A NOVEL AND QUICK EGG YOLK IMMUNOGLOBULIN Y ANTIBODY EXTRACTION METHOD LEVERAGING THE PROTEIN LIQUID-LIQUID PHASE SEPARATION PRINCIPLE

This study presents a novel and efficient method for extracting immunoglobulin Y (IgY) antibodies from egg yolk based on the principle of liquid-liquid phase separation (LLPS) induced by polyethylene glycol 8000 (PEG 8000). Initial delipidation of egg yolk samples with varying PEG 8000 concentrations demonstrated optimal delipidation efficiency and protein recovery at 2.5 % PEG 8000 concentration. Subsequent IgY extraction involved inducing LLPS by raising PEG 8000 concentration to 6.5 %, resulting in turbid solutions and the formation of globular droplet-like condensates observed under a microscope. Unlike the PEG 6000 method that induced aggregation, the method developed here using PEG 8000 does not lead to the appearance of aggregates of IgY. SDS-PAGE analysis confirmed that IgY extracted was no different from the conventional PEG 6000 method, with similar purity levels (77 % vs 79 %). Enzyme-linked immunosorbent assay and western blot analysis confirmed the antigen recognition properties of the isolated IgY. This method significantly reduces the amount of PEG used, leading to substantial cost savings compared to PEG 6000. The method can be completed within one hour. Despite a slightly lower IgY yield by the method, the time- and cost-saving advantages of this method make it a promising alternative for IgY extraction in research. This proposed IgY extraction technique utilizing protein LLPS has the potential to improve the study of the physicochemical properties of IgY and optimized production, while offering a quicker and cost-effective solution for various applications in biomedical research.