DE3645095C2 - antibody - Google Patents

Description

The invention relates to the antibodies specified in claims 1 to 8, which are specific for an Leukocytes are available polypeptide, as well as use this antibody for the detection of the polypeptides according to claim 9.

Leukocytes, both lymphocytes and monocytes were included involved in the inhibition of tumor growth in various Animal tumor models. The increased occurrence of malignant Diseases in immunocompromised people supports the argument that white blood cells play a role in regulation of neoplastic growth. Protein factors, that are produced by these white cells that the Inhibit tumor growth or modulate immune functions, which are already isolated and characterized the interferons, α- and γ-tumor necrosis factor, lymphotoxin,  Interleukin-2 and other lymphokines. Because everyone of these isolated factors a different activity spectrum owns and different in connection with other factors Can continue to exert influence and serious interest in isolation and characterization of all the factors that white cells have in the Produce modulation of cell growth or immune functions. These compounds can be used individually or together in the treatment or diagnosis of cancer, as an accelerator in wound healing or as immunomodulators for the treatment of patients with immune deficiency symptoms, Autoimmunity, organ transplants and the like find.

There are numerous difficulties in locating Isolation, purification or characterization of natural occurring factors can occur. There have to be ways of working for separating and cleaning a factor of interest of other factors in the raw starting material without denaturation the activity of the desired factor. Bioassays need to be developed to identify them of the fractions during the partitions, which one focus on a certain factor, allow. A new factor must be distinguished from factors that are already known are or other factors that may exist and the sought factor either negative or positive in its Can affect effect. The cleaned factor must be characterized will. The cleaned factor must be sufficient Amounts are concentrated to help identify it and enable characterization. With the rising Number of factors that are isolated, each new Factor more difficult to identify because of its role and Function by the numerous other factors present can be covered.  

Beal et al., Cancer Biochem. Biophys. (1979) 3: 93-96 report about the presence of peptides in human urine, which rather transformed the growth and DNA synthesis into than inhibit in normal cells. Holley et al., Proc. Natl. Acad. Sci. (1980) 77: 5989-5992 describe the Purification of epithelial cell growth inhibitors. Letansky, Biosci. Rep. (1982) 2: 39-45 reports that from Rinderplacenta purified peptides the tumor growth and thymidine storage inhibit to a greater extent in DNA in neoplasm than in normal cells. Chen, Trends Biochem. Sci. (1982) 7: 364-365 reports the isolation of a peptide Ascites fluid with a cancer-suppressing property. Redding and Schally, Proc. Natl. Acad. Sci. (1982) 79: 7014-7018 report the isolation of cleaned Peptide or peptides from porcine hypothalamus, which are antimitogenic Activity against multiple normal and tumor cell lines demonstrate. Sone et al., Gann (1984) 75: 920-928 report the production of one or more factors by human macrophages are produced and growth Inhibit certain tumor cells in vitro. Ransom et al., Cancer Res. (1985) 45: 851-862 report isolation of a factor called leukoregulin, which replicates inhibits certain tumor cell lines and lymphotoxin, Interferon and interleukin 1 and 2 appear to differ. Most of these factors are not fully characterized their primary structures are still known.

Aggarwal et al., J. Biol. Chem. (1984) 259: 686-691 and characterized human lymphotoxin (LT) that is produced by a lymphoblastoid cell line, and then clarified the sequence of LT (Aggarwal et al., J. Biol. Chem. (1985) 260: 2334). Gamma interferon (γ-IF), which is produced by lymphoid cells and immunomodulatory and has tumor inhibitory activity has been cloned and expressed. (Gray et al., Nature (1982) 295: 503-508).  The tumor necrosis factor (TNF), which is the growth inhibited by some tumors and by macrophages and certain Leukemia cell lines has been characterized and the TNF cDNA was cloned and in E. coli (Pennica et al., Nature (1984) 312: 724).

The invention provides antibodies that are specific for a polypeptide available from leukocytes are available The polypeptide is used to inhibit proliferation of neoplastic cells is capable of proliferation capable of stimulating normal human fibroblasts, human proliferative and cytotoxic T cell response not inhibited and granulocytic / myelocytic bone marrow colony cell formation not inhibited a molecular weight in the range from 17 to 19 kD, determined by gel exclusion chromatography, and of 28 kD, determined by SDS-PAGE, possesses and relatively insensitive to moderate acid or base and is stable against heat treatment at 56 ° C.

