We report a fully automated sandwich immunoassay for the determination of human insulin using antibody -protein A - bacterial magnetic particle (BMP) complexes and an alkaline phosphatase-conjugated secondary antibody. BMPs bearing protein A - MagA inserted on the external surface of the membrane were prepared in the Magnetospirillum sp. AMB-1 transconjugant for a protein A-magA fusion gene. MagA protein was used as an anchor to attach protein A onto the membrane. Protein A-BMP complexes harvested from transconjugant AMB-1 were subsequently complexed with anti-human insulin antibodies by specific binding between the Z domain of protein A and the Fc component of IgG to form the antibody - protein A - BMP complexes. The complexes were quite monodisperse after the binding of the antibody. The BMPs' monodispersity resulted in high signal and low noise in the immunoassay. The luminescence intensity ((kilocounts/s)/μg of antibody) from antibody - protein A - BMP complexes after immunoreaction was higher than that from BMPs chemically conjugated to an antibody. This was explained by a difference in dispersion. The fully automated sandwich immunoassay system using antibody -protein A - BMP complexes made possible precise assays of human insulin in serum.
|出版ステータス||Published - 2000 8月 1|
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