DE3200657A1 - Process for the preparation of hybrid cells producing an antibody and of antibodies, hybrid cells and antibodies - Google Patents

Abstract

The invention relates to a process for the preparation of hybrid cells producing an antibody and of antibodies which are directed, for example, against Hu-IFN-beta and Hu-IFN-gamma. It furthermore relates to hybrid cells and antibodies and to the use thereof. <IMAGE>

Description

Method of producing an antibody -

the hybrid cells and of antibodies, hybrid cells and antibodies.

The invention relates to a method for producing an antibody producing hybrid cells, in which one (a) immunizes mice with an antigen, (b) a lymphocyte mixture isolated from the mice, (c) B lymphocytes with mouse B myeloma cells fused or hybridized, (d) the mixture of hybrids formed and not hybridized B myeloma cells transferred into a culture medium for the hybrids formed and the hybrids formed are cultured and opposed to the non-hybridized B myeloma cells selected, (e) subcloned for hybrids that produce the antibody that is specifically directed against the antigen, and (f) if applicable isolating the hybrids, the method being characterized in that one is at in step (d) the selection is carried out in a medium containing methyl cellulose and possibly hybrids (which have been selected in step (d)) in injected into the abdominal cavity of mice (previously pretreated with a tumor stimulator have been), increased hybrids removed from the abdominal cavity and then the subcloning and performing isolation steps (e) and (f).

For immunization, for the isolation of a spleen lymphocyte mixture, for Fusion or hybridization, for cultivating the hybrids and for their selection, for subcloning hybrids that produce the antibody they are looking for, and for Isolation of these hybrids or the antibody are relevant to the person skilled in the art Procedure available. For fusion or hybridization and for cultivation formed hybrids can be found, for example, in Köhler et al., Nature (London), 256 (1975) 495-497, Koehler et al., Eur. J. Immunol., 6 (1976) 511-519 and Hochkeppel et al., Eur. J. Biochem., 118 (1981) 437-442.

For selection, subcloning or monocloning and for isolation the hybrid and the antibody are, for example, Secher et al., Nature (London), 285 (1980) 446-450 and Hochkeppel et al., Loc. cit., cited. On the concept of Subcloning or monocloning is finally to Vernon et al. in: Mishell & Shiii, Selected Methods in Cellular Immunology: 373-397, Freeman & Co., San Francisco 1980. Rei the stage of subcloning or monocloning the appearance of a stable cell line with stable antibody production can be observed with the help of an antibody activity test (inhibition of antigen activity>. track, for example according to the method of Havell et al., J. Antimicrob.

Ag. Chemotherap., 2 (1972) 476-484.

For the term myeloma cell see e.g. Milstein, Scientific American, 243 (October 1980) 56-64. Myeloma cells can, for example, according to Groth & Scheidegger, J.

Immunological Methods, 35 (1980) 1-21. Examples of B myeloma cells can be found in the following table.

The person skilled in the art can choose a suitable strain of mice by he tests whether the B myeloma cells he has chosen can interact with B lymphocytes from the test mouse Allow to hybridize. Conveniently, one will use Balb / c mice and B myeloma cells from Balb / c-MSusen, as these mice are generally used in animal experiments. Such mice are commercially available and can, for example, from Zentral institute for laboratory animal breeding / Hanover.

It can be advantageous to use the antigen used for immunization to be used adsorbed on a molecular sieve.

The immunization is preferably carried out more than once. in the In the case of human interferon, the final immunization is preferred with human interferon from which the sugar content has been removed. The sugar content can be removed, for example, according to the method of Bose et al., J. Biol. Chem., 251 (1976) 1659-1662. The final immunization leads preferably about 4 days before isolating the lymphocyte mixture.

The lymphocyte mixture is preferably isolated from female mice, especially female Balb / c mice. Female mice show a faster immune response.

Knowledge Designation of supply source Designation of the supply source (a) FO F.- Miescher-Institut / Basel CNCM / Paris I-153 (b) P3-X 63 Ag 8 Institute for Immunology / Basel Institute for Genetics / Cologne (c) P3-NS 1-1 Institute for Immunology / Basel Institute for genetics / Cologne If you have the selection in a methyl cellulose containing and / or macrophage-free medium carried out, the formed Hybrid cells particularly protected. The term methyl cellulose also includes substituted Methyl celluloses such as carboxymethyl cellulose. An example of methyl cellulose is Methocel MC (4000 cP; Fluka). An example of a suitable medium is that HAT medium; see Hochkeppel et al. loc. cit.

