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IgY Purification

Immunoprecipitation

Described here is an Immunoprecipitation protocol, a procedure used to identify and bind target proteins in solution; These proteins are then available for further characterization by SDS polyacrylamide gel electrophoresis, Western blots or other techniques. For example, your primary Chicken IgY could be mixed with a complex mixture of antigens in solution (often a cell lysate). The antigen-antibody complexes that form can be immunoprecipitated using a solid phase reagent that would bind to the immune complexes. When your primary antibody is a mammalian IgG, the reagent is often Protein A or G coupled to an agarose bead that is used as the immunoprecipitating reagent. IgY does not bind to Protein A or Protein G, so a secondary (anti-IgY) linked to agarose acts as the immunoprecipitating reagent when Chicken IgY is the primary antibody.

Immunoprecipitation

Reagents and Buffers:

50 mM Tris Buffer, pH 8.0

Dissolve 0.6 grams Tris Base in 50 ml distilled water. Adjust the pH to 8.0 with concentrated HCl.

QC to volume of 100 ml. Store in fridge.

Lysis Buffer (NP-40) (50 mM Tris, 1% NP-40, 15 mM NaCl):

To 25 ml of 50 mM Tris buffer, pH 8.0 (see above), add:
0.44 gm NaCl
1.0 ml NP-40

QC to volume of 50 ml with 50 mM Tris buffer, pH 8.0 and mix thoroughly. Store in refrigerator.

Lysis Buffer (RIPA) (50 mM Tris, 1% NP-40, 15 mM NaCl, 0.1% SDS, 0.5% Sodium deoxycholate):

To 25 ml of 50 mM Tris buffer, pH 8.0 (see above), add:
0.44 gm NaCl
1.0 ml NP-40
0.05 gm SDS
0.25 gm Sodium deoxycholate

QC to volume of 50 ml with 50 mM Tris buffer, pH 8.0 and mix thoroughly. Store in refrigerator.

1 M Potassium Phosphate Buffer, pH 7.3

660 ml 1 M K2HPO4
330 ml 1 M KH2PO4
Mix thoroughly. Check pH. Adjust if necessary (to lower pH, add more 1M KH2PO4; to raise pH, add more 1M K2HPO4). Store in fridge.

Phosphate Buffered Saline (PBS)

30 ml 5 M NaCl
10 ml 1 M Potassium Phosphate buffer, pH 7.3

QC to volume of 1 L wtih distilled water and mix thoroughly. Store in fridge.

Phosphate Buffered Saline-Tween, 0.25% (PBS-Tween)

Add 2.5 mL Tween 20 to 1 L PBS.

Procedure

The preparation of your cell lysate will depend on the type of cell used – whether the cell is prokaryotic or eukaryotic, has a cell wall, whether the antigen must retain tertiary structure or biological activity, for example will determine how you prepare your lysate. There are many recipes and experimentation may be required. Using Antibodies, A Laboratory Manual, addresses in detail various protocols. Here, suffice to say that there are 2 common cell lysis buffers: NP-40 and RIPA (see above). Triton X-100 can be substituted for the NP-40 if desired. The former is a less denaturing buffer and might be tried initially to determine if it effectively releases the desired antigen. A variety of protease inhibitors may be added to the lysis buffers to prevent enzyme activity.

Making your Cell Lysate:

  1. Wash cells once with room temperature PBS. Prepare your cell lysate in the cold room, using cold buffers as proteolytic denaturation occurs quickly once the cells lyse. For cultured cells, add cold lysis buffer to the washed cell pellet and gently shake on shaker. Instead of placing on a shaker, bacterial cells require sonication: 4 pulses of between 10-30 seconds should suffice. Recommended for yeast cells is mechanical shearing of the cells: Add an equal volume of glass beads (prewashed twice in 1 N hydrochloric acid and lysis buffer), to your yeast cells suspended in lysis buffer. Vortex vigourously in 30 second pulses until cells are lysed.
  2. Spin for 10 minutes at 10,000g in the cold. Remove the supernatant, careful to not disturb the cell pellet. Supernatants should be kept cold.

