Vol. 9, Special Issue 7, Part J (2025)

This study aimed to develop Chicken Rabies Immunoglobulin (CRIG-Y) as a potential alternative for the prevention and treatment of rabies virus infections. Laying hens were immunized with purified rabies virus protein, and specific IgY antibodies were extracted from egg yolks using polyethylene glycol (PEG) 6000 precipitation. The purified IgY was further processed through reduction and alkylation using dithiothreitol (DTT) and iodoacetamide (IAA) to generate antigen-binding Fab fragments. To enhance antigen recognition, IgY was subjected to pepsin digestion in sodium acetate buffer. IgY was characterized by SDS-PAGE, where non-reducing conditions showed a prominent band at ~180 kDa, indicating intact IgY, while reducing conditions revealed distinct bands at ~63 kDa (heavy chain) and ~27 kDa (light chain). Fab fragments consistently exhibited bands in the 65 kDa range. Indirect ELISA and Western blotting confirmed the rabies virus-specific reactivity of CRIG-Y, with the Western blot detecting a ~65 kDa glycoprotein band characteristic of rabies virus. Protein quantification by the Bradford assay yielded concentrations of 3 mg/mL for purified IgY and 2.1 mg/mL for rabies virus protein. Future work will focus on evaluating the neutralizing antibody titres of CRIG-Y and CRIG-Y Fab using both in vivo (mouse neutralization test) and in vitro (RFFIT) methods.