Purification of IgY

1.

Pack 10 mL of the anti-chicken IgY agarose (ICP9605) into a 50 mL glass column. Wash the Column with 100 mL of PBSt before used.

2.

Crack two chicken eggs of interst, remove the egg albumin. Mix the egg yolks with 70 mL of PBSt and keep stiring for 5 min.

3.

Transfer the egg yolk-PBSt solution to  two 50 mL centrifuge tubes (35 mL each). Add 10 mL chloroform into each tube. Shake thouroughly then centrifuge at 8000 rpm for 10 min.

4.

Collect the supernatant, add 1:1 PBSt to the supernatant and centrifuge at 8000 rmp again.

5.

Collect the supernatant and pass the supernatant through the column at flow rate 2-3mL/min.

6.

Pass the flow through to the column (If it is the second purification) one more cycle.

7.

Wash the column with 100 mL PBSt then with 50 mL PBS only.

8.

Elute the bound chicken IgY with 25 mL 2% acetic acid and collect 25 mL the eluate.

9.

Neutralize the collected elute with 2 mL of saturated Na2CO3T

10.

The eluted anti-Chicken IgY can be firther dialyzed or desalted with G25 Spehadex for protein determination and ELISA titer determination.