Sterzynska Karolina, Budna Joanna, Frydrych-Tomczak Emilia, Hreczycho Grzegorz, Malinska Agnieszka, Maciejewski Hieronim, Zabel Maciej
Wroclaw Research Center EIT+, BioMed, Wroclaw, Poland; Department of Histology and Embryology, Poznan University of Medical Sciences, Poznan, Poland.
Folia Histochem Cytobiol. 2014;52(3):250-5. doi: 10.5603/FHC.2014.0029.
The immobilization of antibodies on various surfaces has been the subject of advanced research in various immunoassay-based diagnostic devices. The physical and chemical stabilities of the immobilized antibodies on a solid surface still cause many problems because upon immobilizing antibody molecules, the antigen-binding ability usually decreases. The silanization of surfaces with organosilanes carrying chemically active groups such as (3-aminopropyl) triethoxysilane (APTES) can accommodate these antigen-binding molecules in an appropriate orientation so that their functionality and binding activity are essentially retained. In this study, n-butyltrimethoxysilane (BMS) and 3-(octafluoropentyloxy)-propyltriethoxysilane (OFPOS) were used as "blocking silanes". The aims of this study were to compare the effectiveness of specific antibody binding of APTES, APTES + BMS and APTES + OFPOS and to characterize the modified surfaces by contact angle measurements and immunofluorescence measurements prior to and after immobilizing proteins. Additionally, we have evaluated the functionality of the immobilized antibodies by their abilities to bind EpCAM-positive human colon adenocarcinoma cell line (LoVo) and EpCAM-negative mouse embryonic fibroblast cell line (3T3). Cell enumeration was conducted on the basis of DAPI-positive signals and recorded using a confocal laser scanning biological microscope. The results of our study showed that the immobilization capability and reactivity of APTES, APTES + BMS and APTES + OFPOS differ. The modification of APTES with unreactive silanes (BMS,OFPOS) is recommended to improve the antibody binding efficiency. However, using OFPOS resulted in more effective antibody and cell binding, and it appears to be the most useful compound in specific antibody-mediated cell recognition.
在各种基于免疫测定的诊断设备中，将抗体固定在各种表面上一直是前沿研究的主题。固定在固体表面上的抗体的物理和化学稳定性仍然引发许多问题，因为在固定抗体分子时，其抗原结合能力通常会下降。用带有化学活性基团的有机硅烷（如（3-氨丙基）三乙氧基硅烷（APTES））对表面进行硅烷化处理，可以使这些抗原结合分子以适当的方向排列，从而基本保留它们的功能和结合活性。在本研究中，正丁基三甲氧基硅烷（BMS）和3-（八氟戊氧基）丙基三乙氧基硅烷（OFPOS）被用作“封闭硅烷”。本研究的目的是比较APTES、APTES + BMS和APTES + OFPOS特异性抗体结合的有效性，并在固定蛋白质前后通过接触角测量和免疫荧光测量对修饰后的表面进行表征。此外，我们通过固定化抗体结合EpCAM阳性人结肠腺癌细胞系（LoVo）和EpCAM阴性小鼠胚胎成纤维细胞系（3T3）的能力来评估其功能。基于DAPI阳性信号进行细胞计数，并使用共聚焦激光扫描生物显微镜进行记录。我们的研究结果表明，APTES、APTES + BMS和APTES + OFPOS的固定能力和反应性有所不同。建议用无反应性硅烷（BMS、OFPOS）对APTES进行修饰，以提高抗体结合效率。然而，使用OFPOS导致更有效的抗体和细胞结合，并且它似乎是特异性抗体介导的细胞识别中最有用的化合物。
