- Email: [email protected]
Establishment of swine interleukin-6 sandwich ELISA
Comparative Immunology, Microbiology & Infectious Diseases 28 (2005) 121–130 www.elsevier.com/locate/cimid
Establishment of swine interleukin-6 sandwich ELISA A. Nuntapraserta,c, Y. Morib, K. Tsukiyama-Koharaa, C. Kaia,* a
Laboratory of Animal Research Center, Institution of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai Minato-ku, Tokyo 108-8639, Japan b National Institute of Animal Health, Tsukuba, Ibaraki 305-0856, Japan c Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand
Abstract We established a sandwich enzyme-linked immunosorbent assay (ELISA) for swine interleukin-6 (SwIL-6), which was applied for detection of SwIL-6 in vitro and in vivo. Anti-SwIL-6 rabbit- and goat-polyclonal antibodies, and monoclonal antibody (mAb) were prepared, conforming that all of the antibodies were reactive with recombinant SwIL-6 by Western blotting and indirect ELISA. A sandwich ELISA was developed using the mAb as a capture antibody and biotinylated goatpolyclonal antibody as a detection antibody. The detection limit of the sandwich ELISA for rSwIL-6 was 49 pg/ml and did not show cross-reactivity with swine IL-1b, IL-4, IL-8, IL-18, IL-12, and IFN-g. Using the ELISA, SwIL-6 was detected in culture medium of the monocytes stimulated with PHA-P and PMA, and the plasma or the bronchoalveolar lavage fluid (BALF) of pigs experimentally infected with Actinobacillus pleuropneumoniae or Mycoplasma hyopneumoniae. This ELISA for SwIL-6 may be useful for understanding the role of this cytokine in various swine diseases. q 2004 Elsevier Ltd. All rights reserved. Keywords: Swine interleukin-6; Sandwich ELISA
Re´sume´ Un sandwich-ELISA a e´te´ e´tabli pour la de´tection de l’interleukine-6 porcine (SwIL-6) in vitro et in vivo. Les anticorps anti-SwIL polyclonaux et monoclonaux ont e´te´ pre´pare´s chez les lapins et che`vres, et leur re´activite´ ont e´te´ confirme´es par Western block et ELISA indirect. Le sandwichELISA a e´te´ exploite´ utilisant un anticorps monoclonal a` capture et un autre anticorps polyclonal biotinyle´ a` de´tection. La limite de de´tection de l’rSwIL e´tait 49 pg /ml sans aucune re´activite´ croise´e * Corresponding author. Tel.: C81 354495497; fax: C81 354495379. E-mail address: [email protected] (C. Kai). 0147-9571/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.cimid.2004.08.003
122
A. Nuntaprasert et al. / Comp. Immun. Microbiol. Infect. Dis. 28 (2005) 121–130
avec IL-1b, IL-4, IL-8, IL-18, IL-12 et IFN-gamma. Avec le sandwich-ELISA l’SwIL-6 e´tait de´tectable dans la culture de monocytes stimule´s par PHA-P ou PMA aussi bien que dans le plasma sanguin et le lavage bronchoalve´olaire (BALF) des porcs expe´rimentalement infecte´s d’Actinobacillus pleuropnemoniae ou Mycoplasma hyopneumoniae. Le sandwich-ELISA est utile pour comprendre le roˆle de l’SwIL chez diverses maladies du porc. q 2004 Elsevier Ltd. All rights reserved. Keywords: Interleukine-6 porcine; Sandwich-ELISA
1. Introduction Cytokine, interleukin-6 is one of the key regulators in mediating the balance between inflammation and the immune response during the bacterial infection or injury process. The cells that produce high level of IL-6 is distributed all over the body as macrophages, monocytes, fibroblasts, endothelial cells, keratinocytes, astrocytes and T- and B-lymphocytes [1]. This cytokine was reported to cause a wide range of biological effects such as induction of all positive mediators of the hepatic acute phase proteins, which may serve as markers of stress and diseases [2–4], and of antibody production from B cells [5,6]. We expressed rSwIL-6 by three recombinant expression systems and examined their biological activities in mouse cells. We also established anti-SwIL-6 polyclonal antibodies from rabbit and goat. These polyclonal antibodies and a monoclonal antibody (mAb) were characterized and utilized for establishment of a sandwich ELISA system which was evaluated by measuring SwIL-6 concentration in various samples such as culture medium, swine plasma or bronchoalveolar lavage fluid (BALF).
