Ruthenium complexes useful as carriers for immunologically active materials

The invention relates to a ruthenium complex having the formula EQU Ru.sup.2+ L.sub.1 L.sub.2 L.sub.3 I wherein L.sub.1, L.sub.2 and L.sub.3 are the same or different are equal to a bi- or polycyclic ligand with at least two nitrogen-containing heterocycles, whereby at least one of these ligands is substituted with at least one group conferring water-solubility, and whereby at least one of these ligands is substituted, optionally via a spacer group, with at least one reactive group, and whereby the ligands L.sub.1, L.sub.2 and L.sub.3 are attached to the ruthenium via nitrogen atoms. The invention is further related to such ruthenium complexes having coupled thereto an immunologically active material, for example, antigens, haptens or antibodies and to the use of said ruthenium complexes in fluorescence spectroscopy. Specific ligands L.sub.1, L.sub.2 and L.sub.3 which are useful in the ruthenium complexes of the invention are, e.g., 2,2'-bipyridine, 1,10-phenanthroline benzbathophenanthroline or bathophenanthroline groups. Groups which are useful for conferring water-solubility on said ligand are, e.g. sulfonic acid groups which are preferably present in the form of their salts. Useful spacer groups are e.g. an alkylene groupcontaining 1-8 carbon atoms and which is optionally substituted with --SO.sub.2 --NH--, --S--, --O--, --COO-- or --CO--NH-- groups. Useful reactive groups to which the immunologically active material is coupled, are e.g. --COOH, --I, --NH.sub.2, --NCS or --SO.sub.2 Hal groups. The ruthenium complexes according to the present invention can be detected with great sensitivity by fluorescence spectroscopy and are thus useful in fluorescense immunoassays.

The present invention is directed to ruthenium complexes to which can be 
coupled an immunologically active material. 
The ruthenium complexes of the invention have the general formula 
EQU Ru.sup.2+ L.sub.1 L.sub.2 L.sub.3 I 
wherein L.sub.1, L.sub.2 and L.sub.3 are the same or different and are 
equal to a bi- or polycyclic ligand with at least two nitrogen-containing 
heterocycles, whereby at least one of the ligands L.sub.1, L.sub.2 or 
L.sub.3 is substituted with at least one group conferring 
water-solubility, and whereby at least one of these ligands is substituted 
optionally via a spacer group, with at least one reactive group, and 
whereby the ligands L.sub.1, L.sub.2 and L.sub.3 are attached to the 
ruthenium via nitrogen atoms. 
The ligands L.sub.1 and L.sub.2 can be the same or different and are equal 
to, for example, 2,2'-bipyridine, 1,10-phenanthroline, 
benzbathophenanthroline or especially bathophenanthroline groups. The 
ligands L.sub.1 and L.sub.2 are preferably the same. 
The ligand L.sub.3 is equal to, for example, a 2,2'-bipyridine, 
1,10-phenanthroline or especially a bathophenanthroline group. 
In the ruthenium complexes of the invention, groups which are useful for 
conferring water-solubility on the ligands L.sub.1 and L.sub.2 are 
sulphonic acid groups which are preferably present in the form of their 
salts. The sodium salts are especially preferred. 
As used herein, the term "spacer group" refers to an alkylene group having 
a maximum of 8 carbon atoms, which optionally can contain --SO.sub.2 
--NH--, --S--, --O--, --COO-- or --CO--NH-- functionalities. 
As used herein, the term "reactive group(s)" refers to the group to which 
is coupled the immunologically active material suitable "reactive groups" 
are for example, --COOH, --I--, --NH.sub.2, --NCS or --SO.sub.2 Halogen 
groups. 
As used herein 2,2'-bipyridine (bpy) refers to a compound of the formula: 
##STR1## 
1,10-phenanthroline refers to a compound of the formula: 
##STR2## 
bathophenanthroline (batho) refers to a compound of the formula: 
##STR3## 
and benzobathophencenthroline (benzobatho) refers to a compound of the 
formula 
##STR4## 
In the case of Ru complexes having 3 identical ligands L.sub.1, L.sub.2 and 
L.sub.3 the ligand must have not only a group conferring water-solubility, 
but also a spacer group with a reactive group. 
A ligand suitable for this purpose is the compound of the formula 
##STR5## 
In the case of Ru complexes which have two different ligand types, one 
ligand type can carry the group or groups conferring water-solubility, 
while the other ligand can substituted by one or more linking groups, 
i.e., a reactive group which is attached to the heterocycle via a spacer 
group. 
Preferred for use herein are ruthenium complexes wherein L.sub.1 and 
L.sub.2 are the same and which have the formula 
##STR6## 
and wherein the ligand L.sub.3 is preferably selected from the following 
group: 
##STR7## 
The synthesis of the ligand L.sub.3, carrying the linking group or groups, 
is carried out according to processes which are described schematically 
hereinafter: 
(1) Preparation of [(SO.sub.2 NH(CH.sub.2).times.COO-t-butyl).sub.2 batho] 
and [(SO.sub.3 Na)(SO.sub.2 NHCH.sub.2 CH.sub.2 COO-t-butyl)]batho. 
Briefly, the synthesis of the ligand L.sub.3, which carries the linking 
group or groups, is carried out using the disodium salt of the 
bathophenanthroline disulphonic acid as the starting material. From this 
there is firstly prepared with PCl.sub.5 the corresponding 
disulphochloride (see F. Muth in "Houben-Weyl, Methoden der Organischen 
Chemie". vol IX, p. 563, 4th edition 1955, G. Thieme Verlag, Stuttgart). 
This is subsequently converted into the corresponding sulphonamide with 
the t-butyl ester of an amino acid (such as e.g .beta.-alanine, glycine, 
4-amino-butyric acid or 6-aminocaproic acid) in accordance with the 
following Scheme (see F. Muth in "Houben-Weyl, Methoden der Organischen 
Chemie", vol IX, p. 609, 4th edition 1955, G. Thieme Verlag Stuttgart): 
##STR8## 
In the above reaction in addition to the disulphonamide there is also 
obtained the monosulphonamide as a byproduct. The saponification of the 
t-butyl ester is carried only after the synthesis of the corresponding Ru 
complex. 
(b) Preparation of [(CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 COOH)batho] or 
[(CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 COOH)batho] 
The synthesis of L.sub.3 ligands having valeric acid or caproic acid as the 
linking group is carried out by means of a Skraup's reaction (see F. H. 
Case and P. F. Strohm; J. Org. Chem. 27, 1641 (1962)) from 
4-phenyl-8-amino-quinoline (see F. H. Case; J. Org. Chem. 16, 1541 (1951)) 
and the p-[.beta.-chloropropionyl] derivatives of the corresponding 
1-phenyl fatty acid methyl esters, whereby the latter are obtained by 
acylating methyl 5-phenylvalerate (Fluka) or methyl 6-phenylcaproate (see 
W. E. Truce and C. E. Olson; J. Amer. Chem. Soc. 75, 1651 (1953)) with 
.beta.-chloropropionyl chloride, in accordance with the following Scheme: 
##STR9## 
(c) Preparation of [(CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 COOH) 
benzobatho]. 
The synthesis of this benzo-bathophenanthrolinepentanoic acid, is carried 
out via several steps from 2-amino-3-naphthoic acid in accordance with the 
following Scheme (see E. Koft and F. H. Case; J. Org. Chem. 27, 865 
(1962)). 
##STR10## 
The synthesis of Ru complexes wherein L.sub.1, L.sub.2 and L.sub.3 ligands 
are identical to each other can be carried out according to either of two 
processes described in the literature. 
According to Braddock and Meyer [J. Am. Chem. Soc. 95, 3158 (1973)] 
water-soluble ruthenium trichloride is heated together with the ligands 
for an extended period of time in DMF, whereby the complex is formed with 
partial decomposition of the solvent. 
However, the process described by Lin et al. [J. Am. Chem. Soc. 98, 6536 
(1976)] is preferably used. In this process potassium 
pentachloroaquoruthenate (K.sub.2 Ru Cl.sub.5.H.sub.2 O) is heated in a 
weak hydrochloric acid environment with a 3-fold stoichiometric amount of 
ligand and the solution is subsequently reduced with sodium hypophosphite. 
