Expression and functional characterization of killer whale (Orcinus orca) interleukin-6 (IL-6) and development of a competitive immunoassay

Veterinary Immunology and Immunopathology 93 (2003) 69–79

Expression and functional characterization of killer whale (Orcinus orca) interleukin-6 (IL-6) and development of a competitive immunoassay Christina Funkea,d,*, Donald P. Kingb, Jim F. McBainc, Dieter Adelungd, Jeffrey L. Stotta a

Laboratory of Marine Mammal Immunology, Department of Pathology, Microbiology, Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA b Department of Exotic Disease Control, Institute for Animal Health, Pirbright GU24 0NF, UK c SeaWorld of California, 500 SeaWorld Drive, San Diego, CA 92109, USA d Institut fuer Meereskunde, Duesternbrooker Weg 20, 24105 Kiel, Germany

Received 11 September 2002; received in revised form 13 February 2003; accepted 20 February 2003

Abstract Interleukin-6 (IL-6) is a cytokine that can reach detectable systemic levels and is a major inducer of the acute phase response. As such, clinical assays to identify this cytokine in mammalian sera are of diagnostic value. A 558 base-pair (bp) fragment of killer whale IL-6 was cloned and expressed as a 21 kDa protein in Escherichia coli. Biological activity of the recombinant killer whale IL-6 (rkwIL-6) was demonstrated using the IL-6-dependent B9 mouse hybridoma cell line; acute phase sera from a killer whale and supernatants from lipopolysaccharide (LPS)-stimulated killer whale peripheral blood mononuclear cells (PBMCs) also supported the proliferation of the B9 hybridoma. Rat anti-mouse IL-6 receptor antibody effectively blocked biological activity of all three sources of IL-6. Polyclonal antisera, specific for the recombinant protein, were obtained by successive immunization of a rabbit with rkwIL-6. The polyclonal antibody was capable of neutralizing the biological activity of both recombinant and native kwIL-6. A competitive enzyme-linked immunosorbent assay (ELISA) was developed using the polyclonal rabbit anti-rkwIL-6 and the recombinant protein; sensitivity of the assay was in the range of 1 ng/ml. The ELISA was subsequently used to identify the presence of native IL-6 in acute phase sera of two species of delphinidae, a killer whale and a bottlenose dolphin. The application of quantitative cytokine assays as diagnostic tools for monitoring cetacean health are becoming feasible as many animals are now being trained for fluke presentation, making blood collection a routine procedure. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Killer whale; Interleukin-6 (IL-6); Inflammation; Recombinant cytokine; Immunoassay

1. Introduction Abbreviations: PMSF, phenylmethansulfonyl fluoride; IPTG, isopropyl-b-D-1-thiogalactopyranoside * Corresponding author. Tel.: þ1-530-752-2543; fax: þ1-530-752-3349. E-mail address: [email protected] (C. Funke).

Interleukin-6 (IL-6) plays a major role in the early phase of the immune response to insults such as bacterial/viral infections, burns, trauma and neoplasia. Major cell sources for IL-6 include macrophages, monocytes, fibroblasts, endothelial cells and lymphocytes.

0165-2427/03/$ – see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0165-2427(03)00055-2

