(131b) Immobilization and Orientation Controll of Anti-CRP Scfv on a Seuface of Polystyrene Latex Beads and Its Application to Latex-Turbidimetoric Assay

Kumada, Y., Kyoto Institute of Technology
Miyamura, Y., Kyoto Institute of Technology
Hirayama, Y., Denka Corporation
Horiuchi, J. I., Kyoto Institute of Technology
 In this study, we investigated immobilization of anti-C-Peactive Protein (CRP) single-chain Fv antibodies onto a surface of polystyrene latex beads for use in latex turbidimetoric assay of CRP. Both Polystyrene-binding peptide (PS-tag) and Poly(methyl methacrylate)-binding peptide-(PMMA-tag) were introduced to its C-terminal region for site-specific attachment and orientation control of scFv, while scFv with D10-tag which was decapeptide only composed of Asp was used as a negative control. Three types of scFvs were over-expressed in an insoluble fraction of E. coli cell, and highly purified via IMAC under the reduced and denatured condition. Conformation of tagged scFvs were refolded by one-step dialysis using 50mM Tris-HCl, pH 8.5 for PMMA-tagged scFv (scFv-PM) and D10-tagged scFv (scFv-D10), and 50mM MES, pH 6.0 for PS-tagged scFv (scFv-PS). Consequently, according to the results of indirect ELISAs and sandwich ELISA, antigen-binding activities of all scFvs tested were fairly recovered by the refolding operation.

 ScFv-immobilized PS latex beads were prepared by the passive adsorption method. Averaged diameter of scFv-PS were significantly increased probably because multi-point interaction of positively-charged PS-tag induced aggregation of PS latex beads which were negatively-charged. On the other hand, latex beads with scFv-PM and scFv-D10 were well-dispersed at the pH between 7.5 and 8.5, while scFv-PM-immobilized latex beads were aggregated at pH 6.5, suggesting that surface charge of scFv-PM-immobilized latex beads might be neutral at this pH which was correspondence to isoelectric point of scFv without PMMA-tag. These results expected that scFv molecules were site-specifically immobilized through the PMMA-tag. ScFv-D10-immobilized latex beads were not aggregated at the pH range between 6.5 and 8.5, suggesting that scFv-D10 molecules were randomly oriented and change in charge density of latex beads was not significant so much because the exposed D10-tag might be caught up on surface charge density which must be reduced by immobilization of scFv.

 Turbidimetoric assays were performed using both scFv-PM and scFv-D10-immobilized latex beads. Consequently, scFv-PM-immobilized latex beads had wider detection range of CRP, compared with scFv-D10-immobilized latex beads.

 Thus, scFv-PM-immobilized latex beads developed in the present study will be useful for economic and sensitive detection of biomarkers using latex-turbidimetric assay.