The synthesis of ruthenium complexes having 2 different ligand types is 
carried out according to known procedure, e.g in accordance with Belser et 
al. Helv. Chim. Acta 63, 1675 (1980). 
When the ligand L.sub.3 is different from the ligands L.sub.1 and L.sub.2, 
a stepwise synthesis of the complex is preferred. (As would be recognized 
by one skilled in the art, the complex can also be prepared by means of a 
statistical synthesis, but this route is not preferred, as it is difficult 
to control.) Thus, there is prepared in a first step with water-soluble 
ruthenium trichloride and double the stoichiometric amount of ligand 
L.sub.1 or L.sub.2 in dimethylformamide (with lithium chloride as the 
catalyst) the intermediate RuL.sub.1 L.sub.2 Cl.sub.2. This intermediate 
is subsequently converted into the desired mixed complex with the ligand 
L.sub.3. 
If there is used as the ligand L.sub.3 a compound whose reactive group is 
present in protected form--e.g. as the t-butyl ester in the case of the 
sulphonamide derivatives of bathophenanthroline--then the cleavage of this 
protecting group is preferably carried out only after the synthesis of the 
corresponding Ru complex. 
The isolation and purification of the ruthenium complexes formed is carried 
out according to conventional methods by reprecipitation, column 
chromatography or preparative thick-layer chromatography. 
Immunologically active materials, which are coupled to the ruthenium 
complexes of the invention, which are for example, antigens, haptens or 
antibodies (inclusive, e.g. Fab fragments). Not only polyclonal 
antibodies, but also monoclonal antibodies can be used herein. 
An especially preferred immunologically active material is an antibody 
against carcinoembryonal antigen. A further especially preferred 
immunologically active material is an antibody against human 
choriongonadotropin, as well as an antibody against a-interferon. 
The coupling of the immunological material to the ruthenium complex is 
carried out in a manner known per se. A preferred coupling method 
comprises treating the ruthenium complex and the immunologically active 
material with a water-soluble carbodiimide derivative, e.g. with 
N-cyclohexyl-N'-(2-morpholinoethyl)-carbodiimide-methyl-p-toluenesulphonat 
e. 
The ruthenium complexes of the invention can be detected very sensitively 
by fluorescence spectroscopy. They are very well suited as label molecules 
for highly sensitive fluorescence immunoassays and they are especially 
suitable for a time-resolved fluorescence immunoassay as is described, for 
example, in DT-OS 26 28 158. By using the ruthenium complexes in 
accordance with the present invention in place of the frequently used FITC 
(fluorescein isothiocyanate) the detection sensitivity in the case of 
fluorescence immunoassays can be improved. This is of advantage especially 
in the determination of small amounts of antigens in body fluids such as 
e.g. plasma and serum. Example of such antigens are e.g carcinoembryonal 
antigen (CEA), .beta.-HCG or a-interferon. 
The time-resolved fluorescence measurement is made using a measurement 
apparatus. Briefly the apparatus works as follows. A pulsating light 
source, i.e., a colour laser-excites the measurement sample to fluoresce, 
with flashes of light of suitable wavelength, i.e., g=453 nm, whereby the 
duration of flash (t=0.7 ns) is much shorter than the decay time of the 
fluorescing label. The fluorescent radiation is then directed with an 
optic through an edge filter (Balzers 610), which transmits the emission 
wavelength of the label, to the photocathode of a photomultiplier. The 
individually detected photons generate pulses of current which, after 
amplification and standardization, are counted digitally (photon counting 
method). By means of a photodiode the exciting flash of light 
simultaneously controls a gate circuit which starts the counter after an 
adjustable time delay (w=2 .mu.s) and which again stops the counting 
process after an adjustable opening time of the measurement aperture (Wt=3 
.mu.s). The time delay (W) is chosen so that during said time delay stray 
light effects as well the background fluorescence have died away almost 
completely. In this manner the number of counted pulses is proportional to 
the intensity of the fluorescence of the label, which is measured 
separately from the background.

EXAMPLE 1 
Preparation of Bathophenanthroline Disulphochloride 
2.2 g of dry bathophenanthroline disulphonic acid disodium salt (Fluka) are 
mixed well with 3.1 g of PCl.sub.5 and 750 .mu.l of POCl.sub.3 in a 500 ml 
round flask. The flask, provided with a reflux condenser and a calcium 
chloride tube, is heated to 110.degree. C. in an oil bath for 2.5 hours. 
Sublimed PCl.sub.5 which has precipitated on the cooler parts of the flask 
is scratched off periodically. Thereafter, the unreacted PCl.sub.5 as well 
as the POCl.sub.3 are removed completely at 110.degree. C. in a water-jet 
vacuum within 5 hours. After cooling to room temperature the crude 
sulphochloride is treated for a short time with 150 ml of benzene, which 
are discarded (in order to remove traces of impurities). The crude 
sulphochloride is thereafter mixed with 150 ml of chloroform and stirred 
at room temperature for 1 hour. The chloroform is removed and the 
procedure repeated. The combined chloroform extracts are concentrated in 
vacuo. The sulphochloride which remains behind is then dried at 
110.degree. C. in vacuo for 4 hours (yield: 1.8 g). 
EXAMPLE 2 
Preparation of sulphonamide derivatives of bathophenanthroline with the 
t-butyl ester of .beta.-alanine[(SO.sub.2 --NH--CH.sub.2 --CH.sub.2 
COO-t-butyl).sub.2 batho] and [(SO.sub.3 Na)(SO.sub.2 NHCH.sub.2 CH.sub.2 
COO-t-butyl)batho] 
1.32 g of .beta.-alanine t-butyl ester of 10.5 ml of triethylamine are 
dissolved in 50 ml of chloroform. To this solution are added, while 
stirring vigorously within 10 minutes, 2.0 g of solid bathophenanthroline 
disulphochloride. After stirring at room temperature for 5 hours the 
reaction mixture is left to stand in the dark for 4 days. Subsequently, 
the solvent as well as the triethylamine are removed at 
40.degree.-50.degree. C. in vacuo. In order to completely remove the 
triethylamine, the residue is treated with 200 ml of chloroform, which is 
thereafter removed in vacuo. This procedure is carried out a total of five 
times until the product no longer has an odour of triethylamine. The 
purification of the residue is carried out by chromatography over 
SiO.sub.2 and leads to two products. 
(a) Isolation of the disulphonamide 
[(SO.sub.2 NHCH.sub.2 CH.sub.2 COO-t-butyl).sub.2 batho] 
The practically pure disulphonamide (Yield: 900 mg) is first eluted from 
the column using 8 l of acetone. 
(b) Isolation of the monosulphonamide 
[(SO.sub.3 Na)(SO.sub.2 NH--CH.sub.2 CH.sub.2 --COO-t-butyl)batho] 
Using 3 l of a mixture of chloroform/methanol/water (7/3/0.5) there is 
eluted a further zone which on the basis of its fluorescence properties is 
identified by NMR as the monosulphonamide of bathophenanthroline 
disulfonic acid (yield: 1.2 g of crude product). The crude monosulfonamide 
product is further purified by dissolving it in 150 ml of chloroform. The 
resulting solution is shaken a total of three times with 100 ml of water 
each time. Thereafter, the monosulphonamide is re-precipitated twice by 
dissolving the compound in 50 ml of methanol/chloroform (3/1) and 
precipitating it by the slow dropwise addition of 200 ml of ether. (Yield: 
360 mg). 