2. Materials and methods 2.1. Production of polyclonal antibodies Anti-SwIL-6 polyclonal antibodies were produced by immunizing rabbit or goat followed by purification using HiTrap rProtein-A and HiTrap Protein-G affinity columns (Amersham Pharmacia Biotech). The goat anti-SwIL-6 antibody was biotinylated with EZ-link Sulfo-NHS-LC-Biotin (Pierce) according to the manufacturer’s instructions to use as a detection antibody in developing a sandwich ELISA. 2.2. Characterization of antibodies to swine IL-6 The antibody binding activity of the polyclonal antibodies against the rSwIL-6 was assayed using an indirect ELISA. Plate was coated with serial concentration of rSwIL-6 (0.2–50 mg/ml) purified from bacteria and detected with the rabbit anti-SwIL-6 antibody (0.8, 1.5, 3, and 6 mg/ml). The anti-SwIL-6 mAb (isotype IgG2) was kindly provided by Dr Hiroyuki Iwata, Yamaguchi University, Japan. The specificity of an anti-SwIL-6 mAb and both polyclonal antibodies (0.001–5 mg/ml) was also determined by this ELISA after
A. Nuntaprasert et al. / Comp. Immun. Microbiol. Infect. Dis. 28 (2005) 121–130
123
coating the rSwIL-6 purified from bacteria at 2.5 mg/ml. Goat anti-mouse-Ig-HRP or goat anti-rabbit-IgG-HRP or rabbit anti-goat-IgG-HRP was used to detect bound mAb or polyclonal antibodies. 2.3. Swine IL-6 sandwich ELISA To develop a sandwich ELISA, the mAb was used as a capture antibody and the biotinylated goat anti-SwIL-6 polyclonal antibody was used as a detection antibody. First, 96-well ELISA microplates (Nunc) were coated with 1 mg/ml of purified anti-SwIL-6 mAb in 100 ml of 0.05 M carbonate buffer, pH 9.6, and incubated overnight at 4 8C. The wells were washed four times with washing buffer (0.15 M NaCl containing 0.05% Tween-20) and then incubated with 100 ml of samples or standard rSwIL-6 purified from bacteria diluted in standard diluent assay buffer (0.01 M Tris–HCl, pH 7.2, 0.15 M NaCl, 0.05% Tween-20, and 0.5% skim milk) for 2 h at room temperature. The plates were rinsed four times and incubated with 100 ml of biotinylated goat anti-SwIL-6 polyclonal antibodies (2.5 mg/ml) for 1 h at 37 8C. After washing, the plates were treated with 0.25 mg/ml of HRP-conjugated streptavidin (ELISA grade) (Biosource International Inc., Camarillo, CA) for 1 h at ambient temperature. After washing four times, 100 ml of component TMB microwell peroxidase substrate (Kirkegaard & Perry Laboratories, Guildford, UK) was added. The reaction was stopped 10 min later with 100 ml of component TMB stop solution (Kirkegaard & Perry Laboratories). The absorbance at 450 nm was determined in MTP-120 microplate reader (Corona Electric, Japan) and a standard curve was obtained. Cross-reactivity was tested against commercially available recombinant swine IL-1b, swine IL-8, and swine IFN-g (BioSource International Inc.), swine IL-12 (Endogen Inc., Woburn, MA), swine IL-4, and swine IL-18 [7]. These cytokines were applied to a sandwich ELISA system described above at a concentration of 1 ng/ml. The ability of this sandwich ELISA assay to detect the rSwIL-6 was tested by using three different kinds of the SwIL-6 produced from bacterial, insect, and COS-7 cells. We also investigated the ability of this sandwich ELISA to detect natural SwIL-6 using the supernatants from swine monocytic culture with PHA-P and PMA mitogens. 2.4. Native swine leukocyte IL-6 IL-6 is mainly secreted by monocytes and macrophages. PBMC were reported to be the main significant source of SwIL-6 compared with fibroblasts or alveolar macrophages [8]. We identified native SwIL-6 produced from monocytes by a sandwich ELISA. Native monocyte SwIL-6 was generated from adherent swine monocyte cells stimulated with mitogens as described [8,9]. Briefly, PBMC were isolated from a 10-week-old normal pig and incubated with the anti-CD14 (M-M9) mAb (1/200 dilution, VMRD Inc., Pullman, WA) and then labeled with the rat anti-mouse IgG1 Microbeads (Miltenyi Biotec, CA). Swine monocytes were then separated in MACS cell separation column (Miltenyi Biotec). Using native monocytic SwIL-6 in a sandwich ELISA, swine monocytes (1!106 cells/ml) after separation from MACS cell column were cultured for 24 h in RPMI-1640 medium supplemented with 10% FCS. Adherent swine monocytes were then stimulated for further 24 h with the range of 0–10 mg/ml of phytohemagglutinin-P
124
A. Nuntaprasert et al. / Comp. Immun. Microbiol. Infect. Dis. 28 (2005) 121–130
Fig. 1. Comparison of Western blotting analysis of rSwIL-6 expressed in bacteria, baculovirus, and mammalian expression systems with (A) the anti-SwIL-6 mAb and (B) the goat-polyclonal antibody against SwIL-6 molecule. (C) Cross-reactivity of the rabbit anti-SwIL-6 antibody against rHuIL-6 by Western blotting analysis. The 20 ml of recombinant proteins purified from bacteria and Tn5 cells, and the supernatant from COS-7 cell culture were analyzed in 12.5% SDS-PAGE followed by Western blotting and visualized by chemiluminescence’s development. M, Size markers (kDa); B, affinity purified rSwIL-6 from bacteria; I, affinity purified rSwIL-6 from insect cells; H, rHuIL-6 produced from bacteria; C, secreted SwIL-6 from COS-7 cells. The arrows indicate the rSwIL-6 proteins.