A good stabilization of selected hybrids can be achieved, when the selected hybrids are injected into the peritoneal cavity of mice that have have previously been pretreated with a tumor stimulator. One can for example select for about 4 days. before placing the selected hybrids in the abdominal cavity injected by mice.

Propagated hybrids taken from the abdominal cavity are then the subcloning and isolation step of the method according to the invention. An example for a customary tumor stimulator is also given below. The professional is familiar with the method to be used; see.

e.g. Secher et al., Nature (London), 285 (1980) 446-450. For example For example, Balb / c mice adopt a hybrid consisting of B lymphocytes from Balb / c mice and B myeloma cells from Balb / c mice.

Furthermore, good results can be obtained using a tumor stimulator or an adjuvant and then eubcloned, the antibody-producing hybrids injected into the abdominal cavity of mice, the multiplied hybrids removed and, if necessary isolated the antibody. Those for injection of selected hybrids above The statements made apply accordingly. Is 2,6,10,14-tetramethylpentadecane provides an example of a common tumor stimulator and Freund's adjuvant an example of a common adjuvant setting.

For the injection of selected hybrids or of the antibodies producing hybrids, female mice in particular are preferably used Balb / c-MXuse.

The invention also relates to an antibody (which is specific against human fibroblast interferon> Hu-IFN-beta is directed) producing Hybrids which can be produced by (a) mice with human fibroblast interferon immunized and furthermore in a manner known per se (b) a lymphocyte mixture from the mice isolated, (c) 8 lymphocytes fused or hybridized with MSuse-B myeloma cells, (d) the mixture of formed hybrids and non-hybridized B myeloma cells in a culture medium for the hybrids formed are transferred and the hybrids formed are cultivated and selected for the non-hybridized B myeloma cells, (e) for hybrids subcloned that produce the antibody specific to human fibroblast interferon is directed, and (f) optionally isolating these hybrids.

The invention also relates to hybrids with those for the production, a monoclonal antibody specific to human fibroblast interferon is directed to essential features of the I-154 deposit and from the deposit derived hybrids.

The invention also relates to antibodies (which are specific against human Immune interferon n Hu-IFN-gamma is directed) producing hybrids, these being Hybrids can be produced by (a) mice with human immune interferon immunized and also in a manner known per se (b) from the mice a lymphocyte mixture isolated, (c) B lymphocytes fused with mouse B myeloma cells or hybridized, (d) the mixture of hybrids formed and non-hybridized B myeloma cells transferred into a culture medium for the hybrids formed Hybrids cultivated and selected from the non-hybridized B myeloma cells, (e) subcloned on hybrids that produce the antibody specific to human immune interferon, and (f) optionally these hybrids isolated.

The invention also relates to antibodies (which are specific against human Fibroblast interferon) or antibodies (which are specific to human Immune interferon is directed) producing hybrids that can be produced by that in step (d) the selection in a methyl cellulose containing Medium carries out and, if appropriate, hybrids (selected in stage (d)) were injected into the abdominal cavity of mice (previously with a tumor stimulator have been pretreated), increased hybrids removed from the abdominal cavity and then carries out the subcloning and isolation stages (e) and (f).

The invention also relates to a monoclonal antibody that specifically directed against human fibroblast interferon (Hu-IFN-beta) and thus producible is to (a) immunize mice with human immune interferon and further in a manner known per se (b) a lymphocyte mixture is isolated from the mice, (c) B lymphocytes fused or hybridized with mouse B myeloma cells, (d) the mixture of formed hybrids and non-hybridized B myeloma cells in transferred to a culture medium for the hybrids formed and the hybrids formed cultivated and selected against the non-hybridized B myeloma cells, (e) subcloned on hybrids that produce the antibody specific to human fibroblast interferon, and (f) optionally the antibody isolated.