EXAMPLE 3 
Preparation of Ru complex-Ru[(SO.sub.3 Na)(SO.sub.2 NH CH.sub.2 CH.sub.2 
COOH)batho].sub.3 (PF.sub.6).sub.2 
18.7 mg of potassium pentachloroaquoruthenate (K.sub.2 RuCl.sub.5.H.sub.2 
O) are dissolved at 60.degree. C. in 2 ml of water to which has previously 
been added 1 drop of 6N HCl. To this solution are added a 3-fold 
stoichiometric amount of ligand (95.8 mg of the tert-butyl ester of the 
monosulphonamide of Example 2 dissolved in 1 ml of DMF) and the mixture is 
heated to reflux under nitrogen for 2.5 hours. After cooling the reaction 
solution, the resulting Ru.sup.3+ complex is reduced with 250 .mu.l of a 1 
molar NaH.sub.2 PO.sub.2 solution to the corresponding Ru.sup.2+ complex 
and then boiled at reflux for a further 2 hours. The solution is 
subsequently filtered, treated with 900 .mu.l of a 10% aqueous ammonium 
hexafluorophosphate solution and left to stand at 4.degree. C. overnight 
whereupon the complex thereby precipitates. After suction filtration the 
complex is purified by means of preparative thick-layer chromatography (4 
silica gel plates, elution agent CH.sub.2 Cl.sub.2 /MeOH/H.sub.2 O 
(7:3:0.5). A red zone in the front is isolated (50 mg of pure product) 
and, for saponification, is treated with 5 ml of trifluoroacetic acid at 
room temperature. After 1 hour the trifluoroacetic acid is removed in a 
water-jet vacuum to give the Ru complex, Ru[(SO.sub.3 Na)(SO.sub.2 
NHCH.sub.2 CH.sub.2 COOH)batho].sub.3 (PF.sub.6).sub.2. 
EXAMPLE 4 
Preparation of Ru[(SO.sub.3 Na).sub.2 batho].sub.2 Cl.sub.2 
627 mg of RuCl.sub.3.3H.sub.2 O, 568 mg of LiCl and 2.36 g of 
bathophenanthroline disulphonic acid diNa salt (Fluka) are boiled at 
reflux in 8 ml of DMF for 6 hours. After cooling to room temperature the 
reaction solution is treated slowly with 60 ml of acetone and then left to 
stand at 4.degree. C. for 20 hours. There thereby precipitates the violet 
crude product. This is filtered off under suction and washed well with 
acetone. A first purification is then carried out by reprecipitation. For 
this purpose, the crude product is dissolved in 50 ml of methanol and 
subsequently again precipitated with 500 ml of acetone/ether (1/1). This 
procedure is repeated. The further purification is carried out by 
chromatography over SiO.sub.2. With 5 l of chloroform/methanol/acetone 
(4/3/3) there are eluted 600 mg of practically pure product (yield: 600 
mg). 
EXAMPLE 5 
Preparation of the mixed complex-Ru[(SO.sub.3 Na.sub.2 batho].sub.2 
[(SO.sub.2 --NH--CH.sub.2 --CH.sub.2 --COO-t-butyl).sub.2 batho]-Cl.sub.2 
The Ru derivative of Example 4 (76.2 mg) is dissolved in a mixture of 1 ml 
of water and 4 ml of methanol and mixed with 41.0 mg of the disulphonamide 
of Example 2 (dissolved in 3 ml of chloroform). This reaction mixture is 
heated at reflux under nitrogen for 3 hours, whereby an orange-red 
solution results. Subsequently, the solvent is distilled off to about 90% 
by blowing in N.sub.2 and warming slightly. Upon cooling to room 
temperature a portion of the product precipitates out. For purification, 
the product is dissolved in 1.5 ml of methanol and 0.5 ml of DMF and 
chromatographed over SiO.sub.2 with the elution agent 
chloroform/methanol/water (7:3:0.5)(yield: 75 mg). 
EXAMPLE 6 
Preparation of the Ru complex-Ru[(SO.sub.3 Na).sub.2 batho].sub.2 
[(SO.sub.2 --NH--CH.sub.2 --CH.sub.2 --COOH).sub.2 batho]Cl.sub.2 
The t-butyl ester of Example 5 is saponified as follows; Ru[(SO.sub.3 
Na).sub.2 batho].sub.2 [(SO.sub.2 --NH--CH.sub.2 --CH.sub.2 
----COO-t-butyl).sub.2 batho]Cl.sub.2 (55 mg) is dissolved in 5 ml of 
trifluoroacetic acid. The reaction mixture is left to stand at room 
temperature for 1 hour and the trifluoroacetic acid is then removed at 
40.degree. C. in a water-jet vacuum. The crude product is firstly purified 
by column chromatography over SiO.sub.2 using the following Elution 
agents: 
300 ml of chloroform/acetone/methanol, (4:3:3) 
500 ml of chloroform/methanol/water, (50:50:2) 
500 ml of chloroform/methanol/water, (50:50:5) 
250 ml of chloroform/methanol/water, (50:50:10) 
100 ml of methanol/water (10/1) 
100 ml of methanol/water (1/1). 
The final purification is carried out by means of preparative thick-layer 
chromatography. For this purpose, 40 mg of the Ru complex are dissolved in 
700 ml of water and applied to 4 PSC plates (SiO.sub.2 plates from Merck). 
These are dried at 60.degree. C. overnight and subsequently 
chromatographed with the elution agent chloroform/methanol/water 
(50:50:2). The main zone is scratched off and extracted three times with 
40 ml of water each time (the silica gel is separated by centrifugation). 
After concentration final traces of SiO.sub.2 are removed from the product 
by dissolving it in a small amount of methanol and centrifuging off the 
undissolved silica gel (yield: 38 mg). 
EXAMPLE 7 
Preparation of the bathophenanthroline disulphonamide with the t-butyl 
ester of glycine--[(SO.sub.2 NHCH.sub.2 --COO-t-butyl).sub.2 batho] 
The above-identified sulphonamide was prepared according to the procedure 
of Example 1 and Example 2 using 10.3 g of glycine-t-butyl ester 
dibenzenesulphimide salt, 28 ml of triethylamine and 4.2 g of 
bathophenanthroline disulphochloride. (Yield: 4.9 g). 
EXAMPLE 8 
Preparation of the mixed complex Ru[(SO.sub.3 Na).sub.2 batho].sub.2 
[(SO.sub.2 NHCH.sub.2 COO-t-butyl).sub.2 batho]Cl.sub.2 
3.6 g of Ru[(SO.sub.3 Na).sub.2 batho]Cl.sub.2 are dissolved in a mixture 
of 160 ml of methanol and 100 ml of water and mixed with 2.01 g of 
[(SO.sub.2 NH--CH.sub.2 COO-t-butyl).sub.2 batho] (dissolved in 80 ml of 
methanol). The reaction mixture is boiled at reflux under N.sub.2 for 5 
hours, whereby a deep red solution results. After cooling the reaction 
solution is concentrated in vacuo to about 70 ml and the mixed Ru complex 
is then precipitated by the slow dropwise addition of 1.4 l of acetone. 
For further purification, the suction filtered crude product is 
reprecipitated twice. For this purpose, the precipitate is dissolved in 
about 150 ml of MeOH/H.sub.2 O/CH.sub.2 Cl.sub.2 (2:1:0.3) and the complex 
is then again precipitated by the slow dropwise addition of 1.5 l of 
acetone. 
Thereafter, the product is chromatographed twice over silica gel with the 
elution agent CH.sub.2 Cl.sub.2 /MeOH/H.sub.2 O (7:3:0.5). After a further 
reprecipitation in a manner analogous to that described above there is 
obtained a pure product. 3.0 g of a red powder identified as the title 
compound. 
EXAMPLE 9 
Saponification of the t-butyl ester of Example 8 to Ru[(SO.sub.3 Na).sub.2 
batho].sub.2 [(SO.sub.2 NHCH.sub.2 COOH).sub.2 batho]Cl.sub.2 
Ru[(SO.sub.3 Na).sub.2 batho].sub.2 [(SO.sub.2 NHCH.sub.2 
COO-t-butyl).sub.2 batho]Cl.sub.2, (500 mg) is dispersed in 40 ml of 
trifluoroacetic acid. After stirring at room temperature for 2.5 hours the 
trifluoroacetic acid is removed in vacuo. The residue is dissolved in a 
mixture 1 ml of DMF and 3 ml of water. The complex is again precipitated 
from the solution by the slow dropwise addition of 500 ml of acetone/MeOH 
(8:2). The reprecipitation procedure is repeated twice and the resulting 
red powder is then dried at 70.degree. C. in vacuo to yield 420 mg of the 
title compound. 
