Fremgangsmåde til elektrokemisk påvisning af bindingsreaktioner

摘要

#CMT# #/CMT# Carrying out homogeneous immunoassay format with electrochemical detection, in a solution, comprises combining two different conjugates as reagents with a sample or a sample-buffer mixture. The first conjugate comprises a redox marker and an analyte molecule. The second conjugate comprises an anti-redox marker antibody or its specific binding fragment, and a molecule binding specifically to the analyte. #CMT# : #/CMT# Independent claims are also included for: (1) a redox marker antibody or its fragment, which binds specifically to the redox marker, comprising ferrocene or its derivatives, osmium-bipyridine-complex or other osmium-based complexes, ruthenium-bipyridyl-complex or other ruthenium-based complexes, p-aminophenol, hexacyanoferrate (II/III), quinone or other redox markers suitable for electrochemical immunoassays, where the redox activity of the bound redox marker is largely suppressed (quenched), preferably upto greater than 90%, preferably greater than 95%, or completely; and (2) a bispecific antibody conjugate for carrying out the above method, comprising the redox marker-antibody or its fragment, and an anti-analyte-antibody or its fragment. #CMT#USE : #/CMT# The method is useful for: washing and separating free immunoassays in diagnosis, preferably for automated and microfluidic immunoassays; inexpensive immunoassay systems and single use test strips; clinical diagnostics; and environmental and food analysis. #CMT#ADVANTAGE : #/CMT# The method: exhibits significantly improved sensitivity, preferably approximately 1000-folds more sensitivity than the conventional immunoassays; is fast since the signal in realtime is generated proportional to the antibody-antigen binding; and has good miniaturization and technical simplicity and thus it can be used for applications outside the laboratory. #CMT#BIOTECHNOLOGY : #/CMT# Preferred Method: The presence of free analyte in the sample causes or promotes the binding of the redox marker to the anti-redox marker antibody, where the redox activity of the bound redox marker is suppressed (quenched) by this binding, and a detectable signal is generated or modified. The required reagents are added in soluble form, and the electrochemical detection of the binding event takes place in realtime. All the required reagents are added as a ready mixture to the sample. All the required reagents are provided as dry reagents in a suitable reaction container, and are dissolved by adding the sample and optionally the buffer, which marks the beginning of the analysis. The immunoassay is carried out in a microfluidic analysis system (Lab-on-a-chip) and/or on a completely automatic analyzer. The method makes use of: a structured electrode e.g. an interdigitated electrode instead of a simple electrode for the detection; and an enzymatically catalyzed cyclic reaction instead of a simple electrochemical redox reaction. Preferred Components: The redox marker comprises ferrocene or its derivatives, osmium-bipyridine-complex or other osmium based complexes, ruthenium-bipyridyl complex or other ruthenium based complexes, p-aminophenol, hexacyanoferrate (II/III), quinone or other redox markers for electrochemical immunoassays. The redox marker is ferrocenium. The anti-redox marker antibody is a monoclonal, anti-ferrocenium-antibody. The molecule binding specifically to the analyte comprises an anti-analyte-antibody, an aptamer, a peptide, a nucleic acid or a chelator, which binds specifically to the analyte, preferably anti-analyte-antibody or its fragment. The two conjugates comprise water-soluble linker molecule, preferably polyethylene glycol-linkers. The redox marker-antibody comprises the anti-ferrocenium antibody or its fragment, which largely or completely suppresses the redox activity of the ferrocenium bound to it. #CMT#EXAMPLE : #/CMT# Acetate buffer (450 mu l) with a pH of 4.4 was filled in an electrochemical measuring cell with a working volume of 1 ml and a potentiostat in the amperometric mode. Then the working electrode was polarized at +100 mV (against silver/silver chloride). Then the bispecific antibody conjugate (10 mu l) and the redox marker progesterone conjugate (10 mu l) was added to the measuring cell. Then hydrogen peroxide/peroxidase (30 mu l) was added and the basic signal was measured. Then undiluted human serum was added (500 mu l). Then the final signal was measured 180 seconds after the addition of the serum.