Isolation Of Anti-HBV Immunoglobulin Of Egg Yolk(IgY)

In recent years, people recognized the yolk is the important source of the antibodiesmore and more, IgY has many merits which the antibodies from mammalian have notwhen used as immune detection reagent, biochemistry detection reagent and medicine.Therefore, many methods purified IgY had been established. Because the process ofpurifying IgY is complex, expensive, IgY cannot be produced on a large scale. So itis not applied widely as perfect reagent. HBV virus was used as the antigen toimmunize laying chickens in our work. Anti-HBV virus immunoglobulin Y (IgY)was isolated from yolks of the eggs laid by these chickens. This article established aconvenient and economical method of purification.The yolk was diluted by 10 times distilled water with pH=5, laid for 6 hours andcentrifuged. And then the supernatant was precipitated with 55% and 33% ammoniumsulfate respectively to obtain the crude sample. Then the crude sample was fractionedby gel filtration chromatogram on Sephadex G150, with using 0.02mol/L pH=7NaH₂PO₄-Na₂HPO₄ with 0.14 mol/L NaCl in it as buffer liquid. The velocity of flowis 10ml/h, volume of sample is 1.0ml, and concentration is 0.3g/ml. The first peak isexpected, and determined by spectra photometry and SDS-PAGE. Conclusion: theIgY is purified and the yield is 10.2mg/ml yolk.Using MALDI-MS,the averagemolecular weights of IgY is 166975Da.The purified IgY was produced, using a two-step salt precipitation and one step gelfiltration protocol. And the yield was large. This method establishes a fundamentalbase for the industrialization because of its convenient and effective.