EXAMPLE 10 
Preparation of the bathophenanthroline disulphonamide with the t-butyl 
ester of 4-aminobutyric acid--[(SO.sub.2 NHCH.sub.2 CH.sub.2 CH.sub.2 
COO-t-butyl).sub.2 batho] 
The disulphonamide was prepared in an analogous manner to that described in 
Example 1 and Example 2 except that 1.57 g of 4-aminobutyric acid t-butyl 
ester, 17 ml of triethylamine and 2.1 g of bathophenanthroline 
disulphochloride was used. Yield: 1.3 g of [(SO.sub.2 NH--CH.sub.2 
CH.sub.2 CH.sub.2 COO-t-butyl).sub.2 batho]. 
EXAMPLE 11 
Preparation of the Mixed Complex-Ru[(SO.sub.3 Na).sub.2 batho].sub.2 
[(SO.sub.2 NHCH.sub.2 CH.sub.2 CH.sub.2 COO-t-butyl).sub.2 batho]Cl.sub.2 
In an analogous manner as the procedure of Example 8, except that 1.28 g of 
Ru[(SO.sub.3 Na).sub.2 batho].sub.2 Cl.sub.2 and 0.775 g of [(SO.sub.2 
NHCH.sub.2 CH.sub.2 CH.sub.2 COO-t-butyl).sub.2 batho] was used, the title 
compound was prepared. Yield: 1.65 g of a red powder. 
EXAMPLE 12 
Preparation of Ru[(SO.sub.3 Na).sub.2 batho].sub.2 [(SO.sub.2 NHCH.sub.2 
CH.sub.2 CH.sub.2 COOH).sub.2 batho]Cl.sub.2 
The t-butyl ester of Example 11 (70 mg) was saponified according to the 
procedure of Example 9 to yield 50 mg of the corresponding acid identified 
as Ru[(SO.sub.3 Na).sub.2 batho].sub.2 [(SO.sub.2 NHCH.sub.2 CH.sub.2 
CH.sub.2 COOH).sub.2 batho]Cl.sub.2. 
EXAMPLE 13 
Preparation of the bathophenanthroline disulphonamide with t-butyl ester of 
6-aminocaproic acid--(SO.sub.2 NHCH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 
CH.sub.2 COO-t-butyl).sub.2 batho] 
In a manner analogous to the procedure described in Example 1 and Example 2 
except that 2.27 g of 6-aminocaproic acid and 2.1 g of bathophenanthroline 
disulphochloride is used the title compound is prepared. Yield: 1.8 g of 
[(SO.sub.2 NHCH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 
COO-t-butyl).sub.2 batho]. 
EXAMPLE 14 
Preparation of the mixed complex Ru[(SO.sub.3 Na).sub.2 -batho].sub.2 
[(SO.sub.2 NHCH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 
COO-t-butyl].sub.2 batho]Cl.sub.2 
Utilizing a similar procedure as that described in Example 8, except that 
2.2 g of Ru[SO.sub.3 Na).sub.2 batho].sub.2 Cl.sub.2 and 1.49 g of 
[(SO.sub.2 NHCH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 
COO-t-butyl).sub.2 batho was used the mixed complex of the title was 
prepared. Yield: 2.8 g of a red powder. 
EXAMPLE 15 
Saponification of the t-butyl ester of Example 14 to Ru[(SO.sub.3 Na).sub.2 
batho].sub.2 [(SO.sub.2 NHCH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 
COOH).sub.2 batho]Cl.sub.2 
Utilizing the procedure of Example 9, 100 mg of the t-butyl ester of 
Example 14 was saponified to yield the title compound. Yield: 85 mg. 
EXAMPLE 16 
Preparation of Ru[(SO.sub.3 Na).sub.2 batho].sub.2 [(SO.sub.3 Na)(SO.sub.2 
NHCH.sub.2 CH.sub.2 COO-t-butyl)batho]Cl.sub.2 
In an analogous manner as the procedure of Example 8, 102 mg of 
Ru[(SO.sub.3 Na).sub.2 batho]Cl.sub.2 and 51 mg of [(SO.sub.3 Na)(SO.sub.2 
NHCH.sub.2 CH.sub.2 COO-t-butyl)batho] were reacted to yield 105 mg of a 
red powder identified as Ru[(SO.sub.3 Na).sub.2 batho].sub.2 [(SO.sub.3 
Na)(SO.sub.2 NHCH.sub.2 CH.sub.2 COO-t-butyl)batho]Cl.sub.2. 
EXAMPLE 17 
Preparation of Ru[(SO.sub.3 Na).sub.2 batho].sub.2 [(SO.sub.3 Na)(SO.sub.2 
NHCH.sub.2 CH.sub.2 COOH)batho]Cl.sub.2 
The t-butyl ester derivative of example 16 (105 mg) was saponified 
according to the procedure of Example 9 to yield crude product. 
Purification of the crude product is carried out by preparative 
thick-layer chromatography (silica gel plates, elution agent CHCl.sub.3 
/MeOH/acetone (4:3:3))to yield 32 mg of Ru[(SO.sub.3 Na).sub.2 
batho].sub.2 [(SO.sub.3 Na)(SO.sub.2 NHCH.sub.2 CH.sub.2 
COOH)batho]Cl.sub.2. 
EXAMPLE 18 
Preparation of methyl p-[.beta.-chloropropionyl]-5-phenylvalerate 
In a stirring flask equipped with a reflux condenser are placed 9.3 g of 
AlCl.sub.3, 10 ml of CS.sub.2 and 1.98 ml of .beta.-chloropropionyl 
chloride. To this mixture are added dropwise at room temperature within 5 
minutes, 3.61 g of 5-phenylvaleric acid (rinsing is carried out with 2 ml 
of CS.sub.2). The reaction mixture is subsequently stirred for a further 
15 minutes, warmed slightly and then cooled. The reaction mixture is then 
pipetted into a stirred mixture of ice, water and ether. The organic phase 
is washed neutral with bicarbonate solution and water. After drying over 
MgSO.sub.4 the solvent is removed and 5.03 g of crystalline product are 
thus obtained. 
EXAMPLE 19 
Preparationn of 5-[p-(7-phenyl-1,10-phenanthrolin-4-yl)phenyl]-pentanoic 
acid--[(CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 COOH)batho] 
Under argon in a stirring flask are placed 2.54 g of 
4-phenyl-8-aminoquinoline, 11.5 ml of H.sub.3 PO.sub.4 (85%) and 2.3 ml of 
arsenic acid solution (80% H.sub.3 AsO.sub.4). The mixture is heated to 
120.degree. C. to dissolve the quinoline and thereafter, 4.56 g of methyl 
p-[.beta.-chloropropionyl]-5-phenylvalerate are added thereto within 5 
minutes. The reaction mixture is subsequently heated, while stirring 
within 10 minutes, to 140.degree. C. and left at this temperature for a 
further 1 hour. The reaction mixture is then cooled and pipetted into a 
stirred mixture of 50 ml of water and 125 ml of CH.sub.2 Cl.sub.2 which is 
cooled well with ice. The pH is brought to 5 by adding sodium hydroxide 
solution to pass the reaction product into the organic phase. After 
evaporation of the solvent there is obtained a crude product which 
contains a mixture of the methyl ester and free acid. By treatment with 
diazomethane (in ether/methanol) the mixture is completely converted into 
the methyl ester. After two-fold chromatography with ethyl acetate on Alox 
III (additionally deactivated with 1.5% H.sub.2 O) there are obtained 
3.022 g of methyl ester, i.e., [(CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 
COOCH.sub.3)batho]. 
For the saponification, the methyl ester is dissolved in 20 ml of ethanol 
the solution is treated with sodium hydroxide solution (0.950 g of NaOH 
dissolved in 5 ml of H.sub.2 O) and heated to 80.degree. C. under argon 
for 2 hours. The cooled solution is added to a CH.sub.2 Cl.sub.2 /H.sub.2 
O mixture, the pH of the solution is adjusted to 4-5 with 85% H.sub.3 
PO.sub.4 and the product is subsequently extracted with CH.sub.2 Cl.sub.2. 
