USE OF THIOL-TERMINAL SILANES AND HETEROBIFUNCTIONAL CROSSLINKERS FOR IMMOBILIZATION OF ANTIBODIES ON SILICA SURFACES
USE OF THIOL-TERMINAL SILANES AND HETEROBIFUNCTIONAL CROSSLINKERS FOR IMMOBILIZATION OF ANTIBODIES ON SILICA SURFACES
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DOI：
10.1016/0003-2697(89)90662-3
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发表时间：
1989-05-01
影响因子：
2.9
通讯作者：
LIGLER, FS
中科院分区：
文献类型：
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作者：
BHATIA, SK;SHRIVERLAKE, LC;LIGLER, FS
使用硫醇末端硅烷和异双功能交联剂开发了一种将功能蛋白共价固定在二氧化硅基质上的方法。使用此程序，高密度的功能性抗体被结合到玻璃盖玻片和二氧化硅纤维上。使用放射性标记抗体确定固定的抗IgG抗体的量在0.66至0.96ng/mm 2 的范围内。固定化抗体结合的 IgG 抗原的相对量（0.37 至 0.55 mol 抗原/mol 抗体）比其他研究人员报道的多三至五倍。此外，通过添加封闭剂，非特异性吸附到抗体包被表面的蛋白质量进一步减少，使得蛋白质抗原的非特异性吸附仅占总抗原结合的2-6%。在这种低背景下，当应用 3 pmol/ml 的抗原浓度时，可以在低至 150 fmol 的水平下测量 IgG 抗原结合。抗体固定化过程简单、易于执行，并且适用于改变生物传感器组件的质量。
A procedure for covalent immobilization of functional proteins on silica substrates was developed using thiol-terminal silanes and heterobifunctional crosslinkers. Using this procedure, a high density of functional antibodies was bound to glass cover slips and silica fibers. The amount of anti-IgG antibody immobilized was determined to be in the range of 0.66 to 0.96 ng/mm2 using radiolabeled antibody. The relative amount of IgG antigen bound by the immobilized antibody (0.37 to 0.55 mol antigen/mol antibody) was three to five times greater than other investigators have reported. In addition, the amount of protein nonspecifically absorbed to the antibody-coated surface was further reduced by the addition of blocking agents so that nonspecific adsorption of protein antigens represented only 2-6% of the total antigen binding. With this low background, IgG antigen binding could be measured at levels at low as 150 fmol when an antigen concentration of 3 pmol/ml was applied. The process for antibody immobilization is straightforward, easy to perform, and adaptable for modifying mass quantities of biosensor components.