Fully Automated Chemiluminescence Immunoassay of Insulin Using Antibody- Protein A-Bacterial Magnetic Particle Complexes

摘要

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 anti-body. BMPs bearing protein A—MagA inserted on the external surface of the membrane were prepared in the Magnetospirillum sp. AMB-1 transconjugant for a pro-tein A—magA fusion gene. MagA protein was used as an anchor to attach protein A onto the membrane. Protein A-BMP complexes harvested from lransconjugantAMB-1 were subsequently complexed with anli-buman 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)/pg of antibody) from antibody—protein A—BMP complexes after inimunoreaction was higher than that from BMPs chemically conjugated to an antibody. This was explained by a difference in dispersion. The fully automated sandwich unmunoassay system using antibody— protein A—BMP complexes made possible precise assays of human insulin in serum.