Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1996-12-23
2000-01-11
Stucker, Jeffrey
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 6, 435 771, 435 791, 435 792, 435 793, 435 794, 435 795, 436518, 436523, 436526, 436528, 436531, 436533, 436536, 436537, G01N 3353
Patent
active
060134577
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method for carrying out an immunoassay in a multiphase system.
Immunological detection methods have become very important in in vitro diagnostics. The reason for this is that they are highly specific and extremely sensitive. In addition, these assays are easy to implement. The detection methods are based on the immunological interaction between the analyte which is to be detected and its binding partner or partners.
In sandwich assays, the analyte is bound, as in a sandwich, by two different antibodies. One of the two antibodies carries a label (marker) which enables its concentration to be determined.
The sandwich method is not appropriate for small analytes since, for example for steric reasons, two different antibodies are not able to bind to the analyte simultaneously. As a rule, the competitive assays are used under these circumstances. In these assays, an analyte and a synthetic derivative of the analyte, for example, compete for the binding sites of the antibody. As a rule, either the analyte derivative (conventional competitive method) or the antibody (e.g. SPALT: solid phase antigen luminescence technique) is labeled. The labeled component is termed a tracer.
A disadvantage of the known competitive methods is that their sensitivity is relatively low as compared with that of sandwich assays since, in contrast to the situation in immunometric assays, the detection reagents cannot be employed in excess, and it is consequently not possible to displace the equilibrium position to the extent desired in favor of the immune complex to be detected.
As a rule, it is necessary to separate off the excess free tracer antibody (in the case of sandwich assays and when carrying out SPALT) or the unbound analyte tracer (in the case of the conventional competitive method) before measuring the signal emitted by the label.
In the methods known as "homogeneous assays", a separation of this nature is not required since the signals from the free and bound tracers differ from each other.
Heterogeneous assays suffer from the disadvantage that, before measuring the signal which correlates with the analyte concentration, one or more separation steps are required in order to separate off the labeled immune complex, which is usually bound to a solid phase, from the free, labeled reagent. This additional step is relatively laborious when an assay is being carried out manually and increases the susceptibility of the method to error; even when carried out on automated analytical equipment, the step of separation is disadvantageous since an additional subassembly is generally required for this method step.
For this reason, "homogeneous assays" were developed at an early stage. The homogeneous assay, which is known under the designation EMIT (enzyme-multiplied immunoassay technology) (Biochem. Biophys. Res. Commun. 47: 846, 1972), has proved to be of value for detecting small molecules, for example of drugs (e.g. steroids). In a modified EMIT, the activity of the enzyme being used as label decreases when the analyte/enzyme conjugate binds to the antibody which is directed against the analyte. This is apparently due to a diminished affinity of the substrate for the active center of the enzyme in the presence of the antibody, or to steric hindrance, or to a confirmational change in the enzyme.
A further variant of EMIT is based on inhibition of the enzymic activity by the analyte derivative which is bound covalently to the enzyme. In this case, the activity is restored when the antibody which is directed against the analyte binds to the enzyme-labeled analyte derivative. Another variant of this method has been developed for relatively large analytes such as, for example, IgG (Anal. Biochem. 102: 167, 1990). However, the sensitivity which is achieved using this method is fairly low.
FETIA (fluorescence excitation transfer immunoassay; J. Biol. Chem. 251: 4172, 1976) is based on the transfer of energy between two fluorescent molecules, one of which is linked to the antibody while the other is linked
REFERENCES:
patent: 4777145 (1988-10-01), Luotola et al.
Zhadin et al. "Spectral Manifestations of the Different Types of Binding of Acriflavine with DNA in the Ultraviolet and Visible Region", Conference: Tezisy Doki.-Vses. Konf. Spektrosk. Biopolim., 2nd (1974), pp. 52-53 (Publisher: Akad. Nauk Ukr. SSR, Fiz.-Tekh. Inst. Nizk. Temp., Kharkov, USSR.) Abstract Only.
Granato "PACE (Probe Assay--Chemiluminescence Enhanced)", Methods Mol. Biol., vol. 28(Protocols for Nucleic Acid Analysis by Nonradioactive Probes), (1994), pp. 209-216. Abstract Only.
Kasmarker Reinhard
Neuenhofer Stephan
Dade Behring Marburg GmbH
Stucker Jeffrey
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