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Hallmarks of IL-6 are its pleiotropic actions including regulation of the acute phase response and augmentation of antigen-specific immune responses. This includes the activation of hepatocytes to produce acute phase proteins, stimulation of the hypothalamus to release adrenocorticotropic hormone and B cells to produce immunoglobulins. Typically, increased systemic concentrations of IL-6 in humans have been correlated to fever, leukocytosis, increase of erythrocyte sedimentation rate, activation of complement and decreased levels of serum iron and zinc (reviewed by Van Snick, 1990). Due to a pivotal role played by IL-6 in inducing an inflammatory response, and its presence in the circulation, techniques have been established to measure IL-6 in serum from multiple species. Standard techniques include quantitation using IL-6dependent established cell lines (Oppmann et al., 1996; Aarden et al., 1987) and enzyme-linked immunosorbent assay (ELISA) (McWaters et al., 2000; Rees et al., 1999). The potential utility of measuring IL-6 for use as an indicator of inflammation in free-ranging marine mammals has been demonstrated in the common seal (Phoca vitulina) and grey seal (Haliochoerus grypus) utilizing an IL-6-dependent B9 hybridoma cell line (King et al., 1993). Unfortunately, cell culture assays are time consuming and not ideal for diagnostic applications. ELISAs represent alternative and practical assays for diagnostic and semi-field applications as they are fast and easy to perform. Commercial kits for IL-6 detection are available for a limited number of species. However, application of molecular biology techniques to the cloning and expression of cytokine genes is a rational approach to developing similar tools for marine mammal species. Sequences for marine mammal IL-6 genes have been reported including killer whale, harbor seal and sea otter (King et al., 1996) as well as beluga whale (St-Laurent and Archambault, 2000). Based upon the value of cetaceans in public displays, development of improved techniques for early identification of a tissue insult are justified. The use of cytokine ELISAs as diagnostic tools for monitoring cetacean health is feasible as many animals are now being trained for fluke presentation, making blood sampling a routine procedure. In this study, we report the expression and purification of recombinant killer whale IL-6 (rkwIL-6), establish its biological activity and specificity, and the generation of polyclonal antisera specific for IL-6.

The rkwIL-6, in concert with the polyclonal antibody, was used to develop a competitive ELISA to measure IL-6 in the serum of clinically ill animals. 2. Materials and methods 2.1. IL-6 expression A pGEM1-T vector (Promega Corp., Madison, WI) containing a kwIL-6 cDNA fragment of approximately 670 bp was used to amplify 558 bp for an expression construct. PCR primers (sense: 50 -AAACATATGCCGGGACCCCTGGGAGAA-30 , antisense: 50 -TTTGGATCCCTACTTGATCCGAATAGC-30 ) with 50 -NdeI and BamHI restriction sites were used for amplification under the conditions previously described (King et al., 1996). The position of the primers in the cDNA fragment of the killer whale IL-6 were 62–79 and 602–619, respectively (GCG accession number L46803). Amplified products were purified (Qiaex1 II, Gel extraction kit, Qiagen Inc., Valencia, CA) per manufacturers instructions. Products were ligated into the expression vector pET-14b (Novagen Inc., Madison, WI) downstream from the T7 promoter and the ribosomal binding site using T4 ligase. The ligated product pET-14b/ kwIL-6 (Fig. 1) was transformed into DH5a Escherichia coli (E. coli, Gibco BRL1, Life Technologies Inc., Rockville, MD) for rapid growth and selection via ampicillin resistance. Plasmid products were purified from overnight cultures using a standard miniprep kit (Qiagen Inc., Valencia, CA). The nucleotide sequence of both strands was determined by DIDEOXY nucleotide methodology using an automated sequencer (Model 373, Applied Biosystems, Foster City, CA). The designed expression vector (pET-14b/kwIL-6) was electroporated (Bio-Rad Gene Pulser II, Hercules, CA, 0.1 cm cuvette, 1.8 V, 25 mF capacitance, 200 O resistance) into E. coli strain BL21(DE3)pLysS (Novagen Inc., Madison, WI) and selected by ampicillin and chloramphenicol resistance. A single colony was used to inoculate 6 ml of Luria– Bertani Medium (LB media) containing ampicillin (50 mg/ml) and chlorampenicol (34 mg/ml) at 37 8C overnight. One milliliter of this culture was further expanded to 1 l and expression was induced with 200 mg/ml IPTG (0.4 mM) when the optical density at 600 nm (DU-20 Spectrophotometer, Beckman Coulter Inc., Fullerton, CA) reached 0.5; a noninduction

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pore Corp., Bedford, MA) at 20 psi and the protein concentration determined by BCATM protein assay (Pierce Inc., Rockford, IL); purity was analyzed by SDS–PAGE (12%) under reducing conditions. 2.3. Animals and blood collection

Fig. 1. Structure of the recombinant expression plasmid pET-14b/ killer whale IL-6. A fragment of cDNA coding for killer whale IL-6, including restriction sites for NdeI and BamHI, was inserted into the pET-14b expression system.

control sample was taken before expression was induced. Both, the induced and the control samples were kept at 37 8C, shaking for 3 h. Cell pellets and supernatants were analyzed for the presence of IL-6 by standard reducing SDS–PAGE (12%) analysis (Laemmli, 1970).