The invention also relates to a monoclonal antibody that specifically directed against human immune interferon (Hu-IFN-gamma) and thus producible is to (a) immunize mice with human immune interferon and further in a manner known per se (b) a lymphocyte mixture is isolated from the mice, (c) B lymphocytes fused or hybridized with mouse B myeloma cells, (d) the mixture from formed hybrids and non-hybridized B myeloma cells in a culture medium for the hybrids formed and the hybrids formed are cultivated and opposite the non-hybridized B myeloma cells selected, (e) subcloned on hybrids, which produce the antibody specific to human immune interferon is directed, and (f) optionally isolating the antibody.

The invention also relates to monoclonal antibodies that specifically against human fibroblast interferon or those specifically against human Immune interferon directed and can be produced by the fact that at stage (d) the selection is carried out in a medium containing methyl cellulose and possibly hybrids (which have been selected in step (d)) into the Injected abdominal cavity of mice (previously pretreated with a tumor stimulator have been), increased hybrids removed from the abdominal cavity and then the subcloning and performing isolation steps (e) and (f).

For further details on the manufacturability of hybrids, those specifically against or specifically against human fibroblast interferon human immune interferon-directed antibodies, or for manufacturability such antibody is based on the general method of manufacture according to the invention referenced by an antibody-producing hybrid cell.

Antibodies are built from a constant common to all of them Area and. a variable area that distinguishes them. at the monoclonal antibodies specific to human fibroblast interferon or specifically directed against human immune interferon, that is variable Human fibroblast interferon or human immune interferon area specific; See e.g. Milstein, Scientific American, 243 (October 1980) 56-64. For example, to identify human fibroblast interferon, see Goeddel et al., Nucleic Acids Research, 8 (18) (1980) 4057-4074, and for identification of human immune interferon see, for example, Yib et al., Proc. Natl. Acad. Sci.

USA, 78 (1981) 1601-1605.

Used B myeloma cells (FO) and hybridoma cells The used Cells were obtained from the Collection Nationale de Cultures de Microorgani.smes / Paris deposited and received the following deposit designations: cells deposit designation (1) B myeloma cells (FO) I-153 (2) Hybridoma cells for Hu (IFN-ß) 1-154 (3) hybridoma cells for Hu- (IFN-gamma) production and description of the cultures: (1) Originally obtained von Groth & Scheidegger, Journal of Immunological Methods, Vol. 35 (1980) 121.

(2) Obtained by fusing (1) with B lymphocytes from Balb / C mbuses, immunized with human fibroblast interferon.

(3) Obtained by the fusion of (1) with B-lymphocytes from Balb / c-MSusen, immunized with human immune interferon.

Culture medium used: Iscove's modified Dulbecco medium (IMDM), Gibco Laboratories Cat. No. 430-2200 with 10 percent. fetal calf serum and 50 / ug / ml Gentamycin; RPMI media can also be used. Sodium hydrogen carbonate concentration 3.024 g NaHCO 3 / liter at pH 7.0 Serum used for cell growth: 10 percent. Fetal Calf serum (Seromed Co.) Composition of solutions for cell dispersions: Likewise IMDM medium with 10 percent. fetal calf serum Optimal growth temperature: 37 oC.

Culture growth in suspension: starting cell concentration is best in the range of 10-3-104 cells / ml, harvests at 106, at the earliest from 105 cells / ml.

Time from the start of cultivation to harvesting approx. 1 week.

Transfer of cells: The cells can be partially on the surface of the tissue culture container. No trypsin should be used for the transfer, rather, the cells should be carefully detached from the surface1 by several drops of the tissue culture medium are dripped onto the surface.

Freezing cells: The cells are centrifuged, IMDM medium poured off and 1 ml of fetal calf serum / DMSO (95% / 5%) added to the pellet. thereafter is slowly frozen in nitrogen vapor or at -70 ° C and for 1 day in nitrogen vapor or kept at -70 ° C., after which it is stored in liquid nitrogen. The composition of the suspension medium is 95% fetal calf serum and 5% DM50, the cell concentration 106 cells / ml.

Thawing cells: The cells are placed in ice-cold IMDM, after which it is centrifuged at 1000 rpm for 10 minutes and in warm IMDM with 10 percent. Calf serum resuspended; after one day the medium should be exchanged again.

