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

白细胞介素超灵敏酶联免疫分析法的开发及应用基础研究

• 批准号: 63580125
• 负责人: HASHIDA Seiichi
• 金额: $ 1.22万
• 依托单位: Miyazaki Medical College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63580125/
• 关键词: Interleukin; Enzyme immunoassay; エンザイムイムノアッセイ; インターロイキン

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

我们开发了一种高灵敏度的双位点酶免疫分析技术，并将其应用于阿托摩尔水平的抗原测量。然而，该技术的灵敏度对于白细胞介素（IL）的测量不够高。例如，hIL-1 α、hIL-1 β和hIL-2的检测限分别为5、30和200 μ g/ml。因此，尝试开发更灵敏的白细胞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，多克隆抗体IgG通常通过在抗原-Sepharose 4B柱上的亲和层析来纯化。不幸的是，如此亲和纯化的抗体IgG制剂含有或多或少的从抗原-Sepharose 4B释放的抗原，这导致高背景，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缀合物偶联至溴化氰活化的琼脂糖4B。 ...更多信息 通过从抗原-生物素化的非特异性兔IgG-琼脂糖4B柱洗脱亲和纯化的抗体IgG通过链霉亲和素-琼脂糖4B柱并进行凝胶过滤。结论：1.以促红细胞生成素为模型抗原，亲和纯化的抗促红细胞生成素IgG包被聚苯乙烯球双位点酶免疫分析的本底显著降低，灵敏度比亲和纯化前提高3倍。夹心转移酶免疫测定法。将抗原与生物素基二硝基苯基抗体IgG和抗体Fab ^1-β-D-半乳糖苷酶缀合物形成的复合物捕获到亲和纯化的抗二硝基苯基IgG包被的聚苯乙烯球上。在消除过量的缀合物后，用二硝基苯基-L-赖氨酸从聚苯乙烯球洗脱复合物，并转移到涂有链霉亲和素的干净聚苯乙烯球上。通过荧光测定法测定与链霉亲和素包被的聚苯乙烯球结合的β-D-半乳糖苷酶活性。当铁蛋白被用作模型抗原时，非特异性结合的β-D-半乳糖苷酶活性显著降低，而特异性结合的β-D-半乳糖苷酶活性降低较少。结果，铁蛋白的检测限降低到3毫阿摩尔.在这些结果的基础上，正在计划开发高灵敏度的双位点酶免疫测定白细胞介素和测量他们的培养上清液中的免疫活性细胞。少

项目成果

Kenji Yone, Seiichi Hashida, Koichiro Tanaka, Y. Ichikawa and Eiji Ishikawa: "Specific and sensitive sandwich enzyme immunoassay for human tumor necrosis factor-alpha." Clinical Chemistry and Enzymology Communications. (1989)

Kenji Yone、Seiichi Hashida、Koichiro Tanaka、Y. Ichikawa 和 Eiji Ishikawa：“针对人类肿瘤坏死因子-α 的特异性且灵敏的夹心酶免疫测定法。”

Yoshiriro Kasai,et al.: "Sensitive sandwich enzyme immunoassay for human erythropoietin." Clinical Chemistry and Enzymology Communications. (1989)

Yoshiriro Kasai 等人：“人促红细胞生成素的灵敏夹心酶免疫测定法。”

Hisanori Umehara,et al.: "Enhanced production of interleukin-1 and tumor necrosis factor-α by cultivated peripheral monocytes in patients with scleroderma." Arthritis & Rheumatism. (1990)

Hisanori Umehara 等人：“通过培养硬皮病和风湿病患者的外周单核细胞来增强白细胞介素 1 和肿瘤坏死因子 α 的产生”（1990 年）。

Yoshihiro Kasai, Seiichi Hashida, Koichiro Tanaka Kenji Chichibu, Hiroyuki Usuki and Eiji Ishikawa: "Sensitive sandwich enzyme immunoassay for human erythropoietin." Clinical Chemistry and Enzymology Communications 1989.

Yoshihiro Kasai、Seiichi Hashida、Koichiro Tanaka Kenji Chichibu、Hiroyuki Usuki 和 Eiji Ishikawa：“人促红细胞生成素的灵敏夹心酶免疫分析。”

Yoshihiro Kasai,et al.: "Sensitive sandwich enzyme immunoassay for human erythropoietin." Clinical Chemistry and Enzymology Communications. (1989)

Yoshihiro Kasai 等人：“人促红细胞生成素的灵敏夹心酶免疫测定法。”