Establishment of swine interleukin-6 sandwich ELISA

• Published in: Comparative immunology, microbiology and infectious diseases microbiology and infectious diseases, 2005-03, Vol.28 (2), p.121-130
• Main Authors: Nuntaprasert, A., Mori, Y., Tsukiyama-Kohara, K., Kai, C.
• Format: Article
• Language: English
• Subjects: Actinobacillus Infections - immunology; Actinobacillus Infections - veterinary; Actinobacillus pleuropneumoniae; Actinobacillus pleuropneumoniae - immunology; Animals; Antibodies, Monoclonal - immunology; Biological and medical sciences; Blotting, Western - veterinary; Bronchoalveolar Lavage Fluid - immunology; Enzyme-Linked Immunosorbent Assay - methods; Enzyme-Linked Immunosorbent Assay - veterinary; Fundamental and applied biological sciences. Psychology; Interleukin-6 - analysis; Interleukin-6 - biosynthesis; Interleukin-6 - immunology; Interleukine-6 porcine; Microbiology; Mycoplasma hyopneumoniae; Mycoplasma hyopneumoniae - immunology; Pneumonia of Swine, Mycoplasmal - immunology; Sandwich ELISA; Swine - immunology; Swine interleukin-6
• ISSN: 0147-9571; 1878-1667
• DOI: 10.1016/j.cimid.2004.08.003

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 goat-polyclonal 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. Un sandwich-ELISA a été établi pour la détection de l'interleukine-6 porcine (SwIL-6) in vitro et in vivo. Les anticorps anti-SwIL polyclonaux et monoclonaux ont été préparés chez les lapins et chèvres, et leur réactivité ont été confirmées par Western block et ELISA indirect. Le sandwich-ELISA a été exploité utilisant un anticorps monoclonal à capture et un autre anticorps polyclonal biotinylé à détection. La limite de détection de l'rSwIL était 49 pg /ml sans aucune réactivité croisée avec IL-1β, IL-4, IL-8, IL-18, IL-12 et IFN-gamma. Avec le sandwich-ELISA l'SwIL-6 était détectable dans la culture de monocytes stimulés par PHA-P ou PMA aussi bien que dans le plasma sanguin et le lavage bronchoalvéolaire (BALF) des porcs expérimentalement infectés d' Actinobacillus pleuropnemoniae ou Mycoplasma hyopneumoniae. Le sandwich-ELISA est utile pour comprendre le rôle de l'SwIL chez diverses maladies du porc.