GB2092614A - Process for the manufacture of hybrid cells producing an antibody in which methylcellulose is used in the selection of hydrides - Google Patents

Abstract

A process for the manufacture of hybrid cells capable of producing an antibody in which (a) mice are immunised with an antigen, (b) a lymphocyte mixture is isolated from the mice, (c) B lymphocytes are fused or hybridised with mouse B myeloma cells, (d) the mixture consisting of formed hybrids and non-hybridised B myeloma cells is conveyed into a culture medium for the formed hybrids and the formed hybrids are cultivated and selected with respect to the non- hybridised B myeloma cells, (e) they are subcloned to provide hybrids which are capable of producing the antibody which is directed specifically against the antigen, and (f) optionally the hybrids are isolated, characterised in that at step (d) the selection process is carried out in a medium containing methylcellulose (as hereinbefore defined), and hybrids selected at step (d) are optionally injected into the abdominal cavity of mice (which have previously been treated with a tumour stimulant), multiplied hybrids are removed from the abdominal cavity and subsequently the subcloning and isolating steps (d) and (f) are carried out. The invention also provides a process for producing monoclonal antibodies, and further provides hybrid cells capable of producing antibodies directed specifically against human interferon and provides such monoclonal antibodies.

Description

SPECIFICATION Process for the manufacture of hybrid cells producing an antibody and for the manufacture of antibodies, hybrid cells and antibodies The present invention relates to the manufacture of hybrid cells adapted to produce specific antibodies, and to monoclonal antibodies.

The invention provides a process for the manufacture of hybrid cells capable of producing an antibody in which (a) mice are immunised with an antigen, (b) a lymphocyte mixture is isolated from the mice, (c) B lymphocytes are fused or hybridised with mouse B myeloma cells, (d) the mixture consisting of formed hybrids and non-hybridised B myeloma cells is conveyed into a culture medium for the formed hybrids and the formed hybrids are cultivated and selected with respect to the non-hybridised B myeloma cells, (e) they are subcloned into hybrids which produce the antibody which is directed specifically against the antigen, and (f) optionally the hybrids are isolated, the process being characterised in that at step (d) the selection process is carried out in a medium containing methylcellulose (as hereinafter defined) and optionally hybrids ( which have been selected at step (d) are injected into the abdominal cavity of mice (which have previously been treated with a tumour stimulant), multiplied hybrids are removed from the abdominal cavity, and subsequently the subcloning and isolating steps (e) and (f) are carried out.

The present invention also provides a process for the manufacture of a monoclonal antibody in which (a) mice are immunised with an antigen, (b) a lymphocyte mixture is isolated from the mice, (c) B lymphocytes are fused or hybridised with B myeloma cells, (d) the mixture consisting of formed hybrids and non-hybridised B myeloma cells is conveyed into a culture medium for the formed hybrids and the formed hybrids are cultivated and selected with respect to the non-hybridised B myeloma cells, (e) they are subcloned to provide hybrids which are capable of producing antibody which is directed specifically against the antigen, and (f) optionally the antibody is isolated, characterised in that at step (d) the selection process is carried out in a medium containing methylcellulose (as hereinbefore defined), and optionally hybrids selected at step (d) are injected into the abdominal cavity of mice (which have previously been treated with a tumour stimulant) and multiplied hybrids are removed from the abdominal cavity.

Relevant processes are available to the man skilled in the art for immunising, for isolating a spleen lymphocyte mixture, for fusing or hybridising, for cultivating the hybrids and for selecting them, for subcloning hybrids which produce the desired antibody and for isolating these hybrids or the antibody.

For fusing or hybridising and for cultivating formed hybrids, reference is made, for example, to Kohler et a/., Nature (London), 256(1975)495-497, K6hlereta!., Eur. J. Immunol., 6(1976)511-519 and Hochkeppel etna!., Eur. J. Biochem., 118 (1981) 437 142.

For selecting, subcloning or monocloning and for isolating the hybrids and the antibody, reference is made, for example to Secher et a/., Nature (London), 285 (1980) 446 a150 and Hochkeppel et a/., loc. cit. With regard to the term subcloning or monocloning, reference is made finally to Vernon etna!., in Mishell and Shiigi, Selected Methods in Cellular Immunology: 373-397, Freeman and Co., San Francisco 1 980. At the subcloning or monocloning step, the occurrence of a stable cell line having stable antibody-production can be observed with the aid of an antibody activity test (inhibition of antigen activity), for example according to the method of Haveli et al., J. Antimicrob. Ag.Chemotherap., 2(1972)476-484.

With regard to the term myeloma cells, see, for example, Milstein, Scientific American, 243 (October 1980) 56-64. Myeloma cells can be obtained, for example, according to Groth and Scheidegger, J. Immunological Methods, 35 (1980) 121. Examples of B myeloma cells are to be taken from the following table.

