IL126245A - Antibodies to ifn-beta2/il-6 receptor - Google Patents

Description

Antibodies to IFN-P2/IL-6 Receptor IFN-p2/IL-6 "710 TIOgHT? D1]! !] T/777 Div.

Yeda Research and Development Co. Ltd.

FIELD OF THE INVENTION This invention relates to antibodies against the soluble extracellular fragment of the human Interferon(P2 Interleukin-6 Receptor (hereinafter (IFN-p2/IL-6-R). The antibodies may be either polyclonal or monoclonal antibodies. IFN- 2/IL-6R is disclosed and claimed in Israel Patent No. 90488, of which this application is a divisional.

BACKGROUND OF THE INVENTION Interferon- β2, now designated Interleukin-6 (hereinafter (IFN-P2/IL-6), is a multifunctional cytokine that regulates the growth and differentiation of various cells and tissues and appears to be one of the important mediators of the response to viral and bacterial infections and to shock. The biological effects now associated with (IFN-P2/IL-6 include: stimulation of immunoglobulin secretion by mature B lymphocytes (BSF-2 activity), growth stimulation of plasmacytomas and hybridomas (HGF activity), activation of T cells, stimulation of hepatic acute phase protein synthesis (HSF activity), stimulation of hematopoiesis, cell differentiation (DIF activity), inhibition of tumor cell growth (AP activity) and other IFN-like effects. As a typical cytokine, IFN- 2/IL-6 is secreted by many cell types and acts in various combinations with other interleukins and interferons. Among its activities viewed as having antitumor potential are : inhibition of cancer cell growth and colony formation, differentiation of malignant cells to more normal phenotypes, stimulation of normal hematopoiesis, stimulation of T-lymphocyte activation, stimulation of antibody secretion by B-cells, stimulation of complement synthesis, and stimulation of antiprotease synthesis.

The cloning of the intact human IFN- 2/IL-6 receptor was reported (Yamasaki et al., Science, Vol. 241, pp. 825-828). However, the natural human IFN-p2/IL-6 receptor 126245/2 soluble extracellular fragment has not been isolated nor described in the literature. The soluble extracellular fragment of the natural human IFN-P2/IL-6 receptor differs from the intact receptor in that it lacks the signal peptide, the transmembranal and intracellular domains, and also by having a different C-terminal than the one assumed by Yamasaki from the cDNA.

SUMMARY OF THE INVENTION The present invention provides antibodies against the substantially purified human Interferon- 2/Interleukin-6 Receptor (herein designated IFN-P2/IL-6-R) soluble extracellular fragment having a molecular weight of about 50 (40-60) Kd when the substantially purified protein is analyzed by SDS-PAGE under non-reducing conditions, moving as a single peak on reversed phase high performance liquid chromatography (HPLC), and containing the following N-terminal amino acid sequence : Leu-Ala-Pro-Arg-Arg-Cys-Pro-Ala-Gln-Glu-Val-Ala-Arg-Gly-Val-Leu-Thr-Ser-Leu-Pro-Gly-Asp-Ser-Val-Thr-Leu-Thr-Cys-Pro-Gly- These antibodies may be polyclonal or monoclonal antibodies.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the reversed-phase HPLC column elution pattern of IFN-P2/ IL-6-R soluble extracellular fragment fractions after partial purification on an immobilized IFN-P2/IL-6 column.

Figure 2 shows the results of analysis of the purified IFN-P2/IL-6-R soluble extracellular fragment fraction by SDS-PAGE under non-reducing conditions, followed 126245/2 by silver staining (lanes 1 and 2), or followed by electroblotting, 125I-rIFN-p2/IL-6 binding, and visualization by autoradiography (lanes 3 and 4).

Figure 3 shows the enhancement of IFN-P2/IL-6 HGF activity by IFN-p2/IL-6-R soluble extracellular fragment on human breast carcinoma T47D cells.