Preferred antibodies according to the invention are the subject of Subclaims 2 to 8.

The attached drawing shows

Fig. 1 shows the amino acid sequence of fragments of the polypeptide of Oncostatin M.

A polypeptide that falls within the definition given and referred to as Oncostatin M is available from leukocytes, e.g. B. from conditioned media from stimulated U937 cells or conditioned media from stimulated normal human peripheral blood lymphocytes (PBL). For fragments and mutants of the polypeptide with the biological activity of intact Oncostatin M, such as cell growth modulation activity  or immunological activity, are those of the invention Antibodies also specific.

The polypeptide fragments are polypeptides with at least 8 Amino acids that are biologically active, at least immunologically cross-active with naturally occurring oncostatin M. Immunologically cross-reactive is understood to mean that a Antibody induced by a polypeptide cross-reacts with intact Oncostatin M, at least if Oncostatin M in one denatured state. These polypeptides are therefore useful for inducing antibodies to oncostatin M, which is used to determine the concentration of Oncostatin M can still be used in a body fluid for binding to Oncostatin M and thus for modulating its Activity, and for purification of Oncostatin M, e.g. B. by Use in an affinity column. A portion of the polypeptides may also affect the cell growth modulation activity of the intact Keep Oncostatin M even though the activity modulates can be, usually reduced compared to the intact Oncostatin M.

Fig. 1 shows the amino acid sequence of poly (amino acid or acids) which are cross-reactive with Oncostatin M, wherein the first sequence represents the N-terminus of Oncostatin M.

The poly (amino acid or acids) contain an amino acid sequence with at least 8 consecutive amino acids which correspond to an amino acid sequence shown in FIG. 1 and differ from this sequence by not more than 3, usually not more than one, amino acid. The difference can either be the insertion of an amino acid, the missing position of an amino acid or the substitution of an amino acid by another, in particular a conservative substitution. The poly (amino acid or acids) normally contain at least 10, in particular at least 12 consecutive amino acids which correspond to the sequence shown in the figure and are distinguished by no more than one amino acid.

For the purposes of illustration, different amino acids are divided into a number of subclasses. The following table shows the subclasses:

Conservative substitution means that amino acids from the same subclass (i.e. either neutral aliphatic, acidic aliphatic, basic aliphatic or aromatic), especially the same polarity for each other can be exchanged. Preferably form amino acids with two to four carbon atoms or five to six carbon atom monomer groups in the aliphatic subclass.

The poly (amino acid) s have no more than about 1000 amino acids in length. Usually they have less than 100 Amino acids, especially less than 50 amino acids. So can the poly (amino acid) s are easily synthesized. If the poly (amino acid) n are over 100 amino acids in length, then the poly (amino acid) n polymers of fragments of Oncostatin M with less than 100 amino acids each be or fusion proteins where the fragment by fusion to an antigen, enzyme, enzyme fragment and the like. the like is bound. In particular the poly (amino acid) s with higher Molecular weights can contain at least one polypeptide fragment  with less than 100 amino acids that is covalent to one large immunogenic polypeptide carrier is bound to immunogenic To create properties. Examples of such protein carriers are bovine serum albumin, keyhole limpet hemocyanin (KLH) u. The like. These conjugated polypeptides are useful for inducing antibodies in a suitable host organism.

U937 cells are a cell line that consists of a histiocytic Lymphoma cell line are derived (Sundstrom and Nilsson, Int. J. Cancer (1976) 17: 565-577) which can be induced differentiate into cells with characteristics of macrophages, followed by treatment with a variety of Means (Harris et al., Cancer Res. (1985) 45: 9-13). For the Oncostatin M can be produced using the U937 cells grow a conventional nutrient medium with serum and treat with a suitable inducer. Conventional phorbols or ingenols can be used, especially 12-0-tetradecanoylphorbol-13-acetate (TPA). Usually about 5-20 ng / ml of the inducer used. The initial cell number is around 10⁵-10⁶ cells / ml.

