Basic Study on Development and Application of Ultrasensitive Enzyme Immunoassay for Interleukins

我们开发了一种高灵敏度的双位点酶免疫测定技术，并将其应用于抗原在原子滴水平的测量。然而，这种技术的灵敏度不足以测量白细胞介素（IL）。例如，hil -1 α、hil -1 β和hIL-2的检出限分别为5、30和200 amol。因此，人们尝试开发更灵敏的白介素酶免疫测定技术。为了高效地将抗原捕获到抗体包被的聚苯乙烯球上，通常在抗原- sepharose 4B柱上通过亲和层析纯化多克隆抗体IgG。不幸的是，这种亲和纯化的抗体IgG制剂或多或少含有抗原sepharose 4B释放的抗原，这导致高本底限制了双位点酶免疫测定的灵敏度。为了消除这些抗原，将抗原生物素化的非特异性兔IgG偶联物与cnbr活化的Sepharose 4B偶联。从抗原生物素化的非特异性兔IgG- sepharose 4B柱洗脱纯化的抗体IgG亲和，通过链亲和素- sepharose 4B柱，并进行凝胶过滤。以促红细胞生成素为模型抗原时，采用亲和纯化抗促红细胞生成素igg包被聚苯乙烯球进行双位点酶免疫分析的背景明显降低，灵敏度较亲和纯化前提高3倍。三明治转移酶免疫测定。抗原与生物素基二硝基苯抗体IgG和抗体Fab^1- β -d -半乳糖苷酶偶联物形成的配合物被捕获在亲和纯化的抗二硝基苯IgG包被的聚苯乙烯球上。去除多余的共轭物后，用二硝基苯赖氨酸从聚苯乙烯球中洗脱配合物，并转移到涂有链霉亲和素的清洁聚苯乙烯球上。用荧光法测定链霉亲和素包被的聚苯乙烯球上的- d -半乳糖苷酶活性。当铁蛋白作为模型抗原时，非特异性结合的β - d -半乳糖苷酶活性显著降低，特异性结合的β - d -半乳糖苷酶活性降低较少。结果表明，铁蛋白的检出限降至3毫微摩尔。在这些结果的基础上，正在计划开发高灵敏度的白细胞介素双位点酶免疫分析法，并在免疫活性细胞的培养上清液中进行检测。少

We developed a highly sensitive two-site enzyme immunoassay technique and applied it to the measurement of antigens at attomole levels. However, the sensitivity by this technique is not sufficiently high for the measurement of interleukins (IL). For example, the detection limits of hIL-1alpha, hIL-1beta and hIL-2 were 5, 30 and 200 amol, respectively. Therefore, attempts were made to develop more sensitive enzyme immunoassay techniques for interleukins.1. In order to trap antigens onto antibody-coated polystyrene balls with high efficiency, polyclonal antibody IgG is usually purified by affinity chromatography on a column of antigen-Sepharose 4B. Unfortunately, the antibody IgG preparations thus affinity-purified contain more or less antigens released from antigen-Sepharose 4B, which cause high background to limit the sensitivity of two-site enzyme immunoassay. To eliminate these antigens, antigen-biotinylated nonspecific rabbit IgG conjugate was coupled to CNBr-activated Sepharose 4B. … More Antibody IgG affinity-purified by elution from a column of antigen-biotinylated nonspecific rabbit IgG-Sepharose 4B was passed through a column of streptavidin-Sepharose 4B and was subjected to gel filtration. When erythropoietin was used as model antigen, the background of two-site enzyme immunoassay using affinity-purified anti-erythropoietin IgG-coated polystyrene balls was significantly lowered, improving the sensitivity 3 fold as compared with that before affinity-purification.2. Sandwich transfer enzyme immunoassay. The complex formed of antigen with biotinyl dinitrophenyl antibody IgG and antibody Fab^1-beta-D-galactosidase conjugate was trapped onto affinity-purified anti-dinitrophenyl IgG-coated polystyrene balls. After eliminating excess of the conjugate, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to clean polystyrene balls coated with streptavidin. beta-D-Galactosidase activity bound to the streptavidin-coated polystyrene balls was assayed by fluorimetry. When ferritin was used as model antigen, nonspecifically bound beta-D-galactosidase activity considerably decreased with less decrease in specifically bound beta-D-galactosidase activity. As a result, the detection limit of ferritin was lowered to 3 milliattomoles.3. On the basis of these results, it is being planned to develop highly sensitive two-site enzyme immunoassay for interleukins and measure them in the culture supernatants of immunocompetent cells. Less