DETAILED DESCRIPTION OF THE INVENTION The receptors of IFN-P2/IL-6 on various human cells are identified by cross-linking experiments with radiolabeled IFN-p2/IL-6. Briefly pure IFN-p2/IL-6 is labeled with [125I] by the chloramine-T method according to published procedures, retaining its intact biological activity. Such labeled IFN-p2/IL-6 is allowed to react with various human cells at 4°C and the resulting IFN-p2/IL-6 receptor complexes are covalently cross-linked and then analyzed by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) followed by autoradiography. After their identification, the receptors are isolated by a method which comprises solubilizing human cells bearing the receptors to obtain a suspension, centrifuging the suspension to obtain a supernatant, applying the supernatant to an immobilized IFN-P2/IL-6 or anti-IFN-p2/IL-6-R monoclonal antibody column, and eluting the bound receptor protein by varying pH conditions, in a state of enhanced purity. If necessary, the eluted fractions may be further purified.

The soluble extracellular fragment of IFN-p2 IL-6-R of the invention was found in human urine. In its substantially purified form, which is substantially free of proteinaceous impurities, it has a molecular weight of about 50 (40-60) Kd when analyzed by SDS PAGE under non-reducing conditions and it moves as a single peak on reversed-phase HPLC.

It is further characterized by the following sequence obtained by N-terminal sequence analysis of the protein: Leu-Ala-Pro- Arg-Arg-Cys-Pro-Ala-Gln-Glu-Val-Ala-Arg-Gly-Val-Leu-Thr -Ser-Leu-Pro-Gly-Asp-Ser-Val-Thr-Leu-Thr-Cys-Pro-Gly- The present invention relates to antibodies against the IFN-p2/IL-6-R soluble extracellular fragment. The functional interaction of the antibodies of the present invention with IFN-p2/IL-6-R provides also a new diagnostic tool, based on immunoassays such as radio immunoassay, ELISA etc. , for the detection of over- or under-production of IFN-p2/IL-6-R by cells in the body in certain disorders.

The antibodies may be either polyclonal or monoclonal. They may be raised in rabbits, mice or other animals or tissue cultured cells derived thereof or can be products of cells of human origin. They may also be produced by recombinant DNA technology either in a form identical to that of the native antibody or as chimeric molecules, constructed by recombination of antibody molecules of man and animal origins. The development of antibody level is followed by the ability of the animal serum to inhibit the hybridoma growth factor (HGF) activity of IFN-p2/IL-6.

For the preparation of the antibodies, either purified IFN-P2/IL-6 receptor or its soluble extracellular fragment or one or more synthetic peptides identical to the known sequence of the proteins, e.g. to the N-terminal protein sequence, may be used to immunize animals. A further possibility is to fuse one of the possible nucleotide sequences coding for a fragment of the receptor to the gene coding for Protein A, to express the fused Protein A-IFN-P2/IL-6-R gene in E. coli, to purify the fused protein by affinity chromatography on IgG Sepharose column and then to use it to immunize animals.

The monoclonal antibodies of the present invention are prepared using conventional hybridoma technique (Kohler et al. (1975) Naturev256:495; Kohler et al. (1976) Eur. J. Immunol. 6:51 1). After immunization, spleen cells alone and/or together with lymph node cells of the immunized animals are isolated and fused with a suitable myeloma cell line. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium and then cloned. The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding IFN-P2 IL-6 receptor or its soluble extracellular fragment. After identification, the desired clones are grown in bulk, either in suspension culture or in ascitic fluid, by injecting the cells into the peritoneum of suitable host mice. The monoclonal antibodies produced by the hybridomas are then isolated and purified.

As mentioned before, the monoclonal antibodies may also be immobilized and used for the purification of the IFN-p2/IL-6 receptor or its soluble extracellular fragment in affinity purification procedure using an immunoadsorbent column.

The invention will now be illustrated by the following non-limiting examples: Example 1; Isolation and purification of IFN-B2/IL-6 receptor from human placenta Placental membranes are prepared according to the method of R.A. Hock and M.D. Holleneberg (1980), J. Biol. Chem., 255, 10731-10736, as follows: placenta pieces are homogenized in a buffer consisting of 25 am Tris-HCI, pH 7.4, 0.25 M sucrose, 0.1 M NaCl, 1.5 mM MgCla, 2 mM EGTA, 1 mM PMSF and 22 Tiu/ml Aprotinin, followed by filtering through gauze and centrifugation (10,000 xg for 30 min. at 4°C). NaCl and MgSO4, are added to the supernatant to a final concentration of 0.1 M and 0.2 mM respectively. The mixture is spun (48,000 xg for 40 min. at 4°C) and the resulting pellet is resuspended in a buffer of 10 mM Hepes, pH 7.4, 150 mM NaCl and 1 mM PMSF and 22 Tiu/ml. The membranes are then solubilized in a solubilization buffer (final concentrations: 10 mM Hepes, pH 7.4, 1-2.10 Triton X-100, 1 mM PMSF and 20 units/ml aprotinin). The suspension is spun first at 10,000 xg for 15 min. and then at 100,000 xg for 60 min. The supernatant is applied to an immobilized IFN-p2/IL-6 column (2,5 mg per 0.8 ml of Affigel-10). Loading is at a flow rate of 0.2-0.5 ml/min. The column is then washed with PBS (50 ml) and the bound material is eluted with a solution of 25 mM citric acid. Fractions of 1 ml are collected and immediately neutralized with 1 M Hepes, pH 8.5. Each fraction is tested for its ability to bind 125I-IFN- 2/IL-6 and for protein content. Protein is determined with fluorescamine.

