Abstract— Chicken egg yolk immunoglobulins (IgY) from immunized hens have considerable advantages for the production of polyclonal antibodies. It reacts with more epitopes and avoids the interference in immunological assays. Regarding the IgY aptitudes, this study was conducted to produce chicken IgY specific for influenza M2 (M2e) protein. Fusion construct harboring C-terminal of bovine heat shock protein 70 (Hsp70) and influenza M2e coding genes was injected to laying hens. Egg yolk antibodies were screened for the presence of anti-M2e antibodies by indirect enzyme-linked immunosorbent assay (ELISA). Anti-M2e antibodies were detected in egg yolks of injected hens from 13 days, with the peak titer at 41 days. Anti-M2e polyclonal, monospecific IgY antibodies could be used for different areas of research, diagnostics, medical application and biotechnology. Keywords—IgY, Influenza, M2e, Hsp70. I. INTRODUCTION HE major low molecular weight serum immunoglobulin in chicken is IgY, which can be obtained easily from the egg yolk. As compared to mammalian IgG, chicken IgY has a slightly larger molecular mass (approximately 167 kDa) immunoglobulin repertoires [1]. IgY is a major specific serum immunoglobulin in chickens. Biologically active IgY antibodies are transmitted vertically from serum into the egg yolk, so IgY could be extracted easily from the egg yolk. IgY has the advantage in that it avoids the interference in immunological assays caused by the complement system, rheumatoid factors, anti-mouse IgG antibodies or human and bacterial Fc receptors [2]. These differences in molecular interaction bring advantages to the application of IgY antibodies; they have been used successfully in a variety of methods in different areas of research, diagnostics, medical application and biotechnology [3]. Influenza is a segmented, negative stranded and enveloped RNA virus of the Orthomyxoviridae family [4]. Influenza A viruses infect humans and a wide range of animals like pigs, horses, wild mammals, and birds [5]. There is a potential Gholamreza Nikbakht, Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran-Iran (corresponding author phone: (98) (21) 61117057; fax: (98) (21) 66427517; e-mail: nikbakht@ut.ac.ir). Monireh Jahantigh, DVM student at the Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran- Iran. Farzaneh Assadian, Ph.D student at the Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran- Iran. threat of animal to human transmission of some subtypes of influenza viruses. The spread of influenza infections in around the world and the consequent risk for occurrence of influenza pandemics and epidemics highlights the necessity to take reliable prophylactic, therapeutic and immunodiagnostic measures suitable for different influenza virus strains. One of the most highly conserved proteins in influenza virus is a transmembrane integral protein, which is called M2 .The 23-amino acid extracellular domain of M2, known as M2e, presents on the surface of influenza virus alongside Hemagglutinin and Neuraminidase [6]. Unlike HA and NA, M2e is highly conserved among different subtypes of influenza A viruses. This makes M2e an attractive target for development of specific antibodies [7]. Since M2e peptide sequence is only 23 amino acids long, for enhancing the immunogenicity of this short peptide, highly conserved heat shock protein, Hsp70, was used as adjuvants. Hsp70 is a member of the heat shock protein family (Hsps). Hsp70 role as an adjuvant in the induction of immune response is highly related to its capacity to present the exogenous antigens through the MHC class I pathway, this mechanism is known as cross-priming [8]. In this study we took advantages of “DNA-designed” egg yolk antibody for production of polyclonal, monospecific antibodies against the recombinant M2e-Hsp70. Production of antibodies based on DNA-designed avian IgY technology is followed by genetic immunization of hens with a plasmid encoding a given antigen. For this purpose, laying hens were injected with a series of test and control plasmids. Egg yolks samples were tested for the presence of anti-M2e antibodies. This could be a useful method for direct generation of antibodies and eliminates the use of costly and laborious antigen preparation and purification in conventional immunization methods. IgYs have been used successfully in a variety of methods in different areas of research, diagnostics, medical application and biotechnology [9]. II. MATERIALS AND METHODS Recombinant pcDNA 3.1 (+) plasmid bearing M2e-Hsp70 coding sequence (pcDNA 3.1-M2e-Hsp70) was kindly provided by Dr. Mahmoud Ebrahimi (Baqiyatallah Medical Sciences University, Tehran, Iran). Chemically competent E. coli strain DH5α was transformed with this vector. Positive transformants were selected on LB-agar plates containing 100 mg/ml Ampicillin. In selected single colonies, the presence of Generation of Egg Yolk Antibodies in Chicken (IgY) Against Influenza M2 (M2e) Protein Gholamreza Nikbakht, Monireh Jahntigh, and Farzaneh Assadian T 3rd International Conference on Bioscience, Biochemistry and Pharmaceutical Sciences (ICBBPS'2014) Feb. 11-12, 2014 Singapore 101