(582ed) Adsorption Characteristics of Whole Antibody and Scfv Onto Various Plastic Surfaces for Improvement of Sensitivity in ELISA

Immunoassay method utilizing antigen-antibody interaction has been widely used for biochemical research as well as clinical diagnosis. Among the immunoassay method, Enzyme-linked immunosorbent assay(ELISA), in which antibody-immobilized polystyrene(PS) plate is used as a solid support, is the most popular in the world. However, the conventional ELISA methods have several disadvantages as follows.

In Sandwich ELISA, whole antibody which is generally produced by hybridoma has been adopted as a ligand antibody. However, production cost is much expensive and thus, diagnosis price became also expensive. Furthermore, whole antibody is greatly huge protein and therefore it is hardly immobilized with high density and suitable orientation on a surface of plastic plate. Most of plastic plates for ELISA are made of PS. PS has been selected of low manufacturing cost as well as easy fabrication. However, PS may not be certainly optimum plastic for antibody immobilization.

In order to improve these problems, we fabricated new microtiter plates that are made of various materials including PS, poly(methyl methacrylate)(PMMA), and the others, and investigated to characterize antibody adsorption phenomena. On the other hand, we also investigated to immobilize a recombinant antibody fragment, especially scFv which consist of V_H and V_L domains connected by flexible linker (G₄S)₃. ScFv rapidly produced by cultivating recombinant E. colicells and easily purified using affinity chromatography. The total production cost for scFv is approximately one-tenth for whole antibody. Although scFv is obtained in insoluble fraction called inclusion body, it can be refolded by dialysis of rapid dilution, and then it can recover original antigen-binding activity. Consequently, it can be immobilized with much higher density onto plastic than conventional whole antibody because its molecular weight is about 30 kDa. In recent years, our research group developed new scFvs that are genetically fused with material binding peptides such as PS-tag and PMMA-tag. Both of them can be successfully immobilized onto the surfaces of plastics, and consequently, they could maintain much high antigen-binding activity with suitable orientation.

Particularly, it was found that PMMA-tag fused scFv(scFv-PM) was efficiently refolded by dialysis and this binding peptide has a potential to recognize not only PMMA but also some other plastic surfaces.

In this study, we selected whole antibody and scFv-PM of anti-Thyroid Stimulating Hormone(TSH) antibody as models, and aimed adsorption characteristics of these antibodies to different plastic surfaces to immobilized then with high density and remaining binding activities. Also, we evaluate adsorption amount as well as signal intensity detected by ELISA for improvement of sensitivity.