A. Nuntaprasert et al. / Comp. Immun. Microbiol. Infect. Dis. 28 (2005) 121–130
125
(PHA-P; Sigma) and 0–100 ng/ml of phorbol myristic acetate (PMA; Sigma). Supernatants were then collected, concentrated and used to detect the SwIL-6 concentration using a sandwich ELISA system. 2.5. IL-6 production in experimental bacteria infections in pigs The role of SwIL-6 in swine diseases was investigated on experimental bacterial infections in pigs. Plasmas and BALF samples were obtained from five normal pigs and five pigs intranasally infected with Actinobacillus pleuropneumoniae (A. pleuropneumoniae serotype 1, strain NZ-62, a field strain isolated in Japan) within 2 days after pleuropneumonia developed as described by Muneta et al. [10]. BALF samples were obtained from five normal pigs and five pigs intranasally inoculated with virulent Mycoplasma hyopneumoniae (M. hyopneumoniae strain E-1, a field strain isolated in Japan). The concentrations of SwIL-6 at protein level in these samples were subjected to a sandwich ELISA as described above. 2.6. Statistical analysis Data are expressed as meanGSD and examined statistically by Student’s t-test.
3. Results 3.1. Characterization of the reactivity of antibodies to rSwIL-6 The antigenicity of rSwIL-6 from three kinds of expression systems was confirmed by Western blotting analysis using these antibodies as described above. To detect the immunoreactivity of rSwIL-6 more accurately, we developed an indirect ELISA for antibody binding reactivity as described in Section 2 by coating plates with purified cytokine from bacteria. The specificity of the purified IgG from mAb or polyclonal antibodies was further analyzed using rSwIL-6 produced from bacteria by an indirect ELISA system. The mAb and goat-polyclonal antibodies recognized rSwIL-6 with highly specific binding activities (Fig. 1). Therefore, these antibodies were considered to be useful for establishment of a sandwich ELISA system. 3.2. Development of sandwich ELISA for Swine IL-6 A sandwich ELISA for SwIL-6 was developed using a combination of the mAb and the goat-polyclonal antibody. The mAb was used as the first antibody to coat the wells of microplates and the goat anti-SwIL-6 was used as the second antibody. The concentration of coating mAb used was 1 mg/ml. The concentration of the biotinylated purified goat anti-SwIL-6 polyclonal antibody was titrated in the ELISA between 1/100 and 1/3200 dilution, and was found to be optimum at 1/800 dilution (2.5 mg/ml). A sandwich ELISA was successfully developed using the mAb as a capture antibody and the biotinylated goat-polyclonal antibody against rSwIL-6 as
126
A. Nuntaprasert et al. / Comp. Immun. Microbiol. Infect. Dis. 28 (2005) 121–130
Fig. 2. The specific antibody response against rSwIL-6. Detection of purified rSwIL-6 from bacteria (50– 0.2 mg/ml) with the purified rabbit anti-SwIL-6 polyclonal antibody was tested using an indirect ELISA (A). The wells were coated with serially diluted rSwIL-6, and were treated with the rabbit anti-SwIL-6 antibody at the concentration of 0.8 mg/ml (6), 1.5 mg/ml (B), 3.0 mg/ml (C), and 6.0 mg/ml (,), respectively. The specificity of mAb (,), and the purified rabbit (B) and goat (C) IgG polyclonal antibodies were also titrated in an indirect ELISA against rSwIL-6 coated at the concentration of 2.5 mg/ml (B).
a detecting antibody. The minimum detectable concentration by this ELISA was 49 pg/ml and the maximum one was 1500 pg/ml (Fig. 2). No cross-reactivity with other swine cytokines, i.e. swine IL-6, IL-1b, IL-4, IL-8, IL-18, IL-12p70, and IFN-g, was recognized in this assay, indicating that this ELISA is specific for SwIL-6. For further validation of the SwIL-6 ELISA, adherent monocytes were stimulated for 24 h with
A. Nuntaprasert et al. / Comp. Immun. Microbiol. Infect. Dis. 28 (2005) 121–130
127
Fig. 3. Adherent monocytes were stimulated for 24 h at the indicated concentration of PHA-P and PMA. Culture supernatants were then colleted and assayed for SwIL-6. Value are the meansGSD from three experiments.
PHA-P plus PMA, and the supernatants were assayed for native SwIL-6 concentration using the sandwich ELISA system. SwIL-6 secretion was inducible in a dose-dependent manner (Fig. 3). The combination of PHA-P (10 mg/ml) plus PMA (100 ng/ml) resulted in maximal SwIL-6 secretion (3-fold, p!0.05). 3.3. IL-6 production in pigs experimentally infected with A. pleuropneumoniae or M. hyopneumoniae To examine the applicability of this sandwich ELISA in clinical cases, we measured the concentration of IL-6 in the plasma and/or BALF samples of control pigs and experimentally A. pleuropneumoniae or M. hyopneumoniae infected pigs. As shown in Fig. 4A, the concentration of IL-6 in the plasma from pre-infected pigs was less than 50 pg/ml, the detection limit, whereas samples from pigs infected with A. pleuropneumoniae showed significant increase (1410G326 pg/ml, p!0.05). The IL-6 concentration in the BALF from A. pleuropneumoniae infected pigs was also higher than that from control pigs (240G101 pg/ml, p!0.05). Time-sequential production of SwIL-6 in BALF after infection with M. hyopeumoniae is shown in Fig. 4. Mean levels of SwIL-6 in BALF of the infected pigs were significantly increased at 2 weeks post-infection compared to those of uninfected or pre-infected ones.
