Biotechnology and Biomedical Engineering Immunology Virtual Lab I Experiments

Extraction of IgG Antibodies from Immunized Hen Egg

Materials Required:

  1. Immunized hen egg
  2. TBS
  3. Dextran sulphate solution
  4. Calcium chloride solution
  5. Saturated sodium sulphate solution
  6. 2% Sodium bicarbonate solution
  7. 1mM EDTA
  8. 005 M Tris Hcl Buffer,pH = 8
  9. PBS Buffer
  10. Ethanol storage solution
  11. DEAE cellulose slurry
  12. Glycerol
  13. Distilled water
  14. Magnetic Stirrir
  15. Stir Bar
  16. Visicooler
  17. Dialysis tubing
  18. Chromatographic column
  19. UV spectrophotometer
  20. Cryovial
  21. Cryobox
  22. -200 CFreezer

Procedure:

  1. Sterilize the egg shell with 70% ethanol.
  2. The yolk is separated from the white and the yolk membrane is cut open.
  3. Pour the yolk into a sterile beaker and dilute it with 1000 µl of Tris-buffered saline (TBS).
  4. Transfer the diluted egg yolk into a sterile centrifuge tube. Centrifuge at 5000 rpm for 10 minutes.
  5. Pipette the precipitate carefully from the centrifuge tube.
  6. Add 1000 µl of Dextran Sulphate solution into diluted egg yolk suspension.
  7. Then add 1000 µl of Calcium chloride solution into the diluted egg yolk and mix it well.
  8. Pipette the precipitate carefully from the centrifuge tube.
  9. Again add 4000 µl of Tris-buffered saline into the diluted egg yolk suspension.
  10. Centrifuge the suspension at 5000 rpm for 10 minutes.
  11. After centrifugation, carefully collect the supernatant into a new beaker. Put a magnetic stirrer (stir bar) in the beaker.
  12. Add 4000 µl of Sodium sulphate into the supernatant solution.
  13. Keep the supernatant in the visicooler for 2 hours at 4oC. Mix the supernatant well with the magnetic stir bar.
  14. Carefully remove the stir bar from the beaker.
  15. Transfer the supernatant to a centrifuge tube labeled concentrated IgG.
  16. Centrifuge the suspension at 13000 rpm for 1 hour at 4oC.
  17. After centrifugation carefully discard the supernatant and the pellet is collected at the bottom of the centrifuge tube.
  18. Add 10 ml of TBS buffer to the pellet and mix it properly. Keep the centrifuge in the ice box.

 

Purification of IgG Fraction by Dialysis Activation of Dialysis Tubing:

  1. Place a small piece of dialysis tubing in a beaker containing 2% sodium bicarbonate solution. Keep it in the water bath for 20 minutes at 100oC.
  2. After that, take the dialyzing tube from the sodium bicarbonate solution and wash it with water.
  3. Keep the dialyzing tube in 1Mm EDTA. Keep it in the water bath for 20 minutes at 100oC.
  4. Then take the dialyzing tube from 1Mm EDTA and wash it with water.
  5. Rinse the dialyzing tube with 005 M Tris Hcl buffer (pH =8).
  6. Place the dialyzing tube on a tissue paper.

 

Purification of IgG Antibodies:

  1. Pipette 10 ml of the IgG pellets suspended in TBS buffer.
  2. Add the resuspended solution into an activated dialyzing tube and clamp it properly.
  3. Wipe the dialyzing tube with tissue paper.
  4. Place the activated dialyzing tube in a beaker containing PBS buffer (pH =7.2). Keep a magnetic stirrer in the beaker.
  5. Place the beaker in a Visicooler for 3 hours at 4oC. Mix the supernatant well with the magnetic stir bar.
  6. Then, pipette the IgG suspension in the dialyzing tubing using a sterile pasteur pipette. Transfer the IgG suspension into a new centrifuge tube labeled dialyzed IgG.

 

Purification of IgG Fraction by Ion-Exchange Chromatography:

  1. Carefully wash the chromatographic column with distilled water.
  2. Transfer DEAE cellulose slurry into the chromatographic column. Then transfer PBS buffer into the column. Later collect PBS buffer into a beaker and then tap the column properly.
  3. Transfer the dialysate IgG into the chromatographic column. Again pour PBS buffer into the column.
  4. After that collect the solution into a sterile centrifuge tube labeled purified IgG.
  5. Take a cuvette labeled Blank (B) and add 1000 µl of PBS buffer into the cuvette. Similarly add 1000 µl of PBS buffer into another cuvette labeled B.
  6. Keep the blank cuvettes on the Spectrophometer and press “Enter” button to start the process. The graph in the monitor indicates the baseline.
  7. Add 1000 µl of purified IgG into a cuvette labeled test (T).
  8. Place the cuvette with purified IgG (T) into the spectrophotometer and press “Enter” button to start the process.
  9. Observe the resultant graph on the monitor.
  10. Transfer 5 ml of 50 % glycerol into a cryovial. Pipette 5 ml of purified IgG and add it into the cryovial containing 50 % glycerol.
  11. Wrap the cryovial with parafilm. Keep it on a cryobox and store it in the -20oC freezer for future purposes.