The man skilled in the art can choose a suitable strain of mouse by testing whether the B myeloma cells chosen by him can be hybridised with the B lymphocytes of the test mouse. Advantageously, Balb/c mice and B myeloma cells of Balb/c mice are used, since these mice are quite common in animal experiments. Mice of that type can be obtained commercially and can be procured, for example, from the Zentralinstitut für Versuchstierzucht (Central Institute for Breeding Laboratory Animals), Hanover.

It may be advantageous for the antigen used for immunisation to be adsorbed over a molecular sieve.

Preferably immunisation is carried out more than once. In the case of human interferon, the final immunisation is carried out preferably with human interferon from which the sugar portion has been removed. The sugar portion can be removed, for example, according to the method of Bose et a/., J. Biol.

Chem., 251(1976)1659-1662. The final immunisation is preferably carried out substantially about 4 days before the lymphocyte mixture is isolated.

Preferably, the lymphocyte mixture is isolated from female mice, especially female Balb/c mice.

Female mice have a relatively rapid immune reaction.

TABLE OF B MYELOMA CELLS Technical Reference Source Source Reference (a) FO F.-Miescher-lnstitut, Basle CNCM, Paris 1-153 (b) P3-X 63 Ag 8 Institut für Immunologie, Basle (Institute for Immunology) Institut für Genetik, Cologne (Institute for Genetics) (c) P3-NS 1-1 Institut für Immunologie, Basle (Institute for Immunology) Institut für Genetik, Cologne (Institute for Genetics) If the selection process is carried out in a medium which contains methylcellulose the formed hybrid cells are especially protected. The expression "methylcellulose" as used herein includes also substituted methylcelluloses such as, for example, carboxymethylcellulose. An example of methylcellulose is Methocel MC (4000 cP; Fluka).An example of a suitable medium is HAT medium; see Hochkeppel et al., loc cit. Further protection may be obtained by using a medium free from macrophages.

A good stabilisation of selected hybrids can be achieved if the selected hybrids are injected into the abdominal cavity of mice which have previously been treated with a tumour stimulant. The selection process can, for example, be carried out for about 4 days before the selected hybrids are injected into the abdominal cavity of mice. The multiplied hybrids removed from the abdominal cavity are then conveyed to the subcloning and isolating step of the process according to the invention. An example of a customary tumour stimulant will be given below. The man skilled in the art is familiar with the methods to be used; see, for example, Secher et a/., Nature (London), 285 (1980) 446-450. For example, Balb/c mice accept a hybrid which has been obtained from B lymphocytes of Balb/c mice and from B myeloma cells of Balb/c mice.

Furthermore, good results are obtained when a tumour stimulant or an adjuvant is injected into the abdominal cavity of mice and then subcloned hybrids producing the antibody are injected, the multiplied hybrids are removed and, optionally, the antibody is isolated. The methods mentioned above for injecting selected hybrids apply as appropriate. 2,6,10,1 4-tetramethylpentadecane is an example of a customary tumour stimulant and Freund's adjuvant is an example of a customary adjuvant.

Preferably female mice, especially Balb/c mice, are used for the injection of selected hybrids or of hybrids producing the antibody.

The invention provides also hybrid cells capable of producing an antibody (which is directed specifically against human fibroblast interferon =Hu-lFN-beta) which hybrids are obtainable by a process wherein (a) mice are immunised with human fibroblast interferon, and furthermore, generally in a manner known per se, (b) a lymphocyte mixture is isolated from the mice, (c) B lymphocytes are fused or hybridised with mouse B myeloma cells, (d) the mixture consisting of formed hybrids and non-hybridised B myeloma cells is conveyed into a culture medium for the formed hybrids, the formed hybrids are cultivated and selected with respect to the non-hybridised B myeloma cells, (e) they are subcloned to provide hybrids which are capable of producing the antibody which is directed specifically against human fibroblast interferon, and (f) optionally these hybrids are isolated.

The term "known" is used herein to mean in actual use in the art or described in the literature of the art, for example, the literature referred to herein.

Furthermore, the invention provides hybrid cells having the features of deposit 1-1 54 necessary for the manufacture of a monoclonal antibody which is directed specifically against human fibroblast interferon and to hybrids derived from the deposit.

Furthermore, the invention provides hybrid cells capable of producing antibody (which is directed specifically against human immune interferon =Hu-lFN-gamma), these hybrids being obtainable by a process wherein (a) mice are immunised with human immune interferon, and furthermore, generally in a manner knownperse, (b) a lymphocyte mixture is isolated from the mice, (c) B lymphocytes are fused or hybridised with mouse B myeloma cells, (d) the mixture consisting of formed hybrids and non-hybridised B myeloma cells is conveyed into a culture medium for the formed hybrids, the formed hybrids are cultivated and selected with respect to the non-hybridised B myeloma cells, (e) they are subcloned to provide hybrids which are capable of producing the antibody which is directed specifically against human immune interferon, and (f) optionally these hybrids are isolated.

