AT397437B - HYBRID CELL LINES FOR PRODUCING MONOCLONAL ANTIBODIES AGAINST BROMNICOTINIC ACID AND 1-METHYL-10ALPHA-METHOXYDIHYDROLYSERGOL; ANTIBODIES AND METHOD FOR THEIR PRODUCTION; YOUR DIAGNOSTIC USE AND DIAGNOSTIC COMPOSITION CONTAINING THIS ANTIBODY - Google Patents

Description

AT 397 437 B

The invention relates to new hybrid cell lines for the production of specific monoclonal antibodies against two haptens, namely bromnicotinic acid (BNS) and 1-methyl-1 Οα-methoxy-dihydrolysergol (NIC), which were derived from nicergoline by chemical cleavage, as well as the monoclonal antibodies formed by these hybrid cell lines Processes for their preparation, preparations containing these antibodies and their diagnostic use.

The possibility to obtain hybrid cell lines by fusion of mouse myeloma cells with spleen cells from immunized mice, which after corresponding cloning permanently produce monoclonal antibodies of desired specificity, was first described by Koehler and Milstein 1975 (Nature 256,495-497). Since then, this method has been used widely.

According to the present invention, monoclonal antibodies are formed which can be used diagnostically for the detection of intact nicergoline molecules. The starting point for the production of these monoclonal antibodies is the cleavage of the nicergoline in BNS and NIC and the coupling of the individual cleavage products to the corresponding carrier substances, e.g. B. bovine serum albumin, for the production of immunogenic conjugates. In the further «follows the appropriate immunization of experimental animals (mice) for the preparation of the cell fusion, u. between basic immunization with BNS or NIC conjugate, in particular bovine serum albumin conjugate, subcutaneously in complete Freund's adjuvant and after three weeks the subsequent immunization with the corresponding conjugate, especially bovine serum albumin conjugate, in incomplete Freund's adjuvant intraperitoneally; 25 pg each / mouse / immunization step. Cell fusion is carried out according to the method cited by Köhler and Milstein. The relevant hybrid cell lines are selected using an ELISA (enzyme-linked immunosorbent assay) method. Here, for example, microtiter plates are coated in parallel with bovine serum albumin and bovine serum albumin conjugate for carrying out the test using standard methods. Only antibodies and the associated cell cultures which give a clearly positive reaction with the conjugate are selected with the carrier, e.g. B. bovine serum albumin carrier, but alone do not give a positive reaction. The specificity of the selected clones for the intact nicergoline is checked by competitive inhibition of the reaction of the respective antibodies against the associated conjugate in the ELIS A test by adding the native nicergoline. Three hybrid cultures with products of the desired antibodies against NIC (named NIC-1, -2, -3) and two cultures with antibody production against BNS (named BNS-1 and -2) were found. Furthermore, it was found that by suitable modification of the ELISA detection system, an unlabeled antibody of the BNS series with a labeled, e.g. B. biotinylated antibodies of the NIC series can be combined to form a measuring system for the intact starting molecule. With the help of monoclonal antibodies, it is now possible for the first time to diagnose the non-metabolized nicergoline.

The invention thus relates to monospecific antibodies, in particular monoclonal mouse antibodies, which a) in an ELISA test with a NIC-OH conjugate, in particular NICOH-bovine serum albumin conjugate (1-methyl-10a-methoxy-dihydrolysergol-17-hemisuccinate-bovine serum albumin conjugate ), react positively, and b) in the same test system with underivatized carrier, e.g. B. bovine serum albumin, do not react.

According to a further feature of the invention, the reactivity with the NIC-OH conjugate, in particular NIC-OH bovine serum albumin conjugate, is inhibited by the addition of native nicergoline in increasing doses to the monospecific antibodies, in particular monoclonal mouse antibodies These monospecific antibodies therefore have a specificity for the native nicergoline. After purification of mice from serum or ascites fluid and subsequent biotinylation using standard methods using avidin-peroxidase conjugates, the antibodies according to the invention react in an ELISA test with NIC-OH conjugate, in particular NIC-OH bovine serum albumin conjugate. The biotinylation is carried out with 200 pg N-biotin succinimide ester / mg of purified antibody in 0.1 molar sodium hydrogen carbonate buffer for 4 hours at room temperature.