Notes: Growing cells should be treated with medium approximately every third day are supplied. A complete exchange of the medium is required after about 1 week. Since not all cells adhere to the surface, the cells have to be centrifuged off when the medium is replaced.

The invention is illustrated below by means of seven figures and two examples explained in more detail.

Legend to. Figure 1: Immunoreaction of "blotted" IFN-ß with monoclonal anti (Hu-IFN-ß) in the ELISA legend to Figure 2: (a) not purified IFN-ß (4 days author's scan) (b) non-purified IFN-ß '(1 day autoradiograph) (c) specifically immunoadsorbed IFN-ß (4 days autoradiography) (d) 14C molecular weight level proteins Legend to Figure 3: Elution profile (Sephadex .G200) of (A) purified anti- (IFN-ß) compared to an IgM control (B) and the neutralization activity (the antiviral Activity of IFN-ß) of the individual fractions Legend for illustration 4: Sephadex.G200 elution profile from monoclonal anti (Hu-IFN-gamma) legend to Figure 5: ELISA reaction of human anti (Hu-IFN-gamma) with Hu-IFN-gamma, that of an SDS polyacrylamide gel (without β-mercaptoethanol pretreatment) on nitrocellulose strips was transferred.

a) Hu-IFN-gamma plus monoclonal anti (Hu-IFN-gamm4) b) Hu-IFN-gamma plus medium (negative control) c) 14C-labeled standard proteins Legend to figure 6: ELISA reaction as in Figure 2, but after previous 8-mercaptoethanol pretreatment from Hu-IFN-gamma to SDS-polyacrylamide gel electrophoresis.

a) Ru-IFN-gamma plus monoclonal anti (Hu-IFN-gamma) b) standard proteins Stained with amido black. Legend to Figure 7: Autoradiogram of an SDS polyacrylamide gale (without ß-mercaptoethanol pretreatment of Hu-IFN-gamma) of iodine-125 labeled and with pansorbin anti- (Hu-IFN-gamma) precipitated m Hu-IFN-gamma a) specifically precipitated Iodine-125-Hu-IFN-gamma b) "Wash", non-specifically adsorbed protein material c) untreated Iodine-125-Hu-IFN-gamma preparation d) 14 C-labeled standard proteins example 1 A female Balb / c mouse was exposed to human fibroblast interferon four times in total (IFN-ß) one.

Dose of 1 / up, immunized per immunization. It became the initial Immunization uses highly purified IFN-ß that has been tested on Blue Sepharose school material was bound. This Blue Sepharose column material / IFN-ß - complex was (mixed with adjuvant) injected subcutaneously into the mouse weekly. In this way, on the one hand, the IFN-ß is stabilized against enzymatic degradation and, on the other hand, the antigenicity of the IFN-ß increased significantly. The last immunization was 4 days before isolation a lymphocyte mixture with non-columnar IFN-ß, its sugar content had been removed.

4 days after the last immunization, the spleen was removed 1 a Lymphocytes mixedly isolated and then B-lymphocytes with ß-mycloma cells (FO) fused and cultured in HAT medium with methyl cellulose for 1 to 2 weeks. Thereafter Growing hybridoma cultures were isolated with sterile capillaries and placed in HAT medium subcloned without methyl cellulose. Colonies that grew out of this were targeted to specific Antibody production tested. Testing was done in an interferon antiviral assay on human FS4 fibroblast cells according to Havell et al., J. Antimibrob.

Ag. Chemotherap., 2 (1972) 4t6 - 484. The inhibition of the Protective effect of fibronlast interferon against VSV virus by the monoclonal Antibodies measured; see table 1.

Several stable mouse hybridoma lines have been established.

There was no cross-reaction with purified leukocyte interferon established; see Table 2. The antiviral effect of interferon can also be observed after Degradation of the zuoker component of the fibroblast interferon by a mixture of sugar-degrading Enzymes are still inhibited by the specific antibody; see Table 2 the reinjection of the stable hybridoma cells (which correspond to the specific monoclonal Excrete antibodies) into the abdominal cavity of female Balb / C mice and the generation an ascites tumor could be found in the abdominal cavity of the Balb / C mice after about 14 to 21 days such an accumulation of specific against human fibroblast interferon Directed antibodies that were able to target the fibroblast interferon can be obtained in an antiviral assay at a dilution of the antibody of 1:; 30 completely to inhibit; see table 3.

