Characterisation of monoclonal antibodies to ovine interleukin-6 and the development of a sensitive capture ELISA

Veterinary Immunology and Immunopathology 73 (2000) 155±165

Characterisation of monoclonal antibodies to ovine interleukin-6 and the development of a sensitive capture ELISA P. McWatersa, L. Hursta,1, P.J. Chaplina, R.A. Collinsb, P.R. Wooda,2, J.-P.Y. Scheerlincka,* a

CRC for Vaccine Technology Unit, CSIRO Animal Health, Private Bag No. 1, Parkville, Vic. 3052, Australia b Institute for Animal Health, Compton, Nr. Newbury, Berkshire, RG20 7NN, UK Received 19 July 1999; received in revised form 19 October 1999; accepted 30 October 1999

Abstract A puri®ed recombinant ovine (rOv) interleukin-6 (IL-6) was used to generate speci®c murine monoclonal antibodies (mAbs) and a polyclonal rabbit antisera to this cytokine. From the 31 initial hybridoma cell lines generated, three stable clones were established which secreted mAbs to rOvIL6, as judged by a direct enzyme-linked immunosorbent assay (ELISA) and Western blotting. Their speci®city was further con®rmed by demonstrating that none of the mAbs recognised any of the six other irrelevant recombinant ovine cytokines tested by direct ELISA. All three mAbs displayed cross-reactivity with human and African green monkey IL-6 as demonstrated by direct ELISA and Western blotting. In contrast, the polyclonal antibodies only cross-reacted with bovine IL-6 and not with either of the human or monkey homologues. By combining a mAb with the polyclonal antisera a sensitive, IL-6-speci®c, capture ELISA was developed that had a sensitivity of 150 pg/ml. This detection system was unequivocally validated by demonstrating that native OvIL-6 could be detected in efferent lymph draining from a stimulated popliteal lymph node. In addition, one of the mAbs was shown to allow the detection of OvIL-6 by intracellular cytokine staining and ¯ow cytometry. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Sheep; Interleukin-6; Monoclonal antibody; Capture ELISA; Flow cytometry

*

Corresponding author. Tel.: ‡61-3-9342-9786; fax: ‡61-3-9342-9830. E-mail address: [email protected] (J.-P.Y. Scheerlinck). 1 Present address: Nephrology Unit, Monash Medical Centre, Clayton, Vic. 3168, Australia. 2 Present address: Veterinary Division, CSL Limited, 45 Poplar Road, Parkville, Vic. 3052, Australia. 0165-2427/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 9 9 ) 0 0 1 5 8 - 0

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1. Introduction Various groups have independently identi®ed interleukin-6 (IL-6) by its ability to induce a vast range of biological effects resulting in the use of a large number of synonyms for this cytokine. These functions include stimulation of B cells, cytotoxic T cells and hepatocytes as well as induction of B cell hybridoma proliferation and possibly also an antiviral activity. While IL-6 is produced by many cell types including ®broblasts, endothelial cells, keratinocytes, monocytes/macrophages, T cells, mast cells and B cells (reviewed by Van Snick, 1990), it is believed that monocytes are the major producers (Aarden et al., 1987). However, lymphocytes can be a major source of IL-6 in certain pathological conditions such as B and T cell in®ltrates in rheumatoid synovial tissue (Hirano et al., 1988). While IL-6 is generally not produced constitutively in any of the cells mentioned above, it can be upregulated by a large number of factors including viral infection, lipopolysaccharide and cytokines such as IL-1, tumour necrosis factor (TNF)-a, interferon (IFN)-g, platelet-derived growth factor, IL-3 and granulocyte/macrophage colony-stimulating factor (GM-CSF) (Van Snick, 1990). Cytokines including IL-6, are key regulators of the immune response and as such their measurement is often essential in unravelling immune mechanisms. A large variety of methods have been used to measure cytokines including RT-PCR, bioassays, intracellular staining followed by FACS analysis and detection of cytokine proteins by speci®c enzyme immunoassays. While the latter method has the advantages of being fast and able to detect the actual cytokine in complex biological ¯uids, it does require the availability of speci®c antibodies. Due to the relatively low level of amino acid sequence identity between cytokines of species belonging to different taxonomic families, it is often necessary to develop monoclonal antibodies (mAbs) to cytokines for at least one species within the family considered. In this study we have generated and characterised a set of mAbs and polyclonal antisera to recombinant ovine (rOv) IL-6, combined these in a sensitive and speci®c capture enzyme-linked immunosorbent assay (ELISA) and demonstrated the ability of one of the mAbs to detect OvIL-6 by ¯ow cytometry. 2. Materials and methods 2.1. Expression of IL-6 A pQE30 vector (Qiagen, Hilden, Germany) encoding OvIL-6 as a poly-histidine fusion protein was provided by Dr. H.-F. Seow. Bacterial cultures (XL1-blue) transformed with the pQE30/IL-6 vector were grown to mid-log phase (optical density (OD) at 600 nm of 0.6) and induced with 2 mM isopropyl-b-thiogalactopyranosidase (Promega, Madison, USA) for 3 h. The bacteria were pelleted at 1600g for 10 min in phosphate buffered saline (PBS, pH 7.2) and lysed using zirconia/silica beads (Biospec Products, USA). The soluble fraction of the lysate was collected by centrifugation at 1000g for 5 min. This soluble lysate was mixed with 250 ml of a 50% slurry of Super¯owTM Ni-NTA resin (Qiagen) for 4 h then washed three times with 10 ml wash buffer