The invention furthermore relates to monospecific antibodies, in particular mouse antibodies, which a) react positively in an ELISA test with a BNS conjugate, in particular BNS-serum albumin conjugate (5-bromnicotinic acid-bovine serum albumin conjugate), and b) in the same test system with underivatized carrier, e.g. B. serum albumin, do not respond.

By adding native nicergoline in increasing doses to the ELIS A test system, the antibodies in their reactivity with the BNS conjugate, z. B. serum albumin conjugate inhibited. These monoclonal antibodies also have a specificity for the native nicergoline.

After cleaning from serum or the ascites fluid from mice, the antibodies bind after their -2-

AT 397 437 B

Immobilization on the surface of plastics (e.g. microtiter plates, magnetic particles) or inorganic supports the intact nicergoline molecule.

The fact that these antibodies according to the invention bind the intact nicergoline molecule can be demonstrated with biotinylated anti-NIC antibodies. 5 According to a further aspect of the invention, there is provided a method for producing monoclonal antibodies - according to the invention, which comprises the following steps: a) Generation of immunologically active NIC-OH or BNS conjugates, e.g. B. Serum Albumin Conjugates; b) basic immunization of mice with a suspension or solution of the corresponding conjugates in 10 complete Freund's adjuvant subcutaneously; c) post-immunization of the treated mice with the same conjugate incomplete Freund's adjuvant intraperitoneally 3 weeks after the primary immunization; d) removing the spleen from said mice and preparing a suspension of spleen cells; e) fusion of said spleen cells with mouse myeloma cells in the presence of a fusion promoter; 15 f) Dilution and growth of the combined cells in separate wells of a microtiter plate in one

Selection medium that only supports the growth of hybrid cells; g) Evaluation of the supernatant in each well of the microtiter plates with positive hybrid growth with regard to the presence of an antibody of the desired specificity by parallel testing of two supernatant aliquots in an ELISA test system with immobilized carrier as negative control and 20 NIC-OH or. BNS conjugate as a positive test substance; h) cloning of the hybrids with the desired reactivity by the "limiting dilution" method; i) recovering the antibody from the supernatant; j) checking the reactivity of the corresponding selected antibodies with the native starting molecule by detecting the competitive inhibition after adding increasing concentrations of the starting molecule in the ELISA test; k) selection and second cloning of the checked hybrid cell lines, as well as the checking of the clones intraperitoneally in Pristan-pretreated mice; and l) harvesting the malignant ascites from said mice which contain the desired antibodies and purifying the antibodies by standard techniques. 30th

The invention also relates to the use of the conjugates, e.g. B. bovine serum albumin conjugates, and the monoclonal antibodies thus generated for the detection and quantitative determination of nicergoline. As a test principle, the competitive inhibition of the reaction between specific monoclonal antibodies and the associated immobilized conjugate is used in an ELIS A system for quantitative determination. Aqueous solutions and plasma or serum samples can be used as samples 35. To increase the sensitivity, the test can also be carried out in the same way with antibodies of the specified specificity iodinated according to standard methods or with iodinated serum albumin conjugates.

The conjugates, e.g. B. bovine serum albumin conjugates, and the associated monoclonal antibodies, which are obtained by the method according to the invention, can be used in similar standard techniques for the quantitative measurement of the substances used as hapten by modifying the antigen or antibody immunization method (paper, plastic ball, magnetic particles, etc.) and the Antibody or antigen detection methods are used.

The invention further consists in a method for the detection and quantitative determination of the non-metabolized nicergoline using purified anti-BNS antibodies (BNS-1 or 2) in combination with anti-NIC antibodies, either the anti-BNS antibodies or the anti-NIC antibodies in a suitable manner, e.g. B. by biotinylation or radioactive labeling, were modified for detection. Here, too, aqueous solutions and plasma or serum samples can be used as samples.

The present invention is illustrated by the following examples: 50 Example 1

Alkaline hydrolysis of nicergoline 5.0 g (16.6 mmol) of nicergoline are boiled for 2 hours in 150 ml of 12% strength aqueous methanolic potassium hydroxide solution with the exclusion of light and reflux.

