Standing out in the field of IgY Immunotechnology

  • You are here:  
  • Home /
  • IgY Purification Record

IgY Purification Record

  • Print
Details
Published: Monday, 09 June 2014 19:05
Hits: 4462

Click here for the IgY Purification Record printout.

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   Lot no. Reagent A: __________________

  3. Place in fridge (at least 2 hours — up to overnight):
    Incubation Time = _________ hr

  4. Centrifuge solution at 2,000 x g ( 4°C) for 15 min.  Collect supernatant into graduated cylinder and discard lipidy pellet. by filtering through gauze inserted into filter paper. The supernatant should be colourless and translucent. If particulates are present, filter through cotton gauze or filter paper. Measure volume of supernatant.
    Volume of supernatant (ml) = _______ (vol. B)     Colourless, translucent supt? ______ yes ______ no

  5. To supernatant (vol. B), 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: ________ hr

  6. Centrifuge 4°C at 2,000 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 1/10 dilutions of the purified IgY in PBS (ie add 100 ul of IgY to 900 ul PBS). Measure the absorbance at 280 nm using PBS as blank.
    Abs#1= _______ Abs#2= _________   Avg. Abs = ________
    Concentration = (Avg Abs x 10)/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 sterilized? ____ yes _____ no
    Preservative? ____ yes _____ no
    If yes, what kind? _____________________    Concentration: _______________

    Click here for the IgY Purification Record printout.