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(50 mM NaH2PO4 pH 8.0, 300 mM NaCl, 20 mM imidazole). The Ni-NTA resin was mixed with 1 ml of elution buffer (50 mM NaH2PO4 pH 8.0, 300 mM NaCl, 250 mM imidazole) overnight at 48C, before the eluent was collected by centrifugation at 1000g for 5 min. The purity of the rOvIL-6 was analysed by SDS-PAGE and found to be >95%. 2.2. Production of mAbs Female, 12±16-week-old BALB/c mice were primed by intraperitoneal (IP) injection of 15 mg of rOvIL-6 emulsi®ed with 50% (v/v) Freund's complete adjuvant (Sigma, St Louis, USA). A second IP injection of 10 mg rOvIL-6 emulsi®ed in 50% Freund's incomplete adjuvant (Sigma) was administered 4 weeks later. After a further 2 weeks, sera from the mice were tested for the presence of anti-rOvIL-6 antibodies. Positive mice were injected IP with a further 10 mg of rOvIL-6 in saline and splenocytes were recovered 4 days later. Splenocytes were fused with NS1/1-Ag-1 cells following the method described by Wood et al. (1990). Brie¯y, the spleen of an immunised mouse was gently ¯ushed using serum-free Dulbecco's Modi®ed Eagle's Medium (DMEM, Gibco BRL, Gaithersburg, USA) at 378C. The splenic lymphocytes were then mixed with NS-1 cells, washed and fused using 50% polyethylene glycol 4000 (Merck, Darmstadt, Germany). Cells were dispensed into 96 well microtiter plates (Nunc, Roskilde, Denmark) with DMEM supplemented with 20% foetal bovine serum (FCS, MultiSer, Trace Biosciences, Castle Hill, Australia), 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 100 mM hypoxanthine, 0.4 mM aminopterin and 16 mM thymidine (all Gibco BRL) for hybridoma selection. Culture supernatants of all hybridomas were screened for anti-rOvIL-6 antibody production by ELISA 10±14 days after fusion and selected hybridomas were cloned twice using limiting dilution. Pristane (Aldrich, Castle Hill, Australia) primed mice were then injected IP with 5106 cloned hybridoma cells for ascites production. The isotype of the mAbs was determined using a commercial isotyping kit (Bio-Rad, Regents Park, Australia) as per manufacturer's instructions. 2.3. Screening and speci®city of antibodies to rOvIL-6 The ELISA used to screen hybridomas for antibodies to rOvIL-6 closely followed the method of Rothel et al. (1997). Brie¯y, 100 ml per well of puri®ed rOvIL-6 (0.5 mg per well), diluted in 50 mM carbonate buffer (pH 9.6), was coated overnight at 48C onto 96 well microtiter plates (Maxisorb, Nunc). Following blocking with 200 ml per well of 0.1% casein in PBS, 100 ml of hybridoma culture supernatants were diluted in PBS containing 0.1% Tween 20 (PBST) and added to the plates for 1 h at room temperature. Plates were then washed ®ve times with PBST. Anti-rOvIL-6 antibody was detected using 100 ml per well of sheep anti-mouse-Ig-horseradish-peroxidase (HRP) conjugate (Silenus, Hawthorn, Australia) diluted in PBST and incubated for 1 h at room temperature then washed as above. Tetramethylbenzidine (TMB, 100 ml per well) substrate (Bos et al., 1981) was added for 30 min before the reaction was stopped with 50 ml per well of 0.5M sulfuric acid and the optical density read at 450 nm on a microplate reader (Titertek-Flow,