Furthermore, the invention provides hybrid cells capable of producing antibody (which is directed specifically against human fibroblast interferon) or antibody (which is directed specifically against human immune interferon), in the production of which hybrids at step (d) the selection process is carried out in a medium containing methylcellulose (as hereinbefore defined), and, optionally, hybrids (which have been selected in step (d)) are injected into the abdominal cavity of mice (which have previously been treated with a tumour stimulant), multiplied hybrids are removed from the abdominal cavity and subsequently the subcloning and isolating steps (e) and (f) are carried out.

The invention provides also a monoclonal antibody which is directed specifically against human fibroblast interferon (Hu-lFN-beta) and which is obtainable by a process wherein (a) mice are immunised with human immune interferon, and furthermore, generally in a manner known per se, (b) a lymphocyte mixture is isolated from the mice, (c) B lymphocytes are fused or hybridised with mouse B myeloma cells, (d) the mixture consisting of formed hybrids and non-hybridised B myeloma cells is conveyed into a culture medium for the formed hybrids and the formed hybrids are cultivated and selected with respect to the non-hybridised B myeloma cells, (e) they are subcloned to provide hybrids which are capable of producing the antibody which is directed specifically against human fibroblast interferon, and (f) optionally the antibody is isolated.

Furthermore, the invention provides a monoclonal antibody which is directed specifically against human immune interferon (Hu-lFN-garnrna) and which is obtainable by a process wherein (a) mice are immunised with human immune interferon, and furthermore, generally in a manner known per se, (b) a lymphocyte mixture is isolated from the mice, (c) B lymphocytes are fused or hybridised with mouse B myeloma cells, (d) the mixture consisting of formed hybrids and non-hybridised B myeloma cells is conveyed into a culture medium for the formed hybrids and the formed hybrids are cultivated and selected with respect to the non-hybridised B myeloma cells, (e) they are subcloned to provide hybrids which are capable of producing the antibody which is directed specifically against human immune interferon, and (f) optionally the antibody is isolated.

Furthermore, the invention provides monoclonal antibodies which are directed specifically against human fibroblast interferon or specifically against human immune interferon in the production of which, at step (d), the selection process is carried out in a medium containing methylcellulose (as hereinbefore defined), and optionally hybrids (which have been selected at step (d)) are injected into the abdominal cavity of mice (which have previously been treated with a tumour stimulant), multiplied hybrids are removed from the abdominal cavity and subsequently the subcloning and isolating steps (e) and (f) are carried out.

With reference to further details about the ability to produce hybrids which produce antibodies directed specifically against human fibroblast interferon or specifically against human immune interferon, or the ability to produce such antibodies, reference is made to the general process according to the invention for the manufacture of hybrid cells producing an antibody.

Antibodies are composed of one constant region which is common to them all and one variable region by which they differ from one another. In the case of monoclonal antibodies which are directed specifically against human fibroblast interferon or specifically against human immune interferon; see, for variable region which is specific for human fibibblast interferon or for human immune interferon; see, for example, Milstein, Scientific American, 243 (October 1980) 56-64. For the identification of human fibroblast interferon see, for example, Goeddel etna!., Nucleic Acids Research, 8 (18) (1 980) 4057-4074, and for the identification of human immune interferon see, for example, Yib et al., Proc.

Natl. Acad. Sci. USA, 78 (1981)1601-1605.

B MYELOMA CELLS (FO) AND HYBRIDOMA CELLS USED The cells used were deposited at the Collection Nationale de Cultures de Microorganismes (National Collection of Microorganism Cultures) Paris and were given the following deposition references: Cells deposition reference (1 ) B myeloma cells (FO) 1-153 (2) Hybridoma cells for Hu-(lFN-ss) 1-154 (3) Hybridoma cells for Hu-(lFN-garnrna) Production and description of the cultures: (1) Originally obtained by Groth and Scheidegger, Journal of Immunological Methods, vol. 35 (1980)121.

(2) Obtained by fusing (1) with B lymphocytes of Balb/c mice which had been immunised with human fibroblast interferon.

(3) Obtained by fusing (1) with B lymphocytes of Balb/c mice which had been immunised with human immune interferon.

Culture medium used: Iscove modified Dulbecco medium (IMDM), Gibco Laboratories Cat. No.430-2200 with 10% foetal calves serum and 50,ug/ml of gentamycin: RPMI medium can also be used. Sodium bicarbonate concentration 3.024 g NaHCO3/litre at pH 7.0.

Serum used for cell growth: 10% foetal calves serum (Seromed Co.).

Composition of solutions for cell dispersion: likewise IMDM medium with 10% foetal calves serum.

