Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-11-24
2001-07-10
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S005000, C435S007710, C435S007910, C435S007920, C435S007930, C435S007940, C435S007950, C436S514000, C436S518000, C436S523000, C436S526000, C436S528000, C436S531000, C436S533000, C436S536000, C436S537000
Reexamination Certificate
active
06258551
ABSTRACT:
The present invention relates to a method for carrying out an immnunoassay in a multiphase system.
Immunological detection methods have become verve 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 o: which is linked to the antibody while the other is linked to the analyte derivative. In this case, the analyte which is to be detected prevents formation of the complex between the labeled antibody and the labeled analyte derivative.
ECIA (enzyme channelling immunoassay; Anal. Biochem. 1056: 223, 1979; Appl. Biochem. Biotechnol. 6, 53-64, 1981) makes use of an antibody and of an analyze tracer each of which carries a different enzyme. The product of the first enzymic reaction constitutes the substrate for the second enzymic reaction. The overall velocity of the two reactions is markedly increased by this co-immobilization.
In SLFIA (substrate-labeled fluorescent immunoassay), an analyte derivative which is labeled with an enzyme substrate competes with the analyte for the binding sites of the anti-analyze antibody. Binding of the substrate-labeled analyte derivative to the antibody prevents the substrate from being reacted enzymically (Burd J. F., Feeney J. E., Carrico R. J., Bogulaski R. C.: Clin. Chem. 23, 1402, 1977; Wong R. C., Burd J. T., Carrico R. J., Buckler R. T., Thoma J., Bogulaski R. C. Clin. Chem. 25, 686, 1979).
If a fluorescent compound is excited in solution with polarized light, the emission which is observed is also polarized. The degree of this polarization depends on the mobility of the excited molecule. The decreasing mobility of a fluorescent tracer when the latter is bound to an antibody is used, in a fluorescence polarization immunoassay, to differentiate between free and bound tracer.
A fluorescence protection immunoassay (H. E. Ullmann: Tokai J. Exp. Clin. Med., Vol. 4, Supplement, pp. 7-32, 1979) is a homogeneous assay which operates in accordance with the competitive method.
In a conventional competitive assay, sufficient anti-analyte antibodies remain free, when analyte concentrations are low, for binding the tracer in such a manner that the label is no longer accessible to an anti-fluorescein antibody and can consequently no longer be quenched. This steric screening can be made even more effective by coupling the anti-analyte antibodies to a sterically demanding component.
In the solid phase antigen technique, the binding of an unwieldy analyte derivative to the tracer antibody prevents, in an analogous manner, it binding simultaneously to the anti-fluorescein antibody.
In a variant of the fluorescence protection immunoassay, the nonspecific absorption of light by active charcoal due to its coupling to the anti-fluorescein antibody is exploited to increase the quenching effect (scavenging effect).
Other techniques have been described, such as, for example, ALFPIA (antigen-labelled fluorescence protection assay; Clin. Chem. 25: 1077, 1979) or SPA (scintillation proximity assay; U.S. Pat. No. 4,569,649; WO 90/11524), in which a signal is generated by means of a radioactive tracer binding close to a scintillator.
The object underlying this invention was to provide an improved method for carrying out an immunoassay, which method is superior to the known methods, particularly as regards sensitivity, lack of susceptibility to potentially interfering influences and implementation.
The object was achieved by employing, in the present novel method, besides the customary components of an r immunoassay which is known from the state of the art, such as, for example, a receptor A which can, for example, be an antigen, an antigen derivative or an antibody, and a tracer which can, independently of the test structure selected, be, for example, a labeled antibody or a labeled antigen, a receptor B as an additional component, which receptor is directed against the label and generates or qualitatively and/or quantitatively alters a signal by interacting with the label. At the same time, suitable immobilization or receptors A and B on one or more phases ensures that the tracer cannot bind simultaneously to receptors A and B. This has the advantage that a tracer which is bound to receptor A and a tracer which is not bound to receptor A can be differentiated directly since they elicit signals which are qualitatively and/o
Kasmarker Reinhard
Neuenhofer Stephan
Dade Behring Marburg GmbH
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Stucker Jeffrey
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