The specificity of the antibody was determined using the well-known ELISA detection method demonstrated. For this purpose, partially purified human fibroblast interferon was used initially on an SDS gel according to Lämmli (Nature, 227 (1970) 680-685) (parallel with standard proteins) separated electrophoretically and then electrophoretically according to Towbin et al. (Proc.

Natl. Sct. USA 76 (1979) 4350-4354) on nitrooellulose strips transferred. While the blot part with the standard proteins is stained with amido black the blot part with fibroblast interferon was subjected to the ELISA-Teohnik and according to Towbin et al. with peroxidase-conjugated anti-mouse IgG antibodies Rabbit and c-disnisidine as enzyme substrate in a sharp brown colored band made visible; see Figure 1.

Table 1 Hybridoma supernatants Antiviral activity of (dilution series) Standard IPN -β (%) 1: 3 20 1: 6 40 1:10 70 1s20 90 control, koino antibody 100 Tabel 2 sample of antiviral protection of FS4 cells against VSV (%) 1 IFN -B 100 2 IFN-ß protein content 100 3 IFN-β + monoclonal. Antibodies against IFN-ß 0 4 IFN-ß + trypsin treated monoclonal antibody against IFN-ß 100 5 IFN-ß-Ü protein content + monoclonal antibody against IFN -B 0 6 IFN-α + monoclonal antibody against IFN.-B 100 table 3 monoclonal hybridoma IgM antiviral activity against IFN-ß of standard IFN-ß (%) 1: 3 30 1: 6 50 1:15 70 1:30 90 Control without antibodies 100 The inventive monoclonal antibodies can be used for the specific separation of human fibroblast interferon (and thus for its purification) from mixtures of substances containing this interferon use.

The monoclonal antibody against IFN-ß was activated on CNBr-Sepharose-4B bound; this antibody column was tested for specific immunoadsorption of IFN-ß tested. For this purpose an unpurified IFN-ß--.

Sample marked with 125.1 (according to Hunter et al., Nature, 194 (1962) 495-496). This 125I-labeled IFN-β sample was then loaded onto the antibody column and then washed thoroughly with neutral TBS (Tris-buffered saline), to remove non-specifically bound protein. The specifically bound 125-I-labeled IFN-ß was then eluted with 0.1 M glycine / HCl buffer (pH 2.3) by lyophilization concentrated and then in parallel with unpurified 125-I-labeled IFN-B and 14-C-labeled molecular weight standard proteins electrophoretically on a SDS polyacrylamide gel separated (Laemmli, Nature (London), 227 (1970) 680-685). The gel was then autoradiographed.

Fig. 2 clearly shows that IFN-ß is specific to the monoclonal Anti- (Hu-IFN-ß) -CNBr-Sepharose column is bound.

Furthermore, the antibody was from the ascites fluid with 35 percent. Ammonium sulfate and a subsequent Sephadex G200 column step. That Sephadex G200 profile showed the monoclonal anti (Hu-IFN-β) to be en IgM; see Fig. 3.

Example 2 A female Balb / c mouse was made according to example 1 except that human immune interferon was used (Hu-IFN-gamma) until a positive antibody titer against Bu-IFN-gamma was found in the serum of the mouse. 4 days before isolation of a lymphocyte mixture the spleen of the immunized Balb / c mouse became a final "booster" immunization performed. 4 days later, the lymphocyte mixture was then isolated from the spleen, and then B lymphocytes with B myeloma cells [FO (Groth and Scheidegger (1980), J.

Immunol. Methods 35, 1 - 25) merged see Hochkeppel et al. Europ. J. Biochem., 118 (1981) 437-442), and 5 days in HAU medium with 1% methyl cellulose and 20% fetal calf serum are cultivated. For better reproduction of survivors The hybrids were then used to stabilize the hybrids against gene loss injected into the abdominal cavity of a Balb / c mouse that had been treated with pristane 2,6,10,14-xetramethylpentadecane had been pretreated. After a week, the ascitic fluid became the abdominal cavity taken sterile and the hybrid cells contained therein were without methyl cellulose in normal Iscove's Dulbeoco Nedium sown that 10% foetales Contained calf serum. The growing hybridoma cultures were then placed in Greiner single clone tissue culture plates subcloned. Colonies growing therefrom were targeted for specific antibody production tested out. Testing occurred after an antiviral neutralization test human CCL23 cells.