The cooled, light yellow solution is extracted twice with 75 ml of chloroform, the combined chloroform extracts are washed with distilled water until the wash water has a neutral reaction. After drying over anhydrous sodium sulfate, the chloroform phase is spun in and the yellowish, resinous residue is crystallized from a little acetone: l-methyl-10a-methoxy-dihydrolysergol. -3-

AT 397 437 B

Yield: 2.85 g (91% of theory, 98.9% pure - HPLC)

Melting point: 214 - 216 ° C (decomp.), Not corrected. UV spectrum: absorption maximum at 225 nm: = 32500

Absorption maximum at 292 nm: = 8340

Infrared spectrum: carbonyl valence vibration (ester) at 1720 cm'1 lacks 13C nuclear magnetic resonance spectrum: 135.20 ppm C16 70/28 ppm C5 130.32 ppm Cll 66.04 ppm 07 126.48 ppm C15 60.75 ppm C7 123.08 ppm 03 49.33 ppm OCH3 on CIO 121.40 ppm C2 43.86 ppm CH3 on N6 115.00 ppm 02 3436 ppm CHt on NI 110.60 ppm C3 32.61 ppm C8 108.68 ppm 04 30.37 ppm C9 73.73 ppm CIO 2239 ppm C4

Mass spectrum: m / e = 300 molecular peak (= base peak) m / e = 285 molecular ion - methyl m / e = 270 molecular ion - two methyl groups Thin layer chromatography on silica gel 60:

Superplasticizer chloroform / methanol / water 90 + 10 + 1 hRf nicergoline - 50 hRfMMD = 13

Eluent methanol hRf nicergoline = 39 hRfMMD = 22 HPLC: on RP18.7 pm with eluent acetonitrile / water / triethylamine 44 + 45 + 1

Retention time nicergoline: 7.98 minutes Retention time MMD: 1.91 minutes

The alkaline aqueous phase combined with the wash water is adjusted to pH 1 with smoking hydrochloric acid, propanol is added and the mixture is concentrated. The mixture is then extracted three times with 100 ml of chloroform each time, the combined chloroform extracts are washed with 0.1 M hydrochloric acid, dried over sodium sulfate and evaporated in a rotary evaporator. The colorless, microcrystalline residue is recrystallized from a little hot water: 5-bromnicotinic acid.

Yield: 251 mg (12% of theory)

Melting point: 176 ° C, not corrected UV spectrum: absorption maximum 273 nm: = 3150

Infrared spectrum: Identical with comparative substance Char.Bands: Carbonylvalence vibration (carboxylic acid) 1675 cm'1

Combination vibration C-Br 1015 cm'1 thin layer chromatography on silica gel 60:

Eluent chloroform / methanol / water 90 + 10 + 1 hRf bromnicotinic acid = 8 eluent methanol hRf bromnicotinic acid = 62 HPLC on RP 18.7 μη »with eluent acetonitrile / water / triethylamine 44 +45 + 1 retention time bromnicotinic acid: 0.91 minutes

Example 2

Antigen synthesis with 5-bromnicotinic acid as hapten

To prepare the hapten solution, 404 mg of 5-bromo nicotinic acid (2 mmol) are suspended in 25 ml of dried dioxane, and 480 μl of tri-N-butylamine (2 mmol) are added to the solution. The solution cooled to 12 ° C is -4-

AT 397 437 B was mixed with 260 [d chlorobisyl isobutyl ester (2 mmol) and stirred at 12 ° C. for 30 minutes

To prepare the carrier solution, 2.0 g bovine serum albumin (0.02 μΜοΙ) are dissolved in 67 ml of distilled water. The pH is adjusted to pH 9.5 with 1M sodium hydroxide solution. After addition of 45 ml of dioxane, the mixture is cooled to 4 ° C. 5 Hapten solution is combined with the carrier solution and the pH during the first two hours

Sodium hydroxide solution adjusted After incubation overnight at 4 ° C, the solution is dialyzed with ten changes against 2 liters of distilled water, then adjusted to pH 4.6, the precipitate is centrifuged at 7500 rpm (SS34) and four times the volume of the supernatant is added. The cloudy solution is added Allow to stand overnight at 4 ° C. and then the precipitate is centrifuged off as above. 10 The combined precipitates are lyophilized.