The concentrated CH.sub.2 Cl.sub.2 extract is recrystallized from benzene, 
to yield 1.35 g of product. A further 0.338 g of product is obtained from 
the concentrated mother liquor by recrystallization from ethanol to give a 
total yield of 1.688 g of [(CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 
COOH)batho] m.p. 234.degree.-235.degree. C. 
EXAMPLE 20 
Preparation of the Ru complex--Ru[(SO.sub.3 Na).sub.2 batho].sub.2 
[(CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 COOH)batho]Cl.sub.2 
The compound of Example 19, i.e., 
5-[p-(7-phenyl-1.10-phenanthrolin-4-yl)-phenyl]-pentanoic acid (34.6 mg) 
dissolved in 2 ml of methanol is added to a solution of 102.4 mg of 
Ru[(SO.sub.3 Na).sub.2 batho].sub.2 Cl.sub.2.2H.sub.2 O in 4 ml of 
methanol and 8 ml of water. This mixture is heated to reflux under N.sub.2 
for 3 hours, whereby an orange-red solution results. Thereafter, the 
solvent is removed by blowing in N.sub.2. The purification of the crude 
product is carried out by means of preparative thick-layer chromatography. 
For this purpose, the complex is dissolved in a small amount of water and 
applied to 6 PSC plates (SiO.sub.2 plates from Merck). After drying the 
plates at 70.degree. chromatography is carried out with the elution agent 
chloroform/acetone/methanol (4:3:3). The main zone product, extracted with 
water, was subjected a further 3 times to a preparative thick-layer 
chromatography, whereby the following elution agents were used: 
acetone/water (9:1), then acetone/water (8.5:1.5) and finally 
chloroform/methanol/water (50:50:2). For the removal of final traces of 
SiO.sub.2, the extracted product was dissolved in a small amount of 
methanol and the undissolved silica gel was centrifuged off. The product 
was then precipitated from 1 ml of methanolic solution with 50 ml of 
acetone. Yield: 42.6 mg (red powder) identified as Ru[(SO.sub.3 Na).sub.2 
batho].sub.2 [CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 COOH)batho]Cl.sub.2. 
EXAMPLE 21 
Preparation of methyl p-[.beta.-chloropropionyl]-6-phenylcaproate 
17.2 g of AlCl.sub.3, 18.5 ml of CS.sub.2 and 3.6 ml of 
.beta.-chloro-propionyl chloride are placed in a stirring flask with a 
reflux condenser. 7.06 g of methyl 6-phenylcaproate are allowed to drop 
into this mixture at room temperature within 10 minutes (rinsing is 
carried out with 4 ml of CS.sub.2). The reaction mixture is subsequently 
stirred for a further 15 minutes, warmed slightly and then cooled. The 
reaction mixture is then pipetted into a stirred mixture of ice, water and 
ether. The organic phase is washed neutral with bicarbonate solution and 
water. After drying over MgSO.sub.4 the solvent is removed, 10.2 g of 
crystalline product identified as methyl 
p-[.beta.-chloropropionyl]-6-phenyl-caproate are obtained. 
EXAMPLE 22 
Preparation of 6-[p-(7-phenyl-1,10-phenanthrolin-4-yl)phenyl]-hexanoic 
acid-[(CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 COOH)batho] 
Under argon in a stirring flask is placed 5.90 g of 
4-phenyl-8-aminoquinoline, 27 ml of H.sub.3 PO.sub.4 (85%) and 5.35 ml of 
arsenic acid solution (80% H.sub.3 AsO.sub.4). The mixture is heated to 
120.degree. C. in order to dissolve the quinoline. Thereafter, 11.15 g of 
methyl p-[.beta.-chloropropionyl]-6-phenylcaproate are added within 5 
minutes. The reaction mixture is subsequently heated while stirring within 
10 minutes to 140.degree. C. and left at this temperature for a further 1 
hour. For the working-up, the reaction mixture is cooled and pipetted into 
a stirred mixture of 100 ml of water and 250 ml of CH.sub.2 Cl.sub.2 which 
is cooled well with ice. The pH is then brought to 5 by adding sodium 
hydroxide solution (about 2/3of a solution of 45 g of NaOH in 200 ml of 
water). Thereby, the substance passes into the organic phase. After 
evaporation of the solvent there are obtained 15.76 g of a viscous oil 
which contains, besides the methyl ester, also free acid. By treatment 
with diazomethane (in ether/methanol) the mixture is again converted 
completely into the methyl ester. This is chromatographed (fractions of 
120 ml) with ethyl acetate on 350 g of Alox III (additionally deactivated 
with 1% H.sub.2 O); fractions 5 to 9 contain pure product. 
Fractions 3, 4 and 10 are, on the other hand, still contaminated. They are 
again chromatographed (as previously described) with ethyl acetate over 
aluminium oxide. From the pure fractions of the two chromatographies there 
are obtained, after removing the solvent, 6.979 g of methyl ester, i.e., 
[(CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 COOCH.sub.3)batho]. For the 
saponification, the methyl ester is dissolved in 45 ml of ethanol, this 
solution is treated with sodium hydroxide solution (2.24 g of NaOH 
dissolved in 11 ml of H.sub.2 O) and heated to 80.degree. C. under argon 
for 2 hours. The ethanol is then removed on a rotary evaporator and the 
residue is taken up in a mixture of CH.sub.2 Cl.sub.2 /water. The pH is 
adjusted to about 3 with 3.75 ml of H.sub.3 PO.sub.4 85% and the product 
is then extracted. The extract is washed neutral, dried and concentrated 
to 300 ml. After the addition of a total of 3.5 g of Norit SX-3 the 
solution is stirred for 40 minutes, filtered and concentrated to a small 
amount of CH.sub.2 Cl.sub.2. 20 ml of benzene are added thereto and the 
crude product is left to crystalline out at room temperature for 48 hours. 
Purification of the crude product yielded [(CH.sub.2 CH.sub.2 CH.sub.2 
CH.sub.2 CH.sub.2 COOH)-batho], m.p. 195.degree. C. 
EXAMPLE 23 
Preparation of the Ru complex Ru[(SO.sub.3 Na).sub.2 batho].sub.2 
[(CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 --COOH)batho]Cl.sub.2 
To 134.2 mg of 6-[p-(7-phenyl-1,10-phenanthrolin-4-yl)-phenyl]hexanoic acid 
dissolved in 5.7 ml of methanol are added of 384 mg of Ru[(SO.sub.3 
Na).sub.2 batho].sub.2 Cl.sub.2.2H.sub.2 O in 20 ml of methanol and 5 ml 
of water. This mixture is heated at reflux for 3 hours under N.sub.2, 
whereby an orange-red solution results. The major part of the solvent is 
subsequently removed by blowing in N.sub.2. The remainder is then removed 
completely with a rotary evaporator. The purification of the product is 
carried out firstly by two-fold column chromatography over SiO.sub.2 with 
the elution agent chloroform/methanol/water (7:3:0.5). The complex is 
subsequently purified further by means of preparative thick-layer 
chromatography (SiO.sub.2 plates from Merck). Elution agent: 
chloroform/methanol/water (7:3:0.5). After scratching-off the main zone is 
extracted with methanol to yield 162 mg of Ru[(SO.sub.3 Na).sub.2 
batho].sub.2 [(CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 
COOH)batho]Cl.sub.2 as a red powder. 
EXAMPLE 24 
Preparation of benzobathophenanthrolinyl-pentanoic acid[(CH.sub.2 CH.sub.2 
CH.sub.2 CH.sub.2 COOH)benzobatho] 
The synthesis of the title compound was carried out starting from 
2-amino-3-napthoic acid by means of a Skraup's reaction analogously to the 
procedure described in Example 18. Total yield: 1.07 g. 
EXAMPLE 25 
Preparation of the Ru complex--Ru[(SO.sub.3 Na).sub.2 batho].sub.2 
[(CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 COOH)benzobatho]Cl.sub.2 
The Ru complex was prepared in an analogous manner to the procedure of 
Example 8 except that 1.28 g of Ru[(SO.sub.3 Na).sub.2 batho].sub.2 
Cl.sub.2 and 0.561 g of [(CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 
COOH)benzobatho] was used. Yield: 1.1 g of a red powder. 