Example 2: Isolation and Purification of IFN-B2/IL-6-R soluble extracellular fragment 2.1 Preparation of the urine concentrate A pool of 200 liter urine from healthy menopausal women was subjected to microfiltration on a Pellicon membrane with a pore size of 0.45 μπι. The filtrate was concentrated by ultrafiltration using a Pellicon membrane with a molecular weight cut off of 10 K to a final volume of 500 ml. The concentrate was dialyzed against phosphate buffered saline containing 1 mM benzamidine and 0.1% sodium azide. 2.2 Ion-exchange chromatography on CM-Sepharose A CM-Sepharose (Pharmacia, Uppsala, Sweden) cation exchange column (2.7x10 cm) was prewashed with 1 M NaCl, 10 mM citric acid pH 5.0, containing 0.02% NaN3 (buffer C) and equilibrated with 10 mM citric acid (pH 5) containing 0.02% NaN3 (buffer A). The concentrate of urinary proteins was dialyzed against buffer A, and centrifuged for min. at 8000 xg. The supernatant was applied at 4°C on the column at a flow rate of 2 ml/min. The column was washed with 1500 ml buffer A and eluted with 250 ml of a solution containing 200 mM NaCl, 10 mM citric acid (pH 5.0) and 0.02% NaN3 (buffer B). A second step of elution was performed with 150 ml buffer C. Fractions of 50 ml were collected and tested for IFN-p2/IL-6 binding activity (binding of 125I-IFN-p2/ IL-6) and their protein concentration was determined. 2.3 Affinity purification on an IFN-p2/IL-6 column IFN- 2/IL-6 was brought to a concentration of 5 mg/ml, then equilibrated with PBS containing 0.02% sodium azide and coupled to Affigel-10 (2.5 mg to 0.8 ml beads). The concentrate of urinary proteins of step 2.1 or 2.2 was equilibrated with phosphate-buffered saline (PBS) and applied to the IFN- 2/IL-6 Affigel-10 column (2.5 mg of protein bound to 0.8 ml of Affigel-10) at a flow rate of 0.2 ml/min. at 4°C. Unbound proteins were washed with PBS and the bound proteins were then eluted at pH 2.5 by applying a solution of 25 mM citric acid, 1 ml fractions were collected into tubes containing 1 M Hepes pH 8.5. The eluted protein was monitored for protein and for binding of 125I-IFN-p2/IL-6 following electroblotting. 2.4 Reversed-phase high Pressure liquid chromatography The reversed-phase HPLC column Aquapore RP-300 4.6 x 30 mm (Brownlee Labs) was prewashed with 0.3% aqueous trifluoroacetic acid (TFA) (Buffer F) until a stable baseline was obtained by the fluorescamine detection system. The protein peak fractions eluted from the affinity IFN- 2/IL-6 columns of step 2.3 were pooled and injected in one 1.8 ml portion onto the column. The column was run with Buffer F at a flow rate of 0.5 ml/minute until the fluorometer did not detect any protein. Elution was performed at a flow rate of 0.5 ml/minute, with a linear gradient of acetonitrile in Buffer F ( — - ) (0-20% for 5 minutes, followed by 20-50% for 60 minutes, and finally 50-80% for 5 minutes). The column was then washed for 15 minutes with 80% acetonitrile. Fractions of 0.5 ml were collected and tested for protein content ( — — ) and for binding of 125I-IFN-p2/IL-6 following electroblotting. As shown in figure 1, the active proteins were found to elute as one protein peak, in fractions corresponding to about 39% acetonitrile. 2.5 SDS-PAGE and binding to '^I-IFN-B2/IL-6 In order to monitor the result of the purification, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed under nonreducing conditions according to the method of Laemmli U.K. et al. (Nature (1970) 227:680). Samples of the active fractions eluting from the reversed phase HPLC, were mixed with 3 x concentrated sample buffer containing no SDS and no 3-mercaptoethanol and loaded on a 12% acrylamide gel. In Figure 2, as a reference for molecular weight, a mixture of molecular weight markers (a lactalbumin 14.4 K, soya bean trypsin inhibitor 20.1 K, carbonic anhydrase 30 K, ovalbumin 43 K, bovine serum albumin 67 K, and phosphorylase b. 94 K) was loaded on lane 1. The gel was run at 150 volt and the protein bands were visualized by silver staining (Oakley, B.R. et al. Anal. Biochem. 105:361). In lane 2, it is shown that the HPLC-purified IFN-P2/IL-6 binding protein moved as a single band, with an apparent molecular weight of 50 (40-60) K. Binding of 125I-IFN-P2/IL-6 (2.2xl07 cprri^g, 1.5xl06 cpm/ml) was done following SDS-PAGE under non-reducing conditions and electroblotting onto nitrocellulose membrane (Schleicher and Schuell 0.45μπι), performed essentially as the Western blotting method (Towbin, H. et al., Proc. Natl. Acad. Sci. USA, 76^4350-4354, 1979). As shown in lane 3, only the 50 (40-60)K protein of the partially purified protein sample from the IFN-P2/ IL-6 affinity column eluate reacted specifically with I-IFN-p2/IL-6. A purified IFN-gamma binding protein sample from IFN-gamma affinity column eluate was used as a negative control (lane 4). Lanes 3 and 4 were visualized by autoradiography. 2.6 N-Terminal Sequence Analysis Samples of the substantially purified IFN-P2/IL-6-R soluble extracellular fragment of the invention (l-5pg, 50-200 pmol each) were applied to pretreated, biobrene-coated glass-fiber discs. The dried discs were subjected to repetitive cycles of Edman degradation in an automated pulsed liquid gas phase protein microsequencer (Model 475) with an on-line HPLC PTH-amino acid analyzer (Model 120) and a data acquisition and processing unit Model 900, (all from Applied Biosystems Inc. Foster City, CA, U.S.A.). The computer-derived sequence was compared with the raw data and was corrected when necessary. Altogether two separate analyses were performed in order to confirm the sequence data. The initial yield was over 40%, indicating that the major protein in the preparation (the 50 K band) is related to the resulting sequence. The N-terminal sequencing of this soluble extracellular fragment of the IFN-P2/IL-6-R gave the following amino acid sequence: Leu-Ala-Pro- Arg-Arg-Cys-Pro-Ala-Gln-Glu-Val-Ala-Arg-Gly-Val-Leu-Thr-Ser-Leu- Pro-Gly-Asp-Ser-Val-Thr-Leu-Thr-Cys-Pro-Gly- Example 3: Preparation of Polyclonal antibodies to IFN-B2/IL-6-R Rabbits were initially injected subcutaneously with 10μg of a pure preparation of the IFN-p2/IL-6-R soluble extracellular fragment emulsified in complete Freund's adjuvant. Three weeks later they were injected again subcutaneously with 10μg of the preparation in incomplete Freund's adjuvant. Three additional injections as solution in PBS were given at 10 day intervals. The rabbits were bled 10 days after the last immunization. The development of antibody level was followed by the ability of the rabbit serum to inhibit the HGF (hybridoma growth factor) activity of IFN-P2 IL-6. The immunoglobulins of the rabbit sera were precipitated by the addition of ammonium sulfate and centrifugation, and purified by dialysis and chromatography.

