457175 Immobilization and Orientation Controll of Anti-CRP Scfv on a Seuface of Polystyrene Latex Beads and Its Application to Latex-Turbidimetoric Assay

Monday, November 14, 2016: 12:50 PM
Mission I (Parc 55 San Francisco)
Yoichi Kumada1, Yohei Miyamura1, Koichi Takahashi2, Yoshiaki Hirayama2, Fumio Gondaira3 and Jun-ichi Horiuchi1, (1)Bio-molecular Engineering, Kyoto Institute of Technology, Kyoto, Japan, (2)Denka Corporation, Tokyo, Japan, (3)Denka-Seiken, Tokyo, Japan

 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.


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