Characterization and measurement of human apolipoprotein A-II by radioimmunoassay.
The development of a radioimmunoassay for apolipoprotein A-II (apo A-II) is described. Initial studies revealed a lack of immunological identity between purified apo A-II used as the standard and serum or HDL. Extensive testing of different buffers, standards, antisera, tracers, utilization of a detergent, and heating of sera failed to resolve the problem. Gel filtration of iodinated and non-iodinated apo A-II on Sephadex G-100 columns showed that apo A-II, in dilute solution, elutes in a higher molecular zone than expected with a broad, assymetrical profile. The use of a subfraction of the tracer in the assay resulted in parallelism in the serum and standard dilution curves. The apo A-II assay was sensitive, specific, and reproducible. Apo A-II added to sera was fully recovered and delipidation did not affect the immunoreactivity of either serum or HDL. Apo A-II contributed approximately 20% to the protein mass of HDL. Comparison of these results with those obtained by radial immunodiffusion, and with previously reported data, indicates that the reactivity of apo A-II in its native and delipidated forms may be markedly influenced by different immunologic methodologies and their specific reagents. Caution should thus be shown at present in assigning absolute concentrations to apo A-II in serum or HDL.
D000328:Adult; D016633:Apolipoprotein A-II; D001053:Apolipoproteins / Q000032:analysis* / Q000302:isolation & purification; D002850:Chromatography, Gel; D004356:Drug Storage; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007460:Iodoacetamide / Q000494:pharmacology; D008075:Lipoproteins, HDL / Q000032:analysis; D008297:Male; D008623:Mercaptoethanol / Q000494:pharmacology; D011863:Radioimmunoassay / Q000379:methods
D016633:Apolipoprotein A-II; D001053:Apolipoproteins; D008075:Lipoproteins, HDL; D008623:Mercaptoethanol; D007460:Iodoacetamide