Example 4: Preparation of monoclonal antibodies to IFN-B2/IL-6-R Female Balb/C mice (3 months old) were first injected with 2.5 μg purified IFN-P2/IL-6-R soluble extracellular fragment in an emulsion of complete Freund's adjuvant, and three weeks later, subcutaneously in incomplete Freund's adjuvant. Three additional injections were given at 10 day intervals, subcutaneously in PBS. A final boost was given intraperitoneally 3 days before the fusion to the mouse showing the highest binding titer in inverted solid phase RIA. Fusion was performed using NSO/1 myeloma cell line and lymphocytes prepared from both the spleen and lymph nodes of the animal as fusion partners. The fused cells were distributed into microculture plates and the hybridomas were selected in DMEM supplemented with HAT and 15% horse serum. Hybridomas that were found to produce antibodies to IFN-P2/IL-6-R were subcloned by the limiting dilution method and injected into Balb/C mice that had been primed with pristane for the production of ascites. Immunoglobulins were isolated from the ascites by ammonium sulfate precipitation (50% saturation), centrifuged, redissolved in water and dialyzed against PBS containing 0.02% azide. The isotypes of the antibodies were defined either with the use of a commercially available ELISA kit (Amersham, U.K.), or by the Ouchterlony method using commercially available antisera against different isotypes.

