A novel use of endpoint nephelometry to standardize the rate nephelometric assay of human and rat plasma apoprotein A.

A sensitive, accurate, and reliable method is described for calibrating the rate immunonephelometric assay of rat and human plasma apolipoprotein A (Apo A). Pure Apo A and high-density lipoprotein (HDL) of known Apo A concentration were used in endpoint nephelometry to determine Apo A concentrations of rat and human plasma pools. The endpoint method had coefficients of variation of 7.96% and 4.35% for rat and human plasma pools, respectively. These plasma pools were then used as secondary standards for the rate nephelometric assay. Excellent agreement (+/- 6%) existed between the plasma Apo A values determined by endpoint nephelometry and rate nephelometry. The Apo A concentration of a frozen human plasma pool determined by endpoint nephelometry was 125.2 +/- 9.6 mg/dl. The value of the same pool determined by rate nephelometry over a 1-year period with the Centers for Disease Control WHO lyophilized plasma standard was 125.4 +/- 21.2 mg/dl. Furthermore, it was found that the rat HDL was also a suitable standard in the rate nephelometric assay of Apo A. In contrast, Apo A, purified to homogeneity, showed different reaction kinetics from that of Apo A in the whole plasma and therefore was not a suitable standard in the rate nephelometric assay. We therefore conclude that primary standard Apo A, purified to homogeneity, can be used by endpoint nephelometry to calibrate plasma pools that can then be used as secondary standards in the rate nephelometric determination of rat and human plasma Apo A. The ready applicability of this method in the accurate determination of plasma Apo A under well-defined experimental conditions such as in chronic ethanol-fed rats and in human subjects with normal lipid levels and those with hyperlipidemia is demonstrated.