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Helsinki, Finland). The speci®cities of the monoclonal and polyclonal antibodies were determined by ELISA after substituting the rOvIL-6 with rOvIL-1b (Seow et al., 1994a), rOvIL-2 (Seow et al., 1997), rOvIL-8 (Seow et al., 1994b), rOvTNFa (Seow et al., 1995), rOvGM-CSF (Entrican et al., 1996) or recombinant bovine (rBov) IFNg coated at 0.5 mg per well. Swine anti-rabbit-Ig-HRP conjugate (Dako, Botany, Australia) was used to detect bound polyclonal antibodies. 2.4. Production of polyclonal antiserum to rOvIL-6 Rabbits were injected subcutaneously with 50 mg rOvIL-6 emulsi®ed in 50% (v/v) Freund's complete adjuvant (Sigma). Four weeks later the rabbits were injected intramuscularly with 50 mg rOvIL-6 emulsi®ed in 50% (v/v) Freund's incomplete adjuvant (Sigma). Rabbits were screened for anti-rOvIL-6 antibodies by ELISA as described above. Once the anti-rOvIL-6 titer had peaked the rabbit immune serum was collected and stored at ÿ208C. 2.5. Capture ELISA for OvIL-6 Ascitic ¯uid from mAb 4B6, used for the development of the IL-6 capture ELISA, was puri®ed on a pilot-scale protein A column (Bioquest, North Ryde, Australia). The puri®ed mAb was diluted in 50 mM carbonate buffer, pH 9.6 and coated at 0.5 mg per well onto 96 well microtitre plates (Maxisorb, Nunc) overnight at 48C. Plates were blocked and washed as described above. Cytokine samples were serially-diluted in PBST containing 1% casein and 100 ml of diluted sample added to wells and incubated for 1 h at room temperature. Rabbit anti-OvIL-6 serum was used to detect captured OvIL-6 (diluted 1:10,000 in PBST). The presence of rabbit antibodies was demonstrated using a swineanti-rabbit-Ig-HRP conjugate followed by development with TMB substrate and sulfuric acid as above. The speci®city of the capture ELISA was established by replacing the rOvIL-6 cytokine with rOvIL-1b, rOvIL-2, rOvIL-8, rOvTNFa, rOvGM-CSF or rBovIFNg at the same concentration as used for rOvIL-6. The ability of this ELISA to detect native IL-6 was assessed by measuring IL-6 in the efferent lymph of sheep vaccinated with an unrelated recombinant vaccine antigen. 2.6. Western blotting A 12% SDS-PAGE was loaded with 1 mg of rOvIL-6, rHuIL-6 (Genzyme, Cambridge, USA) or supernatants derived from CosM6 cells and run under reducing conditions. The recombinant proteins were then electro-blotted onto a nitrocellulose membrane (Hybond super C, Amersham, Buckinghamshire, UK) as described by Towbin et al. (1979). The nitrocellulose was blocked with 0.1% casein in PBST and each lane probed using the antiIL-6 mAbs 4B6, 5A4 and 5B8 or the anti-IL-6 polyclonal antisera. The blot was incubated for 1 h at room temperature with sheep anti-mouse-Ig-HRP (Silenus) or a swine-anti-rabbit-Ig-HRP, then washed and visualised using an enhanced chemiluminescence substrate reagent (Super Signal1, Rockford, Pierce) following the manufacturer's instructions.