4. Discussion In this study, we produced two specific polyclonal antibodies and one mAb against rSwIL-6 and developed indirect ELISA systems. The ELISA system was shown to be
128
A. Nuntaprasert et al. / Comp. Immun. Microbiol. Infect. Dis. 28 (2005) 121–130
Fig. 4. The IL-6 concentrations in the swine plasma and BALF experimentally infected with A. pleuropneumoniae at 2 days after infection were assayed using the sandwich ELISA (A). Control plasma samples were collected before the infection and control BALF was obtained from another healthy pigs with no pneumonia. Concentrations of SwIL-6 in BALF kinetically collected from pigs after infection with M. hyopneumoniae were also determined using the sandwich ELISA (B). Data represent the meanGSD of five pigs. *Significant difference compared with control group at p!0.05.
A. Nuntaprasert et al. / Comp. Immun. Microbiol. Infect. Dis. 28 (2005) 121–130
129
highly sensitive in detecting SwIL-6 protein concentrations at a minimum concentration of 49 pg/ml and to have no cross-reactivity with other swine recombinant cytokines. The ELISA system could also be used to measure the concentration of SwIL-6 in the plasma or BALF from pigs experimentally infected with A. pleuropneumoniae or M. hyopneumoniae. The SwIL-6 protein level in those samples was significantly increased within 2 days in pigs infected with A. pleuropneumoniae serotype 1. Increase in level of SwIL-6 protein was also found in BALF samples after infection with M. hyopneumoniae. Previous studies reported that bioactive pro-inflammatory cytokines such as IL-6 responses and the development of pneumonia were correlated to the severity of diseases from the A. pleuropneumoniae (serotype 1 [11] and serotype 2 [12]) or M. hyopneumoniae [13] infections in swine. Thus this ELISA system may be useful for the study of immunological pathogenesis of bacterial infections in pigs.
Acknowledgements We wish to thank Dr Hiroyuki Iwata, Faculty of Agriculture, Yamaguchi University for the gift of the anti-SwIL-6 monoclonal antibody and Dr Munenori Okuda from Zen-noh Institute of Animal health, Japan for the BALF samples of pigs experimentally infected with Mycoplasma hyopneumoniae. Research was funded in part by grant from Ministry of Education, Science, Sports and Culture, and Recombinant Cytokine Project grant number RCP-3210 from the Ministry of Agriculture, Forestry and Fisheries of Japan. This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN). Dr Athipoo Nuntaprasert (Faculty of Veterinary Science, Chulalongkorn University, Thailand) is supported by Monbusho Scholarship. Authors thank Ms Reiko Satoh and Riyoko Takehara for technical assistance.
References [1] Van Snick J. Interleukin-6: an overview. Annu Rev Immunol 1990;8:253–78. [2] Dobryszycka W. Relevance of haptoglobin in clinical medicine. Folia Histochem Cytochem 1992;30: 197–200. [3] Scott PR, Murray LD, Penny CD. A preliminary study of serum haptoglobin concentration as a prognostic indicator of ovine dystocia cases. Br Vet J 1992;148:351–5. [4] Emery P, Luqmani R. The validity of surrogate markers in rheumatic disease. Br J Rheumatol 1993;32:3–8. [5] Hirano T, Teranishi T, Lin B, Onoue K. Human helper T cell factor(s). IV. Demonstration of a human lateacting B cell differentiation factor acting on Staphylococcus aureus Cowan I-stimulated B cells. J Immunol 1984;133:798–802. [6] Akira S, Taga T, Kishimoto T. Interleukin-6 in biology and medicine. Adv Immunol 1993;54:1–78. [7] Muneta Y, Mori Y, Shimoji Y, Yokomizo Y. Porcine interleukin 18: cloning, characterization of the cDNA and expression with the baculovirus system. Cytokine 2000;12:566–72. [8] Scamurra RW, Arriaga C, Spronger L, Baarsch MJ, Murtaugh MP. J Interferon Cytokine Res 1996;16: 289–96. [9] Manie S, Proudfoot A, Ferrua B. Human interleukin-6: detection of 10 attomoles by colorimetric sandwich ELISA using immunopurified polyclonal anti-IL-6 antibodies. Eur Cytokine Netw 1993;4:51–6.
130
A. Nuntaprasert et al. / Comp. Immun. Microbiol. Infect. Dis. 28 (2005) 121–130
[10] Muneta Y, Mikami O, Shimoji Y, Nakajima Y, Yokomizo Y, Mori Y. Detection of porcine interleukin-18 by sandwich ELISA and immunohistochemical staining using its monoclonal antibodies. J Interferon Cytokine Res 2000;20:331–6. [11] Baarsch MJ, Scamurra RW, Burger K, Foss DL, Mahesqaren SK, Murtaugh MP. Inflammatory cytokine expression in swine experimentally infected with Actinobacillus pleuropneumoniae. Infect Immun 1995;63: 3587–94. [12] Fossum C, Wattrang E, Fuxler L, Jensen KT, Wallgren P. Evaluation of various cytokines (IL-6, IFN-a, IFN-g, TNF-a) as markers for acute bacterial infection in swine—a possible role for serum interleukin-6. Vet Immunol Immunopathol 1998;64:161–72. [13] Asai T, Okada M, Ono M, Irisawa T, Mori Y, Yokomizo Y, Sato S. Increased levels of tumor necrosis factor and interleukin-1 in bronchoalveolar lavage fluids from pigs infected with Mycoplasma hyopneumoniae. Vet Immunol Immunopathol 1993;38:253–60.
