(620aw) Development of ScFv-Immobilized Latex for Detection of CRP By Latex-Enhanced Turbidimetric Immunoassay

Miyamura, Y., Kyoto Institute of Technology
Kumada, Y., Kyoto Institute of Technology
Horiuchi, J. I., Kyoto Institute of Technology

There are many immunoassays utilizing antigen-antibody reaction. Among them, latex-enhanced turbidimetric immunoassay for detection of C-reactive protein (CRP) has been widely used because rapid and quantitative detection is possible by just mixing human serum with latex beads immobilized with anti-CRP antibody. The pentameric antigen, CRP can be interacted with antibody molecules that were immobilized on the surface of latex, and then the latices are aggregated via CRP antigen. Consequently, turbidity of solution increased with CRP concentration. Therefore, the CRP concentration in serum could be quantitatively measured from the turbidity change.

However, there are some problems in the conventional antibody-immobilized latex for turbidimetric immunoassay. Production cost of whole antibody which has been used in this immunoassay is considerably high because it is produced by hybridoma cell or animals. Furthermore, remaining activity of antibodies in adsorption state might be significantly low due to suffering from conformational change after adsorption.

We investigated preparation of latex immobilized with single-chain Fv (scFv) antibody. ScFv is the fusion protein which consists of VH and VL domains via the flexible linker (G4S)3 and can be produced by recombinant E. coli. The material-binding peptide, PMMA-tag (DVEGIGDVDLVNYFEVGATYTFNK) which was originally-developed by this research group was also introduced to the C-terminus of scFv (scFv-PM). PMMA-tag has strong affinity toward poly(methyl methacrylate) (PMMA) and the other plastics.

ScFv-PM could be immobilized on the surface of latex and consequently, remaining activity of scFv-PM on the latex surface became higher than that of scFv without PMMA-tag immobilized, indicating that PMMA-tag prevented conformational change in scFv molecules after immobilization. Approximately 50 ng/ml of CRP was detected by use of the scFv-immobilized latices prepared in this study. Thus, scFv-immobilized latices is useful for economical and sensitive detection for CRP by latex-enhanced turbidimetric immunoassay