Serum samples were obtained from killer whales (Orcinus orca) and a bottlenose dolphin (Tursiops gilli) at SeaWorld Adventure Parks Inc. (San Diego, CA; San Antonio, TX & Orlando, FL) and a beluga whale (Delphinapterus leucas) at the Baltimore Aquarium (MD) in the USA. Minimal serum was available from the beluga whale and precluded extensive analysis. Clinically ill animals were identified by abnormal behavior and classical hematologic changes (complete blood count and clinical chemistries) typically associated with inflammation. Blood was collected from the dorsal sinus in the fluke and serum was stored at 80 8C until tested for IL-6. Whole blood was obtained in cell separation tubes(CPTTM, Vacutainer1 Becton Dickinson,Franklin Lakes, NJ) and shipped on ice overnight for isolation of peripheral blood mononuclear cell (PBMCs). Briefly, samples were centrifuged at 20 8C for 18 min at 1400  g, washed with PBS (pH 7.4) and collected by centrifugation (8 min at 150  g). 2.4. In vitro stimulation of killer whale PBMCs

2.2. Purification of recombinant killer whale IL-6 Bacterial cultures were harvested, centrifuged at 600  g for 5 min, pellets resuspended in 50 mM Tris (pH 8–8.5), 100 mM NaCl, 1 mM EDTA and 1 mM PMSF, homogenized via maceration in a Tenbroeck grinder and sonicated (3  30 s, 50% power). Solubilized bacterial suspensions were centrifuged at 10,000  g (SS34 rotor) for 30 min and washed with 10 mM Tris (pH 8–8.5), 0.5% Triton-X, 1 mM EDTA, 1 mM PMSF, maintained at room temperature for 30 min, sonicated and centrifuged. This process was repeated until most bacterial debris was removed. Inclusion bodies were solubilized in 6 M guanidine, 25 mM Tris (pH 8) and left for 30 min at 37 8C. The solubilized inclusion bodies were diluted in cold phosphate buffered saline (PBS, pH 7.4) and stirred overnight at 4 8C to facilitate IL-6 refolding. The rkwIL-6 was concentrated with a YM 10 filter (Milli-

PBMCs isolated from killer whale blood were incubated with complete media (DMEM, 2 mM glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin) with 10% FBS, stimulated with 10 mg/ml lipopolysaccharide (LPS) from E. coli 0127:38 (Sigma Chemical Co., St. Louis, MO). After 48 h culture, supernatant was collected under sterile conditions and stored at 80 8C for future use. 2.5. Production of rkwIL-6-specific antiserum Polyclonal antiserum to rkwIL-6 was produced in a rabbit. A total of three immunizations were performed. The first immunization of 125 mg was administered subcutaneously (SC) in Freund’s complete adjuvant. Two boosts of 125 mg rkwIL-6 in incomplete Freund’s adjuvant were administered SC at intervals of 3 and 6 weeks after primary immunization.