Yield: 861 mg conjugate (43% of theory) UV difference spectrum: in 0.6% Tris / HCl buffer pH 8.6, which contains 0.3% sodium dodecyl sulfate; 41 mol bromnicotinic acid per mol bovine serum albumin (measuring wavelength: 273 nm) 15 dinitrofluorobenzene Test according to F. SÄNGER, Biochem. J. 45,565 (1949): 26.4 derivatized amino groups per mole of bovine serum albumin

Trinitrobenzenesulfonic acid test according to A.F.S.A. HABEEB, Anal. Biochem. 14.328 (1966), modified 46.2 derivatized amino groups per mole of bovine serum albumin SDS-polyacrylamide gel electrophoiesis under reducing conditions according to U. K. LAEMMLI, Nature 227. 20 680 (1970)

Significant influence on the migration distance (cattle hem albumin 66 kDa, bromnicotinic acid conjugate 57 kDa)

Example 3 25 Synthesis of l-Metvhl-10a-methoxv-dihvdrolvsergol-17-hemisuccinate 300 mg of l-methyl-10a-methoxy-dihydrolysergol (1 mmol) and 400 mg of succinic anhydride (4 mmol) are suspended in 30 ml of dried pyridine and under Sun protection and moisture exclusion heated at 62 ° C under reflux for four hours.

The reaction solution is spun in and dried on a Rotavapor until no more pyridine odor is noticeable. The yellowish residue is taken up in 50 ml of distilled water and shaken out twice with 50 ml of ether in order to remove brownish decomposition products. The aqueous solution is pure, taken up in chloroform and the excess succinic acid filtered off (yield: 382 mg, 95% of theory).

Chromatography on 180 g of silica gel 60 with methanol-water (4 + 1) gives a fat-free, hair-like substance which is recrystallized from acetone-water. 35

Yield: 290 mg (76% of theory)

Melting point: 91 ° C, not corrected. UV spectrum: absorption maximum at 232 nm: Emojar = 18600 - absorption maximum at 295 nm: E ^^ = 5200 40 infrared spectrum: carbonyl valence vibration (saturated esters) 1730 cm '^

Mass spectrum: m / e - 400 molecular peak m / e = 385 molecular ion - methyl m / e = 300 l-methyl-10a-methoxydihydrolysergol 45 m / e = 268 base peak

IO JC core resonance spectrum:

176.53 ppm C4 '68.71 ppm C5 172.93 ppm CI' 66.29 ppm C17 50 135.13 ppm C16 58.92 ppm C7 128.76 ppm Cll 49.37 ppm OCH3 at CIO 126.17 ppm C15 41.96 ppm CHo anN6 123.52 ppm C13 32.70 ppm C8 121.55 ppm C2 30.69 ppm C2 ', C4' 55 115.05 ppm C12 30.19 ppm C9 109.25 ppm C14 21.57 ppm C4 73.56 ppm CIO 5-

AT 397 437 B

Since the signals for C13 and CHj are missing from NI, 100 mg of the hemisuccinate are subjected to alkaline hydrolysis as described under 1) and worked up analogously.

Yield: 85.6 mg (85.6% of theory)

The hydrolysis product (l-methyl-10a-methoxy-dihydrolysergol) gives the following signals: 135.14 ppm C16 70.22 ppm C5 130.23 ppm Cll 65.98 ppm C17 126.40 ppm C15 60.70 ppm C7 123.10 ppm C13 49.32 ppm OCH3 on CIO 121.39 ppm C2 43.85 ppm CH3 on N6 114.98 ppm C12 34.30 ppm CH3 on NI 110.49 ppm C3 32.63 ppm C8 108.70 ppm C14 30, 29 ppm C9 73.68 ppm CIO 22.33 ppm C4 thin layer chromatography on silica gel 60: eluent methanol / water4 +1 eluent hRf 1-methyl-lOa-methoxydihydrolysergol = 12 hRf MMD-17-hemisuccinate = 43 methanol / water 1 + 1 example 4 hRf MMD = 1 hRf MMD-17 hemisuccinate = 50.5

Antigen synthesis with 1-meth vl-1 Οα-methoxvdihvdrolvsergol-17-hemisuccinate as hapten

To prepare the hapten solution, 420 mg of MMD-17 hemisuccinate (1.05 mmol) are suspended in 8.4 ml of dried dioxane; solution is added after the addition of 251 μl of tri-N-butylamine. The solution, cooled to 12 ° C., is mixed with 138 μl isobutyl chloroformate (1.05 mmol) and stirred at 12 ° C. for 30 minutes.

To prepare the carrier solution, 640 mg bovine serum albumin (9.84 pmMol) are dissolved in 20 ml distilled water. The pH is adjusted to 9.0 with 1N NaOH. After adding 13.3 ml of dioxane, the mixture is cooled to 4 ° C.

