Purification of IgY Anti-SARS-CoV-2-Chicken Nucleocapsid and Elimination of Its Low-temperature Storage-induced Aggregates

Abstract

Even though the COVID-19 pandemic has ended, the virus is still circulating. It is important to detect it, as its symptoms are similar to other endemic diseases, e.g. dengue fever. Detection can be done using birds’ specific immunoglobulin Y, IgY Anti-SARS-CoV-2-Chicken Nucleocapsid (IgY anti-N), obtained by immunising chickens with the SARS CoV2 nucleocapsid protein. However, IgY purification requires further research, as commonly used ligands are unsuitable. Another common problem with IgY anti-N is aggregation, even when stored at low temperatures. Thus, this research aims to purify IgY anti-N and eliminate low-temperature storage-induced aggregates. In this study, polyethene glycol (PEG) was used for delipidation. The resulting IgY was divided into 2 parts, one of which was stored at 4 °C and purified within the next 1 or 2 days (labelled as IgY 4 °C), while the other was stored at −20 °C and purified within a period of 1 or 2 years (labelled as IgY −20 °C). The next step involved purifying both samples using hydrophobic chromatography and sequential anion exchange. Peaks resulting from the hydrophobic column were analyzed using High-Performance Liquid Chromatography (HPLC) and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Another purification result was analysed using PAGE. The migration of each separation step result was observed using native, denaturing and non-reducing PAGE. The study showed, qualitatively, that using two-step chromatographic techniques, hydrophobic interaction and ion exchange chromatography significantly increased the purity of IgY during purification. Additionally, the results demonstrated that hydrophobic chromatography purifies IgY and removes aggregates that may arise from storage. Moreover, similar to immunoglobulin G, IgY anti-N was found to have an uncommon migration in native PAGE compared to other proteins. HIGHLIGHTS PEG precipitation for delipidation of egg yolk containing IgY Two-step chromatographic method for IgY purification: hydrophobic interaction and ion exchange Hydrophobic interaction chromatography can purify and also remove the aggregate of the IgY sample Native PAGE cannot be used to calculate antibody molecular weight GRAPHICAL ABSTRACT