Extraction and Purification of Anti-Helicobacter pylori IgY

The aim of this study was to establish an efficient procedure to produce anti-H. pylori urease IgY from the eggs of the laying hens. Four White Leghorn hens (52 weeks old) were immunized intramuscularly with the purified urease of H. pylori with aluminum hydroxide gel adjuvant. To increase the specificity and antibody titer, three boosters were given at 2-week intervals following the first injection. After the final immunization, the eggs were collected daily and stored at 4°C. The yield of yolk proteins were 47.52%, 41.07% and 51.35% for the methods of dextran sulphate, isopropanol and water-soluble fraction (WSF), respectively. Further, IgY was purified successively by ammonia sulfate, affinity chromatography and ion exchange chromatography for each crude protein. Ammonia sulfate salting produced the IgY with a purity of 37-53% and the titer of 22.6-29.5 unit/mg. The purity, relative purification folds and titer characteristics of the purified IgY were 85-90%, 19.63-34.61 and 223.8-273.4 unit/mg protein for affinity chromatography, and 89-92%, 25.33-47.14 and 288.8-372.4 unit/mg protein for ion exchange chromatography, respectively. This anti-Helicobacter pylori IgY was specific against H. pylori usease. Here, we established a simple and inexpensive method to obtain a yield of 90% purity of the anti-H. pylori IgY in combinations of the WSF method, ammonia sulfate precipitation, and affinity chromatography.