Høgåsen K, Mollnes T E, Tschopp J, Harboe M
Institute of Immunology and Rheumatology, National Hospital, University of Oslo, Norway.
J Immunol Methods. 1993 Mar 15;160(1):107-15. doi: 10.1016/0022-1759(93)90014-x.
Vitronectin (S protein) and clusterin (SP-40,40/cytolysis inhibitor) are non-homologous, multifunctional proteins which both inhibit complement lysis. Vitronectin is an adhesive protein which binds strongly to polystyrene by hydrophobic interactions. The current study demonstrated that clusterin adsorbed even more efficiently to polystyrene than did vitronectin. This adsorption increased in the presence of Tween 20 and was not abolished by blocking or by the use of other detergents. In double antibody enzyme immunoassays such non-specific binding might invalidate the results. However, the non-specific binding of both proteins was efficiently abolished by the following experimental format: Dynatech Immulon 2 microtiter plate, acidic sample buffer (pH 6.0) containing 0.2% Tween 20 and high sample dilution. Vitronectin was successfully quantitated using this approach, but the measurement of clusterin was not reliable because of high inter-well variation of binding. However, since few serum proteins adsorb to polystyrene in the presence of detergents, clusterin was successfully quantitated in a single antibody enzyme immunoassay in which samples were coated directly onto Nunc Maxisorp plates in the presence of 0.2% Tween 20. In normal blood donors the serum concentration (median and 2.5-97.5 percentile) of vitronectin was 0.34 g/l (0.24-0.53) and of clusterin 0.34 g/l (0.25-0.42).
玻连蛋白（S蛋白）和簇集素（SP-40,40/细胞溶解抑制剂）是两种非同源的多功能蛋白，它们都能抑制补体溶解。玻连蛋白是一种黏附蛋白，通过疏水相互作用与聚苯乙烯紧密结合。目前的研究表明，簇集素比玻连蛋白更有效地吸附到聚苯乙烯上。在吐温20存在的情况下，这种吸附作用增强，并且不会因封闭或使用其他洗涤剂而消除。在双抗体酶免疫测定中，这种非特异性结合可能会使结果无效。然而，通过以下实验形式可有效消除这两种蛋白的非特异性结合：Dynatech Immulon 2微量滴定板、含有0.2%吐温20的酸性样品缓冲液（pH 6.0）以及高样品稀释度。使用这种方法成功地对玻连蛋白进行了定量，但由于孔间结合差异较大，对簇集素的测量并不可靠。然而，由于在洗涤剂存在的情况下很少有血清蛋白吸附到聚苯乙烯上，因此在单抗体酶免疫测定中成功地对簇集素进行了定量，该测定中样品在0.2%吐温20存在的情况下直接包被在Nunc Maxisorp板上。在正常献血者中，玻连蛋白的血清浓度（中位数和第2.5 - 97.5百分位数）为0.34 g/l（0.24 - 0.53），簇集素为0.34 g/l（0.25 - 0.42）。
