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Yuan X.,University of Tsukuba | Yuan X.,Tsukuba Advanced Research Alliance TARA | Fabregat D.,8186 Llica dAmunt | Yoshimoto K.,Tsukuba Advanced Research Alliance TARA | And 2 more authors.
Colloids and Surfaces B: Biointerfaces | Year: 2012

Rabbit anti-human ferritin (anti-hFT) polyclonal immunoglobulin G (IgG) and poly(ethylene glycol) (PEG) were sequentially co-immobilized onto polystyrene submicroparticles (sMPs) to construct sMP/anti-hFT/PEG (SAP) immunolatex. Chemical immobilization of anti-hFT was performed at different pH levels to evaluate variations in antigen recognition. Basic pH disfavored conjugation of anti-hFT to sMPs, but remarkably increased its antigen recognition in comparison to that at neutral pH. We investigated this intriguing phenomenon further by assessing the kinetics of antibody binding, including the time-dependency of immobilization, antigen recognition, and orientation of bound anti-hFT. Therefore, we attributed high antigen recognition to significant electrostatic repulsion between sMPs and anti-hFT at basic pH, which predominately prevented anti-hFT access to sMPs and concurrently promoted anti-hFT orientations suitable for antigen recognition. Subsequent PEG modification maintained such anti-hFT orientation, without which antigen-accessible orientations would have decreased with time. Thus, properly oriented antibody and immediate PEGylation after antibody immobilization contributed to the formation of a high-performance SAP immunolatex. © 2011 Elsevier B.V.


Yuan X.,University of Tsukuba | Yuan X.,Tsukuba Advanced Research Alliance TARA | Fabregat D.,8186 Llica dAmunt | Yoshimoto K.,Tsukuba Advanced Research Alliance TARA | And 2 more authors.
Colloids and Surfaces B: Biointerfaces | Year: 2012

The chemical surface-modification of carboxylated polystyrene submicroparticles (sMPs) with α-methoxy-poly(ethylene glycol)-pentaethylenehexamine (mPEG-N6), which possesses multiple amino end-groups at one end, was explored with respect to modification efficiency. As a control, a PEG mono-aminated at one end (mPEG-N1) was employed in parallel experiments. Dynamic light scattering (DLS), electrophoretic mobility (μ e), Fourier transform infra-red (FT-IR) absorption, and 3-(p-carboxybenzoyl)quinoline-2-caboxaldehyde (CBQCA) assays were carried out. From reported pK a values of the amino groups, about 25% of the amino groups were protonated at pH9.5 for pentaethylenehexamine (N6), in other words, 1.5 amino groups were protonated and 4.5 amino groups were non-protonated on average for each mPEG-N6 molecule under the conditions. The multiple amino end-groups of mPEG-N6 played two different roles in modifying the sMPs: the protonated part offered electrostatic attraction between mPEG-N6 and the negatively charged sMPs; the non-protonated part covalently reacted with the active ester groups on the sMP surface after EDC-activation treatments. During the PEG-modification process, the former attractive force increased the local mPEG-N6 concentration surrounding the sMPs, which facilitated covalent conjugation to the sMPs. In contrast, protonated mPEG-N1 (80% under the same conditions) tends to cover the negatively charged sMPs, which retards the reaction of non-protonated mPEG-N1. These collaborative actions within each mPEG-N6 chain improved the PEG-modification efficiency, rending mPEG-N6 an ideal PEGylation agent relative to mPEG-N1. © 2011 Elsevier B.V.

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