Preparation And Characterization Of Egg Yolk Immunoglobulin Y Specific To Influenza B Virus

Influenza is one of the most common acute viral diseases, and it causes systemic symptoms of variable degrees, such as mild fatigue, respiratory failure, and even death. Approximately10-20%of the world’s population suffers from seasonal influenza, resulting in250,000-500,000deaths every year. Influenza B virus (IBV) is one of the pathogens involved in localized outbreaks of seasonal influenza. Children recovering from influenza B who receive aspirin treatment may suffer a rare illness, Reye’s syndrome, which can affect the brain and liver.Much effort has been made to prevent and cure influenza B viral infections. Antiviral drugs, such as neuraminidase inhibitors, are widely used to treat influenza. However, influenza B virus is increasingly resistant to these agents. Although vaccination can provide protection, virtually none of the current vaccines is effective enough for use as a therapy. In recent years, based on the principle of passive immunotherapy, specific yolk antibodies (IgY) were used for the treatment of pathogenic bacteria and viruses caused diseases. With a safe, non-resistance and low cost advantages IgY can be used as alternatives to antibiotics.In this study, laying hens were immunized with inactivated influenza B virus, and IgY was isolated from the egg yolk by multiple polyethylene glycol (PEG)6000extraction and ammonium sulfate purification steps. The titers and specificity of the purified antibodies were assessed using ELISA, agar diffusion method and western blot. With hemagglutination inhibition (HI) test and virus plaque reduction assay to study the in vitro antiviral activity, its in vivo antiviral activity was also evaluated using a mouse model. In addition, IgY antibody and quantum dot were used as probes for detection of influenza B virus.The specific IgY titer increased beginning the second week after the first immunization, with the titer peaking at the fifth week. The yield of IgY was5.2mg per milliliter yolk, and the purity was98.2%. The use of western blotting and the hemagglutination inhibition (HI) test demonstrated that IBV-specific IgY binds specifically to influenza B virus proteins, and a plaque reduction assay revealed the neutralization efficacy of IBV-specific IgY at reducing influenza infection in MDCK cells. Furthermore, when mice were treated intranasally prior to or after influenza B virus infection, IBV-specific IgY protected the mice from influenza infection or reduced viral replication in their lungs, respectively. The application of IgY quantum dot probes to detect influenza B virus showed a linear relationship between the virus concentration and the fluorescence intensity, detection limit was2.834×10-/mg/mL.In conclusion, the results presented in this study indicate the possibility of ’universal’ immunotherapy using specific IgY for influenza virus infection. Our results also suggest that egg yolk from immunized laying hens may offer a new large-scale source of low-cost antibodies for immunoassay.