The screening of the anti-IFN-P2/IL-6-R monoclonal antibodies producing hybridomas was performed as follows: Hybridoma supernatants were tested for the presence of anti-IFN-P2/IL-6-R antibodies by an inverted solid phase radioimmunoassay (iRIA). PVC microtiter plates (Dynatech Laboratories, Alexandria, VA) were coated with affinity purified goat anti-mouse serum F(ab)2 antibodies (BioMakor) (l(^g/ml, δθμΐ/well). Following overnight incubation at 4°C the plates were washed twice with PBS containing BSA (0.5%) and Tween 20 (0.05%) and blocked in washing solution for at least 2 hrs at 37°C. Hybridoma culture supernatants (50μ1Λνβ11) were added and the plates were incubated for 4 hrs at 37°C. The plates were then washed three times with the washing solution and 125I-IFN-p2/IL-6-R (50μ1, 105 cpm) was added for further incubation of 16 hrs at 4°C. The plates were washed 3 times and individual wells were cut and counted in a gamma counter. Samples giving counts that were at least 5 times higher than the negative control value were considered positive (Table I). Thirty positive clones were selected, subcloned for further studies and characterized.

Example 5: Western-blotting Western blotting was performed as follows: Samples of partially purified IFN-p2/IL-6-R soluble fragment from human urine were analyzed by SDS PAGE under reducing conditions and electroblotted onto nitrocellulose sheets (BA85, Schleicher and Shuell). Following electroblotting the sheet was incubated overnight with a blocking buffer (5% non-fat milk in PBS containing 0.05% Tween 20 and 0.02% sodium azide) and then for 2 hrs at room temperature with the anti-IFN-p2/IL-6-R monoclonal antibody No. 34-4. Following washing in 0.05% Tween 20 in PBS, the nitrocellulose was incubated for 4 hrs at room temperature with I-goat anti -mouse serum (0.7 x 10 cpm/ml in the blocking buffer). The sheet was then washed, dried and autoradiographed.

Some of the isolated clones and subclones with their isotype and results of binding of IFN-p2/IL-6-R in inverted RIA and Western blotting are listed in Table I.

