IgY Purification Kit- How to Purify IgY Antibodies from Egg Yolk

The steps involved in isolation and purification of IGY are simple and performed at room temperature.

1. Fresh eggs are cracked and yolks are separated using the yolk separator. The yolks are punctured releasing the yolk contents, the membrane can be easily removed using a tooth pick.
2. For fresh yolks, 5 volumes of the yolk weight of reagent A is added with constant stirring. Note: If the yolks are 3 days or older and not frozen, it is recommended to use 6 volumes of Reagent A. It is not recommended to use frozen yolks using this kit.
3. A thick yellow precipitate will form once all the reagent sis added. Continue stirring for 10 minutes and incubate the contents at 4°C overnight. The precipitate will settle and clear supernatant can be noticed within 1-2 hours.
4. The contents are centrifuge in cold swingout bucket centrifuge at 3500-4000Xg for 30 minutes at 4°C.
5. Decant the clear supernatant in a prechilled beaker and slowly add equal volume of Reagent B with constant stirring. Continue to stir for 10 minutes and keep the mixture at 4°C for 2-4 hours to overnight. A clear white precipitate will settle at the bottom within 1-2 hours.
6. Centrifuge the contents at 4°C at 4000xg for 20 minutes to collect the white precipitate.
7. Carefully remove all the liquid and reconstitute the white pellet in antibody reconstitution buffer using the following formula. For 10 grams of yolk, add 2 to 2.5ml of Antibody Reconstitution buffer.
8. Quantitate IgY by contents by measuring the 1:20 diluted OD at 280.
9. The total IgY can be stored in 1:1 ratio of antibody stabilization buffer for long-term storage at -40°C or below.

Storage Conditions

All reagents are stable for up to 1 year when stored at 4oC.

General Recommendations

1. The eggs can be stored at 4°C for long time, but it is recommended that they are cracked fresh before use in this kit for isolation of IgY. The yolks once separated can be stored at 4°C for several days in 0.05% sodium azide without any significant loss in IgY recovery. Yolks should not be stored frozen for use with this kit.
2. The lipid contents in yolks vary significantly, sometimes the dilapidation step does not yield clear supernatant. In such cases one more volume of Reagent A can be added for complete dilapidation of yolks.

This product is for research use only and is NOT intended for use in humans or clinical diagnosis.