Establishment of swine interleukin-6 sandwich ELISA A. Nuntapraserta,c, Y. Morib, K. Tsukiyama-Koharaa, C. Kaia,* a
Laboratory of Animal Research Center, Institution of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai Minato-ku, Tokyo 108-8639, Japan b National Institute of Animal Health, Tsukuba, Ibaraki 305-0856, Japan c Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand
Abstract We established a sandwich enzyme-linked immunosorbent assay (ELISA) for swine interleukin-6 (SwIL-6), which was applied for detection of SwIL-6 in vitro and in vivo. Anti-SwIL-6 rabbit- and goat-polyclonal antibodies, and monoclonal antibody (mAb) were prepared, conforming that all of the antibodies were reactive with recombinant SwIL-6 by Western blotting and indirect ELISA. A sandwich ELISA was developed using the mAb as a capture antibody and biotinylated goatpolyclonal antibody as a detection antibody. The detection limit of the sandwich ELISA for rSwIL-6 was 49 pg/ml and did not show cross-reactivity with swine IL-1b, IL-4, IL-8, IL-18, IL-12, and IFN-g. Using the ELISA, SwIL-6 was detected in culture medium of the monocytes stimulated with PHA-P and PMA, and the plasma or the bronchoalveolar lavage fluid (BALF) of pigs experimentally infected with Actinobacillus pleuropneumoniae or Mycoplasma hyopneumoniae. This ELISA for SwIL-6 may be useful for understanding the role of this cytokine in various swine diseases. q 2004 Elsevier Ltd. All rights reserved. Keywords: Swine interleukin-6; Sandwich ELISA
Re´sume´ Un sandwich-ELISA a e´te´ e´tabli pour la de´tection de l’interleukine-6 porcine (SwIL-6) in vitro et in vivo. Les anticorps anti-SwIL polyclonaux et monoclonaux ont e´te´ pre´pare´s chez les lapins et che`vres, et leur re´activite´ ont e´te´ confirme´es par Western block et ELISA indirect. Le sandwichELISA a e´te´ exploite´ utilisant un anticorps monoclonal a` capture et un autre anticorps polyclonal biotinyle´ a` de´tection. La limite de de´tection de l’rSwIL e´tait 49 pg /ml sans aucune re´activite´ croise´e * Corresponding author. Tel.: C81 354495497; fax: C81 354495379. E-mail address: [email protected] (C. Kai). 0147-9571/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.cimid.2004.08.003
122
A. Nuntaprasert et al. / Comp. Immun. Microbiol. Infect. Dis. 28 (2005) 121–130
avec IL-1b, IL-4, IL-8, IL-18, IL-12 et IFN-gamma. Avec le sandwich-ELISA l’SwIL-6 e´tait de´tectable dans la culture de monocytes stimule´s par PHA-P ou PMA aussi bien que dans le plasma sanguin et le lavage bronchoalve´olaire (BALF) des porcs expe´rimentalement infecte´s d’Actinobacillus pleuropnemoniae ou Mycoplasma hyopneumoniae. Le sandwich-ELISA est utile pour comprendre le roˆle de l’SwIL chez diverses maladies du porc. q 2004 Elsevier Ltd. All rights reserved. Keywords: Interleukine-6 porcine; Sandwich-ELISA
1. Introduction Cytokine, interleukin-6 is one of the key regulators in mediating the balance between inflammation and the immune response during the bacterial infection or injury process. The cells that produce high level of IL-6 is distributed all over the body as macrophages, monocytes, fibroblasts, endothelial cells, keratinocytes, astrocytes and T- and B-lymphocytes [1]. This cytokine was reported to cause a wide range of biological effects such as induction of all positive mediators of the hepatic acute phase proteins, which may serve as markers of stress and diseases [2–4], and of antibody production from B cells [5,6]. We expressed rSwIL-6 by three recombinant expression systems and examined their biological activities in mouse cells. We also established anti-SwIL-6 polyclonal antibodies from rabbit and goat. These polyclonal antibodies and a monoclonal antibody (mAb) were characterized and utilized for establishment of a sandwich ELISA system which was evaluated by measuring SwIL-6 concentration in various samples such as culture medium, swine plasma or bronchoalveolar lavage fluid (BALF).
