Chemistry: analytical and immunological testing – Metal or metal containing – Mn – te – re – fe – ru – os – co – rh – ir – ni – pd – pt
Patent
1997-01-16
1999-11-09
Adams, Donald E.
Chemistry: analytical and immunological testing
Metal or metal containing
Mn, te, re, fe, ru, os, co, rh, ir, ni, pd, pt
436 92, 436172, 436800, 546 10, 556136, 25230116, 25230133, G01N 3320, G01N 2176, C07F 1500, C09K 1106
Patent
active
059812869
DESCRIPTION:
BRIEF SUMMARY
The present invention concerns new hydrophilic metal complexes and their use as luminescent marker groups in immunoassays.
Luminescent metal complexes are known from the state of the art. EP-A-0 178 450 discloses ruthenium complexes that are coupled to an immunologically active material in which the ruthenium complexes contain three identical or different bicyclic or polycyclic ligands with at least two nitrogen-containing heterocycles and at least one of these ligands is substituted with at least one water-solubilizing group such as --SO.sub.3 H or --COOH and at least one of these ligands is substituted directly or via a spacer group with at least one reactive group such as --COOH and the ligands are bound via nitrogen atoms to the ruthenium.
EP-A-0 580 979 discloses the use of osmium or ruthenium complexes as marker groups for electrochemiluminescence. Heterocycles containing nitrogen such as bipyridines are mentioned as ligands for these complexes. WO 87/06706 discloses further metal complexes which are suitable as marker groups for electrochemiluminescence measurements.
The disadvantages of the known metal complexes of the state of the art are a poor quantum yield in electrochemiluminescence measurements due to oxygen quenching and photodissociation or/and a high unspecific binding to proteins.
Therefore the object underlying the present invention was to at least partially eliminate the disadvantages of they state of the art.
Surprisingly it was found that the introduction of C.sub.2 -C.sub.3 alkyleneoxy, C.sub.2 -C.sub.3 alkylenethio or/and C.sub.2 -C.sub.3 alkylene-amino units and in particular ethylene glycol or/and propylene glycol units into luminescent metal complexes reduces the adsorption of conjugates of these complexes with an immunologically reactive substance and thus also improves the stability and recovery of the conjugates in immunoassays. Moreover an increased quantum yield can be achieved.
In addition it was found that the properties of metal complexes can also be improved by introducing polyhydroxy units. These polyhydroxy units can be extended to form dendrimer-like structures with several generations. Moreover incorporation of polyamine structures enables the electron donor required for electrochemiluminescence measurements to be integrated directly into the ligand sphere of the complex.
A further improvement according to the invention concerns metal complexes in the form of a cage or semi-cage in which the ligands are linked together singly or multiply preferably via hydrophilic spacers. This also leads to a substantial improvement of the photostability and to a reduction of the oxygen quenching.
One subject matter of the present invention is thus a metal complex of the general formula (I): or transition metal ions, L.sub.1, L.sub.2 and L.sub.3 are the same or different and denote ligands with at least two nitrogen-containing heterocycles wherein L.sub.1, L.sub.2 and L.sub.3 are bound to the metal cation via nitrogen atoms, X is a reactive or activatable functional group which is covalently bound to at least one of the ligands L.sub.1, L.sub.2 and L.sub.3, n is an integer from 1 to 10, m is an integer from 1 to 6 and is preferably 1 to 3 and A denotes one or several negatively charged groups that are required to balance the charge wherein the complex contains at least one hydrophilic group selected from C.sub.2 -C.sub.3 alkyleneoxy units, C.sub.2 -C.sub.3 alkylenethio units, C.sub.2 -C.sub.3 alkyleneamino units and polyhydroxy units.
The metal complex is preferably a luminescent metal complex i.e. a metal complex which can generate a detectable luminescence reaction. This luminescence reaction can for example be detected by fluorescence or by electrochemiluminescence measurement. The metal cation in this complex is for example a transition metal or a rare earth metal. The metal is preferably ruthenium, osmium, rhenium, iridium, rhodium, platinum, indium, palladium, molybdenum, technetium, copper, chromium or tungsten. Ruthenium, iridium, rhenium, chromium and osmium are particularl
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Journal of the American Chemical Society, vol. 116, No. 8, Apr. 20, 1994, pp. 3399-3404, Seiler, M. et al, "Photoinduced Electron Transfer in Supramolecular Assemblies Composed of . . . ".
Chemical Abstracts, vol. 98, No. 22, May 30, 1993, abstract No. 188896x, Markovitsi, D., et al, "Annelides. VI. Photochemical Properties of Micellar Phases of Metal Ion Complexes" p. 587; pp. 531-537.
Frommberger Bruno
Herrmann Rupert
Issberner Jorg
Josel Hans-Peter
Pappert Gunter
Adams Donald E.
Ponnaluri P.
Roche Diagnostics GmbH
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