Optimal temperature for growth: 370C.

Growth of culture in suspension: The optimum cell concentration at the outset is in the range of from 1 03 to 104 cells/ml, yields at 108, at the eariiest from 105, cells/ml. tme elapsed from the beginning of culture to the yield approximately 1 week.

Transfer of cells: Some of the cells may adhere to the surface of the tissue culture container. No trypsin should be used for the transfer, on the contrary, the cells should be carefully detached from the surface by dropping several drops of the tissue culture medium onto the surface.

Freezing the cells: The cells are centrifuged off, IMDM medium is decanted and 1 ml of foetal calves serum/DMSO (95%/5%) is added to the pellet. Subsequently it is slowly frozen in nitrogen vapour or at -700C and maintained for 1 day in the nitrogen vapour or at -700C, after which it is preserved in liquid nitrogen.

The suspension medium is composed of 95% foetal calves serum and 5% DMSO, the cell concentration is 105 cells/ml.

Thawing the cells: The cells are poured into ice-cold IMAM, then centrifuged for 10 minutes at 1000 rpm and suspended again in warm IMDM with 10% calves serum; after one day the medium should be changed again.

Observations: Growing cells should be supplied with medium approximately every third day. After about 1 week it is necessary to change the medium completely. Since not all cells adhere to the surface, the cells must be centrifuged off when the medium is changed.

The invention is explained in detail below with reference to seven Figures and two Example.

Legend to Figure 1: Immune reaction of "blotted" IFN-ss with monoclonal anti-(Hu-lFN-ss) in ELISA.

Legend to Figure 2: (a) unpurified IFN-ss (4 days autoradiography) (b) unpurified IFN-P(1 day autoradiography) (c) specifically immunoadsorbed IFN-ss (4 days autoradiography) (d) '4C-molecular weight standard proteins.

Legend to Figure 3: Elution profile (Sephadex-G200) of (A) purified anti-(IFN-P) in comparison with an IgM control (B) and the neutralisation activity (of the antiviral activity of IFN-ss) of the individual fractions.

Legend to Figure 4: Sephadex-G200 elution profile of monoclonal anti-(HU-IFN-gamma).

Legend to Figure 5: ELISA reaction of human anti-(Hu-lFN-gamma) with Hu-lFN-gamma which has been transferred from an SDS-polyacrylamide gel (without ss-mercaptoethanol pretreatment) onto nitrocellulose strips.

(a) Hu-lFN-gamma plus monoclonal anti-(Hu-lFN-gamma) (b) Hu-lFN-gamma plus medium (negative control) (c) '4C-labelled standard proteins.

Legend to Figure 6: ELISA reaction as in Figure 2, however after ss-mercaptoethanoi pretreatment of Hu-IFN-gamma for SDS-polyacrylamide gel electrophoresis.

(a) Hu-lFN-gamma plus monoclonal anti-(Hu-IFN-gamma) (b) Standard proteins stained with amido black.

Legend to Figure 7: Autoradiogram of an SDS-polyacrylamide gel (without ss-mercaptoethanol pretreatment of Hu IFN-gamma) of Hu-IFN-gamma labelled with iodine-125 and precipitated with pansorbin anti-(Hu-lFNgamma).

(a) specifically precipitated iodine-1 25 Hu-IFN-gamma (b) "Wash", unspecifically adsorbed protein material (c) untreated iodine-1 25 Hu-lFN-gamma preparation (d) 14C-labelled standard proteins.

EXAMPLE 1 A female Balb/c mouse was immunised four times in total with human fibroblast interferon (IFN-ss) using a dose of 1 y9 per immunisation. In this case, highly purified IFN-p which was bonded to blue sepharose column material was used for the initial immunisation. This blue sepharose column material/lFN-ss complex was injected (mixed with adjuvant) subcutaneously to the mouse weekly. In this manner, on the one hand the IFN-ss was stabilised against enzymatic degradation and on the other hand the antigeneity of the IFN-ss was considerably increased.The final immunisation took place 4 days before the isolation of a lymphocyte mixture using IFN-ss which was not bonded to a column and from which the sugar portion had been removed.