2. Materials and methods 2.1. Production of polyclonal antibodies Anti-SwIL-6 polyclonal antibodies were produced by immunizing rabbit or goat followed by purification using HiTrap rProtein-A and HiTrap Protein-G affinity columns (Amersham Pharmacia Biotech). The goat anti-SwIL-6 antibody was biotinylated with EZ-link Sulfo-NHS-LC-Biotin (Pierce) according to the manufacturer’s instructions to use as a detection antibody in developing a sandwich ELISA. 2.2. Characterization of antibodies to swine IL-6 The antibody binding activity of the polyclonal antibodies against the rSwIL-6 was assayed using an indirect ELISA. Plate was coated with serial concentration of rSwIL-6 (0.2–50 mg/ml) purified from bacteria and detected with the rabbit anti-SwIL-6 antibody (0.8, 1.5, 3, and 6 mg/ml). The anti-SwIL-6 mAb (isotype IgG2) was kindly provided by Dr Hiroyuki Iwata, Yamaguchi University, Japan. The specificity of an anti-SwIL-6 mAb and both polyclonal antibodies (0.001–5 mg/ml) was also determined by this ELISA after
A. Nuntaprasert et al. / Comp. Immun. Microbiol. Infect. Dis. 28 (2005) 121–130
123
coating the rSwIL-6 purified from bacteria at 2.5 mg/ml. Goat anti-mouse-Ig-HRP or goat anti-rabbit-IgG-HRP or rabbit anti-goat-IgG-HRP was used to detect bound mAb or polyclonal antibodies. 2.3. Swine IL-6 sandwich ELISA To develop a sandwich ELISA, the mAb was used as a capture antibody and the biotinylated goat anti-SwIL-6 polyclonal antibody was used as a detection antibody. First, 96-well ELISA microplates (Nunc) were coated with 1 mg/ml of purified anti-SwIL-6 mAb in 100 ml of 0.05 M carbonate buffer, pH 9.6, and incubated overnight at 4 8C. The wells were washed four times with washing buffer (0.15 M NaCl containing 0.05% Tween-20) and then incubated with 100 ml of samples or standard rSwIL-6 purified from bacteria diluted in standard diluent assay buffer (0.01 M Tris–HCl, pH 7.2, 0.15 M NaCl, 0.05% Tween-20, and 0.5% skim milk) for 2 h at room temperature. The plates were rinsed four times and incubated with 100 ml of biotinylated goat anti-SwIL-6 polyclonal antibodies (2.5 mg/ml) for 1 h at 37 8C. After washing, the plates were treated with 0.25 mg/ml of HRP-conjugated streptavidin (ELISA grade) (Biosource International Inc., Camarillo, CA) for 1 h at ambient temperature. After washing four times, 100 ml of component TMB microwell peroxidase substrate (Kirkegaard & Perry Laboratories, Guildford, UK) was added. The reaction was stopped 10 min later with 100 ml of component TMB stop solution (Kirkegaard & Perry Laboratories). The absorbance at 450 nm was determined in MTP-120 microplate reader (Corona Electric, Japan) and a standard curve was obtained. Cross-reactivity was tested against commercially available recombinant swine IL-1b, swine IL-8, and swine IFN-g (BioSource International Inc.), swine IL-12 (Endogen Inc., Woburn, MA), swine IL-4, and swine IL-18 [7]. These cytokines were applied to a sandwich ELISA system described above at a concentration of 1 ng/ml. The ability of this sandwich ELISA assay to detect the rSwIL-6 was tested by using three different kinds of the SwIL-6 produced from bacterial, insect, and COS-7 cells. We also investigated the ability of this sandwich ELISA to detect natural SwIL-6 using the supernatants from swine monocytic culture with PHA-P and PMA mitogens. 2.4. Native swine leukocyte IL-6 IL-6 is mainly secreted by monocytes and macrophages. PBMC were reported to be the main significant source of SwIL-6 compared with fibroblasts or alveolar macrophages [8]. We identified native SwIL-6 produced from monocytes by a sandwich ELISA. Native monocyte SwIL-6 was generated from adherent swine monocyte cells stimulated with mitogens as described [8,9]. Briefly, PBMC were isolated from a 10-week-old normal pig and incubated with the anti-CD14 (M-M9) mAb (1/200 dilution, VMRD Inc., Pullman, WA) and then labeled with the rat anti-mouse IgG1 Microbeads (Miltenyi Biotec, CA). Swine monocytes were then separated in MACS cell separation column (Miltenyi Biotec). Using native monocytic SwIL-6 in a sandwich ELISA, swine monocytes (1!106 cells/ml) after separation from MACS cell column were cultured for 24 h in RPMI-1640 medium supplemented with 10% FCS. Adherent swine monocytes were then stimulated for further 24 h with the range of 0–10 mg/ml of phytohemagglutinin-P
124
A. Nuntaprasert et al. / Comp. Immun. Microbiol. Infect. Dis. 28 (2005) 121–130
Fig. 1. Comparison of Western blotting analysis of rSwIL-6 expressed in bacteria, baculovirus, and mammalian expression systems with (A) the anti-SwIL-6 mAb and (B) the goat-polyclonal antibody against SwIL-6 molecule. (C) Cross-reactivity of the rabbit anti-SwIL-6 antibody against rHuIL-6 by Western blotting analysis. The 20 ml of recombinant proteins purified from bacteria and Tn5 cells, and the supernatant from COS-7 cell culture were analyzed in 12.5% SDS-PAGE followed by Western blotting and visualized by chemiluminescence’s development. M, Size markers (kDa); B, affinity purified rSwIL-6 from bacteria; I, affinity purified rSwIL-6 from insect cells; H, rHuIL-6 produced from bacteria; C, secreted SwIL-6 from COS-7 cells. The arrows indicate the rSwIL-6 proteins.