EXAMPLE 26 
Preparation of the Ru complex--Ru[(SO.sub.3 Na).sub.2 batho].sub.2 
[(COOH).sub.2 bpy]Cl.sub.2 
Following the procedure of Example 8, the title compound was prepared using 
256 mg of Ru[(SO.sub.3 Na).sub.2 batho].sub.2 Cl.sub.2, 49 mg of 
4,4'-dicarboxy-2,2'-bipyridine and 60 mg of NaHCO.sub.3 (for the 
solubilization of the bipyridine) in 35 ml of MeOH/H.sub.2 O (1:2). The 
reaction yielded 290 mg of a red powder identified as Ru[(SO.sub.3 
Na).sub.2 batho].sub.2 [(COOH).sub.2 bpy]Cl.sub.2. 
EXAMPLE 27 
Labelling of anti-CEA with the Ru complex of Example 6 
The coupling of the mixed Ru complex of Example 6 to anti-CEA was carried 
out with the aid of the water-soluble carbodiimide derivative 
N-cyclohexyl-N'-(2-morpholinoethyl)-carbodiimide-methyl-p-toluenesulphonat 
e. As the complex has two reactive groups per molecule, double the molar 
amount of carbodiimide is used. The following stock solutions are prepared 
for the coupling reaction: 
(1) 4.00 mg/ml of Ru complex of Example 6 in water at pH 4.5 
(2) 2.17 mg/ml of anti-CEA from rabbit (DAKO Code No. A 115, Lot 112 B) in 
200 mM NaHCO.sub.3 ; pH 8.5. 
"Stock Solution 1" (136 .mu.l) is diluted with 264 .mu.l of water pH 4.5 
(adjusted with HCl). Thereafter, 0.27 mg of 
N-cyclohexyl-N'-(2-morpholinoethyl)-carbodiimide-methyl-p-toluenesulphonat 
e is added and the mixture mixed using a vortex mixer. After two minutes 
400 ml of the anti-CEA "stock solution 2" are added thereto and the 
mixture is again mixed well in a vortex. The pH is adjusted to 8.5 by the 
addition of a small amount of 1N HCl. The reaction mixture is then left to 
stand at room temperature in the dark for 17 hours. 
For the separation of the labelled anti-CEA, 500 ml of the reaction mixture 
were chromatographed over a column (length 30 cm, diameter 9 mm) with 
acrylamide gel AcA-54 from LKB (elution agent: 150 mM NaCl, 10 mM Na 
phosphate, 0.02% NaN.sub.3, pH 7.0). The fractions with the highest 
content of labelled anti-CEA were combined--a total of 5.2 ml. The content 
of anit-CEA and of Ru complex in this solution was determined by 
UV-spectroscopy (from the optical density at 278 nm--absorption of 
anti-CEA and Ru complex, as well as from the optical density at 445 
nm--absorption of the Ru complex alone). The following concentrations were 
thus obtained: 
0.66.times.10.sup.-6 M/l--Ru complex 
0.58.times.10.sup.-6 M/l--anti-CEA 
This corresponds to a degree of labelling of 1.1. 
EXAMPLE 28 
Performance of a fluorescence immunoassay with CEA standards--sandwich test 
For the quantitative determination of CEA-standards, a sandwich test was 
carried out as follows with a monoclonal CEA antibody and a polyclonal CEA 
antibody (labelled antibody from DAKO): 
Into the requisite number of test tubes (10.times.75 mm) there are in each 
case pipetted 0.250 ml of CEA standard solution (0 ng/ml CEA; 2.5 ng/ml 
CEA; 5 ng/ml CEA; 10 ng/ml CEA and 20 ng/ml CEA in 150 mM NaCl, 10 mM Na 
phosphate with 20 g/l bovine serum albumin), in each case there is added a 
polystyrene bead (diameter 6.5 mm) sensitized with monoclonal anti-CEA and 
incubation is carried out at 37.degree. C. for 24 hours. The polystyrene 
beads are subsequently washed three times with 2-5 ml of distilled water 
each time and then transferred into test tubes each of which contains 
0.250 ml of buffer solution with 1.times.10.sup.-8 M/l labelled rabbit 
anti-CEA (degree of labelling 1.1). 
After a 24 hours incubation at 37.degree. C. the beads are again washed 
three times with 2-5 ml of distilled water each time and subsequently 
transferred into test tubes with in each case 2 ml of sulphuric acid 
(0.09N). After 30 minutes the sulphuric acid solution is pipetted into 
measurement cuvettes and the content of Ru complex is measured by 
fluorescence spectroscopy (excitation wavelength 453 nm, emission 
wavelength 612 nm). 
The measurement was carried out with the apparatus described earlier using 
an edge filler B 610 from Balzers, a periodic delay of the fluorescence 
measurement of 2 .mu.sec (based on the excitation pulse) and an opening of 
the measurement aperture of 3 .mu.sec. 
In Table I there are given the values of a CEA determination which was 
carried out with a series of CEA standards. 
The sensitivity in the case of this two-step test amounts to 60 pg/ml CEA. 
TABLE I 
______________________________________ 
Determination by fluorescence spectroscopy of CEA 
standards (two-step procedure) 
Rel. fluorescence 
Concentration of CEA 
intensity 
______________________________________ 
0 ng/ml CEA 0.071 
2.5 ng/ml CEA 0.532 
5 ng/ml CEA 1.041 
10 ng/ml CEA 1.846 
20 ng/ml CEA 4.121 
______________________________________ 
EXAMPLE 29 
Performance of a fluorescense immuno assay with CEA standards--one--pot 
procedure 
The CEA test can also be carried out in a one-pot procedure in accordance 
with the following method. 
To the requisite number of test tubes (10.times.75 mm) there are in each 
case added 0.125 ml of CEA standard solution (0 ng/ml CEA; 2.5 ng/ml CEA; 
5 ng/ml CEA; 10 ng/ml CEA; 20 ng/ml CEA in foetal calf serum) as well as 
in each case 0.125 ml of a solution with 2.times.10.sup.-8 M/l rabbit 
anti-CEA which is labelled with the Ru complex (degree of labelling 1.25; 
dilution buffer pH 7.1 containing 0.1M/l Tris, 20% foetal calf serum 0.05% 
Thimerosal and 0.02% Tween 20). Then, in each case there is added thereto 
a polystyrene bead (diameter 6.5 mm sensitized with monoclonal anti-CEA) 
and incubation is carried out at 37.degree. C. for 24 hours. The beads are 
subsequently washed three times with 2 to 5 ml of distilled water each 
time and then transferred into test tubes with in each case 2 ml of 
sulphuric acid (0.09N). After 30 minutes the sulphuric acid solution is 
pipetted into measurement cuvettes and the content of Ru complex is 
measured by fluorescence spectroscopy (the measurement conditions are 
identical with those of Example 28. The results of this CEA determination 
are compiled in Table II. A sensitivity of 430 pg/ml CEA is found in the 
one-pot procedure. 
TABLE II 
______________________________________ 
Concentration of CEA 
Rel. fluorescence 
(Roche standard solutions) 
intensity 
______________________________________ 
0 ng/ml CEA 0.27 
2.5 ng/ml CEA 1.21 
5 ng/ml CEA 2.66 
10 ng/ml CEA 5.13 
20 ng/ml CEA 9.74 
______________________________________ 
EXAMPLE 30 
Labelling of anti-HCG 
The labelling of anti-HCG with the Ru complex of Example 6 was carried out 
in the same manner as that of anti-CEA in accordance with Example 27. The 
following stock solutions were prepared for the coupling reaction: 
(1) 4.00 mg/ml of Ru complex of Example 6 in water at pH 4.5 
(2) 10.83 mg/ml of anti-HCG from rabbit (DAKO Code No. A 231 Lot 032 A) in 
200 mM NaHCO.sub.3 ; pH 8.5. 