The corresponding volumes of hapten and carrier solution (see table) are combined and the pH is kept between 7.0 and 8.0 during the first 2 hours by adding 1N sodium hydroxide solution. It is then left to stand at 4 ° C. overnight. After dialysis against distilled water (ten changes), the solution is lyophilized

Molar ratio H2 N carrier / hapten μΐ carrier solution μΐ water dioxane (3 + 2) μΐ hapten solution mol hapten / mol carrier (* 1: 0.5 5120 2589 381 12.0 1: 1 5120 2209 761 13.7 1 : 1.5 5120 1829 1141 32.8 1: 2 5120 1448 1522 36.4 1: 2.5 5120 1067 1903 46.8 1: 3 5120 687 2283 19.9 (*: Determination by UV differential spectroscopy at 295 nm )

The occupancy rate can therefore be controlled by varying the molar ratio of the reactants to one another.

A conjugate in a ratio of 1: 1.3 is used for the immunization (yield: 62%).

Allocation with hanten: a) Difference spectrum: 23.2 mol hapten / mol carrier b) Dinitrofluorobenzene test: 25.4 mol / mol carrier -6-

AT 397 437 B c) SDS-polyacrylamide gel electrophoresis: ^ conjugate ~ ™ a coupling rate of 30 mol hapten / mol carrier

To demonstrate that the hapten is not changed by the coupling reaction, a reaction 5 is carried out in accordance with Example 4, wherein instead of bovine serum albumin as a carrier, double the molar amount of 6-

Aminocaproic acid is used.

For working up, the reaction mixture is neutralized after standing overnight at 4 ° C with 1M hydrochloric acid and rotated in after addition of propanol.The residue is taken up in chloroform / methanol 1 + 1 and purified by preparative thin layer chromatography on silica gel 60 with chloroform / water 1 + 1 as the mobile phase 10 The band is eluted with the chloroform-methanol mixture (hRf = 23). 21 mg of the amide with 6-aminocaproic acid are subjected to alkaline hydrolysis as described under 1)

Yield: 9.7 mg of oily residue. Thin-layer chromatography on silica gel 60: in the flow agents methanol and methanol / water 4 + 1, the hydrolysis product and 1-methyl-1 Οα-methoxydihydrolysergol show the same Rf value 15. The antigen synthesis is shown in the following formula: 20 25 30

35 1-methyl-lOa-methoxy-dihydrolysergol 40 45 50 5-bromnicotinic acid

5-bromnicotinic acid-bovine serum albumin conjugate 1-methyl-10a-methoxy-dihydrolysergol-17-hemisuccinate

z -7- 55

AT 397 437 B

l-methyl-10a-methoxy-dihydrolysergol-17-hemisuccinate-bovine serum albumin conjugate

Example 5

Selection of a specifically reactive hybrid against the NIC bovine serum albumin konugate 25 mg of NIC bovine cramalbumin conjugate is taken up in 1 ml of phosphate-buffered saline (PBS) and the wells of a microtitre plate (ΙΟΟμΙ / well) are coated with an appropriate amount of this solution , 4 ° C). As a control plate, the wells of a microtiter plate are coated with the non-derivatized bovine serum albumin. After washing the plates with PBS, one well of the test and control plate is incubated with 50 μl of the same hybrid supernatant (2 hours, 37 ° C.). Excess antibody is washed out again (3 × PBS) and the plates are incubated with anti-mouse (IgG + IgM) • goat anti-peroxidase conjugate ναι as above. The amount of bound enzyme is detected by the substrate reaction with diamino-benzidine / hydrogen peroxide as a dye. Only those hybrid lines are cultivated which show a positive reaction only with the hapten-bovine serum albumin conjugate, but not with the bovine serum albumin.

Example 6

Evidence of the specificity of the selected antibodies for the intact nicergoline

The ELIS A test is carried out as described in Example 5 with BNS bovine serum albumin conjugate. An additional sample set contains, in addition to the tissue culture supernatant with the hybrid supernatant to be examined, a large excess of the intact starting substance (saturated solution of nicergoline in PBS, 50 μΐ hybrid supernatant with 50 μΐ nicergoline solution). Only hybrid lines are used which, when the intact nicergoline is added in the ELISA test, show a reduced reactivity against the corresponding bovine serum albumin conjugate and must therefore also be reactive with the nicergoline.