After giving the inducer a sufficient amount of time on the cells , usually three to six days, the Supernatant removed, the cells are covered with serum-free nutrient medium washed, attached cells are again serum-free Medium washed and then the cells during at least 12 hours, usually not more than about 48 hours, in a serum-free nutrient medium e.g. B. RPMI-1640 incubated. Supernatants are then collected and the cells removed by centrifugation. The cell-free supernatants are tested for cell growth inhibition activity (GIA). The supernatant contains approximately 50 to 500 units of GIA / ml.  

Oncostatin M can also be obtained from mitogen-stimulated normal human peripheral blood lymphocytes (PBL's). PBL's can be isolated from leuco fractions by dilution of the fractions and centrifugation using Ficoll gradients. Cells collected from the intermediate gradient layer are washed and shocked to the red To remove blood cells. The remaining cells are made up the solution separated by centrifugation, in serum and Buffer containing thrombin resuspended, shaken, and then the platelet-like aggregate is left for one stop for a short period of time. The suspended cells will transpered, recovered by centrifugation, in serum resuspended and on a column containing nylon wool transferred. The column is incubated to attach Allow monocytes and B lymphocytes, and then washed. Most peripheral blood T lymphocytes do not adhere and are eluted from the column. These cells are at 37 ° in culture medium, e.g. B. RPMI-1640 medium cultured and with a suitable inducer, e.g. B. Phytohemagglutinin (approx. 1 to 5 mg / l) for about 100 hours, after which the Supernatants are collected. The supernatants are centrifuged, to remove cells and by ultrafiltration or Dialysis focused.

After isolating the cell-free supernatant from either U-937 cells or normal PBL’s, that is conditioned Medium concentrated, conventionally using a hollow fiber system or an ultrafiltration membrane followed by Dilution with acetic acid (up to a concentration of 0.1N acetic acid), followed by concentration on the about ten times and repeated dilution and concentration. The concentrates can be lyophilized and directly can be used, or the freeze-dried product can be used for further cleaning.  

The Oncostatin M can be obtained by a gel permeation chromatography method be cleaned using aqueous 40% acetonitrile-0.1% trifluoroacetic acid as isocratic mobile phase on a Bio-sil TSK250 column, under supervision the activity of each of the factions. By cleaning a composition is obtained which is at least about 0.5-5 × 10⁴-GIA units / ml in the active fractions.

The partially purified product from gel permeation chromatography can be further cleaned by application reverse phase high pressure liquid chromatography Apply a linear gradient, being the primary solvent 0.1% trifluoroacetic acid in water and that secondary solvent is acetonitrile, which is 0.1% trifluoroacetic acid contains. The scheme can be varied, whereby the chromatography generally takes about 3-4 hours and the main time expenditure of more than approx. 50% of the time period and no more than about 80% of the time in the range of 30-45% of the secondary solvent. Under these Conditions elute the active fractions at approx. 41-42% Acetonitrile.

The pooled fractions can be further cleaned by Repeat the reverse phase HPLC, taking a faster one Change in gradient conditions and one made slower flow rate is applied. Under these conditions the activity dips at approximately 40.5-41.5% acetonitrile on.

The reverse phase HPLC can then be repeated using the Solution system is changed, being the secondary solvent n-propanol-0.1% trifluoroacetic acid. A linear one Gradient is applied, with the gradient in the range between 23 and 35% n-propanol is slowly changed. The Main activity is in the range of 25.5-27.5% propanol  observed and obtained a substantially homogeneous product with a specific activity greater than 10 GIA units / ng protein. Usually the product is cleaned to a specific activity of at least about 100 GIA units / ng protein to achieve, especially 150 GIA units / ng.

As described, the polypeptides are characterized by a molecular weight of about 17 to 19 kilodaltons (kD), in particular approx. 18 kD, determined by size exclusion chromatography. The connections are still identified by an apparent molecular weight of approximately 28 kD, determined by polyacrylamide gel electrophoresis under reducing or non-reducing conditions.

The amino acid sequence of fragments of purified Oncostatin M was analyzed. Oncostatin essentially has the amino acid sequences given in Figure 1. In Figure 1, the first sequence illustrates the N-end of Oncostatin M, while the remaining sequences represent inner fragments of the polypeptide.