4 days after the final immunisation, the spleen was removed, a lymphocyte mixture was isolated and subsequently B lymphocytes were fused with B myeloma cells (FO) and cultivated for from 1 to 2 weeks in HAT medium having methylcellulose. Growing hybridoma cultures were then isolated with sterile capillary tubes and subcloned in HAT medium without methylcellulose. Colonies growing therein were tested for specific antibody production. The testing took place in an antiviral interferon assay on human FS4-fibroblast cells according to Havell petal., J. Antimicrob. Ag. Chemotherap., 2 (1972) 476-484. In that test, the inhibition by the monoclonal antibodies of the protective action of fibroblast interferon against VSV-virus was measured; see Table 1. Several stable mouse hybridoma lines were established.No cross-reaction with purified leucocyte interferon was found; see Table 2. The antiviral action of interferon can still be inhibited by the specific antibody also after the sugar portion of the fibroblast interferon has been degraded by a mixture of sugar-degrading enzymes; see Table 2. By reinjecting the stable hybridoma cells (which secrete the specific monoclonal antibody) into the abdominal cavity of female Balb/c mice and by the production of an ascitic tumour, it was possible to obtain from the abdominal cavity of the Balb/c mice, after about 14 to 21 days, such an enrichment of antibodies directed specifically against human fibroblast interferon which were able to inhibit completely the fibroblast interferon in an antiviral assay even when the antibody was diluted to 1:30; see Table 3.

The specificity of the antibody was shown by means of the known ELISA indicator method. For this purpose, partially purified human fibroblast interferon was first of all split up on an SDS gel according to Lämmli (Nature, 227 (1970) 680-685) (parallel with standard proteins) by electrophoresis and then transferred by electrophoresis according to Towbin ear as (Proc. Navi. Sci. USA, 76 (1979)4350-4354) onto nitrocellulose strips.While the blot portion having the standard proteins was stained with amido black, the blot portion having fibroblast interferon was subjected to the ELISA technique and rendered visible in a sharp brown colour band according to Towbin eft at using peroxidase-conjugated anti-mouse-lgG-antibody from rabbits and o-dianisidine as enzyme substrate; see Figure 1.

TABLE 1 Hybridoma supernatents Antiviral activity of (dilution series) standard IFN-p (%) 1:3 20 1:6 40 1:10 70 1:20 90 Control, no antibodies 100 TABLE 2 Antiviral protection of Sample FS4-cells against VSV (%) 1 IFN-p 100 2 IFN-/3-protein portion 100 3 IFN-p + monoclonal antibody against IFN-p O 4 IFN-p + monoclonal antibody, treated with 100 trypsin, against IFN-p 5 IFN-B-protein portion + monoclonal 0 antibody against IFN-P 6 IFN-a + monoclonal antibody against IFN-P 100 TABLE 3 Monoclonal hybridoma-lgM Antiviral activity of against IFN-P standard IFN-P (%) 1:3 30 1:6 50 1:15 70 1:30 90 Control without antibodies 100 The monoclonal antibody according to the invention can be used for the specific separation of human fibroblast interferon (and thus for the purification thereof) from mixtures of substances containing this interferon.

The monoclonal antibody against IFN-B was bonded to CNBr-activated Sepharose-4B; this antibody column was tested for specific immunoadsorption of IFN-B. For this purpose, an unpurified IFN-p sample was labelled with 1251 (according to Hunter et aA, Nature, 194 (1962) 495-496). This '251-labelled IFN-p sample was then loaded onto the antibody column and subsequently washed thoroughly with neutral TBS (tris-buffered saline) to remove unspecifically bonded protein. The specifically bonded 1251-labelled IFN-P was then eluted with 0.1 M glycine HCI buffer (pH 2.3), concentrated by lyophilisation and subsequently, parallel with unpurified 1251-labelled IFN-P and 14Clabelled molecular weight standard proteins, was split up by electrophoresis on an SDS-polyacrylamide gel (Laemmli, Nature (London), 227 (1970) 680-685). The gel was subsequently autoradiographed.

Figure 2 shows clearly that IFN-P is specifically bonded by the monoclonal anti-(Hu-lFN-p)-CNBr- Sepharose column.

Furthermore, the antibody from the ascitic liquid was purified with 35% ammonium sulphate and a subsequent Sephadex-G200 column step. The Sephadex-G200 profile showed that the monoclonal anti-(Hu-lFN-p) is an IgM; see Fig. 3.