"Stock Solution 1" (150 ml) is diluted with 250 ml of water pH 4.5 
(adjusted with HCl). Thereafter, 0.30 mg of 
N-cyclohexyl-N'-(2-morpholinoethyl)-carbodiimidemethyl-p-toluenesulphonate 
is added to this solution and the mixture is mixed using a vortex, mixer. 
After 2 minutes there is added thereto the anti-HCG solution (222 ml of 
stock solution 2 diluted with 178 .mu.l of a 200 mM NaHCO.sub.3 solution 
pH 8.5) and the mixture is mixed well in a vortex. The pH-value is 
adjusted to 8.5 with a small amount of HCl. The reaction mixture is then 
left to stand at room temperature in the dark for 17 hours. For the 
separation of the labelled anti-HCG, 500 ml of the reaction mixture are 
chromatographed over a column (length 30 cm, diameter 9 mm) with 
acrylamide gel AcA-54 (from LKB) (elution agent: 150 mM NaCl, 10 mM Na 
phosphate, 0.02% NaN.sub.3, pH 7.0). The fractions with the highest 
content of labelled anti-HCG are combined--a total of 5.2 ml. The content 
of anti-HCG and of Ru complex is determined in this solution by 
UV-spectroscopy. The following concentrations are thus obtained: 
2.01.times.10.sup.-6 M/l Ru complex, 
2.04.times.10.sup.-6 M/l anti-HCG. 
This corresponds to a degree of labelling of 1.0. 
EXAMPLE 31 
Performance of a fluorescence immunoassay with .beta.-HCG standards 
For the quantitative determination of .beta.-HCG standards, a sandwich test 
was carried out as follows with a monoclonal .beta.-HCG antibody and a 
polyclonal HCG antibody (labelled antibody from DAKO). To the requisite 
number of test tubes (10.times.75 mm) there are added 0.050 ml of 
.beta.-HCG standard solution (0 mIU/ml; 10 mIU/ml; 25 mIU/ml; 50 mIU/ml; 
100 mIU/ml; 200 mIU/ml). Thereafter, 0.050 ml of a solution containing 
5.12.times.10.sup.-8 M/l anti-HCG which is labelled as described in 
Example 30 in dilution buffer, pH 7.1, (0.1M/l Tris, 20% foetal calf 
serum, 0.05% Thimerosal and 0.02% Tween 20). An additional 0.150 ml of 
buffer solution (150 mM NaCl, 10 mm Na phosphate with 20 g/l bovine serum 
albumin) is added to each test tube and finally a polystyrene bead 
(diameter 6.5 mm sensitized with monoclonal anti-.beta.-HCG) is added to 
each tube and incubation is carried out at 37.degree. C. for 16 hours. The 
beads are subsequently washed three times with 2 to 5 ml of distilled 
water each time and then transferred into test tubes with in 2 ml of 
sulphuric acid (0.09N). After 30 minutes the sulphuric acid solution is 
pipetted into measurement cuvettes and the content of Ru complex is 
measured by fluorescence spectroscopy as described in Example 28. The 
results of the .beta.-HCG determination are summarized in Table III. A 
sensitivity of 2.2 mIU/ml .beta.-HCG is found. 
TABLE III 
______________________________________ 
Determination by fluorescence spectroscopy of .beta.-HCG 
standards (one-pot procedure) 
Rel. fluorescence 
Concentration of .beta.-HCG 
intensity 
______________________________________ 
0 mIU/ml 0.44 
10 mIU/ml 0.34 
25 mIU/ml 0.80 
50 mIU/ml 1.56 
100 mIU/ml 2.78 
200 mIU/ml 5.81 
______________________________________ 
EXAMPLE 32 
Labelling of anti-a-interferon 
The labelling of monoclonal anti-a-interferon (from Roche Diagnostica) with 
the Ru complex of Example 6 was carried out in the same manner as that of 
anti-CEA in accordance with Example 27. The following stock solutions were 
prepared for the coupling reaction: 
(1) 3.75 mg of Ru complex of Example 6 in water at pH 4.5. 
(2) 6.0 mg/ml of monoclonal anti-a-interferon in 200 mM NaHCO.sub.3 ; pH 
8.5. 
A Water-soluble carbodiimide derivative (0.81 mg) is added to 400 .mu.l of 
"Stock Solution 1" and mixed for a short time in a vortex. After 2 minutes 
there are added thereto 400 .mu.l of the anti-a-interferon Stock Solution 
2 and the mixture is mixed using a vortex mixer. The reaction mixture is 
then left to stand at room temperature in the dark for 17 hours. For the 
separation of the labelled anti-a-interferon, 500 ml of the reaction 
mixture are chromatographed over a column (length 30 cm, diameter 9 mm) 
with acrylamide gel AcA-54 from LKB (elution agent: 150 mM NaCl, 10 mM Na 
phosphate, 0.02% NaN.sub.3, pH 7.0). The fractions with the highest 
content of labelled anti-a-interferon are combined--a total of 5.2 ml. The 
content of anti-a-interferon and of Ru complex of Example 6 is determined 
in by UV-spectroscopy. The following concentrations are obtained: 
11.5.times.10.sup.6 M/l Ru complex, 
1.8.times.10.sup.-6 M/l anti-a-interferon (monoclonal). 
This corresponds to a degree of labelling of 6.4. 
EXAMPLE 33 
Performance of a fluorescence immunoassay with a-interferon standards 
For the quantitative determination of interferon-raA standards (recombinant 
leucocyte interferon), there is used a sandwich test developed by Roche 
Diagnostica as a EIA. In place of the enzyme-labelled second antibody 
there is, however, used monoclonal anti-interferon which is labelled with 
the Ru complex of Example 6 (degree of labelling 6.4). 
The quantitative determination of the various interferon-raA standards was 
carried out according to the following procedure (one-step test). 
To the requisite number of test tubes (10.times.75 mm) there are in each 
case added 0.100 ml of interferon-raA standard solution (0 U/ml r IFNaA; 
25 U/ml r IFNaA; 50 U/ml r IFNaA; 100 U/ml r IFNaA; 150 U/ml r IFNaA; 200 
U/ml r IFNaA in normal human serum with Thimerosal) as well as in each 
case 0.50 ml of a solution with 5.26.times.10.sup.-10 M/l monoclonal 
anti-a-interferon which is labelled with the Ru complex of Example 6 
buffer system: 150 mM NaCl, 10 mM Na phosphate pH 7.5). There is then 
added thereto in each case a polystyrene bead (diameter 6.5 mm; sensitized 
with monoclonal anti-interferon) and incubation is carried out at room 
temperature (26.degree. C.) for 24 hours. The beads are subsequently 
washed three times with 2-5 ml of distilled water each time and then 
transferred into test tubes with in each case 2 ml of sulphuric acid 
(0.09N). After 30 minutes the sulphuric acid solution is pipetted into 
measurement cuvettes and the content of Ru complex is measured by 
fluorescence spectroscopy. (The measurement conditions are identical with 
those of Example 28). The results of the r IFNaA determinations which are 
summarized in Table IV illustrate that in the range of 0-200 U/ml there is 
an approximately linear relationship between the fluorescence intensity 
and the r IFN.alpha.A concentration. The sensitivity amounts to 0.46 U/ml 
r IFNaA. 
TABLE IV 
______________________________________ 
Determination by fluorescence spectroscopy of r IFN.alpha.A 
standards 
Concentration of r IFN.alpha.A 
(Roche standard solutions) 
Rel. fluorescence intensity 
______________________________________ 
0 U/ml 0.14 
25 U/ml 0.51 
50 U/ml 0.89 
100 U/ml 1.80 
150 U/ml 2.77 
200 U/ml 4.01 
______________________________________ 
EXAMPLE 34 
Labelling of polyclonal anti-HCG with the Ru complex of Example 20 
The following stock solutions are prepared for the coupling reaction: 
(1) 5.4 mg/ml of Ru complex of Example 20 in water at pH 4.5. 
(2) 6.0 mg/ml of polyclonal anti-HCG from rabbits (DAKO Code No. A 231, Lot 
032A) in 200 mM NaHCO.sub.3 ; pH 8.5. 