Preclearing your Lysate

Preclearing is a technique used to optimize the immunoprecipitation reaction by clearing the supernatant of proteins that may non-specifically bind to immune complexes, increasing the background of your reaction. It is done by mimicking the immunoprecipitation procedure but using a pre-immune or non-specific irrelevant antibody from the primary antibody producing animal. In some immunoprecipitation reactions, this step may not be necessary.

  1. Add Normal Chicken IgY (approximately 0.01 x lysate volume) to your cell lysate and incubate for 1 hour in the cold.
  2. Now add 0.1 x lysate volume of packed (or 0.2 ml resuspended agarose) Donkey anti-IgY agarose to the mixture and incubate for another 30 minutes in the cold.
  3. Centrifuge mixture at 10,000g for 15 min in the cold, and then carefully collect the supernatant. Now your sample is ready for your Primary Chicken IgY antibody.

Preparing your Immune Complexes

The most efficient immunoprecipitation reaction is one that uses an affinity purified IgY. This is because all of the IgY present will be antigen-specific and therefore most or all of the immune complexes precipitated will contain antigen. Using a total IgY preparation will result in only between 0.5 and 5% of the precipitated immune complexes containing antigen.

  1. In the cold room (or on ice), add your lysate aliquot into your 1.5 ml microtubes. Bring the volume up to 0.5 ml with lysis buffer. Add between 0.5 and 5 ug of your primary IgY antibody per 100 ug cell lysate. Incubate in the cold for 1 hour.
  2. Add 200 ul (50% slurry) Donkey anti-IgY agarose (20% vol/vol in lysis buffer) to each tube. Incubate again for 1 hour in the cold, rocking.
  3. Centrifuge the agarose bead immunoprecipitates at 10,000 x g for 15 seconds. Discard supernatant by aspiration and wash agarose pellet 3 times with lysis buffer.
  4. After the final wash and complete aspiration, your antigen-containing immune complexes are available for further analysis.
  5. If you want to free the antigen from the immune complexed agarose, you may want to add elution buffer to the complexes (please see Affinity Purification of IgY).

References

Ed Harlow & David Lane, Eds. Using Antibodies : A Laboratory Manual. Chapter 7. Cold Spring Harbor Laboratory Press, New York, 1999.

Dai, L, Taylor, MS, O'Donnell, KA and JD Boeke. 2012. Poly (A) Binding Protein C1 is Essential for Efficient L1 Retrotransposition and Affects L1 RNP Formation. Mol Cell Biol. 32:4323-4336.

Other IgY Resources:

Western Blots

Described here is a Western Blot protocol, sometimes called Immunoblotting, a procedure which will identify your immunizing antigen (via a blotted SDS Polyacrylamide gel) using your primary Chicken IgY antibody as a probe.  This is the protocol that we use at Gallus Immunotech.  There are many variations and a more detailed description of the procedure can be found here.

Reagents and Buffers:

Polyacrylamide Gel Electrophoresis Buffers:

1 M Tris Buffer, pH 6.8

Dissolve 12.1 grams Tris Base in 50 ml distilled water.  Adjust the pH to 6.8 with concentrated HCl.

QC to volume of 100 ml. Store in fridge.

Sample Buffer:

2.0 ml Glycerol
0.2 g Sodium Dodecyl Sulphate (SDS)
0.1 g Bromophenol Blue
0.6 ml 1 M Tris buffer, pH 6.8
1.0 ml 1 M Dithiothreitol (DTT) (added fresh from frozen stock), if you want a reduced protein profile

QC to volume of 10 ml with distilled water and mix thoroughly. Sample buffer without DTT may be refrigerated.

10X SDS Polyacrylamide Gel Running Buffer

30.3 g Tris base
144.0 g Glycine
10.0 g Sodium Dodecyl Sulphate (SDS)

QC to volume of 1 L with distilled water and mix thoroughly.  Store in fridge.

Coomassie Blue Gel Stain (0.1%)

0.25 g Coomassie Briliant Blue R-250 powde
100 ml Methanol
25 ml Glacial Acetic Acid

QC to volume of 250 ml with distilled water and filter (paper, Whatman #1).  Label and store at room temperature.

Destain Solution

150 ml Methanol
50 ml Acetic Acid

QC to volume of 500 ml with distilled water.  Label and store at room temperature.