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Immunoglobulin from rabbit serum was purified with caprylic acid followed by precipitation with saturated ammonium sulfate (SAS). Briefly, immune serum was diluted with acetate buffer (60 mM, pH 4.0) and adjusted to a pH of 4.5 before caprylic acid was added dropwise to the solution. After 30 min at RT, the solution was centrifuged at 1400  g for 30 min. Supernatant was subjected to prefiltration (Millipore Corp., Bedford, MA) and diluted in PBS (pH 7.4). SAS (4 8C) was added slowly to a final saturation of 45% and the precipitate collected by centrifugation at 1400  g for 30 min at 10 8C. Precipitate was resuspended in PBS (pH 7.4) and passed through a PD-10 column (SephadexTM G-25M, Amersham Pharmacia, Sweden) to desalt. The protein concentration of purified immunoglobulin was determined by BCATM protein assay and the purity analyzed by SDS–PAGE (12%). 2.6. Biological activity of killer whale IL-6 Biological activity of purified rkwIL-6, LPS-stimulated PBMC supernatants and killer whale serum was determined with the IL-6-dependent B9 mouse hybridoma cell line as previously described (King et al., 1993; Mosmann, 1983). In summary, 2  104 cells/ml (RPMI 1640, 5% FBS, 2 mM glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 5  105 M 2-mercaptoethanol) were incubated (5% humidified CO2, 37 8C) in 96-well plates with serial three-fold dilutions of each of the following: recombinant human IL-6 (rhIL-6, generously provided by DNAX, Palo Alto, CA), supernatant from LPS-stimulated PBMCs, purified rkwIL-6 and killer whale serum. Killer whale serum was heat inactivated at 57 8C for 30 min and filtered (0.22 mm, Millipore Corp., Bedford, MA) prior to use. After 72 h, MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl-tetrazolium bromide, Sigma Chemical Co., St. Louis, MO) was added to proliferating cells. The reaction was stopped after 3 h by adding lysis solution (20% SDS, 0.02N HCl) overnight and absorbance at 600 nm measured (Emax1, Molecular Devices Corp., Sunnyvale, CA). All samples were run in triplicate when sufficient sample was available. 2.7. Functional characterization of killer whale IL-6 Specificity of killer whale IL-6 in the bioassay was demonstrated as previously described (Swiderski

et al., 2000). IL-6 activity was blocked with rat antimouse IL-6 receptor antibody (Rt a Ms IL-6R, BD PharMingen, Franklin Lakes, NJ, clone D7715A7) to establish that rkwIL-6 and native killer whale IL-6 were responsible for proliferation of the IL-6-dependent B9 cells. B9 cells (2  104 cells/ml) were preincubated (5% humidified CO2, 37 8C for 1 h) with Rt a Ms IL-6R antibody (4 mg/ml), an irrelevant control antibody (Rat a Ms IL-10, PharMingen, Franklin Lakes, NJ; 4 mg/ml) and a media control. Serial three-fold dilutions of IL-6 (recombinant, PBMC supernatants or serum) were applied to the cells. After 72 h the degree of proliferation was measured as described previously. All samples were assayed in duplicate with the exception of the killer whale serum; this sample was assayed in a single well due to limited sample availability. Polyclonal antibody specific for the expressed IL-6 protein was utilized to further define the specificity of the cytokine assay. Recombinant and native kwIL-6 were incubated as three-fold dilutions with either hyper immune rabbit serum (anti-rkwIL-6) or control rabbit serum (preinoculation) prior to addition of B9 cells; cell proliferation was assessed as described above. Serum from the beluga whale, which became available at the conclusion of this study, was similarly tested but with a purified preparation of the hyper immune rabbit serum. 2.8. ELISA for killer whale IL-6 (kwIL-6) A competitive ELISA was used to detect IL-6 in whale serum. A volume of 100 ml recombinant killer whale IL-6 at 1 mg/ml was adsorbed to a solid phase (Pro-Bind, Falcon, Beckton Dickinson, Lincoln Park, NJ) in carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, 3 mM NaN3, pH 9.6) overnight at 4 8C. Plates were washed three times with 200 ml PBSTween (PBS-T: 500 mM NaCl, 7.5 mM Na2HPO4, 2.5 mM NaH2PO4, 0.1% Tween 20, pH 7.2) and blocked with bovine serum albumin (1% BSA in PBS-T) for 1 h. Meanwhile, 100 ml rkwIL-6 standard (1 mg/ml–1 ng/ml, PBS-T) and serial dilutions (PBS-T) of 100 ml killer whale and bottlenose dolphin serum samples were each incubated in duplicate with 100 ml diluted (1:18,000) rabbit anti-rkwIL-6 for 1 h at 57 8C. After the plate was washed three times with PBS-T, the incubated samples were applied to the plate for 1 h. Plates were washed and rabbit anti-rkwIL-6 bound to

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antigen on the solid phase was detected with 100 ml biotinylated goat anti-rabbit antibody specific for immunoglobulin heavy and light chain (Vector Laboratories Inc., Burlingame, CA), washed, and 100 ml HRP–streptavidin conjugate (Zymed, South San Francisco, CA) diluted to 0.8 mg/ml in PBS-T was added. After a final wash step, 100 ml of O-phenylenediamine dihydrochloride (OPD, Sigma Chemical Co., St. Louis, MO) substrate was added to each well and left to incubate in the dark for 10 min. The enzymatic reaction was stopped using 150 ml of 1 M H2SO4. Colorimetric changes of the substrate were determined with spectrophotometric detection at 490 nm.

