particles. Because interaction between large lipoproteins and immunocomplexes of high-density lipoproteins increases light scattering nonspecifically, false estimations may be obtained in serum with excessive hyperlipoproteinemia. Furthermore, the accessibility of antigenic sites of apo A-1 in intact high-density lipoproteins is limited. Immunonephelometry of apo A-1 in serum necessitates therefore the elimination of various interferents, which we have achieved by a single one-step extraction of lipids in a two-phase liquid system. With n-hexanol/polyfluoro-polychloro-polyethylene, about 90% of the serum lipids are extracted and only apolipoprotein B is precipitated at the interphase. This pretreatment eliminates the interferences caused by excessive hypertriglyceridemia and therefore greatly facilitates the endpoint immunonephelometry of apo A-1 in normal and pathological serum samples.

We investigated the immunoprecipitation of apolipoprotein B-binding lipoproteins, or of apo A-I- and apo C-binding lipoproteins, by delipidated antiserum for measuring cholesterol in the nonprecipitated lipoprotein fractions. After immunoprecipitation of serum with delipidated anti-apo B, we determined by immunoelectrophoresis that no beta- or pre-beta lipoproteins were present, whereas alpha-lipoproteins remained in the supernate. Conversely, after immunoprecipitation with an antiserum against apo A-I + apo C, only lipoproteins with beta-mobility were detected and no apo B from beta-lipoproteins was in the precipitate. The concentration of cholesterol in the supernate after immunoprecipitation with anti-apo B correlated highly (r = 0.93, n = 118) with cholesterol measured after precipitation with phosphotungstic acid/MgCl2. The cholesterol concentration after immunoprecipitation with anti-apo AI and anti-apo C correlated similarly well (r = 0.94, n = 145) with the LDL-cholesterol calculated by Friedewald's formula in serum specimens reflecting moderate hyperlipoproteinemia.

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