Example 7

Quantitative determination of intact nicergoline with the help of the described monoclonal antibodies

Microtiter plates are coated with purified anti-BNS antibody as described above. The washed microtiter plates (3 x PBS) are incubated with a standard dilution series of nicergoline in PBS (1 ng -10 pg / ml) and with suitable sample dilutions (hem) for 2 hours at 37 ° C. After washing again, the microtiter plates are incubated with a 1: 100 Dilution of the biotinylated anti-NIC antibody (0.5 mg / ml) incubated for 2 hours at 37 ° C. After the next washing step, the mixture is incubated again with a suitable dilution of a commercially available avidin-peroxidase conjugate and then the substrate reaction is carried out with diammdbenzidine / hydrogen peroxide. The optical density is determined at 490 nm. The calibration curve is calculated from the values for the standard concentrations and thus the nicergoline content in the serum samples is determined by comparison

Example 8

Production of polyclonal antibodies

Basic immunization: 1 mgNIC-OH or BNS conjugate in 1 ml 0.9% saline are mixed with 1 ml complete Freund's adjuvant and injected subcutaneously at 10 to 12 places on the back of a male rabbit (approx. 4 kg) -

Post-immunization:

After 4 weeks, 0.5 mg conjugate in 0.5 ml 0.9% saline solution is mixed with an equal volume of incomplete Freund's adjuvant and administered intramuscularly in each case on the hind legs in the vicinity of the poliplietal lymph nodes. The post-immunization is repeated four times in total -8-

Claims (14)