3. Results 3.1. Expression and purification of recombinant killer whale IL-6 Killer whale IL-6 was expressed at high levels in a prokaryotic system. The cDNA, corresponding to nucleotides 62–619 of the killer whale IL-6 gene

Fig. 2. Expression of rkwIL-6 in E. coli. Proteins were separated using 12% SDS–PAGE and visualized by coomassie brilliant blue stain. Lane 1: molecular weight standards; lane 2: cell lysate from un-induced bacterial culture; lane 3: cell lysate from IPTG-induced bacteria; lane 4: rkwIL-6 purified from bacterial inclusion bodies (indicated by arrow).

Fig. 3. Dose–response proliferation of B9 hybridoma cells to rkwIL-6 (*), supernatant from stimulated killer whale PBMCs (!), acute phase killer whale serum (5), and rhIL-6 (*). All samples were run in triplicate wells and represent the mean value. Results from a single representative assay are illustrated.

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(King et al., 1996), was cloned into the pET-14b expression vector (Fig. 1); the cDNA was lacking the signal peptide cleavage sites and an additional nine nucleotides encoding three amino acids. DNA

sequencing confirmed the correct orientation and reading frame of the plasmid construct prior to transformation into BL21(DE3)pLysS E. coli for protein expression. Cell pellets and supernatant, from both

Fig. 4. Utilization of rat anti-mouse IL-6 receptor antibody to block IL-6 activity on B9 hybridoma cells. B9 hybridoma cells were pretreated with rat anti mouse IL-6 receptor antibody prior to addition of rkwIL-6 (A), supernatant from stimulated killer whale PBMCs (B), and acute phase killer whale serum (C). Cultures treated with rat anti-mouse IL-6 receptor antibody (5) were controlled by comparison to cultures pretreated with irrelevant antibody (*). All samples were run in duplicate wells and represent the mean value. Acute phase killer whale serum was run as a single well. Results from a single representative assay are shown.

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induced and noninduced transformed cultures, were analyzed by SDS–PAGE for visible protein bands. A protein of slightly greater than 21.5 kDa molecular weight, corresponding in molecular weight to the

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predicted product of the IL-6 construct (Fig. 2), was identified in the cell pellet of the culture, but not in the supernatant (data not shown). This was not unexpected as many proteins expressed in transformed

Fig. 5. Neutralization of IL-6 activity with rabbit anti-rkwIL-6. IL-6 preparations included rkwIL-6 (A), supernatant from LPS-stimulated killer whale PBMCs (B), and acute phase killer whale serum (C). IL-6 preparations were incubated with either preinoculation serum (*) or rabbit immune serum (!). All samples were run in duplicate wells and represent the mean value. Results from a single representative assay are shown.

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microorganisms accumulate in inactive inclusion bodies (Rudolph and Lilie, 1996). Compared to other proteins, the recombinant protein appeared overexpressed in the induced culture. The recombinant killer whale IL-6 was purified (Fig. 2) at a final concentration of 240 mg/ml. 3.2. Biological activity of killer whale IL-6 The purified recombinant killer whale IL-6, cell culture supernatants from LPS-stimulated killer whale PBMCs and serum samples from a clinically ill killer whale all stimulated proliferation in the IL-6-dependent B9 cell line. All samples produced an S-shaped dose–response curve characteristic of IL-6 (Fig. 3) (Mire-Sluis and Thorpe, 1998). Biological activity from all three sources was blocked to varying degrees by rat anti-mouse IL-6 receptor antibody. The signal (optical density) in the IL-6 bioassay was reduced when the B9 indicator cells were pretreated with anti-IL-6R antibody as compared to those pretreated with either an irrelevant antibody or media (Fig. 4A–C).

