Sensitive amplification immunoassay for human IgG and anti-human IgG by using an enzyme cascade system |
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Authors: | Atsushi Seki Eiichi Tamiya Isao Karube |
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Institution: | Research Centre for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153 Japan |
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Abstract: | A sensitive heterogeneous immunoassay for human IgG and anti-human IgG was developed using an enzyme cascade system in limulus amoebocyte as a signal amplification system. Lipopolysaccharide (LPS) was conjugated to human IgG and anti-human IgG was adsorbed on polystyrene beads. The LPS-labelled human IgG mixed with unlabelled human IgG was allowed to react in a competitive manner with the immobilized anti-IgG on the polystyrene bead surface. After B/F separation, the LPS activity in the supernatant (free) and LPS activity on the bead (bound) were measured by using the chromogenic limulus test. IgG could be measured in the range 10?7-10?11 g ml?1. LPS-labelled anti-IgG and IgG absorbed on polystyrene beads were prepared, and LPS-labeled anti-IgG mixed with unlabelled anti-IgG was allowed to react again in a competitive manner with solid-phase IgG. The LPS activity specifically bound to the bead was then measured. Anti-IgG could be measured in the range 10?7-10?11 g ml?1. |
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