In this study, immobilization of single-chain Fv (scFv) antibodies on the surfaces of polystyrene (PS) latex beads via material-binding peptides was investigated for sensitive immuno-turbidimetric assay of C-reactive protein (CRP). Anti-CRP scFvs fused with polystyrene-binding peptide (PS-tag) and poly(methylmethacrylate)-binding peptide (PMMA-tag) were over-expressed in Escherichia coli cells and recovered in the active form following refolding. The beads with PMMA-tag-fused scFv (scFv-PM) were successfully suspended with sufficient dispersion at pH 8.0. Three types of alternative scFv-PMs with a penta-asparatic acid tag (D5-tag) introduced at different positions were then designed. All of the D5-tagged scFv-PMs were successfully immobilized on the surfaces of beads with no significant change in the diameter of the latex beads at pH levels ranging from 6.0 to 8.0. According to the results of turbidimetric assay for the detection of CRP, 13 ng/ml of CRP was detectable using beads with D5-tagged scFv-PMs at 400 ng/cm³, and no turbidity change was observed in the absence of antigen. When the density of scFv-PM was 250 ng/cm², which was 63% of the maximum density, the beads were dispersed well and reactive with the antigen at a concentration range comparable to those with D5-tagged scFv-PMs. These results indicate that controlling charge density on the surface of beads after site-directed immobilization is definitely important in order to maintain high levels of dispersion and reactivity. Thus, the usefulness of the scFv-PM as well as D5-tagged scFv-PMs developed in the present study should be significant when used as ligand antibodies in the preparation of immuno-latex beads.

在这项研究中，研究了通过材料结合肽将单链Fv（scFv）抗体固定在聚苯乙烯（PS）乳胶珠表面的方法，以用于C反应蛋白（CRP）的敏感免疫比浊测定。与聚苯乙烯结合肽（PS-tag）和聚（甲基丙烯酸甲酯）结合肽（PMMA-tag）融合的抗CRP scFvs在大肠杆菌中过表达细胞并在重折叠后以活性形式回收。具有PMMA标签融合的scFv（scFv-PM）的珠子已成功悬浮，并在pH 8.0下具有足够的分散性。然后设计了三种类型的替代scFv-PM，它们在不同位置引入了五-天冬氨酸标签（D5-标签）。所有D5标签的scFv-PMs已成功固定在珠子的表面上，在pH值为6.0至8.0的范围内，胶乳珠子的直径没有明显变化。根据比浊法检测CRP的结果，使用带有D5标签的scFv-PMs浓度为400 ng / cm ^3的珠子可检测到13 ng / ml CRP ，并且在没有抗原的情况下未观察到浊度变化。scFv-PM的密度为250 ng / cm ^2时在最大密度的63％的条件下，微珠分散良好，并在与D5标记的scFv-PMs相当的浓度范围内与抗原反应。这些结果表明，定点固定后控制珠子表面的电荷密度对于保持高水平的分散性和反应性绝对重要。因此，当在制备免疫胶乳珠子中用作配体抗体时，本研究中开发的scFv-PM以及带有D5标签的scFv-PM的实用性应该很重要。