3.3. Neutralizing activity of rabbit polyclonal anti-rkwIL-6 antibody Rabbit a-rkwIL-6 immune serum was tested in the B9 bioassay for its capability to neutralize the biological activity of both recombinant and native kwIL-6 (IL-6 present in serum samples and LPS-stimulated PBMC supernatant). As illustrated in Fig. 5, immune serum neutralized the biological activity of rkwIL-6 (Fig. 5A), native kwIL-6 present in LPS-stimulated PBMC supernatants (Fig. 5B) and native IL-6 in killer whale serum (Fig. 5C); preimmunization serum had no neutralizing effect. A purified preparation of this polyclonal anti-kwIL-6 antibody also neutralized IL-6 activity in beluga whale serum (Fig. 6). Again, preimmunization serum did not neutralize the biological activity. 3.4. Competitive IL-6 ELISA A competitive ELISA for cetacean IL-6 was developed using purified polyclonal rabbit antibody raised against recombinant killer whale IL-6. A single limiting

Fig. 6. Neutralization of beluga whale IL-6 activity with rabbit anti-rkwIL-6. Acute phase serum from a beluga whale (&) was compared to beluga whale serum preincubated with preinoculation rabbit serum (*) or rabbit immune serum (!). The beluga whale serum was run in duplicate wells and represents the mean value.

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Fig. 7. Competitive IL-6 ELISA. Samples include rkwIL-6 (*), acute phase killer whale serum (&), acute phase bottlense dolphin serum (~), and serum from one of three apparently healthy killer whale (^). All samples were preincubated with rabbit anti-rkIL-6 before being applied to rkwIL-6 adsorbed to a solid phase. Samples were run in duplicate and presented as the mean value.

dilution of the antibody was pretreated with decreasing concentrations of rkwIL-6 to establish a standard curve based upon signal reduction in the ELISA (i.e. unbound anti-IL-6 antibody was able to bind the rkwIL-6 on the solid phase). The detection limit of the assay was determined to be in the range of 1 ng/ml. The assay was successfully applied to the detection of native IL-6 in the serum of two clinically ill cetaceans (O. orca, T. gilli) (Fig. 7); serum IL-6 concentrations extrapolated from the standard curve in the ELISA were roughly in the range of 10–100 ng/ml. IL-6 was also detected in these serum samples using the B9 bioassay. Serum samples from three apparently healthy animals were negative in both the ELISA and bioassay. Insufficient serum was available from the beluga whale to analyze with the ELISA.

4. Discussion Timely diagnosis of inflammatory responses in cetaceans is often problematic, and as such, initiation of treatment can be delayed. Animals can die rapidly while exhibiting minimal outward signs of disease and

currently available diagnostic tools are often insufficiently sensitive to detect the disturbance of homeostasis at an early stage. Thus, the development and application of assays that capitalize on identification of inflammatory cytokines in marine mammals is warranted. Initial studies directed at defining the potential utility of identifying IL-6 as an indicator of inflammation in a clinical setting were based upon IL-6-dependent cells lines (Shenkin et al., 1989). Cytokine-dependent cell lines are typically not species specific and thus have broad applicability. However, they offer limited clinical utility as they are slow and labor-intensive (House, 1999). ELISAs have been utilized as an additional tool in measuring systemic levels of IL-6 in humans (Muller Kobold et al., 2000; Saladino et al., 1992). Thus, use of recombinant cytokines for the development of antibody-based competitive binding assays represent a viable approach for developing similar diagnostics in veterinary medicine. The current study employed a cloned fragment of killer whale IL-6 corresponding to nucleotides 71–753 of the human transcript; this clone lacked nine residues of the hydrophobic secretory signal (King et al., 1996). PCR primers were used to amplify and clone a

