Generation and Characterization of Chicken Egg Yolk Antibodies against Bovine Interleukin-6

Abstract

Abstract. Interleukin-6 (IL-6), a multifocal cytokine, has been widely regarded as a potential biomarker of diagnosis of disease. Thus, the antibody against IL-6 became more important in detecting IL-6. So far, the anti-human, rat, sheep and porcine IL-6 antibody has been reported. We aimed to produce chicken anti-bovine IL-6 immunoglobulin Y (IgY) polyclonal antibody. In the present study, the gene of bovine IL-6 were synthesized by company (Luoyang Laipson Information Technology Co., Ltd). Then the gene was cloned and expressed with pET-32a expression vector, and then followed by purification of recombinant bovine IL-6 (rbIL-6) protein with Ni-NTA resin. Protein mass of about 45 kDa was found with SDS-PAGE. Egg yolk IgY against rbIL-6 was extracted from egg yolk by the method of PEG precipitation after using the purified rbIL-6 to immune chicken. The egg yolk IgY polyclonal antibody was evaluated by indirect ELISA and Western blot followed by Checkerboard titration for optimal dilutions ratio of antigen and IgY/IgG titers. The results showed that the titer of anti-rbIL-6 IgY gradually raised after the first booster immunization. Booster immunizations were administered at 2-week intervals, and IgY titers were monitored to determine the immune response. The peak titer of anti-rbIL-6 IgY was 1:64 000 after the fifth booster immunization. The Western blot showed that the IgY could specific bind bovine IL-6. Conclusion: We successfully prepared anti-bovine IL-6 IgY polyclonal antibody that would contribute to bovine disease diagnostic test development for improving herd sanitary states of cattle and dairy farms. Keywords: Bovine interleukin-6, Egg yolk antibodies, Immunoglobulin Y, Indirect ELISA.