藉由不同粒徑奈米粒子證明免疫球蛋白辨識粒徑差異性

Abstract

免疫球蛋白(IgG)具有彈性鉸鏈可以自由的旋轉與移動Fab片段和 Fc片段。具備自由旋轉與移動的能力可以提供抗原抗體具備更佳的辨識能力。然而，用傳統的生物分子替代抗原，作為免疫球蛋白(IgG)辨識抗原決定部位的研究是有困難的，因為大多數的生物分子(例如:蛋白質)有結構的變化和序列辨識構造問題。因此我們提出一個假說應用不同粒徑大小的奈米金粒子(GNP)作為抗原抗體彈性辨識研究的引子，經由奈米金粒子(GNP)的使用可作為量測Fab片段與抗原結合彈性大小的一把尺。利用酵素連結免疫吸附分析進行實驗將抗5奈米金粒子(GNP)的抗血清和已結合3.5奈米、4.5奈米、5奈米、6奈米、8奈米、12奈米、17奈米金粒子(GNP)的表面進行分析。實驗結果發現4.5奈米與5奈米金粒子(GNP)與抗血清中蛋白有最大的結合，3.5奈米與6奈米金粒子(GNP)與抗血清中蛋白有部分結合。然而，在8奈米或是更大粒徑的奈米金粒子(GNP)則無結合現象。雖然在免疫球蛋白(IgG)中的兩個Fab片段可以自由的旋轉與移動是已證實的結果，但經由我們的實驗結果發現免疫球蛋白(IgG)的鉸鏈當進行抗原辨識過程中彈性與自由度是受到限制的。 我們也同時證明奈米金粒子(GNP)與免疫球蛋白(IgG)複合體構造的關係。利用抗血清與5奈米奈米金粒子(GNP)和量子點(QD)共同沉澱，形成IgG(QD-IgG)進而形成GNP-IgG-QD複合體經由低溫電子顯微鏡進行觀察。經由穿透式電子顯影鏡證實GNP-IgG-QD結合在一起形成三個一組。經由量測結果得知GNP-IgG-QD的夾角範圍由35度到120度，由上述結果應證了Fab-Fc具有彈性鉸鏈。總結上述實驗結果我們發現在Fab片段進行抗體抗原辨識過程是彈性是受到限制的，而Fab-Fc的彈性是比較大的。

Immunoglobulin contains flexible hinges that allow free rotation and movement of Fab and Fc fragments. This free rotation and movement would provide broader search for the antibody-antigen recognition; however, the flexibility of immunoglobulin regarding the epitope binding is difficult to measure using traditional biomolecules as antigen. This is in parts due to the dynamic and sequence-specific structure of most biomolecules, i.e. protein. The hypothesis underlined the current study is to probe the flexibility of antigen-antibody recognition by applying a wide range of gold nanoparticles (GNPs). The collection of GNPs thus will serve as molecular ruler to measure the functional flexibility of Fab fragment upon serching for antigen. Enzyme-Linked Immunosorbent Assay (ELISA) was performed using anti-5 nm GNP antiserum to bind microwells coated with 3.5 nm, 4.5 nm, 5 nm, 6 nm, 8 nm, 12 nm, and 17 nm GNPs. Maximal binding was observed at 4.5 nm and 5 nm GNPs. Partial binding activity was observed for 3.5 nm and 6 nm GNP. Nevertheless, the antiserum does not bind to 8 nm or larger GNPs. Although free rotation and movement of the two Fab fragments are expected, our result indicated that the hinges allowed only a very limited freedom during the antigen recognition process. We also examined structure of GNP-immunoglobulin complex. The antiserum was co-precipitated with 5 nm GNP and quantum dots-conjugated IgG (QD-IgG) to form a GNP-IgGs-QD complex and examined under cryo-electron microscopy. EM image confirmed the composition of GNP-immunoglobulins- QD trio. The obtained angles of GNP-immunoglobulins-QD ranged from 35 to 120 degrees which indicated that the flexibility of Fab-Fc hinge was comparable to previous reports. In summary, during the antibody-antigen recognition the Fab arms showed only a very limited flexibility while Fab-Fc shows much bigger degrees of freedom.