EXAMPLE 2 A female Balb/c mouse was immunised in accordance with Example 1, except that human immune interferon was used (Hu-IFN-gamma) and immunisation was continued until a positive antibody titer against Hu-IFN-gamma was found in the mouse serum. 4 days before the isolation of a lymphocyte mixture from the spleen of the immunised Balb/c mouse, a final "booster" immunisation was carried out. 4 days later, the lymphocyte mixture was then isolated from the spleen, and then B lymphocytes were fused (see Hochkeppel et a!,, Europ. J. Biochem., 118 (1981) 437-442) with B myeloma cells [FO (Groth and Scheidegger (1980), J. Immunol. Methods 35, 1-25)] and cultivated for 5 days in HAT medium with 1% methylcellulose and 20% foetal calves serum.For a better multiplication of surviving hybrids and to stabilise the hybrids against loss of genes, the hybrids were subsequently injected into the abdominal cavity of a Balb/c mouse which had been pretreated with pristane 2,6,10,1 4-tetramethylpentadecane. After a week, the ascitic liquid was removed from the abdominal cavity in a sterile manner and the hybrid cells contained therein were sown without methylcellulose in normal Iscove Dulbecco medium which contained 10% foetal calves serum. The growing hybridoma cultures were then subcloned in Greiner single-clone tissue-culture plates. Colonies growing therein were tested for specific antibody production. The test was carried out according to an antivirai neutralisation test on human CCL23-cells.In that test, first of all, an antibody from rabbit directed against mouse immunoglobulins was adsorbed for 1 6 hours onto the surface of the holes of an immunomicrotiter plate, then the supernatents of each hybridoma culture were added likewise for 1 6 hours in order to bond possible Hu-IFN-gamma antibodies contained therein onto the adsorbed antibody, and finally 10 antiviral units per ml of Hu-lFN-gamma were added (4 hours). The supernatent was then tested, in comparison with an untreated Hu-IFN-gamma sample, for remaining antiviral activity against vesicular stomatitis virus (VSV) on human CCL23-cells. In this manner, several stable mouse hybridoma lines were established which secrete specifically monoclonal anti-(Hu-lFN-gamma).

To identify the type of immunoglobulin of the monoclonal antibody, the antibody from hybridoma supernatents was purified firstly using a 40% strength ammonium sulphate precipitation and then using a Sephadex-G200 column chromatography step. In that process the anti-(Hu-lFN-gamma) was identified as IgM which was eluted in the void volume of the Sephadex-G200 column in a manner identical to the elution profile of an IgM control (Figure 4). The specificity of the monoclonal anti-(Hu lFN-gamma)-lgM purified in that manner was then shown clearly using the following specificity tests.

First of all, partially purified Hu-IFN-gamma was split up by electrophoresis on an SDS-polyacrylamide gel according to Lämmli (Nature (London) 227 (1970) 680-685) withoutp-mercaptoethanol pretreatment parallel with standard proteins and then transferred by electrophoresis according to Towbin eft at (Proc. Natl. Sci. Acad. USA, 76 (1979) 4350--4354) onto nitrocellulose strips. While the blot portion having the standard proteins was stained with amido black, the blot portion having Hu-lFNgamma was subjected to the ELISA technique according to Hochkeppel etna!. (Europ. J.Biochem., 1 18 (1981) 437-442), the blot being incubated firstly with monoclonal mouse anti-(Hu-lFN-gamma), then with IgM-antibody from rabbits directed against mouse IgM and finally with IgM-antibody from goats directed against rabbit IgM (which was peroxidase-conjugated), whereupon the Hu-IFN-gamma was rendered visible in a sharp band around 46000 Daltons using o-dianisidine as enzyme substrate (Figure 5). The same experiment was repeated with Hu-IFN-gamma which had been pretreated with ,B-mercaptoethanol/SDS. With ELISA blot, a specific band was rendered visible around 22000 Daltons using o-dianisidine (Figure 6).The monoclonal anti-(Hu-lFN-gamma) is thus directed specifically either against two different (Hu-lFN-gamma)-subspecies or once against the dimer (Figure 5) and once against the monomer (Figure 6) of the Hu-IFN-gamma molecules, the displacement of the molecular weight of the immuno-reacting band after ,B-mercaptoethanol pretreatment pointing rather to the latter case of Hu-lFN-gamma monomer and Hu-lFN-gamma dimer.

In a further specificity test, partially purified Hu-lFN-gamma which had previously been labelled with iodine-1 25 according to Hunter eft at [Nature, 124 (1 962) 495-496] was incubated firstly with monoclonal mouse anti-(Hu-lFN-gamma) and then with IgG from rabbits directed against IgM. The complex was then precipitated with IgG-specific Staphylococcus aureus protein A (Pansorbin). After the complex had been washed thoroughly several times and the '251-Hu-lFN-gamma precipitated specifically with monoclonal anti-(Hu-IFN-gamma) had then been separated by treatment with SDS, the immuno-precipitated material was split up by electrophoresis without P-mercaptoethanol pretreatment on an SDS-polyacrylamide gel according to Lämmli (Nature (London), 227 (1970) 680-685) parallel with 14C-labelled standard proteins. After it had been dried, the gel was then autoradiographed. The autoradiogram (Figure 7) shows clearly that '251-lFN-gamma can be precipitated specifically using monoclonal anti-(Hu-IFN-gamma) and can be rendered visible as a clean radioactive band around 46000 Daltons on the autoradiogram of an SDS-polyacrylamide gel.

References: ConA-, Sephacryl-S-200- and Sephadex-G-200 columns: Deutsche Pharmacia GmbH, 6000 Frankfurt/Main 50.

Iscove (Modified) Dulbecco Medium: BCK-Biokult-Chemie, Postfach 21 02 65, 7500 Karlsruhe.

