Chemistry: analytical and immunological testing – Metal or metal containing – Mn – te – re – fe – ru – os – co – rh – ir – ni – pd – pt
Patent
1996-06-20
1999-09-28
Achutamurthy, Ponnathapura
Chemistry: analytical and immunological testing
Metal or metal containing
Mn, te, re, fe, ru, os, co, rh, ir, ni, pd, pt
436518, 436172, 436536, 436537, 556136, 25230116, 25230133, 546 10, G01N 3320, G01N 2176, C07F 1500, C09K 1106
Patent
active
059587830
DESCRIPTION:
BRIEF SUMMARY
DESCRIPTION
The present invention concerns new metal complexes with a charged linker 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 and the ligands are bound via nitrogen atoms to the ruthenium.
The groups capable of coupling are introduced in a very complicated manner by activation and consecutive reactions on the solubilizing groups of the ligands. The production of monoactivated compounds which enable coupling to biological substances such as antibodies without cross-linking also proves to be particularly complicated.
EP-A-0 580 979 discloses the use of osmium and 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.
Further 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 the state of the art.
Surprisingly it was found that the introduction of free positive or/and negative charge carriers into the linker which links the reactive coupling group of the metal complex to one of the ligands 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.
Furthermore it was found that luminescent metal complexes with a charged linker can be produced in a surprisingly simple manner.
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 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 the counterions which may be required to balance the charge wherein the linker contains at least one positive or/and negative charge carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1-5 show preferred compounds of the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
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 particularly preferred. Ruthenium is most preferred.
The ligands L.sub.1, L.sub.2 and L.sub.3 are ligands containing at least two nitrogen-containing heterocycles. Aromatic heterocycles are preferred such as bipyridiyl, bipyrazyl, terpyridyl and phenanthrolyl. The ligands L.sub.1, L.sub.2 and L.sub.3 are preferably selected from bipyridi
REFERENCES:
patent: 4745076 (1988-05-01), Muller et al.
patent: 5075447 (1991-12-01), Muller et al.
Terpetschnig et al., Biophysical journal., vol. 68., pp. 342-350, Jan. 1995.
International Patent Application No. WO 86/02734 published May 9, 1986.
Hoss Eva
Josel Hans-Peter
Ofenloch-Hahnle Beatus
Seidel Christoph
Upmeier Barbara
Achutamurthy Ponnathapura
Ponnaluri P.
Roche Diagnostics GmbH
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