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2.7. Flow cytometry Peripheral blood mononuclear cells (PBMC) were isolated from ovine heparinised blood using Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) density gradient (1.077 g/l speci®c gravity). The method used to detect IL-6 using ¯ow cytometry was essentially as described by Jung et al. (1993). Brie¯y, PBMC (2.5106/ml) were cultured in DMEM Glutamax-I Medium (Gibco-BRL) supplemented with 10% FCS, 1 mM sodium pyruvate, 50 mM 2-mercaptoethanol, 200 U/ml penicillin, and 10 mg/ml streptomycin, in the presence of 10 mg/ml Brefeldin A (Sigma), 1 mg/ml ionomycin (Sigma) and 10 ng/ml phorbol-12myristate 13acetate (PMA, Sigma). Control cells were cultured in similar conditions without Brefeldin A, ionomycin and PMA. After overnight culture at 378C in an atmosphere of 9% CO2 in air, the cells were washed twice in PBS and the cell pellet resuspended in 5 ml 1% paraformaldehyde (w/v in PBS) for 10 min. The cells were then centrifuged for 5 min at 800g and permeabilised by washing twice with PBS-S (PBS with 1% w/v BSA, 0.1% w/v NaN3 and 0.1% w/v saponin (Sigma)) and adjusted to a cell concentration of approximately 1107/ml. Cells (1106) were incubated with a mAb (1 mg) to OvIL-6, or an isotype matched irrelevant control mAb. A FITC-conjugated anti-mouse IgG1 isotype-speci®c antibody (Caltag, San Fransisco, USA) was added for 1 h at 48C. Cells were then washed twice in 200 ml PBS-S and analysed on a FACSortTM (Becton Dickinson, USA). 2.8. Transfection of Cos-M6 cells Cos-M6 cells were transfected with plasmid DNA encoding ovIL-6 according to the method of Linsley et al. (1991). Brie¯y, cells (106 per 10 cm culture dish) were cultured 18±24 h prior to transfection with DNA (37.5 mg per dish) in a 5 ml volume of serum free DMEM, containing 0.1 mM chloroquine and 600 mg/ml DEAE dextran. After 3 h of culture at 378C, the cells were washed with PBS containing 10% dimethyl sulfoxide and then cultured at 378C in the presence of DMEM containing 10% FCS. After 24 h the medium was replaced with serum-free DMEM and the medium harvested after 3 days of culture at 378C. 3. Results 3.1. Production and characterisation of antibodies to rOvIL-6 Thirty-one of the 219 hybridoma supernatants screened by ELISA reacted strongly with rOvIL-6, while at the same time failed to react to rOvIL-2 produced in a similar E. coli expression system. Three of these hybridomas were cloned twice and the resultant mAbs (4B6, 5A4, 5B8) were further characterised by direct ELISA, Western blot, and ¯ow cytometry. The isotypes of mAbs 4B6 and 5B8 were found to be IgG1, while mAb 5A4 was an IgG2a (data not shown). Using rOvIL-6 at 5 mg/ml in an ELISA, puri®ed ascites from mAb 4B6 gave an endpoint titer (mean optical density without mAb plus ®ve standard deviations) of 1107

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Fig. 1. Binding of mAbs 4B6 (^), 5A4 (*), 5B8 (&) and polyclonal rabbit serum (&) to puri®ed rOvIL-6. The antibodies were titrated in an ELISA against rOvIL-6 coated at 5 mg/ml.

corresponding to 100 pg/ml. The end-point titer for unpuri®ed 5A4 was 5106 and for unpuri®ed 5B8 ascites was 6.6105. Polyclonal rabbit serum raised against OvIL-6 had an end-point titer of 1.5106 (Fig. 1). At a dilution of 1:200 none of the three mAbs or the polyclonal antiserum reacted with rOvIL-1b, rOvIL-2, rOvIL-8, rOvTNFa, rOvGMCSF or rBoIFNg by ELISA, demonstrating the speci®city of the anti-OvIL-6 mAbs. Western blot analysis using the mAbs 4B6, 5A4 and 5B8 against rOvIL-6 revealed that each mAb detected a protein band with an apparent relative molecular mass (Mr) of 26,000±27,000 (Fig. 2). This compared well with the predicted molecular weight of OvIL-6 (23.5 kDa).