Greinertissue-culture plates (384-fold for cellclones): Greinerand Sone, 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 Hochkeppel etna!., 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, B-3000 Leuven, Belgien, and (b) Flow Laboratories, Mühlengrabenstr. 10, 5309 Meckenheim/Bonn.

Claims (41)

1. A process for the manufacture of hybrid cells capable of producing an antibody in which (a) mice are immunised with an antigen, (b) a lymphocyte mixture is isolated from the mice, (c) B lymphocytes are fused or hybridised with mouse B myeloma cells, (d) the mixture consisting of formed hybrids and non-hybridised B myeloma cells is conveyed into a culture medium for the formed hybrids and the formed hybrids are cultivated and selected with respect to the non-hybridised B myeloma cells, (e) they are subcloned to provide hybrids which are capable of producing the antibody which is directed specifically against the antigen, and (f) optionally the hybrids are isolated, characterised in that at step (d) the selection process is carried out in a medium containing methylcellulose (as hereinbefore defined), and hybrids selected at step (d) are optionally injected into the abdominal cavity of mice (which have previously been treated with a tumour stimulant), multiplied hybrids are removed from the abdominal cavity and subsequently the subcloning and isolating steps (e) and (f) are carried out.

2. A process according to claim 1, wherein B lymphocytes from Balb/c mice and/or B myeloma cells from Balb/c mice are used.

3. A process according to claim 1 or claim 2, wherein immunisation is carried out more than once and/or the final immunisation is carried out substantially 4 days before the lymphocyte mixture is isolated.

4. A process according to any one of the preceding claims, wherein an antigen which is adsorbed over a molecular sieve is used for immunisation.

5. A process according to any one of the preceding claims, wherein a human interferon is used for immunisation.

6. A process according to claim 5, wherein the human interferon is Hu-lFN-p or Hu-IFN-y.

7. A process according to claim 5 or claim 6, wherein the final immunisation is carried out with human interferon after the sugar portion thereof has been removed.

8. A process according to any one of the preceding claims, wherein the lymphocyte mixture is isolated from female mice.

9. A process as claimed in claim 8, wherein the lymphocyte mixture is isolated from female Balb/c mice.

10. A process according to any one of the preceding claims, wherein at step (d) the selection process is carried out in a medium free from macrophages.

11. A process according to any one of the preceding claims, wherein at step (d) the selection process is carried out for 4 days before the selected hybrids are injected into the abdominal cavity of mice.

12. A process according to any one of the preceding claims, wherein additionally, at step (e), a tumour stimulant or an adjuvant is injected into the abdominal cavity of mice and then hybrids producing the antibody are injected, the multiplied hybrids are removed and optionally the antibody is isolated.

13. A process according to any one of claims 1, 11 and 12, wherein injections are made into the abdominal cavity of female mice.

14. A process as claimed in claim 13, wherein injections are made into the abdominal cavity of female Balb/c mice.

1 5. A process for the manufacture of a monoclonal antibody in which (a) mice are immunised with an antigen, (b) a lymphocyte mixture is isolated from the mice, (c) B lymphocytes are fused or hybridised with B myeloma cells, (d) the mixture consisting of formed hybrids and non-hybridised B myeloma cells is conveyed into a culture medium for the formed hybrids and the formed hybrids are cultivated and selected with respect to the non-hybridised B myeloma cells, (e) they are subcloned to provide hybrids which are capable of producing the antibody which is directed specifically against the antigen, and (f) optionally the antibody is isolated, characterised in that at step (d) the selection process is carried out in a medium containing methylcellulose (as hereinbefore defined), and optionally hybrids selected at step (d) are injected into the abdominal cavity of mice (which have previously been treated with a tumour stimulant) and multiplied hybrids are removed from the abdominal cavity.

1 6. A process according to claim 15, wherein the provision of any one of claims 2 to 13 are used.

17. Use of the hybrid cells capable of producing antibody a) for an injection according to claim 9, 12 or 13, or b) for antibody-isolation according to any one of claims 12 to 16.

1 8. Hybrid cells capable of producing antibody which is directed specifically against human fibroblast interferon which hybrid cells are obtainable by a process wherein (a) mice are immunised with human fibroblast interferon, (b) a lymphocyte mixture is isolated from the mice, (c) B lymphocytes are fused or hybridised with mouse B myeloma cells, (d) the mixture consisting of formed hybrids and non-hybridised B myeloma cells is conveyed into a culture medium for the formed hybrids, the formed hybrids are cultivated and selected with respect to the non-hybridised B myeloma cells, (e) they are subcloned to provide hybrids which are capable of producing the antibody which is directed specifically against human fibroblast interferon, and (f) optionally these hybrids are isolated.

1 9. Hybrid cells having the features of deposit 1-1 54 necessary for the manufacture of a monoclonal antibody which is directed specifically against human fibroblast interferon and hybrids derived from the deposit.

