Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Chemiluminescent
Reexamination Certificate
2000-12-04
2003-07-29
Le, Long V. (Department: 1641)
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Chemiluminescent
C436S536000, C436S526000, C436S172000, C436S805000, C436S806000, C435S968000, C435S007100, C204S402000, C204S403010, C205S775000, C205S777500
Reexamination Certificate
active
06599473
ABSTRACT:
The invention refers to a method for the analysis of a sample with regard to a substance contained therein.
The analysis of a liquid sample is generally concerned with the determination of the concentration of a substance (analyte) contained therein (quantitative analysis). In some cases, it is sufficient simply to determine whether the analyte is present (in a concentration exceeding a threshold value) in the sample or not (qualitative analysis). In medical applications for which the present invention is of particular importance, the analysis of body fluids (primarily blood, blood serum and urine) with regard to the analytes contained therein, such as hormones, antibodies, antigens or drugs, plays an important role.
The invention refers to the improvement of a certain type of analytic procedure which may be designated as an electrochemiluminescence binding reaction analysis (subsequently referred to as ECL-BBA standing for electrochemiluminescence biochemical binding analysis). Such a method has the following characteristic features.
a) The analytic selectivity is based on a specific biochemical binding reaction using biochemical substances which selectively can only bind to each other. Primary examples are immunological chemical binding reactions between antibodies and antigens or haptens with which the antibodies bind specifically. Other biochemical binding reactions are protein binding, in particular between avidine and biotin, the lectine carbohydrate binding, binding between receptors and ligands and the hybridization of nucleic acids.
Such specific biochemical binding reactions have been used for some time for analytic purposes. There are a plurality of differing one or multi-step reaction processes (test protocols) which finally lead, through the participation of the analyte and at least one specifically binding substance contained in the reagent system (binding reagents), to the formation of a complex characteristic for the analysis. This complex normally (but not necessarily) contains the analyte.
b) In order to render the complex, whose concentration constitutes a measure of the analytic result sought, detectable, a marking substances (label) is normally used which is coupled to a binding reagent of the reagent system, e.g. an antibody. The species comprising the marking substance and the binding reagent is designated as a conjugate.
The invention refers to cases in which the marking substance is capable of effecting an ECL-reaction. When such a substance is subjected to a suitable electrical potential on a voltametric electrode, it emits light which can be measured photometrically. A second electrochemically active substance, designated as a precursor, normally contributes to this reaction. In practice, primarily a ruthenium complex (ruthenium-tris [bipyridyl]) is used as ECL-label in combination with TPA (tripropylamine) as precursor. The two electrochemically active substances react on the electrode each releasing an electron and thereby forming a strongly reducing or oxidizing species. The subsequent redox reaction brings the ECL-label into an excited state from which it returns to the ground state with the emission of a photon. The ECL-label reaction is preferably a circular reaction so that a single label molecule emits a plurality of photons after application of a voltage to the electrode.
c) In the methods to which the invention refers, the ECL-marked complex molecules characteristic for the analysis are fixed to magnetic microparticles (beads). In practice, magnetized polystyrol balls having a diameter of typically 2 to 3 &mgr;m are used. Fixing is effected by means of a pair of specific biochemical binding partners. The pair streptavidin biotin has turned out to be particularly advantageous. The beads are coated with a streptavidin polymer. Biotin is bound to the complex molecule.
The beads with the bound marked complex are introduced into the measuring cell of a measuring apparatus. The cell is equipped the electrodes (normally a working electrode, a counter electrode and, in particular for the case of a potentiometric measurement scheme, a reference electrode) which are, necessary for generating the electrical field required for triggering the ECL-reaction. The beads are drawn onto the surface of the working electrode in the magnetic field of a magnet disposed below the working electrode. Since this normally occurs in flow-through cells with continuously flowing sample fluids, the magnetic deposition of the beads is designated as “capturing”.
Generally after the capturing step a washing step is carried out during which a washing fluid flows by the working electrode to remove unwanted components. An electric potential required for triggering the ECL-reaction is then applied to the working electrode and the resulting luminescence light is measured using a suitable optical detector. The intensity of the luminescence light is a measure for the concentration of the marked beads on the surface of the working electrode which, in turn, is a measure of the concentration of the analyte in the sample. A calibration allows calculation of the sought concentration from the measured luminescence signal.
A plurality of different variations of this type of ECL-BBA-method have been discussed and described in the literature. Such variations may refer to each of the individual aspects mentioned.
With regard to aspect a), the tests are distinguished from each other by different test protocols (for example sandwich tests and competitive tests, each with a plurality of different sub-variations). A fundamental difference obtains between homogeneous tests which do not require separation between the formed complex molecules and the non-complexed conjugate and heterogeneous tests which require such a bound/free separation. The present invention can be used for very differing test protocols as long as they include a reaction sequence which comprises at least one specific chemical binding reaction and which leads to the formation of a complex which is characteristic of the analysis and which is marked with an ECL-label.
Also with regard to aspect b) the invention is universally applicable, i.e. it is independent of the ECL-label used and possible additional components of the ECL-reaction. The invention has turned out to be particularly usefully for test methods using the mentioned ruthenium complex in combination with TPA.
With regard to aspect c), the invention is solely directed to tests in which the complex characteristic for the analysis is bound to magnetic microparticles and in which these microparticles are deposited on the surface of a working electrode in the magnetic field of the magnet. The invention is otherwise independent of variations of aspect c) and can e.g. be used with differing bead materials and sizes as well as differing methods for fixing the complex to the beads.
More detailed information concerning the ECL-BBA-method can be taken from the extensive literature. Towards this end in particular the following publications are cited, the complete disclosure of which is hereby incorporated by reference:
1) G. F. Blackburn et al. “Electrochemiluminescence Detection for Development of Immunoassays and DNA Probe Assays for Clinical Diagnostics”, Clin. Chem. 37 (1991), 1534-1539
2) J. K. Leland and M. J. Powell: “Electrogenerated Chemilumenescence: An Oxidative-Reduction Type ECL Reaction Sequence using Triprolyl Amine”, J. Electrochem. Soc., 137(1990), 3127-3131
3) J. H. Kenten et al.: “Improved Electrochemiluminescent Label for DNA Probe Assays: Rapid Quantitative Assays of HIV-1 Polymerase Chain Reaction Products”, Clin. Chem. 38 (1992), 873-879
4) N. R. Hoyle: “The Application of Electrochemiluminescence to Immunoassay-based Analyte Measurement” in “Bioluminescence and Chemilumenescense”; Proceedings of the 8
th
International Symposium on Bioluminescence and Chemilumenescence, Cambridge, September 1994, A. K. Campbell et al. (edit.), John Wiley & Sons
5) WO 89/10551
6) WO 90/11511
As mentioned, the measurement of the ECL-light is normally carried out in
Egger Martin
Mueller Guenter
Pauselius-Fuchs Ursula
Punzmann Gabriele
Cheu Jacob J.
Le Long V.
McDonnell & Boehnen Hulbert & Berghoff
Roche Diagnostics GmbH
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