It is important that Reagents A and B and your eggs are at 4°C before use.
Separate cold egg yolk from the white using the egg separator. Rinse yolk with distilled water and roll on paper towel to remove adhering egg white. Puncture yolk membrane with Pasteur pipette and allow yolk to drain into tared beaker. Note the weight of the yolk. Assume that 1 ml of yolk is equal to 1 gram.
Add 5 volumes of cold reagent A to the yolks very slowly while stirring gently and continuously until well mixed. Avoid foaming.
Allow diluted yolk to stand for at least 2 hours or up to 24 hours at 4°C. Mix gently before adding to centrifuge tubes. Centrifuge at 4000 x g (or higher) for 15 min. at 4°C. Use longer centrifugation time for lower speed.
Collect supernatant into graduated cylinder. The supernatant should be colourless and translucent. If particulates are present, centrifugation step should be repeated and if necessary, filtered. Measure volume.
Transfer supernatant into a beaker and add equal volume of cold Reagent B while stirring with stir bar. Continue stirring for 2 min. and allow suspension to stand in the cold for 1 hour.
Centrifuge at 4000 x g (or higher) for 15 minutes at 4°C and discard the supernatant. Use longer centrifugation time for lower speed.
Dissolve precipitate in volume of PBS equal to original egg yolk volume and filter sterilize. IgY concentration will be between 4 and 7 mg/ml with purity of 90% or greater.
Store your IgY in the refrigerator for a year (or longer) with no loss of activity.