Oncostatin M is still characterized by its activity against certain cell strains. The polypeptide is missing cytotoxic activity against WI26 and WI38 human fibroblasts and mice L929 cells, which are sensitive to tumor necrosis factor and are sensitive to γ-interferon human tumor cell line. It also turned out that it is the proliferation of normal human T lymphocytes not inhibited and also not granulocytic / myelocytic Colony formation in bone marrow cells Concentrations up to 100 GIA units / ml. Still stimulated Oncostatin M proliferation from normal human Fibroblasts, as shown using the example of WI38 and WI26 cells, and inhibits the proliferation of tumor cells such as A375, HBT10, A549 and SK-MEL28, and can the growth of  Multiply colony forming cells from normal bone marrow. Oncostatin M does not suppress human proliferative or cytotoxic T cell response in mixed leukocyte culture reactions (MLC) at concentrations of 500 GIA units / ml.

It has been found that the polypeptide according to the invention is stable against moderate acid or base and also against heat treatment at 56 ° C.

The amino acid sequence of the polypeptides can be determined completely become more commercially available using Sequence analyzers. The polypeptide can be synthesized are made in accordance with known techniques, using automated synthesis facilities that are also commercially available.

Alternatively, the polypeptides can be obtained by recombinant DNA techniques getting produced. From a partial amino acid sequence samples can be derived, which are then used for the screening of a human genome bank can be used can. The bank can be a cDNA bank or a chromosome bank be. Are the clones identified as responsive to the sample, then the fragments which contain the gene of interest in different ways identified and manipulated in a variety of ways. The fragment can be sized by endonuclease restriction can be reduced, the fragments obtained being cloned and be checked for the presence of the desired genes. Cells, that produce the peptides or increased amounts of the Peptides can be used to produce messenger RNA be used. Single-strand cDNA can be obtained from the messenger RNA getting produced. The cDNA can then be linked to total messenger RNA from a cell that, if anything, little produced by the polypeptide. They don't  responsive or attached cDNA can then be isolated and used Preparation of ds cDNA can be used with the samples can be screened.

Alternatively, DNA fragments can be used in λgt11, so that the coding of the fragments down from and in the frame can be done with the β-galactosidase gene. Antibodies can to the Oncostatin M polypeptide and for screening of the fused proteins obtained for cross-reactivity be used. In this way, fragments that the Encode polypeptide or a fragment thereof and used to identify the desired gene will.

Once the complete gene is identified, either as cDNA or as chromosome DNA, it can be different Manipulated to produce expression. When the gene is expressed in a host that is the wild-type transcriptional and translational regulatory regions of Oncostatin M then recognizes the full gene with its Wild type 5'- and 3'-regulatory regions in a suitable Expression vector are introduced. Different expression vectors exist using replication systems from mammalian viruses, such as Simian virus 40, adenovirus, Bovine Papilloma Virus, Vaccinia Virus, Insect Baculo Virus u. The like. These replication systems were developed to markers to create, which allow a selection of transfectants, as well as creating convenient restriction sites in which the genes can be used.

If the gene is to be expressed in a host, which one the naturally occurring wild-type transcriptional and translational regulatory regions are not recognized further manipulations required. A variety of 3'-transcriptional Regulatory regions are known and can  be used outside or below the stop codon. The 5 'non-coding region above the structural gene can be by endonuclease restriction, Bal31 resection or the like. be removed. On the other hand, where can a convenient restriction site present near the 5'-end of the structural gene is the structural gene to be restricted and an adapter are used to bind the structural genes to the Promoter region, the adapter being the lost nucleotides of the structural gene. Different strategies can be used to create an expression cassette, which in the 5'-, 3'-direction of transcription one has a transcriptional and translational initiation region, which are also regulatory sequences for inducing the Regulation, the structural gene under the transcriptional and translational control of the initiation region and a transcriptional and translational termination region can include.

