Performing a Purification of IgY Antibodies with HiTrap™ IgY Purification HP

Extracted from Affinity Chromatography Vol. 1: Antibodies, GE Healthcare, 2016

HiTrap™ IgY Purification HP 5 ml columns are prepacked with a thiophilic adsorption medium (2-mercaptopyridine coupled to Sepharose® High Performance). The interaction between the protein and the ligand has been suggested to result from the combined electron donating- and accepting-action of the ligand in a mixed-mode hydrophobic-hydrophilic interaction.

Figure 3.34 shows the purification of α–Hb IgY from 45 ml of egg yolk extract (corresponding to one quarter of a yolk) and the SDS-PAGE analysis indicating a purity of over 70%.

purification of a–Hb IgY

Fig 3.34. (A) Purification of IgY on HiTrap™ IgY Purification HP. (B) SDS-PAGE of nonreduced samples on PhastSystem using PhastGel 4–15, Coomassie staining.

Sample preparation

Refer to Column packing and preparation for general considerations.

As much as possible of the egg yolk lipid must be removed before purification. Water or polyethylene glycol can be used to precipitate the lipids. Precipitation with water is described below.


Buffer preparation

Binding buffer: 20 mM sodium phosphate, 500 mM potassium sulfate, pH 7.5

Elution buffer: 20 mM sodium phosphate, pH 7.5

Wash buffer: 20 mM sodium phosphate, pH 7.5 with 30% isopropanol

To improve recovery of total IgY or a specific IgY antibody, replace 500 mM potassium sulfate with 600 to 800 mM sodium sulfate in the binding buffer. The sample should have the same concentration of sodium sulfate as the binding buffer. Using more than the recommended salt concentration in the binding buffer will reduce the purity of the eluted IgY.

  1. Separate the egg yolk from the egg white.
  2. Add nine parts of distilled water to one part egg yolk.
  3. Mix and stir slowly for 6 h at 4°C.
  4. Centrifuge at 10 000 × g, at 4°C for 25 min to precipitate the lipids.
  5. Collect the supernatant containing the IgY.
  6. Slowly add potassium sulfate to the sample, stirring constantly, to a final concentration of 500 mM.
  7. Adjust pH to 7.5.
  8. Pass the sample through a 0.45 µm filter immediately before applying it to the column.



See Affinity Purification Using HiTrap™ Columns for general instructions for purification using HiTrap™ columns.

  1. Fill the syringe or pump tubing with distilled water. Remove the stopper and connect the column to the syringe (use the connector supplied).
  2. Snap off the tab on the column outlet.
  3. Wash out the ethanol with 25 ml of distilled water.
  4. Equilibrate the column with 25 ml of binding buffer. The recommended flow rate is 5 ml/min*.
  5. Apply the sample using a syringe fitted to the Luer connector or by pumping it onto the column. For optimal results, use a flow rate of 0.5 to 5 ml/min during sample application 6. Wash with at least 50 ml of binding buffer or until the absorbance reaches a steady baseline or no material remains in the effluent. Maintain a flow rate of 5 to 10 ml/min for washing.
  6. Elute with 50 ml of elution buffer using a one-step or linear gradient though larger volumes are sometimes required to break the interaction.
  7. After elution, regenerate the column by washing it with 35 ml of wash buffer and re-equilibrate the column with 25 ml of binding buffer. The column is now ready for a new purification.

* 5 ml/min corresponds to approximately 120 drops/min when using a 5 ml HiTrap™ column.

The purity of the eluted IgY can be improved by using gradient elution with, for example, a linear gradient 0% to 100% elution buffer over 10 column volumes, followed by 100% elution buffer for several column volumes.

To increase binding capacity, connect several HiTrap™ IgY Purification HP columns in series. A HiTrap™ column can be used with a syringe, a peristaltic pump, or connected to a liquid chromatography system.

Reuse of HiTrap™ IgY Purification HP depends on the nature of the sample. To prevent cross-contamination, it should only be reused when processing identical samples.



Store in 20% ethanol at 2°C to 8°C.


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