Fig. 2. Western blot of rOvIL-6 detected with mAbs 5A4 (lane 1), 4B6 (lane 2) and 5B8 (lane 3). The presence of antibodies bound to rOvIL-6 was visualised using an anti-mouse-Ig-HRP conjugated antibody followed by enhanced chemiluminescence development.

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Fig. 3. Intracellular staining of ovine PBMC with an anti-OvIL-6 mAb. PBMC cultured in the presence (Ð) or absence (. . .) of Brefeldin A, ionomycin and PMA were permeabilised and stained with the mAb 4B6 or an isotype matched control antibody (- - -). The marker on the graph represents the positive population referred to in the text.

The polyclonal antiserum raised against rOvIL-6 cross-reacted with supernatants from CosM6 cells transfected with a plasmid encoding BoIL-6, but failed to react with the control non-transfected CosM6 supernatants, or recombinant human (rHu)IL-6 (data not shown). In contrast, the three mAbs recognised rHuIL-6, CosM6 supernatants containing rBoIL-6, as well as the control CosM6 supernatants by direct ELISA. Moreover, all three mAbs were shown to react in a Western blot with a protein with a Mr in the range 26,000± 27,000 in supernatants harvested from CosM6 cells derived from African green monkey. The anti-rOvIL-6 polyclonal antiserum failed to detect this protein in CosM6 cells by Western blot (data not shown). The ability of the three mAbs to cross-react with rBoIL-6 expressed in CosM6 cells could not be determined due to the reactivity of the mAbs to monkey IL-6 present in the CosM6 cell supernatants. 3.2. Flow cytometry Ovine PBMC were stimulated with ionomycin and PMA in the presence of Brefeldin A and stained for intracellular IL-6 using the mAb 4B6. As illustrated in Fig. 3 intracellular IL-6 could be detected in approximately 40% of the lymphocytes. In contrast IL-6 could not be detected in non-stimulated PBMC. Furthermore, 4B6 was the only mAb found to be suitable for the detection of IL-6 by ¯ow cytometry. 3.3. IL-6 capture ELISA Using the mAbs and polyclonal antisera described above we optimised a capture ELISA for OvIL-6. The most sensitive detection of rOvIL-6 by capture ELISA was achieved when the mAbs were used as capture antibody for IL-6 and the polyclonal antiserum for detection of the captured cytokine. Of the three mAbs, 4B6 provided the greatest sensitivity for IL-6 detection. By testing a range of dilutions of mAb and polyclonal antisera the optimal conditions for the capture ELISA were determined. The

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Fig. 4. Detection of rOvIL-6 in a capture ELISA using mAb 4B6 (*), 5A4 (&) and 5B8 (~) each coated at 5 mg/ml. Rabbit polyclonal antiserum (1:10,000) was used to detect the captured cytokine. Bound rabbit antibodies were visualised using an anti-rabbit-Ig-HRP conjugated antibody.