20. Hybrid cells capable of producing antibody which is directed specifically against human immune interferon which hybrids are obtainable by a process wherein (a) mice are immunised with human interferon, (b) a lymphocyte mixture is isolated from the mice, (c) B lymphocytes are fused or hybridised with mouse B myeloma cells, (d) the mixture consisting of formed hybrids and non-hybridised B myeloma cells is conveyed into a culture medium for the formed hybrids and the formed hybrids are cultivated and selected with respect to the non-hybridised B myeloma cells, (e) they are subcloned to provide hybrids which are capable of producing the antibody which is directed specifically against human immune interferon, and (f) optionally these hybrids are isolated.

21. Hybrid cells according to claim 1 8 or claim 20, wherein at step (d) the selection process is carried out in a medium containing methylcellulose (as hereinbefore defined), and hybrids selected at step (d) are optionally injected into the abdominal cavity of mice (which have previously been treated with a tumour stimulant), multiplied hybrids are removed from the abdominal cavity and subsequently the subcloning and isolating steps (e) and (f) are carried out.

22. Monoclonal antibody which is directed specifically against human fibroblast interferon and is obtainable by a process wherein (a) mice are immunised with human fibroblast interferon, (b) a lymphocyte mixture is isolated from the mice, (c) B lymphocytes are fused or hybridised with mouse B myeloma cells, (d) the mixture consisting of formed hybrids and non-hybridised B myeloma cells is conveyed into a culture medium for the formed hybrids and the formed hybrids are cultivated and selected with respect to the non-hybridised B myeloma cells, (e) they are subcloned to provide hybrids which are capable of producing the antibody which is directed specifically against human fibroblast interferon, and (f) optionally the antibody is isolated.

23. Monoclonal antibody which is directed specifically against human immune interferon and is obtainable by a process wherein (a) mice are immunised with human immune interferon, (b) a lymphocyte mixture is isolated from the mice, (c) B lymphocytes are fused or hybridised with mouse B myeloma cells, (d) the mixture consisting of formed hybrids and non-hybridised B myeloma cells is conveyed into a culture medium for the formed hybrids and the formed hybrids are cultivated and selected with respect to the non-hybridised B myeloma cells, (e) they are subcloned onto hybrids which produce the antibody which is directed specifically against human immune interferon, and (f) optionally the antibody is isolated.

24. An antibody according to claim 22 or claim 23, wherein at step (d) the selection process is carried out in a medium containing methylcellulose (as hereinbefore defined), and hybrids selected at step (d) are optionally injected into the abdominal cavity of mice (which have previously been treated with a tumour stimulant), multiplied hybrids are removed from the abdominal cavity and subsequently the subcloning and isolating steps (e) and (f) are carried out.

25. A hybrid according to anyone of claims 1-8 to 21, wherein in the processthe provision of-any one of claims 2 to 13 is used.

26. A monoclonal antibody according to any one of claims 22 to 24, wherein in the process the provision of any one of claims 2 to 13 is used.

27. A process for the production of hybrid cells as defined in claim 18, which comprises carrying out a process as defined in claim 18, claim 21 or claim 25.

28. A process for the production of hybrid cells as defined in claim 20, which comprises carrying out a process as defined in claim 20, claim 21 or claim 25.

29. A process for the production of a monoclonal antibody as defined in claim 22, which comprises carrying out a process as defined in claim 22, claim 24 or claim 26.

30. A process for the production of a monoclonal antibody as defined in claim 23, which comprises carrying out a process as defined in claim 23, claim 24 or claim 26.

31. A process as claimed in claim 1, carried out substantially as described in Example 1 or Example 2 herein.

32. A process as claimed in claim 15, carried out substantially as described in Example 1 or Example 2 herein.

33. A process as claimed in claim 27 or claim 29, carried out substantially as described in Example 1 herein.

34. A process as claimed in claim 28 or claim 30, carried out substantially as described in Example 2 herein.

35. Hybrid cells as defined in claim 1, whenever produced by a process as claimed in any one of claims 1 to 13 and claim 31.

36. A monoclonal antibody as defined in claim 15, whenever produced by a process as claimed in any one of claims 15, 16 and 32.

37. Hybrid cells as defined in claim 18, whenever produced by a process as claimed in claim 27.

38. Hybrid cells as defined in claim 20, whenever produced by a process as claimed in claim 28.

39. A monoclonal antibody as defined in claim 22, whenever produced by a process as claimed in claim 29.

40. A monoclonal antibody as defined in claim 23, whenever produced by a process as claimed in claim 30.

41. Use of a hybrid as claimed in claim 1 8 or claim 20 for a) an injection as claimed in claim 21 or for b) isolation of an antibody as claimed in claim 22 or claim 23.