Yoshioka M, Mukai Y, Matsui T, Udagawa A, Funakubo H
Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan.
J Chromatogr. 1991 May 31;566(2):361-8. doi: 10.1016/0378-4347(91)80252-8.
When preparing an affinity column and a biosensor, it is desirable to immobilize a unimolecular layer of pure protein on a matrix. In this work, we tried to immobilize a monoclonal antibody on a surface of a glass test-tube as a model, to confirm the stability of this ultra-thin layer by an enzyme immunoassay, and to estimate the thickness of the layer on a slide glass by Fourier transform infrared reflection spectrometry. A new test-tube was washed and dried. The tube was filled with 5% 3-aminopropyltriethoxysilane. The 3-aminopropylsilylated surface was treated with glutaraldehyde and 5.6.10(-2) mg/ml solution of a normal mouse monoclonal antibody. The Schiff base between glutaraldehyde and the antibody was further reduced with 7.9.10(-3)% NaBH4. The tube was washed with 0.05% Tween 20 to block non-specific binding. The antibody immobilized on the surface was measured by an enzyme immunoassay based on a reaction of anti-mouse immunoglobulin G labelled with alkaline phosphatase, with which p-nitrophenol was produced from p-nitrophenylphosphate as a substrate. Meanwhile, various amounts of the antibody were immobilized on slide glasses in the same manner. The antibody on each surface was measured by Fourier transform infrared reflection spectrometry. The antibody immobilized under the final conditions was detectable by the enzyme immunoassay, and stable at 4 degrees C for ten days. The antibody on the slide glass was a unimolecular layer, as judged from the Fourier transform infrared spectra referred to -CONH- band semiquantitatively. Thus, we found the optimal conditions for immobilizing an ultra-thin layer of the monoclonal antibody on the glass surface.(ABSTRACT TRUNCATED AT 250 WORDS)
在制备亲和柱和生物传感器时，期望将单分子层的纯蛋白质固定在基质上。在本研究中，我们尝试将单克隆抗体固定在玻璃试管表面作为模型，通过酶免疫测定法确认该超薄层的稳定性，并通过傅里叶变换红外反射光谱法估算载玻片上该层的厚度。将新的试管清洗并干燥。向试管中加入5%的3-氨丙基三乙氧基硅烷。用戊二醛和5.6×10⁻²mg/ml的正常小鼠单克隆抗体溶液处理3-氨丙基硅烷化表面。戊二醛与抗体之间的席夫碱用7.9×10⁻³%的NaBH₄进一步还原。用0.05%的吐温20洗涤试管以阻断非特异性结合。基于碱性磷酸酶标记的抗小鼠免疫球蛋白G的反应，通过酶免疫测定法测量固定在表面的抗体，该反应以对硝基苯磷酸为底物产生对硝基苯酚。同时，以相同方式将不同量的抗体固定在载玻片上。通过傅里叶变换红外反射光谱法测量每个表面上的抗体。在最终条件下固定的抗体可通过酶免疫测定法检测到，并且在4℃下稳定十天。从傅里叶变换红外光谱中半定量参考-CONH-带判断，载玻片上的抗体为单分子层。因此，我们找到了将单克隆抗体超薄层固定在玻璃表面的最佳条件。（摘要截短至250字）