Exemplary transcriptional initiation reactions or Promoters include for bacteria, β-gal promoters, TAC promoters, Lambda left and right promoters, etc .; for yeast, glycolytic Enzyme promoters such as ADH-I and II promoters, GPK promoters and PGI promoters and TRP promoters, etc .; for mammalian cells, SV40 early and late promoters, adenovirus major late promoters etc. As already indicated, the expression cassette within an episomal replication system Maintenance in a suitable cellular host exist or exist without a replication system where it can be integrated into the host genome. The DNA can be introduced into the host using known techniques be, e.g. B. by transformation using with Calcium phosphate of precipitated DNA, transfection by contacting the cells with the virus, microinjection of the DNA in the cells u. the like  

Once the gene is introduced into the appropriate host, then the host can grow and the structural gene is expressed. In individual cases it may be desirable to have one Signal sequence (secretion ladder) above and in the reading frame to provide with the structural gene, which increases secretion of the structural gene and the cleavage of the secretion ladder is enabled so that the mature polypeptide in Supernatant is provided. If no secretion is planned, then the host cells can be harvested, under conventional ones Conditions lysed and the desired product isolated and cleaned by known techniques, such as. B. by chromatography, electrophoresis, solvent extraction u. the like

The polypeptides described can be used to produce more specific Antibodies are used for the polypeptides that in turn can find application in vivo or in vitro. According to the invention Use are the subject of claim 9. The antibodies can be prepared in a conventional manner e.g. B. by using the polypeptide as an immunogen and injecting the polypeptide into a mammalian host, e.g. B. a mouse, a cow, a goat, a sheep, a rabbit and the like. Like., In particular together with an aid, for. B. complete Freunds Excipient, Aluminum Hydroxide Gel u. Like. The The host can then be bled and the blood can then become Isolation of polyclonal antibodies can be used, or in the case of the mouse, the peripheral blood lymphocytes or spleen lymphocytes (B cells) for fusion with a suitable one Myeloma cell used to make up the chromosomes the monoclonal expression of substances specific for the substances To immortalize antibodies.

Both polyclonal and monoclonal antibodies can be used are produced, which are then used for diagnosis or detection the polypeptides in a sample, such as cells or physiological  Liquid, e.g. B. blood can be used. The Antibodies can also be used in affinity chromatography Purification of the polypeptides and their isolation from natural or synthetic sources can be used. The antibodies can also be used for control purposes the amount of polypeptides associated with cells in a culture or find application in vivo, with cell growth can be modified.

The following examples serve to explain the invention, particularly preferred embodiments, but not for limitation.

Experimental Materials and methods 1. Preparation of a polypeptide Oncostatin M isolated from U937 cells Production of a tumor cell growth inhibitor from a histiocytic lymphoma cell line

U937 cells, a histiocytic lymphoma cell line (Sunstrom and Nilsson, Int. J. Cancer (1976) 17: 565-577 Roller bottles at a concentration of 4 × 10⁵ cells / ml in a total volume of 300 ml RPMI-1640 medium, that with 10% fetal calf serum (FCS), penicillin / streptomycin (PS), L-glutamine and 10 ng / ml 12-0-tetradecanoylphorbol-13-acetate (TPA) was added. Four days the supernatants containing FCS and TPA were later away. The roller bottles were cleaned five times with serum  RPMI-1640 washed, and the detached cells (1 × 10⁵ cells / ml) were washed three times with serum-free medium and returned to the bottles, with a final volume of Obtain 125 ml of serum-free RPMI-1640 medium per roller bottle has been. The supernatants were collected a day later for Centrifugal removal of cells by 0.45 micron (µ) Nalgen filter and filtered using a Hollow fiber system on one volume of 150 ml (initial volume 1500 ml) concentrated. Oncostatin M was also made from supernatants from serum-free, TPA-treated U937 cells isolated in 150 cm² tissue culture flasks. The supernatants were washed with an Amicon Diaflo membrane PM-10 concentrated, cut off 10 kD and dialyzed. In connection to the dialysis was the concentrate with acetic acid diluted to a final concentration of 0.1N acetic acid in 500 ml and to 50 ml using an Amicon PM-10 filter concentrated. The 50 ml concentrate was on 400 ml diluted with 0.1N acetic acid and with the same filter concentrated to 40 ml. The concentrate was washed with 1N acetic acid diluted and the precipitate obtained was centrifuged away. The concentrate obtained was frozen and freeze-dried. The freeze-dried material was used for cleaning levels.

