Fully automated chemiluminescence immunoassay of insulin using antibody-protein A-bacterial magnetic particle complexes.
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)/microg 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.
Tanaka
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T
Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Japan.
Matsunaga
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T
D000469:Alkaline Phosphatase / Q000276:immunology; D001331:Automation; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology*; D027682:Cation Transport Proteins; D015870:Gene Expression; D006801:Humans; D007118:Immunoassay; D007328:Insulin / Q000032:analysis*; D008163:Luminescent Measurements; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D013205:Staphylococcal Protein A / Q000235:genetics / Q000276:immunology*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D007328:Insulin; C097085:MagA protein, Magnetospirillum; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D000469:Alkaline Phosphatase