Brandt R, Hedlöf E, Asberg S, Gustavsson J G, Carlsson J
J Immunol Methods. 1986 Dec 24;95(2):169-75. doi: 10.1016/0022-1759(86)90403-5.
A competitive enzyme immunoassay has been developed for the determination of beta 2-microglobulin in undiluted human serum. In the assay a beta 2-microglobulin-beta-galactosidase conjugate competes with beta 2-microglobulin from the sample for the binding to anti-beta 2-microglobulin antibodies bound to small size agarose particles (micro-Sepharose) via a double antibody. The conjugate is made by providing beta 2-microglobulin with 'reactive disulphide structures.' This is achieved by the heterobifunctional reagent SPDP and the subsequent linkage of the formed derivative to beta-galactosidase by a thioldisulphide exchange reaction. This procedure gives conjugates with high immunoreactivity and enzyme activity. The assay is fast and simple. The reagents are incubated together for 60 min in the wells of a Millititer plate and separation is performed by filtration. Bound enzyme-labelled beta 2-microglobulin is measured by incubation with substrate for 15 min. Both incubations are performed at room temperature without agitation. Due to the high capacity of the micro-Sepharose no sample predilution is needed. The use of the Millititer filtration system gives a rapid and efficient separation and the advantages of access to equipment for dispensing and reading adapted to the microtitre format.
已开发出一种竞争性酶免疫测定法，用于测定未稀释人血清中的β2-微球蛋白。在该测定法中，β2-微球蛋白-β-半乳糖苷酶结合物与样品中的β2-微球蛋白竞争结合通过双抗体结合到小尺寸琼脂糖颗粒（微琼脂糖）上的抗β2-微球蛋白抗体。该结合物通过为β2-微球蛋白提供“反应性二硫键结构”制成。这通过异双功能试剂SPDP来实现，随后通过硫醇二硫键交换反应将形成的衍生物与β-半乳糖苷酶连接。该方法得到具有高免疫反应性和酶活性的结合物。该测定法快速且简单。将试剂在微量滴定板的孔中一起孵育60分钟，通过过滤进行分离。通过与底物孵育15分钟来测量结合的酶标记的β2-微球蛋白。两次孵育均在室温下进行，无需搅拌。由于微琼脂糖的高容量，无需对样品进行预稀释。使用微量滴定过滤系统可实现快速高效的分离，并具有使用适合微量滴定形式的分配和读取设备的优点。