Western Blot Buffers

Transfer Buffer

3.0 g Tris base
14.4 g Glycine
200 ml Methanol

QC to volume of 1 L with distilled water. Label and store in refrigerator.

Amido Black Stain

40 ml Methanol
10 ml Acetic Acid
0.1 g Amido Black Stain

QC to volume of 100 ml with distilled water. Label and store at room temperature.

1 M Potassium Phosphate Buffer, pH 7.3

660 ml 1 M K2HPO4
330 ml 1 M KH2PO4

Mix thoroughly. Check pH. Adjust if necessary (to lower pH, add more 1M KH2PO4; to raise pH, add more 1M K2HPO4). Store in fridge.

Phosphate Buffered Saline (PBS)

30 ml 5 M NaCl
10 ml 1 M Potassium Phosphate buffer, pH 7.3

QC to volume of 1 L wtih distilled water and mix thoroughly. Store in fridge.

Phosphate Buffered Saline-Tween, 0.05% (PBS-Tween)

Add 0.5 mL Tween 20 to 1 L PBS.

Blocking Buffer (5% milk in PBS)

Dissolve 5 g non fat dry milk powder in 100 ml PBS. Store in fridge for up to a week.

Diluent Buffer

Dissolve 1.0 g non fat dry milk powder in 100 ml PBS-Tween Store in fridge for up to a week.

Procedure

  1. Prepare your experiment by noting what protein samples are going in what wells on your gel, the % of acrylamide of your gel, whether it’s a gradient gel and the amount of protein you are adding per well.  For a mini gel, it is recommended to not add more than 5 ug and if the protein sample consists of few proteins, it is advisable to add even less protein. You may want to include a well with pre-stained molecular weight marker and by running a duplicate gel, you can stain one gel (or transferred gel) for protein and immunoblot the second.
  2. Run your polyacrylamide gel electrophoresis according to the instructions of the manufacturer.  We use BioRad’s Mini Protean System: http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4006157B.pdf
  3. Once it is finished running, remove the gel and equilibrate immediately in Transfer Buffer along with nitrocellulose (or polyvinylidene difluoride (PVDF) membrane), 2 absorbent filter papers and 2 fibre support pads for at least 30 minutes, ensuring that the gels and membranes are fully immersed in the buffer.
  4. Meanwhile, place the duplicate gel in Coomassie blue for an hour shaking gently.  Destain overnight shaking gently. You may also choose to transfer both gels and protein stain the duplicate blot with Amido Black.
  5. To assemble the sandwich for protein transfer onto the membrane, place the open transfer cassette in a shallow vessel containing Transfer buffer.  In this order, place fibre support pad, filter paper, equilibrated gel, membrane (making sure there are no air bubbles), filter paper and fibre pad.  Roll carefully with the side of a beaker to remove air bubbles and ensure maximal contact. Carefully close the cassette.
  6. Place the cassette in the transfer tank containing transfer buffer with the membrane closest to the red (positive) electrode (anode). When the cassette is in the tank, the buffer should completely cover the cassette.  If possible, add ice pack to prevent overheating.  Hook up to power pack and transfer for 60 minutes at 100 volts or overnight at 25 volts.
  7.  Remove the blot from the cassette and rinse thoroughly with water. Divide the blot into the sections that will be immunostained or stained for total protein (Amido Black). Mark the membrane pieces by notching one corner
  8. For total protein staining, immerse the rinsed blot into the Amido black stain and shake gently for 10 minutes.  Destain with distilled water
  9. To immunostain your blot, first block the protein binding sites by transferring the blot to Blocking Buffer, shaking gently for 1 hour at room temperature or in the refrigerator overnight.
  10. After blocking, rinse the blot twice in PBS for five minutes each. In a shallow tray, add the primary antibody diluted in Diluent Buffer. The recommended antibody concentration should be in the range of 1 to 50 ug/ml. Incubate at least 1 hr at room temperature, gently shaking.  Sensitivity may increase with a longer (overnight) incubation.
  11. Wash the blot 3 times with PBS-Tween and once with PBS for 5 minutes each.
  12.  Now, prepare the labeled anti-IgY secondary antibody.  We use our Horseradish Peroxidase (HRP)-labeled Donkey anti-IgY  (Cat. # DAIgY-HRP) at a 1:15000 dilution (0.07 ug/ml).  A ball park figure for diluting secondary conjugated antibodies is between 0.5 and 5 ug/ml.  Incubate for 1 hr at room temperature, gently shaking.
  13. Wash the blot 3 times with PBS-Tween and once with PBS for 5 minutes each.
  14. Your immunoblot is ready to be developed now.  The enzyme (HRP)-conjugated  secondary antibody in the presence of its substrate (we use 3,3,5,5-Tetramethylbenzidine or TMB) catalyzes the conversion of that substrate to a coloured product. These substrates are available commercially, for example: https://www.lifetechnologies.com/order/catalog/product/37574