TABLE I Clones producing monoclonal antibodies to IFN-P2/IL-6 receptor Screening Western blot with iRIA Clone number [CPMI] +M -M Isotype 4.4 20,455 + + IgG! 1,085 + + IgM 17.1 36,565 + IgG2a .2 31,450 + IgG! 22 1 1,465 + + IgG2 24.2 8,850 + + IgG, 2,000 IgG2a 28.7 1,645 IgG, 29 4,165 .8 1,755 + IgM 31 3,060 32.5 31,465 + + IgGi 33.2 14,875 IgG, 34.4 33,480 + k, IgG, .2 35,495 + + k, IgG3 36 1,445 + IgM 37 9,640 IgG, 38.4 35,975 + IgG, 39.1 5,195 + + IgG2 40 1,415 + IgG, 41 1,870 + IgG, 42.5 33,565 IgG, 43 1,255 IgG, 46 6,090 48 18,000 IgG, 49 8,000 + IgM 50.3 28,440 + + IgG, 51 1,075 IgGi 52 3,945 IgM 53.4 3,440 IgG, x M: mercaptoethanol (reducing agent) Example 6: Affinity chromatography of IFN-P2/IL-6-R soluble fragment preparations with monoclonal antibodies Antibodies against IFN-P2/IL-6-R are utilized for the purification of the soluble fragment by affinity chromatography. The monoclonal antibody No. 34-4 was used in this example for affinity chromatography, after testing its binding capacity for the radiolabeled antigen in inverted solid phase radioimmunoassay (iRIA). Ascitic fluid containing the monoclonal antibody secreted by hybridoma No. 34-4 was purified by ammonium sulfate precipitation at 50% saturation followed by extensive dialysis against PBS. About lOmg of immunoglobulins were bound to 1 ml polyacrylhydrazide agarose as specified by Wilchek and Miron, Methods in Enzvmology 34:72-76, 1979. 250 ml of human urine, partially purified on carboxymethyl sepharose (CMS) column (equivalent to 250 1 of crude urine) were loaded on 0.5 ml of the anti-IFN-p2/IL-6-R antibody column at 4°C at a flow rate of 0.25 ml/min. The column was washed with PBS until no protein was detected in the washings. IFN-P2/IL-6-R soluble fragment was eluted by 25 mM citric acid buffer, pH 2.2 (8 x 1 column volume fractions) and immediately neutralized by I M Hepes buffer, pH 8.5, with a total recovery of 88% IFN-P2/IL-6-R soluble fragment. Silver stain analysis of SDS PAGE of the eluted fractions revealed a major band of M.W. of 50,000 and another major band of 150,000 (contaminants). Further purification of this preparation was obtained by RP-300 HPLC, and the soluble receptor fragment eluted at 39% acetonitrile in a pattern similar to that of Fig. 1.

TABLE II Immunoaffinitv Purification of IL-6 receptor from urine (CMS) FLUORESCAMINE ELISA McAb purity yield column Sample ml μg/ml μ^πιΐ % 34.4 Load 250 2200 550,000 0.38 95 Efluent 250 2000 500,000 0.06 15 Elution 1 1.2 20 24 7.7 9 38 Elution 2 1.2 45 54 30.4 36.5 67 Elution 3 1.2 18 21.6 12 14.4 80 Elution 4 1.2 1 1 13 a 9.6 87 Total eluted 69.5 88 Example 7; ELISA test Microtiter plates (Dynatech or Maxisorb by Nunc) were coated with anti -IFN-p2/IL-6-R monoclonal antibody No. 34-4 (immunoglobulin fraction, 120 μΐ, 20 μg/ml in PBS), overnight at 4°C. The plates were washed with PBS containing BSA (0.5%) and Tween 20 (0.05%) and blocked in the same solution for at least 2 hrs at 37°C. The tested samples were diluted in the blocking solution and added to the wells (100 μΐ/well) for 4 hrs at 37°C. The plates were then washed 3 times with PBS containing Tween 20 (0.05%) followed by the addition of rabbit anti-IFN- 2/IL-6-R serum (1 : 1000, 100 μΐ/well) for further incubation of 4 hrs at 37°C. The plates were washed 3 times and a conjugate of goat-anti-rabbit horseradish peroxidase (HRP, Biomakor, 1 :2000, 100 μΐ/well) was added for 2 hrs at 37°C. The plates were washed 4 times and the color was developed by ABTS (2,2'-azino-bus (3-ethylbenzthiazoline-6sulfonic acid), Sigma) substrate. The plates were read by an automatic ELISA reader. Alternatively, the ELISA could be performed by replacing the rabbit polyclonal anti-IFN-p2 IL-6-R antibodies with a monoclonal antibody No. 22-1 either conjugated to HRP or biotinylated. 126245/2

Claims (3)

1. An antibody against the substantially purified human Interferon- 2/Interleukin-6 Receptor (herein designated IFN-p2/IL-6-R) soluble extracellular fragment having a molecular weight of about 50 (40-60) Kd when the substantially purified protein is analyzed by SDS-PAGE under non-reducing conditions, moving as a single peak on reversed phase high performance liquid chromatography (HPLC), and containing the following N-terminal amino acid sequence : Leu-Ala-Pro-Arg-Arg-Cys-Pro-Ala-Gln-Glu-Val-Ala-Arg-Gly-Val- Leu-Thr-Ser-Leu-Pro-Gly-Asp-Ser-Val-Thr-Leu-Thr-Cys-Pro-Gly-

2. An antibody according to claim 1 which is a polyclonal antibody.

3. An antibody according to claim 1 which is a monoclonal antibody.