Accessing functional flexibility of immunoglobulin by nanoparticles- the size matters

• Main Authors: Yu-shiun Chen, 陳昱勳
• Other Authors: Dr.Guewha Steven Huang
• Format: Others
• Language: en_US
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/40465371139975269164

碩士 === 國立交通大學 === 奈米科技研究所 === 94 === 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.