Determining the Efficacy of Sourcing IgY Antibodies from Chicken Eggs Using the PierceTM Chicken IgY Purification Kit

by Sachi Kishinchandani

ABSTRACT

IgY is known to be an immunoglobulin that is formed by the maternal immune system and passed down to offspring, in reaction to certain foreign substances. It is hypothesized that IgY can be used to boost human immune systems. Thus, to ask how we can easily gather these proteins for increased human immunity, we analyzed the purification of IgY via the Thermo ScientificTM PierceTM Chicken IgY Purification Kit. Through a Bradford assay, SDS PAGE, and a Densitometry, we determined that it is possible to purify around 40-80 mg of IgY per egg using similar methods. Since each hen produces hundreds of eggs annually, and each egg contains such a large portion of IgY, it is reasonable to conclude that mass purification of IgY is feasible.

INTRODUCTION/BACKGROUND

Our immune system plays a vital role in protecting our body from foreign diseases, viruses, and harmful bacteria. Therefore,the study of its mechanisms and functions is extremely important. One important feature of the immune system across a variety of species is how antibodies are passed from mothers to their newborn offspring [1]. Over time, natural selection has led to adaptation of antibodies to be passed down to offspring for survival, whether it is in the form of the IgG antibodies passing through the placenta in mammals, or birds passing down IgY antibodies in their egg yolk [1].

By studying the IgY antibodies found in chicken eggs, we can study and potentially introduce useful IgY antibodies into humans (for example, via the consuption of IgY Antibodies), in order to bolster our immune systems. IgY antibodies can be used to create immunization in a form that does not specifically require a vaccine [2]. People can be passively immunized against respiratory illness with the help of IgYs to help neutralize respiratory pathogens and disease spread. IgY is an ideal candidate for this antibody insertion due to its structural similarity to the IgG antibodies common in humans, as well as its ability to target specific antigens [3]. IgY has been found to bind specifically to influenza virus Nucleoprotein (NP) by detecting multiple virus subtypes in swine [4]. These traits make IgY a worthwhile subject of study, especially one whose potential outcomes are so beneficial. 

In this series of experiments, we focused on investigating IgY protein concentrations, abundance, and purity in potential sources for IgY protein purification, as the extent of these properties are not widely available in current literature. We used the Thermo ScientificTM PierceTM Chicken IgY Purification Kit to extract the IgY protein from the egg yolk [5]. The extraction of the IgY protein allowed us to conduct a Bradford assay, SDS PAGE, and a Densitometry in order to determine the concentration, abundance and purity of the protein. With the results of this experiment, we determined that we can effectively purify IgY from store-bought chicken eggs.

MATERIALS AND METHODS

PHYSICAL PURIFICATION OF IgY PROTEIN

We purified IgY from store-purchased Eggland Best eggs using the Pierce™ Chicken IgY Purification Kit from Thermo Fisher to physically separate the antibody rich yolk from the white using a series of suspensions and centrifugations with reagents. We conducted this by first separating the egg yolk from the egg whites. We then rolled the egg yolk on a paper towel to remove excess egg-white protein, pricked the yolk sac and then drained an approximate 8 mL of the yolk into a beaker. This step is essential to remove any contaminant protein from the egg white. We then added five times the egg yolk volume of cold Delipidation Reagent (40 mL) from the Thermo ScientificTM PierceTM Chicken IgY Purification Kit to the 8 mL egg yolk. The mixture was stirred until well incorporated and then stored at 4 ºC for a week. 

The mixture was then centrifuged for 15 min at 6,000 × g in a refrigerated centrifuge. The colorless and translucent supernatant was decanted and placed in a secondary conical tube, to which the precipitation reagent solutions from the Thermo ScientificTM PierceTM Chicken IgY Purification Kit were added. The mixture was placed in a 4 ºC incubator overnight, and then centrifuged for 15 min at 6000 rpm in a 4 ºC refrigerated centrifuge. We then isolated the pellet from the conical tube.

Our final step in the purification of the protein was to resuspend the pellet in Phosphate buffered saline (PBS) (100 mM Na3PO4, 150 mM NaCl, pH 7.2). We added the PBS to the pellet and stirred the mixture until it was well incorporated. 

BRADFORD ASSAY

Our next step was determining the concentration of the IgY protein using a Bradford assay. The assay uses acidic Coomassie dye to bind to proteins and result in a visible color change from brown to blue, the intensity depending on the amount of protein present. We measured out 5 mL of Bradford reagent into 8 glass test tubes. We pipetted Bovine Serum Albumin (BSA) protein into the first six test tubes, and IgY into the remaining two test tubes. With the BSA protein test tubes, we measured the absorbance at 595 nm for each solution with a spectrophotometer, and created a standard curve of the absorbance in relation to BSA protein amounts. We then measured the absorbance of our protein to be 0.383. 

SDS PAGE

Because current literature information on IgY proteins states that two chains of IgY should occur around 70 kDa and 30 kDa [6], we decided to make our gel a 12% acrylamide. With our SDS PAGE, we first created a standard mixture of 1 L Running buffer, 10 mL of 1x Separating buffer and 10 mL 1x Stacking buffer. We also created a 1 mL of 10% APS solution for activating the polymerization of the gel by dilution of stock APS available in the lab. We then added 100 uL of APS and 10 uL of TEMED into both the Stacking and Separating buffers. We then began to pipette the Separating buffer solution in between the glass plates until a marked line, allowing the Separating buffer to set with isopropyl alcohol before adding the Stacking buffer on top. While the Stacking buffer was still liquid, we inserted the comb to create the wells in the gel. 

