Development of a novel antigen capture-ELISA using IgY against porcine interleukin-6 and its application.
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.
Lee
Deog Yong
DY
Department of Infectious Diseases, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Seoul, 151-742, Korea.
Cho
Young Wook
YW
Kang
Sang Gyun
SG
Shin
Sung Jae
SJ
Yoo
Han Sang
HS
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*
D015415:Biomarkers; D018076:DNA, Complementary; C016512:IgY; D007136:Immunoglobulins; D015850:Interleukin-6; D011994:Recombinant Proteins