Mustafaoglu Nur, Alves Nathan J, Bilgicer Basar
Department of Chemical and Biomolecular Engineering, ‡Department of Chemistry and Biochemistry, §Advanced Diagnostics and Therapeutics, ∥Mike and Josie Harper Cancer Research Institute, and ⊥Center for Rare and Neglected Diseases, University of Notre Dame , Notre Dame, Indiana 46556, United States.
Langmuir. 2015 Sep 8;31(35):9728-36. doi: 10.1021/acs.langmuir.5b01734. Epub 2015 Aug 24.
Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.
由于致力于将诊断和传感设备的尺寸缩小至微型尺寸，以提高终端用户的可及性，抗体和抗体片段的定向固定变得越来越重要。传感设备尺寸的减小要求固定化抗体保留其抗原结合活性以确保正常运行。Fab片段因其尺寸小且易于制造，在小型诊断设备中越来越常用。在本研究中，我们使用先前描述的UV-NBS（生物素）方法，利用UV能量引发Fab片段上的核苷酸结合位点（NBS）与IBA-生物素分子之间的光交联反应，用IBA-EG11-生物素连接体对Fab片段进行功能化。我们的结果表明，与其他典型固定方法相比，通过UV-NBS（生物素）方法固定生物素化的Fab片段在表面产生的固定化Fab水平最高，同时保留了抗原结合活性。UV-NBS（生物素）方法分别比物理吸附、NHS-生物素和ε-NH3（+）方法提高了432倍、114倍和29倍的抗原检测灵敏度。此外，利用通过UV-NBS（生物素）方法固定的Fab片段检测PSA的检测限（LOD）明显低于其他固定方法，PSA的LOD为0.4 pM。总之，Fab片段的位点特异性生物素化在不造成结构损伤或丧失抗原结合活性的情况下，为UV-NBS固定技术在众多诊断设备和纳米技术中的广泛应用潜力提供了支持。