A. Nuntaprasert et al. / Comp. Immun. Microbiol. Infect. Dis. 28 (2005) 121–130
125
(PHA-P; Sigma) and 0–100 ng/ml of phorbol myristic acetate (PMA; Sigma). Supernatants were then collected, concentrated and used to detect the SwIL-6 concentration using a sandwich ELISA system. 2.5. IL-6 production in experimental bacteria infections in pigs The role of SwIL-6 in swine diseases was investigated on experimental bacterial infections in pigs. Plasmas and BALF samples were obtained from five normal pigs and five pigs intranasally infected with Actinobacillus pleuropneumoniae (A. pleuropneumoniae serotype 1, strain NZ-62, a field strain isolated in Japan) within 2 days after pleuropneumonia developed as described by Muneta et al. [10]. BALF samples were obtained from five normal pigs and five pigs intranasally inoculated with virulent Mycoplasma hyopneumoniae (M. hyopneumoniae strain E-1, a field strain isolated in Japan). The concentrations of SwIL-6 at protein level in these samples were subjected to a sandwich ELISA as described above. 2.6. Statistical analysis Data are expressed as meanGSD and examined statistically by Student’s t-test.
3. Results 3.1. Characterization of the reactivity of antibodies to rSwIL-6 The antigenicity of rSwIL-6 from three kinds of expression systems was confirmed by Western blotting analysis using these antibodies as described above. To detect the immunoreactivity of rSwIL-6 more accurately, we developed an indirect ELISA for antibody binding reactivity as described in Section 2 by coating plates with purified cytokine from bacteria. The specificity of the purified IgG from mAb or polyclonal antibodies was further analyzed using rSwIL-6 produced from bacteria by an indirect ELISA system. The mAb and goat-polyclonal antibodies recognized rSwIL-6 with highly specific binding activities (Fig. 1). Therefore, these antibodies were considered to be useful for establishment of a sandwich ELISA system. 3.2. Development of sandwich ELISA for Swine IL-6 A sandwich ELISA for SwIL-6 was developed using a combination of the mAb and the goat-polyclonal antibody. The mAb was used as the first antibody to coat the wells of microplates and the goat anti-SwIL-6 was used as the second antibody. The concentration of coating mAb used was 1 mg/ml. The concentration of the biotinylated purified goat anti-SwIL-6 polyclonal antibody was titrated in the ELISA between 1/100 and 1/3200 dilution, and was found to be optimum at 1/800 dilution (2.5 mg/ml). A sandwich ELISA was successfully developed using the mAb as a capture antibody and the biotinylated goat-polyclonal antibody against rSwIL-6 as
126
A. Nuntaprasert et al. / Comp. Immun. Microbiol. Infect. Dis. 28 (2005) 121–130
Fig. 2. The specific antibody response against rSwIL-6. Detection of purified rSwIL-6 from bacteria (50– 0.2 mg/ml) with the purified rabbit anti-SwIL-6 polyclonal antibody was tested using an indirect ELISA (A). The wells were coated with serially diluted rSwIL-6, and were treated with the rabbit anti-SwIL-6 antibody at the concentration of 0.8 mg/ml (6), 1.5 mg/ml (B), 3.0 mg/ml (C), and 6.0 mg/ml (,), respectively. The specificity of mAb (,), and the purified rabbit (B) and goat (C) IgG polyclonal antibodies were also titrated in an indirect ELISA against rSwIL-6 coated at the concentration of 2.5 mg/ml (B).
a detecting antibody. The minimum detectable concentration by this ELISA was 49 pg/ml and the maximum one was 1500 pg/ml (Fig. 2). No cross-reactivity with other swine cytokines, i.e. swine IL-6, IL-1b, IL-4, IL-8, IL-18, IL-12p70, and IFN-g, was recognized in this assay, indicating that this ELISA is specific for SwIL-6. For further validation of the SwIL-6 ELISA, adherent monocytes were stimulated for 24 h with
A. Nuntaprasert et al. / Comp. Immun. Microbiol. Infect. Dis. 28 (2005) 121–130
127
Fig. 3. Adherent monocytes were stimulated for 24 h at the indicated concentration of PHA-P and PMA. Culture supernatants were then colleted and assayed for SwIL-6. Value are the meansGSD from three experiments.
PHA-P plus PMA, and the supernatants were assayed for native SwIL-6 concentration using the sandwich ELISA system. SwIL-6 secretion was inducible in a dose-dependent manner (Fig. 3). The combination of PHA-P (10 mg/ml) plus PMA (100 ng/ml) resulted in maximal SwIL-6 secretion (3-fold, p!0.05). 3.3. IL-6 production in pigs experimentally infected with A. pleuropneumoniae or M. hyopneumoniae To examine the applicability of this sandwich ELISA in clinical cases, we measured the concentration of IL-6 in the plasma and/or BALF samples of control pigs and experimentally A. pleuropneumoniae or M. hyopneumoniae infected pigs. As shown in Fig. 4A, the concentration of IL-6 in the plasma from pre-infected pigs was less than 50 pg/ml, the detection limit, whereas samples from pigs infected with A. pleuropneumoniae showed significant increase (1410G326 pg/ml, p!0.05). The IL-6 concentration in the BALF from A. pleuropneumoniae infected pigs was also higher than that from control pigs (240G101 pg/ml, p!0.05). Time-sequential production of SwIL-6 in BALF after infection with M. hyopeumoniae is shown in Fig. 4. Mean levels of SwIL-6 in BALF of the infected pigs were significantly increased at 2 weeks post-infection compared to those of uninfected or pre-infected ones.
