Development of a novel antigen capture-ELISA using IgY against porcine interleukin-6 and its application.
- Author:
Deog Yong LEE
;
Young Wook CHO
;
Sang Gyun KANG
;
Sung Jae SHIN
;
Han Sang YOO
- Publication Type:Original Article ; Research Support, Non-U.S. Gov't
- Keywords:
Antigen capture-ELISA;
porcine interleukin-6;
IgY;
protein expression
- MeSH:
Animals;
Biological Markers/blood;
Blotting, Western/veterinary;
Chickens;
Cloning, Molecular;
DNA, Complementary/genetics/isolation&purification;
Electrophoresis, Polyacrylamide Gel/veterinary;
Enzyme-Linked Immunosorbent Assay/methods/*veterinary;
Female;
Immunoglobulins/*blood;
Interleukin-6/*immunology;
Mice;
Mice, Inbred ICR;
Recombinant Proteins/immunology;
Swine/*immunology
- From:Journal of Veterinary Science
2004;5(4):337-343
- CountryRepublic of Korea
- Language:English
-
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.