Gel permeation chromatography

A Bio-Sil TSK-250 column (600 x 21.5 mm) was made connected to a high pressure liquid chromatography system. The crude fraction (10 mg / ml) was in 40% acetonitrile in 0.1% aqueous trifluoroacetic acid (0.1% TFA) dissolved. A 2 ml aliquot of the mixture was injected and the elution isocratic with a mobile phase of 40% acetonitrile in 0.1% TFA. The flow rate was 2.5 ml / min and the rate of displacement was set at 0.25 cm / min. 5 ml fractions were collected. Chromatography was carried out at room temperature  carried out. Aliquots of each fraction were made evaporated and tripled to growth inhibitor activity (GIA) examined on A375 cells.

The active fractions 21 and 22) from six Runs were pooled. The pooled material had one Total of approximately 4.8 × 10⁵ GIA units. It was found that the factor was an apparent molecular weight (apparent molecular weight) of 18 kD, determined by Size exclusion chromatography (Bio-Sil TSK-250 column).

Reverse phase high pressure chromatography (HPLC) of TSK-250 fractions

Pooled TSK-250 fractions 21 and 22 described above were diluted twice with 0.1% TFA. The Mixture was applied to a µ-Bondapak-C18 column (7.8 × 300 mm) (designated C18¹) isocratically injected at room temperature. The flow rate was set at 2.0 ml / min and the flow rate to 0.25 cm / min. The linear gradient was used between the primary Solvent 0.1% TFA and the secondary solvent Acetonitrile TFA 0.1%. The gradient conditions were 0-30% in 20 min; then 30-45% in 150 min; 45-55% in 20 min; and 55-100% in 10 min. All solvents were of HPLC quality. 4 ml fractions were collected and equal proportions of each fraction were inhibited on growth Activity examined. It was found that the Fractions 72-75 contained the main activity. The active ones Fractions were between 41 and 52% of the acetonitrile concentration eluted.

Fractions 72-75 were pooled. 16 ml of 0.1% TFA were added to the pooled fractions. The mixture was into a µ-Bondapak C18 column (3.9 × 300 mm) (as C18² referred to) injected at room temperature. The flow rate  was set to 1 ml / min and the displacement or Flow rate to 0.25 cm / min. The gradient conditions were 0-35% in 10 min; 35-45% in 100 min; and 45-100% in 10 min. The fractions were collected and equal shares were taken and examined for GIA. The most of the activity came from the column between 40.7 and 41.3% acetonitrile concentration (residence time 83-86 min).

Active fractions were pooled and duplicated with 0.1% TFA diluted and isocratic on a µ-Bondapak-C18 column (3.9 x 300 mm) (referred to as C18³) at room temperature. The flow rate was 1 ml / min and the flow rate 0.25 cm / min. A linear gradient was used between the primary solvent 0.1% TFA and the secondary solvent n-propanol-TFA (0.1%). The gradient conditions were 0-23% in 20 min and 23-35% in 120 min. Fractions were collected and equal proportions each faction was checked for GIA. Most of the Activity appeared between 25 and 26.5% propanol concentration (Dwell time 59 min). This obviously homogeneous Fraction contained approximately 300 ng protein and approximately 4000 GIA units.

2. Antibodies to a synthetic peptide from Oncostatin M reacts in ¹²⁵I-labeled Oncostatin M in radioimmune precipitation a) Peptide synthesis

The peptide corresponded to residues 6-19 of the Oncostatin M protein and was by solid phase technique, as described, on a Beckman automated Device synthesized (Gentry et al., J. Biol. Chem. (1983) 258: 11-219). The peptide was removed from the resin using the "low-high" RF method (Tam et al., J. Amer. Chem. Soc. (1983) 105: 6442-6445).  

b) production of antibodies

The peptide was derived from bovine γ-globulin coupled as described (Genty and Lawton, Virology (1986) 152: 421-431). White New Zealand rabbits were primed and strengthened five times, subcutaneously at 4 points as described (Gentry and Lawton, Virology (1986) 152: 421-431). Antisera were 2 weeks received after the fifth boost.

