Efficient inhibition of interfacial nonspecific interaction to create practically utilizable high ferritin-response immunolatex.
Immunolatex particles (LAmP-s), which were prepared by covalently co-immobilizing antiferritin and a mixture of pentaethylenehexamine-ended poly(ethylene glycol) (N6-PEG) of two different chain lengths onto the surface of polystyrene submicroparticles, were formulated with various antiferritin loads. Following the quantification of the bound antiferritin, as well as the differentiation of the physically adsorbed antiferritin from chemically bound antiferritin, using the copper reduction/bicinchoninic acid reaction (the Micro BCA method), dynamic light scattering (DLS) and electrophoretic mobility (mu(e)) measurements were performed to characterize the size, homogeneity, and surface charge of the complex. Compared to the control immunolatex particles, which were prepared similarly but traditionally using bovine serum albumin (BSA) as a blocking agent (LAB-s), the LAmP-s complex showed a difference only in the surface charge property, because of the altered surface treatment in the case of the LAmP-s (PEGylation) and LAB-s complexes (BSA covering). However, the LAmP-s complex was much more reactive than the LAB-s complex, not only in phosphate buffer (PB, 10 mM, pH 7.4) but also in 100% fetal bovine serum (FBS), as measured by the turbidimetric monitoring method. The electrical repulsion between the negatively charged LAB-s complex and the antigen was the primary obstacle in the former case, and the overwhelming nonspecific deposition of contaminants from FBS onto the LAB-s complex was the main reason in the latter case. Moreover, the PEGylation procedure allowed the LAmP-s complex to possess invariable size and reactivity for at least one month at 4 degrees C without salt, and colloidal stability at high salt concentrations up to 2.0 M for at least one week, obviously demonstrating that the PEG surface modification technique described in this paper is a promising method of constructing efficient immunoassay systems.
Yuan
Xiaofei
X
Graduate School of Pure and Applied Sciences, University of Tsukuba, Ten-noudai 1-1-1, Tsukuba, Ibaraki 305-8577, Japan.
Fabregat
Dolça
D
Yoshimoto
Keitaro
K
Nagasaki
Yukio
Y
D000906:Antibodies / Q000737:chemistry* / Q000276:immunology; D000937:Antigen-Antibody Reactions; D005293:Ferritins / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000276:immunology; D007118:Immunoassay; D010316:Particle Size; D011073:Polyamines / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D011137:Polystyrenes / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000494:pharmacology*; D013499:Surface Properties
D000906:Antibodies; D011073:Polyamines; D011137:Polystyrenes; D012710:Serum Albumin, Bovine; D011092:Polyethylene Glycols; D005293:Ferritins; C031122:pentaethylenehexamine