4. Discussion In this study, we produced two specific polyclonal antibodies and one mAb against rSwIL-6 and developed indirect ELISA systems. The ELISA system was shown to be
128
A. Nuntaprasert et al. / Comp. Immun. Microbiol. Infect. Dis. 28 (2005) 121–130
Fig. 4. The IL-6 concentrations in the swine plasma and BALF experimentally infected with A. pleuropneumoniae at 2 days after infection were assayed using the sandwich ELISA (A). Control plasma samples were collected before the infection and control BALF was obtained from another healthy pigs with no pneumonia. Concentrations of SwIL-6 in BALF kinetically collected from pigs after infection with M. hyopneumoniae were also determined using the sandwich ELISA (B). Data represent the meanGSD of five pigs. *Significant difference compared with control group at p!0.05.
A. Nuntaprasert et al. / Comp. Immun. Microbiol. Infect. Dis. 28 (2005) 121–130
129
highly sensitive in detecting SwIL-6 protein concentrations at a minimum concentration of 49 pg/ml and to have no cross-reactivity with other swine recombinant cytokines. The ELISA system could also be used to measure the concentration of SwIL-6 in the plasma or BALF from pigs experimentally infected with A. pleuropneumoniae or M. hyopneumoniae. The SwIL-6 protein level in those samples was significantly increased within 2 days in pigs infected with A. pleuropneumoniae serotype 1. Increase in level of SwIL-6 protein was also found in BALF samples after infection with M. hyopneumoniae. Previous studies reported that bioactive pro-inflammatory cytokines such as IL-6 responses and the development of pneumonia were correlated to the severity of diseases from the A. pleuropneumoniae (serotype 1 [11] and serotype 2 [12]) or M. hyopneumoniae [13] infections in swine. Thus this ELISA system may be useful for the study of immunological pathogenesis of bacterial infections in pigs.
Acknowledgements We wish to thank Dr Hiroyuki Iwata, Faculty of Agriculture, Yamaguchi University for the gift of the anti-SwIL-6 monoclonal antibody and Dr Munenori Okuda from Zen-noh Institute of Animal health, Japan for the BALF samples of pigs experimentally infected with Mycoplasma hyopneumoniae. Research was funded in part by grant from Ministry of Education, Science, Sports and Culture, and Recombinant Cytokine Project grant number RCP-3210 from the Ministry of Agriculture, Forestry and Fisheries of Japan. This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN). Dr Athipoo Nuntaprasert (Faculty of Veterinary Science, Chulalongkorn University, Thailand) is supported by Monbusho Scholarship. Authors thank Ms Reiko Satoh and Riyoko Takehara for technical assistance.
References [1] Van Snick J. Interleukin-6: an overview. Annu Rev Immunol 1990;8:253–78. [2] Dobryszycka W. Relevance of haptoglobin in clinical medicine. Folia Histochem Cytochem 1992;30: 197–200. [3] Scott PR, Murray LD, Penny CD. A preliminary study of serum haptoglobin concentration as a prognostic indicator of ovine dystocia cases. Br Vet J 1992;148:351–5. [4] Emery P, Luqmani R. The validity of surrogate markers in rheumatic disease. Br J Rheumatol 1993;32:3–8. [5] Hirano T, Teranishi T, Lin B, Onoue K. Human helper T cell factor(s). IV. Demonstration of a human lateacting B cell differentiation factor acting on Staphylococcus aureus Cowan I-stimulated B cells. J Immunol 1984;133:798–802. [6] Akira S, Taga T, Kishimoto T. Interleukin-6 in biology and medicine. Adv Immunol 1993;54:1–78. [7] Muneta Y, Mori Y, Shimoji Y, Yokomizo Y. Porcine interleukin 18: cloning, characterization of the cDNA and expression with the baculovirus system. Cytokine 2000;12:566–72. [8] Scamurra RW, Arriaga C, Spronger L, Baarsch MJ, Murtaugh MP. J Interferon Cytokine Res 1996;16: 289–96. [9] Manie S, Proudfoot A, Ferrua B. Human interleukin-6: detection of 10 attomoles by colorimetric sandwich ELISA using immunopurified polyclonal anti-IL-6 antibodies. Eur Cytokine Netw 1993;4:51–6.
130
A. Nuntaprasert et al. / Comp. Immun. Microbiol. Infect. Dis. 28 (2005) 121–130
[10] Muneta Y, Mikami O, Shimoji Y, Nakajima Y, Yokomizo Y, Mori Y. Detection of porcine interleukin-18 by sandwich ELISA and immunohistochemical staining using its monoclonal antibodies. J Interferon Cytokine Res 2000;20:331–6. [11] Baarsch MJ, Scamurra RW, Burger K, Foss DL, Mahesqaren SK, Murtaugh MP. Inflammatory cytokine expression in swine experimentally infected with Actinobacillus pleuropneumoniae. Infect Immun 1995;63: 3587–94. [12] Fossum C, Wattrang E, Fuxler L, Jensen KT, Wallgren P. Evaluation of various cytokines (IL-6, IFN-a, IFN-g, TNF-a) as markers for acute bacterial infection in swine—a possible role for serum interleukin-6. Vet Immunol Immunopathol 1998;64:161–72. [13] Asai T, Okada M, Ono M, Irisawa T, Mori Y, Yokomizo Y, Sato S. Increased levels of tumor necrosis factor and interleukin-1 in bronchoalveolar lavage fluids from pigs infected with Mycoplasma hyopneumoniae. Vet Immunol Immunopathol 1993;38:253–60.