title: Development of a novel antigen capture-ELISA using IgY against porcine interleukin-6 and its application.
abstract: Interleukin-6 (IL-6) is introduced as a marker of disease. At present, a variety of method may be used to quantify expression of this protein. Antigen capture-ELISA is a sensitive and accurate quantification method previously used with ovine, rat, and human IL-6 proteins. However, it has never been reported to quantify porcine IL-6 protein using capture ELISA. In this study, we generated and characterized a set of IgY and mono-specific polyclonal antibodies to recombinant porcine IL-6 (rpIL-6), and combining these with a sensitive and specific capture-ELISA for a diagnostic purpose. cDNA encoding the mature protein coding region of porcine IL-6 was cloned and expressed with pQE-30UA expression vector. rpIL-6 was then expressed and purified by using Ni-NTA resin. Protein mass of 24 kDa was found with SDS-PAGE and the identity of the protein was confirmed by Western-blot. Production of polyclonal antibodies against rpIL-6 was performed using the purified rpIL-6 in mice and hens. An antigen capture-ELISA was developed with the antibodies after their extraction. To compare the IL-6 level in the different sanitary state of farms, pig sera were randomly collected and concentration of IL-6 in the sera was measured with the antigen capture-ELISA. The capture-ELISA with the optimal concentration of antibodies, in this study, was able to detect about 10 ng/ml of rpIL-6. IL-6 levels determined with the capture-ELISA in pig sera showed positive correlation with the sanitary states of the farms. These results suggested that the developed antigen capture-ELISA could be a good tool for the screening of microbial infection in pig farms.
authors.affiliation: Department of Infectious Diseases, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Seoul, 151-742, Korea.
authors.forename: Deog Yong
authors.initials: DY
authors.lastname: Lee
authors.forename: Young Wook
authors.initials: YW
authors.lastname: Cho
authors.forename: Sang Gyun
authors.initials: SG
authors.lastname: Kang
authors.forename: Sung Jae
authors.initials: SJ
authors.lastname: Shin
authors.forename: Han Sang
authors.initials: HS
authors.lastname: Yoo
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000097:blood; D015153:Blotting, Western / Q000662:veterinary; D002645:Chickens; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D005260:Female; D007136:Immunoglobulins / Q000097:blood*; D015850:Interleukin-6 / Q000276:immunology*; D051379:Mice; D008813:Mice, Inbred ICR; D011994:Recombinant Proteins / Q000276:immunology; D013552:Swine / Q000276:immunology*
chemical_list: D015415:Biomarkers; D018076:DNA, Complementary; C016512:IgY; D007136:Immunoglobulins; D015850:Interleukin-6; D011994:Recombinant Proteins