The coupling reaction is carried out according to the procedure described 
in Example 27. For this purpose there are used in each case 400 .mu.l of 
the Stock Solutions 1 and 2 as well as 0.56 mg of water-soluble 
carbodiimide. The labelled antibodies were separated from excess Ru 
complex by gel chromatography as described in Example 32. The 
UV-spectroscopic determination of the content of anti-HCG and of Ru 
complex gave the following values: 
3.81.times.10.sup.-6 M/l Ru complex, 
1.87.times.10.sup.-6 M/l anti-HCH (polyclonal). 
This corresponds to a degree of labelling of 2.0. 
EXAMPLE 35 
Labelling of polyclonal anti-CEA with the Ru complex of Example 23 
The following stock solutions are prepared for the coupling reaction: 
(1) 1.71 mg/ml of Ru complex of Example 23 in water at pH 4.5. 
(2) 2.17 mg/ml of polyclonal anti-CEA from rabbits (DAKO Code No. A115, Lot 
112B) in 200 mM NaHCO.sub.3 ; pH 8.5. 
The coupling reaction is carried out according to the procedure described 
in Example 27. For this purpose there are used in each case 400 .mu.l of 
the stock solutions 1 and 2 as well as 0.19 mg of water-soluble 
carbodiimide. The labelled antibodies were separated from excess Ru 
complex by gel chromatography as described in Example 27. The 
UV-spectroscopic determination of the content of anti-CEA and of Ru 
complex gave the following values: 
0.43.times.10.sup.-6 M/l Ru complex, 
0.94.times.10.sup.-6 M/l anti-CEA. 
This corresponds to a degree of labelling of 0.5. 
EXAMPLE 36 
Labelling of h-IgG with the Ru complex of Example 17 
The following stock solutions are prepared for the coupling reaction: 
(1) 3.6 mg/ml of Ru complex of Example 17 in water at pH 4.5. 
(2) 6.0 mg/ml of h-IgG in 200 mM NaHCO.sub.3 ; pH 8.5. 
The coupling reaction is carried out according to the procedure described 
in Example 27. There are used thereby in each case 400 .mu.l of the stock 
solutions 1 and 2 as well as 0.37 mg of water-soluble carbodiimide. The 
labelled antibodies are separated for excess Ru complex by gel 
chromatography as described in Example 27. The UV-spectroscopic 
determination of the content of h-IgG and of Ru complex gave the following 
values: 
2.31.times.10.sup.-6 M/l Ru complex, 
1.62.times.10.sup.-6 M/l h-IgG. 
This corresponds to a degree of labelling of 1.4. 
EXAMPLE 37 
Labelling of h-IgG with the Ru complex of Example 3 
The following stock solutions are prepared for the coupling reaction: 
(1) 4.0 mg/ml of Ru complex of Example 3 in water at pH 4.5. 
(2) 6.0 mg/ml of h-IgG in 200 mM NaHCO.sub.3 ; pH 8.5. 
The coupling reaction is carried out according to the procedure described 
in Example 27. There are thereby used in each case 400 .mu.l of the stock 
solutions 1 and 2 as well as 1.11 mg of water-soluble carbodiimide. The 
labelled antibodies are separated from excess Ru complex by gel 
chromatography as described in Example 22. The UV-spectroscopic 
determination of the content of H-IgG and of Ru complex gave the following 
values: 
10.4.times.10.sup.-6 M/l Ru complex, 
1.8.times.10.sup.-6 M/l h-IgG. 
This corresponds to a degree of labelling of 5.8. 
EXAMPLE 38 
Labelling of h-IgG with the Ru complex of Example 26 
The following stock solutions are prepared for the coupling reaction: 
(1) 29.0 mg/ml of Ru complex of Example 26 in water at pH 4.5. 
(2) 6.0 mg/ml of h-IgG is 200 mM NaHCO.sub.3 ; pH 8.5. 
The coupling reaction is carried out according to the procedure described 
in Example 27. There are thereby used 526 .mu.l of the stock solution 1 
and 400 .mu.l of the stock solution 2 as well as 7.45 mg of water-soluble 
carbodiimide. The labelled antibodies are separated from excess Ru complex 
by gel chromatography as described in Example 22. The UV-spectroscopic 
determination of the content of h-IgG and of Ru complex gave the following 
values: 
0.81.times.10.sup.-6 M/l Ru complex, 
0.78.times.10.sup.-6 M/l h-IgG. 
This corresponds to a degree of labelling of 1.0. 
EXAMPLE 39 
Labelling of h-IgG with the Ru complex of Example 9 
The following stock solutions are prepared for the coupling reaction: 
(1) 3.7 mg/ml of Ru complex of Example 9 in water at pH 4.5. 
(2) 6.0 mg/ml of h-IgG in 200 mM NaHCO.sub.3 ; pH 8.5. 
The coupling reaction is carried out according to procedure described in 
Example 27. There are thereby used in each case 400 .mu.l of the stock 
solutions 1 and 2 as well as 0.75 mg of water-soluble carbodiimide. The 
labelled antibodies are separated from excess Ru complex by gel 
chromatography as described in Example 27. The UV-spectroscopic 
determination of the content of h-IgG and of Ru complex gave the following 
values: 
4.41.times.10.sup.-6 M/l Ru complex, 
1.21.times.10.sup.-6 M/l h-IgG. 
This corresponds to a degree of labelling of 3.6. 
EXAMPLE 40 
Labelling of h-IgG with the Ru complex of Example 12 
The following stock solutions are prepared for the coupling reaction: 
(1) 3.81 mg/ml of Ru complex of Example 12 in water at pH 4.5. 
(2) 6.0 mg/ml of h-IgG in 200 mM NaHCO.sub.3 ; pH 8.5. 
The coupling reaction is carried out according to the procedure described 
in Example 27. There are thereby used in each case 400 .mu.l of the stock 
solutions 1 and 2 as well as 0.75 mg of water-soluble carbodiimide. The 
labelled antibodies are separated from excess Ru complex by gel 
chromatography as described in Example 27. The UV-spectroscopic 
determination of the content of h-IgG and of Ru complex gave the following 
values: 
5.1.times.10.sup.-6 M/l Ru complex, 
1.17.times.10.sup.-6 M/l h-IgG. 
This corresponds to a degree of labelling of 4.70. 
EXAMPLE 41 
Labelling of h-IgG with the Ru complex of Example 15 
The following stock solutions are prepared for the coupling reaction: 
(1) 3.87 mg/ml of Ru complex of Example 15 in water at pH 4.5. 
(2) 6.0 mg/ml of h-IgG in 200 mM NaHCO.sub.3 ; pH 8.5. 
The coupling reaction is carried out according to the procedure described 
in Example 27. There are thereby used in each case 400 .mu.l of the stock 
solutions 1 and 2 as well as 0.75 mg of water-soluble carbodiimide. 
The labelled antibodies are separated from excess Ru complex by gel 
chromatography as described in Example 27. The UV-spectroscopic 
determination of the content of h-IgG and of Ru complex gave the following 
values: 
11.7.times.10.sup.-6 M/l Ru complex, 
1.04.times.10.sup.-6 M/l h-IgG. 
This corresponds to a degree of labelling of 11.2. 
EXAMPLE 42 
Labelling of h-IgG with the Ru complex of Example 25 
The following stock solutions are prepared for the coupling reaction: 
(1) 3.45 mg/ml of Ru complex of Example 25 in water at pH 4.5. 
(2) 6.0 mg/ml of h-IgG in 200 mM NaHCO.sub.3 ; pH 8.5. 
The coupling reaction is carried out according to the procedure described 
in Example 27. There are thereby used in each case 400 .mu.l of the stock 
solutions 1 and 2 as well as 0.375 mg of water-soluble carbodiimide. The 
labelled antibodies are separated from excess Ru complex by gel 
chromatography as described in Example 27. The UV-spectroscopic 
determination of the content of h-IgG and of Ru complex gave the following 
values: 
5.69.times.10.sup.-6 M/l Ru complex, 
0.45.times.10.sup.-6 M/l h-IgG. 
This corresponds to a degree of labelling of 12.8.