References

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489

Ed Harlow & David Lane, Eds. Using Antibodies : A Laboratory Manual. Chapter 8. Cold Spring Harbor Laboratory Press, New York, 1999.

Other IgY Resources:

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:

Table II:

Manufacturer Chemical Activation Linkage group on Protein/Peptide
CNBr-Activated Sepharose Cyanogen Bromide Amino (-NH2)
Pierce  SulfoLink Coupling Resin Iodoacetyl Sulfhydryl (-SH2)

Pierce NHS-Activated Agarose,
Sigma NHS-Sepharose 4 Fast Flow

Hydroxysuccinimide Amino (-NH2)
Sigma Tresyl Chloride Activated Agarose Tresyl Chloride Amino (-NH2)
Sulfhydryl (-SH2)
     

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)

testtubes wide

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:

References

Overview of Affinity Purification

Ed Harlow & David Lane, Eds, Using Antibodies: A Laboratory Manual. Chapter 4. Cold Spring Harbor Laboratory Press, New York, 1999.

Comparison of IgG, IgE, IgY

Enzyme Linked Immunosorbent Assay (ELISA)

Described here is a Direct ELISA, a procedure which will provide you with a titre of your primary IgY antibody:

Reagents and Buffers:

Coating Buffer (0.05 M Carbonate Buffer, pH 9.6)
1.9 g Na2CO3*H2O
2.9 g NaHCO3

QC to volume of 1 L wtih distilled water and mix thoroughly. Check the pH, adusting if necessary. Store in fridge, indicating a 2-week expiry date.

1 M Potassium Phosphate Buffer, pH 7.3

660 ml 1 M K2HPO4
330 ml 1 M KH2PO4
Mix thoroughly. Check pH. Adjust if necessary (to lower pH, add more 1M KH2PO4; to raise pH, add more 1M K2HPO4). Store in fridge.

Phosphate Buffered Saline (PBS)

30 ml 5 M NaCl
10 ml 1 M Potassium Phosphate buffer, pH 7.3

QC to volume of 1 L wtih distilled water and mix thoroughly. Store in fridge.

Phosphate Buffered Saline-Tween, 0.05% (PBS-Tween)

Add 0.5 mL Tween 20 to 1 L PBS.

Blocking Buffer (2% milk in PBS-Tween)

Dissolve 2 g non fat dry milk powder in 100 ml PBS-Tween. Store in fridge for up to a week.

Diluent Buffer

Dissolve 0.3 g non fat dry milk powder in 100 ml PBS-Tween Store in fridge for up to a week.

0.1M Citric Acid

2 g citric acid

QC to volume of 100 mL wtih distilled water and mix thoroughly. Store in fridge.

0.1M Sodium Phosphate Dibasic (Na2HPO4)

14.2 g Na2HPO4

QC to volume of 100 mL wtih distilled water and mix thoroughly. Store in fridge.

Citrate Phosphate Buffer, pH 4.0

Combine equal amounts of 0.1M Citric Acid and 0.1M Na2HPO4.