c) iodination of Oncostatin M

A sample of partial purified Oncostatin M was applied with iodine 122 published procedure radiolabeled (Linsley et al., PNAS (1985) 82: 356-360). An aliquot Amount of labeled preparation containing 100,000 cpm was mixed with rabbit antiserum which against the N-terminal 17 amino acids of Oncostatin M was directed (final dilution 1:20) in the absence or presence of the N-terminal peptide (the N-terminal 17 amino acids of Oncostatin M) (2 µg) and subjected to immunoprecipitation analysis, as described (Linsley et al., Biochemistry (1986) 25: 2978-2986).

Specifically, a tube containing 5 µl was preincubated with 2 µg of the N-terminal peptide in 10 ml TNEN (20 mM Tris pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.05% Nonidet P-40), containing 0.1% BSA for 30 minutes at 4 ° C before adding I¹²⁵ Oncostatin M in 85 µl TNEN containing 0.1% BSA and 40 mM dithiothreitol (DTT) took place. Seven tubes containing 5 µl antisera were with I¹²⁵ Oncostatin M in 85 ul TNEN, the Contained 0.1% BSA and 40 mM DTT for 30 minutes Incubated 4 ° C before adding 50 µl 10% formalin fixed Staphylococcus aureus took place.  

Following an additional incubation for 30 minutes at 4 ° C the tubes were microfuged and the pellets were Washed four times with 1 ml of TNEN before doing the PAGE analysis have been subjected. A diffuse band of Mr = 32 kD was found observed after SDS / PAGE analysis of the immunoprecipitate. The precipitation of this species was by inclusion an excess of unlabeled peptide inhibits the N-terminal 17 amino acids of Oncostatin M corresponded to what indicates that the precipitation is specific for this peptide is.

The intact polypeptide or fragment thereof described used as immunogens to prevent antibody formation to induce. The induced antibodies are found according to the invention Use for titering in a body fluid existing Oncostatin M or to modulate the activity of the factor by binding to it. Furthermore, these antibodies together with purified Oncostatin M or fragments of which used as a component of diagnostic kits are used in conjunction with other reagents, especially antibodies, to track down and quantify Oncostatin M.

Although the foregoing invention is for the purpose of illustration and has been described in detail for good understanding it is clear to the person skilled in the art that deviations and modifications can be carried out without the scope of the claims leave.

Claims (9)

1. Antibody specific for a polypeptide consisting of Leukocytes are available to inhibit proliferation of neoplastic cells is capable of proliferation stimulate by normal human fibroblasts capable of human proliferative and cytotoxic T cell response not inhibited and granulocytic / myelocytic Bone marrow colony cell formation does not inhibited, a molecular weight in the range of 17 to 19 kD determined by gel exclusion chromatography, and of 28 kD as determined by SDS-PAGE, owns and relative insensitive to moderate acid or base and is stable against heat treatment at 56 ° C.   2. Antibody specific for a polypeptide according to claim 1, which is available from leukocytes that mitogen activated normal human peripheral blood lymphocytes are. 3. Antibody specific for a polypeptide according to claim 1, which is available from leukocytes that induced phorbol diester human histiocytic lymphoma cells are. 4. Antibody specific for a polyamino acid with at least eight amino acids that are immunologically cross-reactive is with the polypeptide according to any one of claims 1 to 3. 5. Antibody specific for a polyamino acid according to claim 4, characterized in that the polyamino acid has an amino acid sequence with at least ten consecutive amino acids, which corresponds to one of the following amino acid sequences and deviates from this sequence by no more than three amino acids. 6. Antibody according to claim 5, characterized in that the polyamino acid contains twelve consecutive amino acids, which corresponds to one of the following amino acid sequences and deviates from this sequence by no more than one amino acid, which deviation is either a blank or a conservative substitution. 7. Antibody according to one of claims 1 to 3, wherein the Polypeptide is part of a composition that is in the is essentially free of cell components. 8. Antibody according to one of the preceding claims monoclonal antibodies. 9. Use of the antibodies according to one of claims 1 to 8 for the detection of a polypeptide obtainable from leukocytes characterized according to claims 1 to 6.