AT 397 437 B Serum production: By incision of the ear vein, plasma is obtained 1 week after the last post-immunization, centrifuged after coagulation at 2000 g and the serum obtained in this way is used for analysis. 5 PATENT CLAIMS 10 1. Monospecific antibodies, in particular monoclonal mouse antibodies, which a ) react positively in an ELISA test with an NIC-OH conjugate, in particular NICOH-bovine serum albumin conjugate (1-methyl-10a-methoxy-dihydrolysergol-17-hemisuccinate-bovine serum albumin conjugate), and b) in the same test system with underivatized carrier, e.g. . B. bovine serum albumin, do not react. 15 2. Monospecific antibodies, in particular monoclonal mouse antibodies according to claim 1, which are inhibited by adding native nicergoline in increasing dosage to the ELISA test system in their reactivity with the NIC-OH conjugate, in particular NIC-OH bovine serum albumin conjugate. 3. Monospecific antibodies, in particular monoclonal mouse antibodies according to claims 1 and 2, which after purification from serum or ascites fluid of mice and subsequent biotinylation according to standard methods using avidin-peroxidase conjugates in an ELISA test with NIC-OH conjugate, in particular react NIC-OH bovine serum albumin conjugate. 4. Monospecific antibodies, in particular mouse monoclonal antibodies, which a) react positively in an ELISA test with a BNS conjugate, in particular BNS serum albumin conjugate (5-bromo nicotinic acid-bovine serum albumin conjugate), and b) in the same test system with underivatized carrier, z. B. serum albumin, do not respond. Monospecific antibodies, in particular monoclonal mouse antibodies according to claim 4, which by adding native nicergoline in increasing dosage to the ELISA test system in their reactivity with the BNS conjugate, e.g. B. BNS serum albumin can be inhibited. 6. Monospecific antibodies according to claim 5, which bind the intact nicergoline molecule after cleaning from serum or ascites fluid of 35 mice after their immobilization on the surface of plastics (eg microtiter plates, magnetic particles) or inorganic carriers. 7. A process for the production of polygonal antibodies, characterized in that NIC-OH or BNS conjugate is mixed with Freund's complete adjuvant and rabbits are injected subcutaneously and intramuscularly several times after at least 40 4 weeks, and then the serum is obtained. S. Hybrid cell lines which produce monoclonal antibodies according to one of Claims 1 to 6 and are formed by fusion of cells from a mouse previously immunized with BNS or NIC-OH conjugate, in particular serum albumin conjugate, and cells from a mouse myeloma line. 45 9. A method for producing monoclonal antibodies according to one of claims 1 to 6, characterized by the following steps: a) Generation of immunologically active NIC-OH or BNS conjugates, for. B. Serum Albumin Conjugates; b) basic immunization of mice subcutaneously with a suspension or solution of the corresponding conjugates in complete Freund's adjuvant; c) post-immunization of the treated mice with the same conjugate in incomplete Freund's adjuvant intraperitoneally 3 weeks after the primary immunization; d) removing the spleen from said mice and preparing a suspension of spleen cells; e) fusion of said spleen cells with mouse myeloma cells in the presence of a fusion promoter; 55 f) diluting and multiplying the combined cells in separate wells of a microtiter plate in a selection medium which only supports the growth of hybrid cells; g) Evaluation of the supernatant in each well of the microtiter plates with positive hybrid growth with regard to the presence of an antibody of the desired specificity by parallel testing of two supernatant aliquots in an ELISA test system with immobilized carrier as negative control and NIC-OH or BNS conjugate from positive test substance; h) cloning of the hybrids with the desired reactivity by the "limiting dilution" method; 5 i) recovering the antibody from the supernatant, 10. A method for producing monoclonal antibodies according to one of claims 1 to 6, characterized by the following steps: a) Generation of immunologically active NIC-OH or BNS conjugates, for. B. Serum Albumin Conjugates; 10 b) basic immunization of mice with a suspension or solution of the corresponding conjugates in complete Freund's adjuvant subcutaneously; c) post-immunization of the treated mice with the same conjugate in incomplete Freund's adjuvant intraperitoneally 3 weeks after the primary immunization; d) removing the spleen from said mice and preparing a suspension of spleen cells; 15 e) fusion of said spleen cells with mouse myeloma cells in the presence of a fusion promoter; f) diluting and multiplying the combined cells in separate wells of a microtiter plate in a selection medium which only supports the growth of hybrid cells; g) evaluating the supernatant in each well of the microtiter plates with positive hybrid growth with regard to the presence of an antibody of the desired specificity by parallel testing of two supernatant aliquots 20 in an ELISA test system with immobilized carrier as negative control and NIC-OH or BNS conjugate from positive test substance; h) cloning of the hybrids with the desired reactivity by the "limiting dilution" method; i) recovering the antibody from the supernatant; j) checking the reactivity of the corresponding selected antibodies with the native starting molecule 25 by weaving the competitive inhibition after adding increasing concentrations of the starting molecule in the ELISA test; k) Selection and second cloning of the checked hybrid cell lines, as well as the testing of the clones by intraperitoneal means in pristine-pretreated mice; and l) harvesting the malignant ascites from the above-mentioned mice, which contain the desired antibodies, and purifying the antibodies according to standard techniques. 11.5-bromnicotinic acid carrier conjugates, in particular bovine serum albumin conjugates of the formula: 35 0 H ΒΓγ ^ γ '%' χ · '^ ΤΓ! * ΒΓ 40 where X is a hydrocarbon solid with 1 to 6 C atoms, which is optionally represented by S, Ο, N can be interrupted. 45 12. 1-methyl-lOa-methoxy-dihydrolysergol conjugates, in particular 1-methyl-lOa-methoxy-dihydrolysergol-17-hemisuccinate-serum albumin conjugates, of the formula 50 -10- 55 AT 397 437 B where X is a hydrocarbon radical with 1 to 6 carbon atoms, which can optionally be interrupted by S, Ο, N. 13. A process for the preparation of 5-bromnicotinic acid conjugates, in particular serum albumin conjugates, characterized in that 5-bromnicotinic acid is reacted with the carrier (serum albumin). 14. A process for the preparation of l-methyl-10a-methoxy-dihydrolysigol-17-hemisuccinate-carrier conjugate (serum albumin conjugate), characterized in that 1-methyl-1 Οα-methoxy-dihydrolysergol with succinic anhydride and the l- Methyl-10a-methoxydihydrolysergol-17-hemisuccinate is implemented with the respective haptic wearers. Process for the quantitative determination of nicergoline with antibodies, characterized in that the competitive inhibition of the reactivity between specific antibodies according to claim 2 or 5 and the associated immobilized conjugate is determined in an ELISA system. 16. A method for the detection and quantitative determination of the non-metabolized nicergoline, characterized in that purified anti-BNS antibodies (BNS-1 or -2) are used in combination with anti-NIC antibodies, either the anti-BNS antibodies or the anti-NIC antibodies in a suitable manner, e.g. B. by Biotinyliemng or radioactive labeling, are modified for detection. 17. Diagnostic preparation, characterized in that it contains anti-BNS antibodies according to claim 3 and / or anti-NIC antibodies according to claim 6 in a separate manner, wherein either the anti-BNS antibodies or the anti-NIC Antibodies in a suitable manner, e.g. B. by Biotinyliemng od «reactive labeling, modified for detection. -11-