(620au) Development of PMMA Plate Immobilized with Fab Antibodies Possessing High Antigen-Binding Activity

Authors: 
Inoue, M., Kyoto Institute of Technology
Kumada, Y., Kyoto Institute of Technology
Horiuchi, J. I., Kyoto Institute of Technology

Fab antibody is a recombinant protein which consists of Fd region of heavy chain (Fab H) and full-length of light chain (Fab L). Fab antibody can be produced at lower cost compared with conventional monoclonal antibody. Since Fab antibody is useful for economical diagnosis. In general, Fab antibody is prepared from IgG type antibody (Whole Ab) by enzymatic digestion, while it can also be produced as a soluble protein in recombinant E. coli cells. However, their productivity levels are generally low.

The aim of this study is development of production process for Fab antibody with high yield and high antigen binding activity by refolding. We prepared Fab antibody by mixing Fab H and Fab L fragments, both of which was separately produced in insoluble fraction of E. coli cells and refolded. In addition, we successfully prepared Fab antibody-immobilized poly (methyl methacrylate) (PMMA) plate by immobilization of mixture of PMMA-tag-fused Fab H (Fab H-PM) and Fab L (Fab L-PM). According to the results of sandwich ELISA, we successfully demonstrated preparation of PMMA plate immobilized with highly-activated Fab antibody. We further investigated preparation of Fab antibody-PMMA plate with a wide variety of antigen-binding specificities. By use of 5 different Fab H-PM and Fab L-PM as models, we tried to prepare total 25 types of Fab antibodies on the surface of PMMA plate. The results indicated that Fab antibodies on the PMMA plate show different binding affinity against the antigen, suggesting that they originally possess different antigen specificities. Thus, the developed method in this study will be useful for efficient preparation of antibody library on a PMMA plate with a wide variety of antigen-binding specificity.