IgY Purification Record

Name of Operator: ___________________ Date: ____________

Chicken No.: _______

Immunizing Antigen: ____________ Lot size (# eggs): _______

Date of Eggs: _______________________

Yield IgY per ml egg yolk (mg) __________

IgY Concentration: _____________ mg/ml

Total IgY Yield: ____________ Total vol. (ml) _______

Instructions

1. Assume that 1 gram of yolk is equal to 1 ml.
   • volume of egg yolks = _________ ml (vol. A)
2. Add 5 times the egg yolk volume (A) of cold Reagent A to the container and stir gently:
   • volume of Reagent A = 5 x (A) = _________ ml (vol. B)
   • lot no. Reagent A: __________________
3. Place in fridge (at least 2 hours — up to overnight).
   • volume of Reagent A = 5 x (A) = _________ ml (vol. B)
   • lot no. Reagent A: __________________
4. Centrifuge solution at 10,000 x g ( 4 degrees C) for 15 min. Discard lipidy pellet. Collect supernatant into graduated cylinder by filtering through gauze inserted into filter paper. The supernatant should be colourless and translucent. If particulates are present, centrifugation step should be repeated. Measure volume of supt.
   • volume of supernatant (ml) = _______ (vol. C)
   • centrifugation step repeated? _____ yes _____ no
   • colourless, translucent supt? ______ yes ______ no
5. To supernatant (vol. C), add the same volume of cold Reagent B and mix gently. Let sit for at least 1 hr in the fridge (up to overnight).
   • volume Reagent B added (vol. C): ______________ ml
   • lot no. Reagent B: ___________________
   • incubation time: ____________________
6. Centrifuge 4 degrees C at 10,000 x g for 15 min. Resuspend the pellet in the original egg yolk volume (vol. A) with PBS:
   • final IgY volume (vol. A): __________________ ml
7. To determine the IgY conc. and yield, prepare two 1/20 dilutions of the purified IgY in PBS (ie add 50 ul of IgY to 950 ul PBS). Measure the absorbance at 280 nm using PBS as blank.
   • Abs#1= _____ Abs#2= _______ Avg. Abs = ________
   • concentration = (avg Abs x 20)/1.35 = ________ mg/ml
   • yield = concentration x final
   • volume (vol. A) = ________ mg
8. Filter sterilize or add a preservative and refrigerate. IgY activity will remain unchanged for at least one year.
   • Filter sterilize? ____ yes _____ no
   • Preservative? ____ yes _____ no
   • If yes, what kind? ____________ Concentration: ________

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