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558 bp fragment that included the binding site for the IL-6 receptor, gp80, and the signal transduction unit, gp130 (Brakenhoff et al., 1996; Ehlers et al., 1996; Fontaine et al., 1993; Kruttgen et al., 1990). Despite the fact that the lack of glycosylation in an E. coli expression system can be seen as a disadvantage, a prokaryotic expression system was used in this study as it has been demonstrated in other species that glycosylation is not necessary to produce a biologically active recombinant interleukin-6 (Rees et al., 1999; Braciak et al., 1996; Van Snick, 1990). The recombinant IL-6, purified from the bacterial inclusion bodies, had an approximate molecular weight just greater than 21.5 kDa as determined by SDS–PAGE. This correlates well with the theoretical molecular weight of 21 kDa obtained using The Expert Protein Analysis System (ExPASy, http://www.expasy.ch). This size of the recombinant kwIL-6 is consistent with molecular weights reported for purified murine IL-6 ranging from 21.5 to 30 kDa (Bauer et al., 1988; May et al., 1988). The rkwIL-6 was further characterized by establishing receptor-specific biological activity. The recombinant protein supported the growth of the IL-6dependent B9 hybridoma in a similar fashion (produced the classical S-shaped dose–response curve) to that provided by recombinant human IL-6, supernatant derived from LPS-stimulated killer whale PBMCs and serum derived from killer whales experiencing an acute phase reaction. The specificity of these IL-6 activities was confirmed by the ability of monoclonal antibody specific for the murine IL-6 receptor to block activity. This approach was the same as that described by Swiderski et al. (2000) to establish the specificity of recombinant equine IL-6. Upon expression and characterization of the recombinant killer whale IL-6, efforts were directed towards development of an IL-6 ELISA. The necessity of developing a cetacean-specific assay was suggested by the inability of human IL-6-specific antibodies to cross-react with killer whale IL-6 (Funke et al., unpublished data). Polyclonal antibodies specific for killer whale IL-6 were produced in rabbits using the purified recombinant protein as antigen. The immune serum was characterized by its ability to bind the recombinant protein in an ELISA and neutralize the biological activity of both native and recombinant killer whale IL-6. The neutralizing activity of the polyclonal antibody was not as dramatic using killer whale serum,

apparently the result of background created by the low rabbit serum dilutions employed. However, the ability of the antibody to neutralize the native cytokine was dramatic using LPS-stimulated killer whale PBMC supernatants. Nonspecific inhibitors were apparently present in the killer whale serum as the lowest serum dilutions incubated with irrelevant antibody did not give peak values in the B9 assay (Figs. 4C and 5C). The dramatic neutralization of beluga whale IL-6 activity (Fig. 6) was probably facilitated by the use of a purified preparation of the polyclonal rabbit antirkwIL-6. The slight reduction in IL-6 activity, using the control rabbit serum, was also apparently due to factors inhibitory to the B9 assay. The fact that the curve for the control rabbit antibody fell below that of the anti-rkwIL-6 at high dilutions would support the presence of such inhibitors; the anti-rkwIL-6 had been purified and inhibitors apparently removed. A competitive ELISA for rapid identification of IL6 in cetacean serum was successfully developed. Serum IL-6 was identified by its ability to reduce the binding of a limiting concentration of the polyclonal antibody to recombinant IL-6 in an ELISA plate. ELISA sensitivity for the rkwIL-6 was determined to be in the range of 1 ng/ml, a value not inconsistent with those reported for commercial kits, the latter ranging in sensitivity from picogram to nanogram levels. The IL-6 ELISA was able to identify the presence of the cytokine in serum obtained from two clinically ill animals, a killer whale and a dolphin (Fig. 7). Three samples were analyzed from killer whales that were clinically normal and no IL-6 was detected. Unfortunately, serum samples from additional cetaceans with systemic inflammation were not available for this study and thus a comparison to the bioassay would be premature. Thus, additional animals will need to be analyzed to establish sensitivity and clinical utility of the competitive ELISA. However, the reagents and preliminary data described in this report demonstrate the potential utility of employing cytokine analysis for identifying inflammation in cetacean species.

Acknowledgements The authors would like to thank the veterinary and technical staff of SeaWorld (California, Florida, Ohio

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and Texas) and the National Aquarium Baltimore, MD for providing the samples used in this study. The rhIL-6 was a generous gift from Dr. Robert Coffman, DNAX (Palo Alto, CA).

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