Initially, an antibody directed against mouse immunoglobulins was used from rabbits for 16 hours on the surface of the wells of an immunomicrotiter plate adsorbed, then the supernatants of the respective hybridoma cultures also for 16 hours are added to remove any Hu-IFN-gamma antibodies contained in the adsorbed To bind antibodies, and finally 10 antiviral units per ml of Hu-IFN-gamma added (4 hours). The supernatant was then checked for residual antiviral activity against vesicular stomatitis virus (VSV) on human CCL23 cells against a untreated Hu-IFN-gamma sample tested. In this way several became stable Mouse Rybridoma lines established, which specifically monoclonal anti (hu-IFN-gamma) eliminated.

To identify the immunoglobulin type of the monoclonal antibody the antibody was initially obtained from hybridoma supernatants by means of 40% ammonium sulfate precipitation and a subsequent Serhadex G200 column chromatography step. The anti (Hu-IFN-gamma) was identified as IgM, which was in the exclusion volume the Sephadex-G 200 column is eluted identically to the elution profile of an IgM control (Figure 4).

The specificity of the monoclonal anti (Ru-IFN-gamma) IgM purified in this way was then clearly proven with the following specificity tests. First was partially purified Hu-IFN-gamma on an SDS polyacrylamide gel according to Lämmli (Nature (London) 227 (1970) 680-685) without B-mercapto-ethanol pretreatment in parallel with Standard proteins are separated by electrophoresis and then separated electrophoretic according to Towbin et al. (Proc. Natl. Sci.

Acad. USA, 76 (1979) 4350-4354) transferred to nitrocellulose strips. While the blot part with the standard proteins was stained with amido black, the blot part with Hu-IFN-gamm of the ELISA-Teehnik according to Hochkeppel et al. (Europ. J. Biochem. 118 (1981) 437-442), the blot first being with monoclonal Mouse anti (Hu-IFN-gamma) and subsequently with IgM antibodies directed against mouse IgM from rabbits and finally with IgM antibodies directed against rabbit IgM from goat (which was peroxidase conjugated), after which the Hu-IFN-gamma using o-dianisidine as enzyme substrate in a sharp band around 46,000 Daltons was made visible (Figure 5). The same experiment was done with Hu-IFN-gamma repeated that had been pretreated with ß-mercaptoethanol / SDS. With ELISA blot a specific band around 22,000 daltons was visualized using o-dianisidine (Figure 6). So the monoclonal anti (Ru-IFN-gamma) is specific to either against two different (Hu-IFN-gamma) subspecies or once against the dimer (Figure 5) and once directed against the monomer (Figure 6) of the Hu-IFN-gamma molecule, wherein the shift in molecular weight of the immunoreactive band according to ß-mercaptoethanol pretreatment more indicates that it is more likely to be Ru-IFN-gamma monomer and dimers.

In a further specificity test, initially partially purified Hu-IFN-gamma, previously described by Hunter et al. [Nature, 124 (1962) 495-961 with iodine-125 labeled with mouse monoclonal anti (Hu-IFN-gnmm) as well as subsequently incubated with rabbit IgG directed against IgM. The complex was then with IgG-specific Staphylococcus aurus protein A (pansorbin) precipitated. After several thorough washing of the complex and subsequent detachment of the monoclonal Anti- (Ru-IFN-gamma) specifically precipitated 125I-Hu-gamma by SDS treatment the immunoprecipitated material was electrophoretically without ß- Mercaptoethanol pre-treatment on an SDS polyacrylamide gel according to Lämmli (Nature (London), 227 (1970) 680 - 685) separated in parallel with 14C-labeled standard proteins.

The gel was then autoradiographed after drying. That Autoradiogram (Figure 7) clearly shows that 125I-IFBN-gamma specifically means monoclonal anti (Hu-IFN-gamma) can be precipitated and radioactive as a clean Band around 46,000 daltons visible on the autoradiogram of an SDS polyacrylamide gel can be made.

