Department of Biomolecular Engineering, Kyoto Institute of Technology, 606-8585, Japan.
J Immunol Methods. 2013 Dec 31;400-401:70-7. doi: 10.1016/j.jim.2013.10.006. Epub 2013 Oct 28.
In this study, we demonstrated the successful preparation of a Fab antibody-immobilized hydrophilic polystyrene (phi-PS) plate via one- and two-step solid-phase refolding methods. Both polystyrene-binding peptide (PS-tag)-fused Fd fragment of heavy chain (Fab H-PS) and full-length of light-chain (Fab L-PS) were individually produced in insoluble fractions of Escherichia coli cells, and they were highly purified in the presence of 8M of urea. Antigen-binding activities of Fab antibody immobilized were correctly recovered by the one-step solid-phase refolding method that a mixture of Fab H-PS and Fab L-PS was immobilized in the presence of 0.5-2M urea, followed by surface washing of the phi-PS plate with PBST. These results indicate that by genetic fusion of a PS-tag, a complex between Fab H and Fab L was efficiently immobilized on the surface of a phi-PS plate even in the presence of a low concentration of urea, and was then correctly refolded to retain its high antigen-binding activity via removal of the urea. A two-step solid-phase refolding method whereby Fab H-PS and Fab L-PS were successively refolded on the surface of a phi-PS plate also resulted in Fab antibody formation on the plate. Furthermore, both the binding affinity and the specificity of the Fab antibody produced by the two-step method were highly maintained, according to the results of sandwich ELISA and competitive ELISA using Fab antibody-immobilized plate via two-step solid-phase refolding. Thus, the solid-phase refolding method demonstrated in this study should be quite useful for the preparation of a Fab antibody-immobilized PS surface with high efficiency from individually produced Fab H-PS and Fab L-PS. This method will be applicable to the preparation of a large Fab antibody library on the surface of a PS plate for use in antibody screening.
在这项研究中，我们通过一步法和两步固相复性方法成功制备了 Fab 抗体固定化亲水性聚苯乙烯（phi-PS）板。重链 Fd 片段（Fab H-PS）和全长轻链（Fab L-PS）的聚苯乙烯结合肽（PS-tag）融合的 Fd 片段都在大肠杆菌细胞的不可溶性部分中产生，并且在 8M 脲存在下高度纯化。通过一步固相复性方法，正确回收了 Fab 抗体固定化的抗原结合活性，其中 Fab H-PS 和 Fab L-PS 的混合物在 0.5-2M 脲存在下固定在 phi-PS 板上，然后用 PBST 对 phi-PS 板进行表面洗涤。这些结果表明，通过 PS-tag 的遗传融合，即使在低浓度脲存在下，Fab H 和 Fab L 之间的复合物也能有效地固定在 phi-PS 板的表面，然后通过去除脲正确折叠以保留其高抗原结合活性。两步固相复性方法，其中 Fab H-PS 和 Fab L-PS 相继在 phi-PS 板表面复性，也导致板上形成 Fab 抗体。此外，根据使用两步固相复性法制备的 Fab 抗体固定化板进行夹心 ELISA 和竞争 ELISA 的结果，通过两步法制备的 Fab 抗体的结合亲和力和特异性都得到了高度保持。因此，本研究中展示的固相复性方法应该非常有助于从单独生产的 Fab H-PS 和 Fab L-PS 高效制备 Fab 抗体固定化 PS 表面。该方法将适用于在 PS 板表面制备用于抗体筛选的大型 Fab 抗体文库。
