Development of DNA-Designed Avian IgY Antibodies for Quantitative Determination of Bovine Interferon-Gamma

Abstract

Interferon-gamma (IFN-γ), a cytokine produced by sensitized T lymphocytes, is one of the key elements in defining T helper 1 lymphocyte immune responses. Quantitative evaluation of IFN-γ expression could provide an important analytical tool for measurement of cell-mediated immunity and investigating immune responses to infectious diseases. Method of DNA-designed avian IgY antibodies was used for production of monospecific polyclonal antibodies that allows quantification of the recombinant bovine IFN-γ protein. IFN-γ cDNA was subcloned and expressed in mammalian expression plasmid (pcDNA3.1(+)) under the control of the human cytomegalovirus promoter. Chickens were immunized by plasmid DNA, and egg yolk antibodies extracted from eggs were collected after immunization. IgY-specific antibodies were evaluated by an antigen capture enzyme-linked immunosorbent assay (ELISA) using recombinant IFN-γ. Based on the results, developed bovine IFN-γ capture ELISA could detect up to 1 ng/ml of IFN-γ by 64-fold diluted IgY. Monospecific anti-bovine IFN-γ antibodies generated in chickens are useful for quantifying different concentrations of recombinant bovine IFN-γ, which is expressed in cell culture.

Candidaaccommodates non-culturableHelicobacter pylori inits vacuole - Koch’s postulates aren’t applicable

“…Paragraph 4: All the immunologic methods for production of IgY-Hp and purification of antibodies were performed according to standard protocols, mentioned with the relevant reference in the original paper[ 15 ]. IgY-Hp perfectly and specifically interacted with H. pylori antigens, as described in the reference papers we used[ 16 - 18 ].…”

Stimulation of IgY responses in gene gun immunized laying hens by combined administration of vector DNA coding for the target antigen Botulinum toxin A1 and for avian cytokine adjuvants

“…Despite most studies being conducted in mammals, several reports involving DNA immunization of avians (ducks, chickens) have been published (Rollier et al 2000, Kazimierczuk et al 2004, Brujeni et al 2011. Plasmids coding for the target antigen were usually administered intramuscularly, resulting in one case in strong antibody responses to Helicobacter pylori urease (Kazimierczuk et al 2004) and in another, in rather weak responses to bovine interferon-gamma (Brujeni et al 2011). In contrast, Witkowski and coworkers (2009) achieved high titers of antibodies specific for poxviral proteins by using a gene gun to deliver the plasmids.…”

“…Customary intramuscular injection requires high doses of DNA to achieve robust transfection and sufficient antigen production in the immunized organism (Cho et al 2004, Kazimierczuk et al 2005, Brujeni et al 2011. In contrast, low doses of DNA applied via gene gun can efficiently induce high antibody titers against the antigen encoded (Witkowski et al 2009).…”

Endocytotic uptake of FITC-labeled anti-H. pylori egg yolk immunoglobulin Y in Candida yeast for detection of intracellular H. pylori

“…Briefly, IgY-Hp was raised in hens by immunization with whole cell lysate of H. pylori and extracted from egg yolk according to Nikbakht Brujeni et al (2011). FITC-IgY-Hp was prepared by adding FITC solution to the antibody and removing unbound FITC using Sephadex G25 column (Saniee et al, 2013b).…”