Abstract
An automated enzyme-linked immunosorbent assay (ELISA) system was developed using beads in a single tip (BIST) technology. Sandwich ELISA was performed on glass beads functionalized with a capture antibody via the fusion of antibody-binding streptococcal protein G and a silica/glass-binding protein Si-tag. Our system enables rapid, sensitive detection of antigens.
Highlights
Automated enzyme-linked immunosorbent assay using beads in a single tip (BIST) technology coupled with a novel anchor protein for oriented antibody immobilization
Without horseradish peroxidase (HRP)-conjugated mouse IgG1, the beads showed less than 200 RLU. These results clearly indicate that Si-tagged streptococcal protein G (SpG) binds to both silica/glass surfaces and immunoglobulin G (IgG) and functions as an anchor protein suitable for antibody immobilization
A er washing three times with 200 mL of phosphate-buffered saline (PBS), the beads were incubated with gentle horizontal rotation overnight at 4 C in a 200 mL aliquot of 10 mg mLÀ1 solution of capture antibody in 50 mM citrate buffer containing 0.15 M NaCl.[16]
Summary
Automated enzyme-linked immunosorbent assay using beads in a single tip (BIST) technology coupled with a novel anchor protein for oriented antibody immobilization By using Si-tagged SpG as an anchor protein, antibodies can be immobilized on a silica/glass surface in an oriented manner.
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