Mix thoroughly. Check pH. Adjust if necessary (to lower pH, add more citric acid, to raise pH, add more Na2HPO4).

elisa

Procedure

  1. Prepare your experiment by noting on your 96-well ELISA plate, which IgY lots are being tested. Make sure to include a negative control (wells with no antigen, just coating buffer) as well as a positive control (which may be an antibody that you know is positive OR your primary antibody diluted similarly to your antigen).
  2. Dilute the antigen to 2.0 ug/ml (peptide, 5 ug/ml) in Coating Buffer. Add 100 ul to each well, cover the plate and incubate overnight at 4° C.
  3. Next day, flick out contents of wells and wash once with PBS-Tween. Pat the plate dry on a paper towel.
  4. Block the rest of the protein binding sites on your plate by adding 200 ul Blocking Buffer to each well. Cover plate and incubate for 2 hours at room temperature. Wash plates 3 time with PBS-Tween.
  5. To each well add 100 ul Diluent Buffer. Dilute test antibodies to 300 ug/ml (in diluent buffer) and add 50 ul to appropriate wells in row A only. Serial dilute the antibody by mixing with micropipette contents of wells in row A, 8 times and removing 50 ul and adding to row B. Repeat down to row G, discarding the last 50 ul/well from row G. Leave row H blank. Incubate plate(s) for 1-2 hours at room temperature.
  6. Wash plates 3 times with PBS-Tween. To each well add 100 ul Horseradish Peroxidase (HRP) Donkey anti-IgY (Cat. # DAIgY-HRP; Gallus Immunotech), diluted 1 :5000 in Diluent Buffer. Incubate plate(s) for 1 hour at room temperature.
  7. Wash plates 3 times with PBS-Tween. Pat the plate dry on a paper towel. Now, develop the plates by adding the enzyme substrate. There are a number of substrates available commercially. We use ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) (Sigma), adding 0.4 mg to Citrate Phosphate buffer. Just before adding 100 ul to each well, add 10 ul 30% hydrogen peroxide (H2O2) per 100 ml substrate solution. Incubate approximately 20 minutes or until green colour develops. Measure colour at 410 nm setting in ELISA reader.

Other IgY Resources:

 

IgY Purification from Chicken Egg Yolks

Once you've immunized your hens for IgY antibody production and collected immune eggs, the next step is to purify the IgY from the egg yolks. Unlike serum, which contains a number of immunoglobulin classes – IgG, IgM, IgD, IgA and IgE, yolk contains only one class of antibody – IgY. However, the egg yolk is a complex mixture of water (50%) lipids (32-35%) and proteins (16%)1 and for most experimental purposes, it is advisable to, at least, partially purify the IgY protein, by removing the bulk of the lipids and lipoproteins. Proteins residing within the yolk are of 4 types: lipovitellins, phosphorous-containing lipoproteins (40%), apovitellenins, containing less phosphorous but more lipid (37.3%), phosvitin, a phosphoprotein (13.4%) and the livetins (9.3%), of which IgY is part1. Removal of the yolk lipids and lipoproteins leaves a water soluble fraction, containing IgY along with other proteins, which crudely could be compared to an animal serum, in terms of useability in immunoassays. A number of methods which remove most of the lipids/lipoproteins have been described (reviewed in Refs. 1, 2):

  • Polyethylene Glycol precipitation3
  • Dextran Sulphate Precipitation4
  • Organic Solvent Lipid Solubilization5
  • Natural Gums, Xantham/Carageenan Precipitation6
  • Lipid Dilution/Ultrafiltration7

Following the delipidation of your egg yolk, the almost lipid-free solution can now be treated in a number of ways to concentrate/purify the IgY fraction:

  • Polyethylene Glycol Precipitation3
  • Sodium Sulphate Precipitation4
  • Ultrafiltration/Ammonium Sulphate Precipitation7
  • Preparative Electrophoresis8

There are a number of IgY Purification Kits commercially available that use one or more of the methods described above (Gallus Immunotech also provides an IgY purification service.):

Manufacturer IgY Purification Kit Name IgY Yield
(mg IgY /gram yolk)
IgY Purity
(% Purity by SDS PAGE)
Cost**
(per mg IgY purified)
Gallus Immunotech IgY Eggspress Purification Kit 4-9 85-90 a
Pierce Biotechnology/ Thermoscientific Chicken IgY Purification Kit 4-9 85-90 b
Agro-Bio EggsPure 2-5 90 c
GE Healthcare HiTrap IgY Purification Information not available Information not available Information not available
Affiland IgY Purification Kit Information not available 98 Information not available

*75% for 2-step protocol, 90% purity for 3-step protocol a = cheapest, c = most expensive 

Other IgY Resources:

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