Production, purification and serological determination of immunoglobulin-y (IgY) from chicken eggs against Clostridium tetani toxoid

Tetanus is a bacterial disease caused by the tetanospasmin (TeNT) neurotoxin produced by Clostridium tetani, which is life-threatening for humans and many animal species. It uses monoclonal antibodies produced in the mouse for the diagnosis and treatment of the disease, the use of which has several advantages, such as homogeneity and specificity. In contrast, a remarkable feature of polyclonal antibodies, especially IgY extracted from poultry, is that they are obtained in greater quantities than mammalian (IgG) antibodies. In this thesis study, breeding studies have been carried out within the Transitional Zone Agricultural Research Institute since 2015 and in 2020, pure lines of 'Anatolian-T' breed broiler chickens, breed registered by the Republic of Türkiye Ministry of Agriculture and Forestry, were used. Randomly selected chickens at the age of 22 weeks, C. tetani toxoid + adjuvant (FCA/FIA) was immunized by injection from the chest muscle and two reminder injections were given at intervals of 4 weeks and the immunization process was completed. Blood was taken from the wing veins of vaccinated animals before and 14 days after vaccination, and total antibody titers were measured by the ELISA method. It was observed that total antibody titers in the serum of blood samples taken 14 days after the last vaccination reached the highest level. IgY antibodies were isolated from the eggs of immunized and nonimmunized chickens non-invasively using the PEG (6000) extraction protocol. Purified anti-C. tetani molecular weight was determined by SDS-PAGE analysis and displayed in the appropriate and expected position on the gel. Then, the images obtained as a result of western blot analysis of confirmed the presence of IgY antibodies. Agar gel immunodiffusion (AGID) and rapid slide agglutination test were performed to prove that the confirmed IgY antibodies were anti-C. tetani IgY. The precipitation lines were formed between isolated C. tetani-specific IgYs and tetanus toxoid as a result of the AGID test, and the formation of typical aggregation in the quick slide agglutination test, serologically determined that the egg yolk antibodies obtained were produced specifically for C. tetani toxoid.