Enhancement of binding avidity by bivalent binding enables PrPSc-specific detection by anti-PrP monoclonal antibody 132

Anti-prion protein (PrP) monoclonal antibody 132, which recognizes mouse PrP amino acids 119–127, enables us to reliably detect abnormal isoform prion protein (PrPSc) in cells or frozen tissue sections by immunofluorescence assay, although treatment with guanidinium salts is a prerequisite. Despite the benefit of this mAb, the mechanism of PrPSc-specific detection remains unclear. Therefore, to address this mechanism, we analyzed the reactivities of mono- and bivalent mAb 132 to recombinant mouse PrP (rMoPrP) by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). In ELISA, binding of the monovalent form was significantly weaker than that of the bivalent form, indicating that bivalent binding confers a higher binding stability to mAb 132. Compared with other anti-PrP mAbs tested, the reactivity of bivalent mAb 132 was easily affected by a decrease in antigen concentration. The binding kinetics of mAb 132 assessed by SPR were consistent with the results of ELISA. The dissociation constant of the monovalent form was approximately 260 times higher than that of the bivalent form, suggesting that monovalent binding is less stable than bivalent binding. Furthermore, the amount of mAb 132 that bound to rMoPrP decreased if the antigen density was too low to allow bivalent binding. If two cellular PrP (PrPC) are close enough to allow bivalent binding, mAb 132 binds to PrPC. These results indicate that weak monovalent binding to monomeric PrPC diminishes PrPC signals to background level, whereas after exposure of the epitope, mAb 132 binds stably to oligomeric PrPSc in a bivalent manner.抗朊蛋白（prion protein, PrP）单克隆抗体132可识别小鼠PrP的119至127位氨基酸。尽管需以胍盐处理作为前提条件，但该抗体可通过免疫荧光试验，实现在细胞或冰冻组织切片中可靠检测异常型朊蛋白（prion protein scrapie, PrPSc）。尽管该单克隆抗体具备上述优势，但其特异性识别PrPSc的分子机制仍未阐明。为此，本研究通过酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）与表面等离子体共振（surface plasmon resonance, SPR）技术，分析单价与二价形式的132号单克隆抗体对重组小鼠PrP（recombinant mouse PrP, rMoPrP）的反应活性。ELISA结果显示，单价形式抗体的结合活性显著弱于二价形式，表明二价结合可赋予132号单克隆抗体更高的结合稳定性。与其他受试抗PrP单克隆抗体相比，二价形式的132号单克隆抗体的反应活性极易受抗原浓度降低的影响。通过SPR测定的132号单克隆抗体结合动力学结果与ELISA实验结果一致。单价形式抗体的解离常数约为二价形式的260倍，这表明单价结合的稳定性低于二价结合。此外，当抗原密度过低而无法实现二价结合时，与rMoPrP结合的132号单克隆抗体的数量会减少。当两个细胞型PrP（cellular PrP, PrPC）间距足够近以支持二价结合时，132号单克隆抗体可与PrPC结合。上述结果表明，与单体PrPC的弱单价结合会将PrPC的信号强度降至背景水平；而当表位暴露后，132号单克隆抗体可通过二价方式与寡聚型PrPSc稳定结合。