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Dynamic reaction in a homogeneous HDL-cholesterol assay visualized by electron microscopy
1Department of Laboratory Medicine, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu City, 431-3192 Japan. akikondo@hama-med.ac.jp
Background:
Measurement of HDL-cholesterol (HDL-C) by homogeneous assays with automated analyzers is replacing precipitation methods. However, in this reaction-type assay, interactions between the reagents and lipoproteins remain unknown.
Methods:
Electron microscopy was used to investigate the reactions in a homogeneous HDL-C assay. Negative staining with 10 g/L uranyl acetate was performed for lipoprotein visualization by electron microscopy. Observations of the interactions between lipoproteins and the reagents of a polyanion-polymer/detergent assay were achieved by cooling the reaction mixture in ice water. This treatment also allowed observation of the time course of the reaction.
Results:
In the first-reagent reaction (polyanion-polymer), every lipoprotein aggregated almost completely. In the second-reagent reaction (enzymes and detergent), only HDL in the lipoprotein aggregates was selectively resolved and reacted enzymatically. Reagent 1 contains two important substances: polyanion and synthetic polymer. Using x-ray microanalysis, we confirmed that aggregation of lipoproteins in the first reaction occurred through interaction with the phosphotungstate of the polyanion.
Conclusion:
Electron microscopy morphologically revealed the dynamic reaction in a homogeneous HDL-C assay.