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Affinity Purification of IgY
The purified egg yolk IgY fraction, as well as containing IgY directed against the immunizing antigen, contains all the other antibody specificities that were present in the animal\'s circulation at the time the eggs were formed. The IgY fraction, depending on the purification method, is approximately 85-90% pure (as determined by SDS polyacrylamide gel electrophoresis).
The specific activity (percentage of immunoglobulin Y that is directed against your antigen) of this IgY fraction depends on the immunogenicity of your antigen and the genetic make-up of the immunized hen. It is reasonable to expect between 2 and 10% specific activity of IgY from a chicken immunized with a protein greater than 50 kilodaltons. Expect less than 1% specific activity to a peptide from a hen immunized with a peptide-KLH conjugate (there will be a much higher specific activity directed to the carrier KLH portion of the immunogen). For many applications, this non-affinity purified antibodiy fraction will work just fine and it is unnecessary to further purify your IgY. In fact, further purification may result in the loss of your highest avidity antibodies.
However, sometimes, it is necessary to “affinity purify” your IgY fraction, ie to remove the IgY antibodies that are not antigen-specific, leaving only the IgY that recognizes the immunizing antigen. You might want to perform this extra step, especially if you’re finding that there is “non-specific” or cross-reactivity occurring in your immunoassays. To perform affinity purification, it is first necessary to bind your immunizing protein to a solid phase.
Preparing your "Immunizing Antigen" Solid Phase
If enough of your immunizing protein antigen is available, the most common solid phase support is an agarose bead. Per 1 ml agarose bead, the amount of protein antigen (or peptide) required for an efficient column would be approximately 10 mg. (If immunizing protein is limited in availability, please see the end of this section for an alternative analytical method, using nitrocellulose as your solid phase. ) Agarose resins, already chemically activated to bind to your protein or peptide immunogen via a free amino or sulfhydryl group, are available commercially. Some examples are listed below:
|Manufacturer||Chemical Activation||Linkage group on Protein/Peptide|
|Pharmacia CNBr-Activated Sepharose||Cyanogen Bromide||Amino (-NH2)|
|Pierce SulfoLink Coupling Resin||Iodoacetyl||Sulfhydryl (-SH2)|
|Sigma Tresyl Chloride Activated Agarose||Tresyl Chloride||Amino (-NH2)
For the binding of your antigen to the activated resin, follow the instructions of the resin’s manufacturer, making sure that you measure the protein concentration of your sample (optical densitiy at 280 nm) before binding and after binding, to ensure that the binding to the agarose has taken place.
Applying your IgY Sample to the Affinity Column
Measure the concentration of your IgY fraction (purified using one of the procedures outlined in Table I), using the extinction coefficient 1.36. Make a 1:10 dilution of your IgY and measure the absorbance at 280 nm. Calculate the IgY concentration by using the following formula:
IgY concentration (mg/ml) = Absorbance (280 nm) x 10 (IgY dilution factor 1.36 (extinction coefficient)
Dilute your IgY fraction to approximately 1 mg IgY / ml PBS. Apply the diluted IgY to your antigen column (use about 80 mg IgY per ml antigen-bound agarose), at a very slow drip, using a peristaltic pump. When the sample has finished, apply PBS to the column, until the PBS coming through the column has an absorbance (at 280 nm) of 0. Now apply a PBS solution to which NaCl (0.5 M) has been added, again until the absorbance (280 nm) is 0. Now, it’s time to elute the antigen-specific IgY.
Eluting your Antigen-Specific IgY
The most common method to dissociate your IgY antibody from its ligand is by an extreme change in the pH. The antigen-specific IgY is then eluted off, into a neutralizing buffer. Other elution buffers may contain high molarity salts and/or chaotropic agents. You may make up your own buffers or purchase them. Pierce Biotechnology has a good selection of buffers that you may need. Eluting your antigen-specific IgY may require some experimentation to determine the optimal elution buffer. The following method will work in most circumstances:
Apply at a slow rate, a 0.1 M glycine buffer, between pH 2.0 and 3.0 (this may require testing for optimization; try pH 2.3 to start), making sure that the eluted IgY is dripping into a neutralizing buffer (0.2 M sodium phosphate buffer, pH 8.0). Continue until the IgY eluate absorbance is 0. The ratio of neutralizing buffer: elution buffer should be about 1:4. As soon as the column eluate has a 0 absorbance (280 nm), neutralize the column by running PBS through, until the pH of the eluate is neutral.
Now, you want to determine whether you still have specific IgY remaining on your column, that would be eluted in an alkaline environment. Apply at a slow rate, 0.1 M triethylamine or triethanolamine, pH 11 -11.5, making sure that the eluted IgY is dripping into a neutralizing buffer (1.0 M sodium phosphate buffer, pH 5.0). Continue until the IgY eluate absorbance is 0. The ratio of neutralizing buffer: elution buffer should be about 1:25-30. As soon as the column eluate has a 0 absorbance (280 nm), neutralize the column by running PBS through, until the pH of the eluate is neutral. If no IgY was eluted at the alkaline pH, it is not necessary to perform alkaline elution on the next run.
Your column is now ready for another affinity purification procedure. To store your column, add 0.1% sodium azide and store in refrigerator.
Modification of Affinity Purification Procedure, when immunizing antigen is limiting
If only analytical quantities of affinity-purified IgY are required and antigen is limiting, you may want to blot your antigen on nitrocellulose and let the blot dry (or alternatively, transfer onto nitrocellulose from an SDS gel). Incubate a small amount of your diluted IgY fraction with the antigen blot and place on a shaker for an hour. Wash blot thoroughly with a 0.05% Tween 20 PBS solution until the washing solution has an absorbance of 0 (280 nm). Add your elution buffer (as described above), shake briefly and quickly neutralize your eluate (as described above). Wash your nitrocellulose blot in PBS until eluate is neutral and store in 0.1% sodium azide in refrigerator.
Concentrating your Antigen-Specific IgY
You may want to concentrate your affinity purified IgY – it will be more stable and take up less room in the fridge. If you decide to store it in a diluted form (less than 1 mg/ml), consider adding BSA, to a concentration of 1%.
Unlike mammalian IgG, IgY does not bind to protein A or protein G, so concentration of your affinity-purified IgY using protein A or protein G columns is not an option. Ultrafiltration and ultra-centrifugal filtration are effective, albeit expensive procedures that will concentrate your specific IgY. A cheaper alternative is to precipitate the IgY in your sample with 20% ammonium sulphate. After 1 hour incubation in the fridge, spin out your IgY and resuspend in PBS. Add sodium azide to a concentration of 0.1 percent and store at 4°C.
Other IgY Resources:
- Hen Immunization Protocol
- IgY Purification from Chicken Egg Yolks
- Enzyme Linked Immunosorbent Assay (ELISA)
- Western Blots
Ed Harlow & David Lane, Eds, Using Antibodies: A Laboratory Manual. Chapter 4. Cold Spring Harbor Laboratory Press, New York, 1999.