Yazynina E V, Zherdev A V, Dzantiev B B, Izumrudov V A, Gee S J, Hammock B D
Immunobiochemistry Laboratory, Russian Academy of Sciences, Moscow, Russia.
Anal Chem. 1999 Aug 15;71(16):3538-43. doi: 10.1021/ac990072c.
Linear water-soluble polyelectrolytes, i.e., poly(methacrylate) polyanion and poly(N-ethyl-4-vinylpyridinium) polycation, were used as carriers for the reactants in immunoassay. The strength of ionic forces through distance and the cooperative binding of oppositely charged chains, the carriers interact with each other at an extremely high rate and affinity. These properties of the polyelectrolytes made it possible to carry out the immunochemical steps of the assay in true solution and then to quickly separate the resulting products from the reaction mixtures. The above approach was applied to an assay for the herbicide simazine. Both enzyme-linked immunosorbent assay (ELISA) and dot blot formats of the immunoassay were evaluated. In the ELISA format, the polycation was adsorbed on the surface of a microtiter plate. A tracer antigen (simazine) was allowed to interact in solution with components of the reaction mixture containing simazine-peroxidase conjugate, specific antibodies, and staphylococcal protein A conjugated with the polyanion, and then the mixture was added to the immobilized polycation. Quick separation of the immunoreactants was achieved due to formation of interpolyelectrolyte complexes between polycation and polyanion molecules. After washing, the microplate wells were filled with a solution of substrate, and the optical density of the reaction products was measured. In the second format, a solution of the same reaction mixture (after incubation) was filtered through a porous membrane, with the polycation adsorbed. The subsequent addition of substrate led to the development of colored spots. Sensitivity of the dot blot format was close to that of the traditional ELISA format using the same reactants, i.e., 0.5 ng/mL. However, the assay was much faster (assay time decreased from 100-120 to 45 min). Sensitivities of the dot immunoassay were 1 ng/mL for densitometric detection and 10 ng/mL for visual detection with a duration of 20 min. The techniques developed here were used for simazine determination in water, milk, and juices.
线性水溶性聚电解质，即聚（甲基丙烯酸酯）聚阴离子和聚（N-乙基-4-乙烯基吡啶鎓）聚阳离子，被用作免疫分析中反应物的载体。通过距离的离子力强度以及带相反电荷链的协同结合，这些载体以极高的速率和亲和力相互作用。聚电解质的这些特性使得在真溶液中进行分析的免疫化学步骤成为可能，然后能够快速从反应混合物中分离出所得产物。上述方法应用于除草剂西玛津的分析。评估了免疫分析的酶联免疫吸附测定（ELISA）和斑点印迹形式。在ELISA形式中，聚阳离子吸附在微量滴定板的表面。使示踪抗原（西玛津）在溶液中与含有西玛津-过氧化物酶结合物、特异性抗体以及与聚阴离子结合的葡萄球菌蛋白A的反应混合物成分相互作用，然后将混合物加入固定化的聚阳离子中。由于聚阳离子和聚阴离子分子之间形成聚电解质间复合物，实现了免疫反应物的快速分离。洗涤后，向微量滴定板孔中加入底物溶液，并测量反应产物的光密度。在第二种形式中，将相同反应混合物的溶液（孵育后）通过吸附有聚阳离子的多孔膜过滤。随后加入底物导致出现有色斑点。斑点印迹形式的灵敏度与使用相同反应物的传统ELISA形式相近，即0.5 ng/mL。然而，该分析速度快得多（分析时间从100 - 120分钟减少到45分钟）。斑点免疫分析的灵敏度对于光密度检测为1 ng/mL，对于目视检测为10 ng/mL，持续时间为20分钟。此处开发的技术用于测定水、牛奶和果汁中的西玛津。