optimal coating concentration for mAb 4B6 was 5 mg/ml, whereas a 1:10,000 dilution was optimal for the polyclonal antiserum. This protocol resulted in a reliable detection limit of 150 pg/ml of rOvIL-6 as de®ned by the mean OD of eight replicate negative wells plus ®ve standard deviations (Fig. 4). To determine the speci®city of the IL-6 ELISA, irrelevant recombinant cytokines were used as negative controls. At a concentration of 1 mg/ml (i.e. more than 5000 the detection limit for rOvIL-6), rOvIL-1b, rOvIL-2, rOvIL-8, rOvTNFa, rOvGM-CSF and rBovIFNg were not detected when using the standardised capture ELISA (data not shown). To further validate the IL-6 ELISA, physiological samples were tested to determine whether this capture ELISA could detect native OvIL-6. Initially, supernatants from PBMC stimulated with a range of mitogens (Concanavalin A, phytohemagglutinin or PMA plus ionomycin) were used and all shown to be negative for IL-6 using the capture ELISA. Using the ELISA the amount of IL-6 in efferent lymph collected from a cannulated popliteal lymph node following immunisation with a recombinant vaccine was estimated. While pre-immunisation levels of IL-6 were below 10 ng/ml of lymph, a sharp peak of IL-6 lasting 3 days and reaching 160 ng/ml, was observed after vaccination (data not shown). 4. Discussion This paper describes the generation of antibodies to OvIL-6 and the development of a sensitive capture ELISA for this cytokine. The polyclonal rabbit anti-IL-6 serum had a reduced cross-reactivity compared to the mAbs raised against OvIL-6 and only recognised the bovine homologue. This is probably not unexpected given that there is a much higher identity between the predicted amino acid sequences between sheep and cattle IL-6 (88%) than between the sheep and human homologues (53%). However, even

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though the identity with HuIL-6 is low, there are obviously highly conserved epitopes between different species, given that all three mAbs raised against OvIL-6 cross-reacted with the human and monkey homologues. Indeed, the detection of a protein with a similar Mr to rOvIL-6 in CosM6 supernatants indicates that IL-6 is constitutively produced by this monkey cell line, which has also been reported for ®broblasts in other species (Van Snick, 1990). The use of a bioassay to quantify cytokines in complex biological ¯uids such as serum, culture supernatants or lymph are hampered by the overlapping biological activities of cytokines, which cross-react in many of these bioassay systems. Indeed, one such example is the IL-6-dependent B9 myeloma cell line, which has also been shown to be sustained with murine IL-4 (Ebrahimi et al., 1995), clearly jeopardising the speci®city of this assay for the measurement of IL-6 in biological ¯uids. Furthermore, although there are a number of cytokines which cross-react on cell lines derived from a different species (Scheerlinck et al., 1998), this is often due to an incomplete cross-reactivity making the assay unsuitable to quantify the levels of cytokines in biological samples. Indeed, while OvIL-6 can sustain the proliferation of the murine 7TD-1 cells, the detection limit for OvIL-6 is only approximately 125 ng/ml (unpublished result) compared to a reported sensitivity of about 1 ng/ml for mouse IL-6 (Van Snick et al., 1986). Given that the percentage identity at the amino acid level between murine and ovine IL-6 is only 42%, it is probably not surprising that there appears to be only a partial cross-reactivity between these two species. Indeed, sequence analysis has revealed that when the amino acid identity of a cytokine from two different species is less than 60%, most cytokines do not cross-react on cells from that different species (Scheerlinck, 1999). Due to this low degree of speci®city and the poor sensitivity of the bioassay for OvIL6, we needed to develop a sensitive capture ELISA for this cytokine. Using one of the mAbs to capture the cytokine and the polyclonal antisera to detect this protein proved the most sensitive ELISA for the detection of IL-6. This feature of an increased sensitivity when the cytokine is captured with a mAb, compared to the polyclonal sera, has been reported for other cytokines (Egan et al., 1994; Rothel et al., 1997) and is probably due to the increased ef®ciency in which a puri®ed mAb would bind to the ELISA plate compared to the unpuri®ed polyclonal antisera. Interestingly, the capture ELISA failed to detect IL-6 production by stimulated PBMC, whilst under similar conditions transcripts for IL-6 could readily be detected in bovine PBMC using RT-PCR (Covert and Splitter, 1995) and in ovine PBMC by ¯ow cytometry in the present study. The detection of transcripts is likely due to the increased sensitivity of RT-PCR, but also illustrates that detection of mRNA does not always re¯ect the production of protein. However, the demonstration that IL-6 was detected in 40% of stimulated lymphocytes, but was not present in the culture supernatants could indicate that the IL-6 represented an intracellular store of IL-6, which has been reported for other cytokines, such as IL-15 (Tagaya et al., 1997). Alternatively, IL-6 may be produced and secreted by lymphocytes in response to mitogen, but rapidly used by other lymphocytes and/or possibly inactivated by soluble receptors that have been described for HuIL-6 (Lust et al., 1992). Indeed, given that IL-6 is a pro-in¯ammatory cytokine and is often associated with pathological conditions (Hirano et al., 1988), it is likely that the production and longevity of this cytokine is tightly regulated. The inability to detect IL-6