Notes: ConA, Sephacryl-S-200 and Sephadex-G-200 columns: German Pharmacia GmbH, 6000 Frankfurt / Main 50 Iscoves (modified) Dulbecco-Nedium: BCK-BIokult-Chemie, Postfach 21 02 65, 7500 Karlsruhe Greiner tissue culture plates (384-fold for cell clones): Greiner & Söhne, Postfach 13 20, 7440 Nürtingen IgM, IgG, goat and rabbit antibodies or IgM antibodies: Byk-Mallinckrodt, Von-Hevesystr. 1-3, 6057 Dietzenbach 2 SDS-polyacrylamide gel: see Laemmli, U. K., Nature (London), 227 (1970) 680-685 and ochkeppel et al., Eur. J. Biochem., 118 (1981) 437-442 pansorbin (Staphylococcus aureus protein A): calbiochem, Postfach 11 03 60, 6300 Giessen CCL23 cells: (a) Regainstitut, Minderbroederstraat 10, R-3000 Leuven, Belgium, and (b) Flow Laboratories, Mühlengrnbenstr. 10, 5309. Meckenheim / Ronn Blank page

Claims (20)

PATENT CLAIMS: 1. Method for producing an antibody producing hybrid cells in which one (a) immunizes mice with an antigen, (b) a lymphocyte mixture isolated from the mice, (c) B lymphocytes with mouse B mycloma cells fused or hybridized, (d) the mixture of hybrids formed and not hybridized B myeloma cells are transferred to a culture medium for the hybrids formed and the hybrids formed are cultured and opposed to the non-hybridized B myeloma cells selected, (e) subcloned for hybrids that produce the antibody that is specifically directed against the antigen, and (f) optionally isolating the hybrids, thereby G e k e n n nz e i c h ne t that one in stage (d) the selection in one Performs methyl cellulose-containing medium and selected in step (d) Hybrids may be injected into the abdominal cavity of mice (previously with a tumor stimulator have been pretreated), increased hybrids taken from the abdominal cavity and then performs the subcloning and isolation steps (e) and (f). a. Method according to claim 1, characterized in that g e k e n n-X e i c h n e t, that one has 9 lymphocytes from Balb-C mice and / or B myclomal cells from Balb-C mice used. 3. The method according to claim 1 or 2, characterized g e k e n nz e i c h n e t that one immunizes more than once and / or that one has the last immunization carried out about 4 days before isolation of the lymphocyte mixture. 4. The method according to any one of the preceding claims, characterized g e does not indicate that immunization is carried out with an antigen attached to a molecular sieve is adsorbed. 5. The method according to any one of the preceding claims, characterized g e no indication that immunization is carried out with a human interferon, e.g. Ru-IFN-ß or HuIFN-gamma, and possibly the last immunization with the human interferon after removing its sugar content. 6. The method according to any one of the preceding claims, characterized g e no indication that the lymphocyte mixture from female mice, preferably female Balb / c mice isolated. 7. The method according to any one of the preceding claims, characterized g e it is not indicated that in stage (d) the selection is carried out in one carries out macrophage-free medium. 8. Procedure according to. one of the preceding claims, characterized g e it is not stated that in stage (d) one selects for about 4 days, before initiating the selected hybrids into the peritoneal cavity of mice. 9, the method according to any one of the preceding claims, characterized g e it is not stated that a tumor stimulator is additionally used in stage (f) or an adjuvant and then the antibody producing hybrids into the abdominal cavity injected by mice, the multiplied hybrids removed and ggi. isolated the antibody. 10. The method according to claim 1, 8 or 9, characterized g ek e n n z e i c h.n e t that one can enter the abdominal cavity of female mice, preferably female Injected into Balb-C mice. 11. Method for the preparation of a monoclonal antibody, at by immunizing (a) mice with an antigen, (b) a lymphocyte mixture from the mice isolated (c) B lymphocytes fused or hybridized with mouse B mycloma cells, (d) the mixture of hybrids formed and non-hybridized B mycloma cells transferred to a culture medium for the hybrids formed and the hybrids formed cultivated and selected from the non-hybridized B myeloma cells, (e) subcloned on hybrids that produce the antibody specific to the antigen is directed, and (f) ggi. the antibody is isolated, thereby g e k e n nz e i c h n e t that one in step (d) the selection in a methyl cellulose Carries out containing medium and optionally selected hybrids in step (d) injected into the abdominal cavity of mice (previously pretreated with a tumor stimulator been are) and extracts increased hybrids from the abdominal cavity. 