Expression of recombinant porcine interleukin-2 and application of its antibody to immunoassays.
Interleukin-2 plays an important role in T lymphocyte proliferation and immune response regulations. In this study, porcine IL-2 cDNA was cloned from peripheral blood mononuclear cells, and recombinant porcine IL-2 (rpIL-2) was expressed in Escherichia coli. The size of rpIL-2 without signal peptides was about 15 kDa when determined by SDS-PAGE and Western blotting analysis. Anti-rpIL-2 antibody was produced from mice immunized with the purified rpIL-2, and its specificity was examined by Western blotting and ELISA. In the Western blotting assay, anti-rpIL-2 and anti-recombinant human IL-2 (rhIL-2) antibodies specifically recognized rpIL-2 and rhIL-2, respectively. However, anti-rpIL-2 antibody did not recognize rhIL-2, and anti-rhIL-2 antibody also did not react with rpIL-2 in the same assay. In ELISA, anti-rpIL-2 antibody strongly interacted with both rpIL-2 and rhIL-2, and anti-rhIL-2 antibody also efficiently recognized both proteins. Taken together, the specificity of anti-rpIL-2 antibody for rpIL-2 was demonstrated by Western blotting and ELISA. It was also shown that ELISA is more efficient than Western blotting in determining the species cross-reactivity of anti-rpIL-2 antibody.
Choi
In-Soo
IS
Department of Infectious Diseases, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Korea.
Yoo
Han-Sang
HS
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D000918:Antibody Specificity; D015153:Blotting, Western / Q000662:veterinary; D003001:Cloning, Molecular; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000662:veterinary; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D013045:Species Specificity; D013552:Swine
D000906:Antibodies; D007376:Interleukin-2; D011994:Recombinant Proteins