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in efferent lymph draining a resting lymph node (Paul Chaplin, personal communication) would also indicate that the production of IL-6, like other cytokines, is tightly regulated. Moreover, the detection of IL-6 in efferent lymph draining a stimulated lymph node suggests that this cytokine plays a role in the early inductive phases of an immune response at the local lymph node. The reagents produced and characterised in this study have resulted in the generation of two complementary ways of detecting OvIL-6 and these will greatly enhance our ability to investigate the role this cytokine plays in immune regulation. Acknowledgements This work was supported by the Cooperative Research Centre for Vaccine Technology, Australia. The authors are grateful to Dr. R. Windon for allowing access to efferent lymph and Dr. H.-F. Seow for providing the rOvIL-6 construct. References Aarden, L.A., De Groot, E.R., Schaap, O.L., Lansdorp, P.M., 1987. Production of hybridoma growth factor by human monocytes. Eur. J. Immunol. 17, 1411±1416. Bos, E.S., van der Doelen, A.A., van Rooy, N., Schuurs, A.H.W.M., 1981. 3,30 ,5,50 -Tetramethylbenzidine as an Ames test negative chromogen for horse-radish peroxidase in enzyme-immunoassay. J. Immunoassay 2, 187±204. Covert, J., Splitter, G., 1995. Detection of cytokine transcriptional pro®les from bovine peripheral blood mononuclear cells and CD4(‡) lymphocytes by reverse transcriptase polymerase chain reaction. Vet. Immunol. Immunopathol. 49, 39±50. Egan, P.J., Rothel, J.S., Andrews, A.E., Seow, H.F., Wood, P.R., Nash, A.D., 1994. Characterization of monoclonal antibodies to ovine tumor necrosis factor-a and development of a sensitive immunoassay. Vet. Immunol. Immunopathol. 41, 259±274. Ebrahimi, B., Roy, D.J., Bird, P., Sargan, D.R., 1995. Cloning, sequencing and expression of the ovine interleukin 6 gene. Cytokine 7, 232±236. Entrican, G., Deane, D., MacLean, M., Inglis, L., Thomson, J., McInnes, C., Haig, D.M., 1996. Development of a sandwich ELISA for ovine granulocyte/macrophage colony-stimulating factor. Vet. Immunol. Immunopathol. 50, 105±115. Hirano, T., Matsuda, T., Turner, M., Miyasaka, N., Buchan, G., Tang, B., Sato, K., Shimizu, M., Maini, R., Feldmann, M., Kishimoto, T., 1988. Excessive production of interleukin 6/B cell stimulatory factor-2 in rheumatoid arthritis. Eur. J. Immunol. 18, 1797±1801. Jung, T., Schauer, U., Heusser, C., Neumann, C., Rieger, C., 1993. Detection of intracellular cytokines by ¯ow cytometry. J. Immunol. Methods 159, 197±207. Linsley, P.S., Brady, W., Grosmaire, L., Aruffo, A., Damle, N.K., Ledbetter, J.A., 1991. Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation. J. Exp. Med. 173, 721±730. Lust, J.A., Donovan, K.A., Kline, M.P., Greipp, P.R., Kyle, R.A., Maihle, N.J., 1992. Isolation of an mRNA encoding a soluble form of the human interleukin-6 receptor. Cytokine 4, 96±100. Rothel, J.S., Hurst, L., Pepin, M., Berton, P., Corner, L.A., Wood, P.R., 1997. Analysis of ovine IL-1b production in vivo and in vitro by enzyme immunoassay and immunohistochemistry. Vet. Immunol. Immunopathol. 57, 267±278. Scheerlinck, J.-P.Y., Chaplin, P.J., Wood, P.R., 1998. Ovine cytokines and their role in the immune response. Vet. Res. 29, 369±383.

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