12. The method according to claim 11, characterized in that g e k e n n -z e i c h n e t that one applies the measures of one of the preceding claims 2 to 10. 13. Use of the antibody producing hybrids for one Injection according to claim 9 or 10 or an antibody isolation according to claim 9, 10, 11 or 12. 14. Antibody specific to human fibroblast interferon is directed, producing hybrids by h e r s t e 1 1 b a r, that one (a) Mice immunized with human fibroblast interferon and further in itself in a known manner (b) a lymphocyte mixture isolated from the mice, (c) B lymphocytes iused or hybridized with MSuse-B myeloma cells, (d) the mixture of Hybrid and non-hybridized B myeloma cells in a culture medium for the formed Hybrids transferred, the hybrids formed cultivated and compared to the non-hybridized B-uyeloma cells selected, (e) subcloned on hybrids that produce the antibody, which is specifically directed against human fibroblast interferon, and (f) if necessary, these hybrids are isolated. 15. Hybrids with those for the production of a monoclonal antibody which is specifically directed against human fibroblast interferon are essential Deposit Features 1-154 and Deposit Deriving Hybrids. 16. Antibody specific to human immune interferon is directed, producing hybrid, by h e r 5 t e 1 1 b a r that one (a) Mice immunized with human immune interferon and also known per se Way (b) isolated a lymphocyte mixture from the mice, (c) with B-lymphocytes Mouse B myeloma cells fused or hybridized, (d) the mixture of Hybrid and non-hybridized B myeloma cells in a culture medium for the formed Hybrids Transferred and cultivated the hybrids formed and, on the other hand, not hybridized B myeloma cells are selected, (e) subcloned for hybrids which have the Produce antibodies specifically directed against human immune interferon and (f) optionally isolating these hybrids. 17. Hybrid according to claim 14 or 16, characterized in that h e r -s t e 1 1 b a r that one in step (d) the selection in a methyl cellulose containing Medium carries out and in stage (d) selected hybrids in injected into the naval cavity of mice (previously pretreated with a tumor stimulator have been), increased hybrids removed from the abdominal cavity and then the subcloning and performs isolation steps (e) and (f). 18. Monoclonal antibody specific to human fibroblast interferon is directed, by h e r s t e 1 1 b a r that one (a) mice with human Fibroblast interferon immunized and furthermore in a manner known per se (b) A lymphocyte mixture isolated from the mice, (c) B lymphocytes with M: use R myeloma A cells fustontert or hybridized, (d) the mixture of hybrids formed and non-hybridized R-myeloma cells in a culture medium for the formed Hybrids are transferred and the hybrids formed are cultivated, and not compared to the hybrids hybridized B myeloma cells are selected, (e) subcloned for hybrids which have the Produce antibodies that are specific to human fibroblast interferon is directed, and (f) optionally isolating the antibody. 19. Monoclonal antibody specific to human immune interferon is directed, by h e r -5 t e 1 1 b a r that one (a) mice with human Immunized interferon and also immunized in a manner known per se (b) from the mice a lymphocyte mixture isolated, (c) B lymphocytes fused with mouse B myeloma cells or hybridized, (d) the mixture of hybrids formed and non-hybridized B myeloma cells transferred into a culture medium for the hybrids formed and the cultivated hybrids formed and compared to the non-hybridized B myeloma cells selected, (e) subcloned for hybrids that produce the antibody that specifically directed against human immune interferon, and (f) optionally isolated the antibody. 20. Antibodies according to claim 18 or 19, characterized in that they are 1 1 b a r that in step (d) the selection is made in a methyl cellulose containing Medium carried out and in stage (d) selected hybrids in injected into the abdominal cavity of mice (previously pretreated with a tumor stimulator have been), increased hybrids removed from the abdominal cavity and then the subclnation and performing isolation steps (e) and (f).