We stored the gel for four days in a moist paper towel, then conducted SDS PAGE with 5 uL of our protein and 5 uL of sample buffer in each of our wells, with the Broad Range 5 Microliters NEB in one of the wells for reference. We let the SDS PAGE run for 90 min --the first 10 min at 120 V and the rest of the time at 180 V--until the bands reached the bottom of the gel, and then stained the gel with Coomassie blue solution. We then destained the gel using the premade solutions Destain #1 (50% methanol, 10% acetic acid) and Destain #2 (7% methanol, 10% acetic acid). 

DENSITOMETRY

This part of the experiment was to determine the concentration of the IgY bands. The densitometry was conducted in a similar fashion to the SDS PAGE. In order to address the possible presence of a dimer in our previous SDS-PAGE gel, we increased the concentration of BME in two of the wells with our IgY protein in the hopes of removing any remaining disulfide bonds. Instead of creating a gel for this experiment, we used a BIO-TEC premade gel. We created 5 solutions of different concentrations of BSA: 0.2 ug, 0.5 ug, 1 ug, 1.5 ug, and 2 ug. We then added 5 uL of these solutions, mixed with 5 uL 2x Laemmli buffer into separate wells. We also added our purified IgY protein into the gel, with two wells made with 5 uL of protein and 5 uL 2x Laemmli buffer each, and two wells with 5 uL protein and 5 uL 10% BME Laemmli buffer each. We ran the gel at 300 V for 15 min, until the dye front hit the bottom of the gel. We stained and destained the gel exactly as we did for the SDS PAGE. We then used a machine to analyze the concentrations and Molecular Weight (MW) of the bands of IgY in comparison to BSA.

RESULTS & DISCUSSION

The overall purpose of the experiments was to determine the practicality of extracting large quantities of protein from store-bought chicken eggs. From the experiments, we were able to determine the following:

PHYSICAL IgY PURIFICATION

The weight of our purified protein match the expected result of using the kit. We were able to purify 70.2756 mg of IgY solution from a single store-bought egg (Table 1).

Table 1. The total mass of IgY protein solution as a result of the initial purification of IgY protein with Thermo ScientificTM PierceTM Chicken IgY Purification Kit. 

BRADFORD ASSAY

Once our protein was purified, we were able to determine the concentration of the protein from a Bradford assay, comparing the absorbance of our protein to the absorbance of BSA. 

Figure 1. The absorbance of BSA over the concentration of BSA measured via a spectrophotometer, used as a standard curve to determine that the concentration of our IgY protein is 2.43 ug/uL.

Table 2. Calculated concentrations of our IgY solution as determined by a Bradford Assay. Conducted by measuring the absorbance of different volumes of the IgY solution in a spectrophotometer. These data were used to find the average concentration of the IgY protein to be 2.43 ug/uL. 

With these calculated concentrations (Table 2), we found that the average concentration of our IgY sample is 2.43 ug/uL. This matched expected results as previous studies have estimated that the concentration of IgY purified from egg yolk should be 2-4 mg/mL [7]. 

SDS-PAGE

Knowing the concentration of the protein helped us conduct SDS-PAGE, which would help us find out the size and purity of our purified IgY protein. 

Figure 2. SDS-PAGE of our IgY protein with clear bands around 27 kDa and 130 kDa. Done in a 12% acrylamide gel that was stained with Coomassie blue solution. The left-most well on the right end of the figure indicates standard protein molecular weights from the NEB Protein standard solution. The remaining wells have 5uL of 2.34 ug/uL of IgY and 5uL of 2X Laemmli buffer, as indicated at the bottom of each column in the figure.  

Figure 3. Graph of the log (molecular weight) of NEB Protein Standard as a function of relative distance. The curve between the distances 2.5 and 4.4 cm was used to find the MW of the heavy band to be 117.7 kDa. The curve between the distances 14.7 and 18.6 cm were used to find the MW of the second band to be 27.28 kDa.

Once we conducted our SDS-PAGE and stained/destained it, we found that the gel showed significant IgY purity because the most significant bands were at around 27 kDa and 130 kDa. We also found a light band around 43 kDa (Figure 2), which was confirmed to be the contaminant ovalbumin by our instructors (see acknowledgments). 

We calculated the MW of the bands to be 27.28 kDa and 117.7 kDa. In order to do so, we calculated the equation of the line between the points 2.5 and 4.4 cm to be logMW = -0.7169 x + 2.2932. When we plug 3.1 in for x, the MW ends up being 117.7 kDa. We also calculated the equation of the line between points 14.7 and 18.6 cm to be logMW = -0.02987x + 1.971. Plugging in 17.9 for x, we get MW = 27.28 kDa (Figure 3). 

We expected to have the band at 27.28 kDa for the IgY light chain, but our other expected value for the IgY proteins was around 70 kDa for the heavy chain, not 117.7 kDa, based on current literature on IgY. Because this 117.7 kDa band was almost exactly twice the weight of our expected heavy chain weight, we hypothesized that our protein’s disulfide bonds had not broken down completely. In order to understand whether this high molecular weight was a result of the small amount of BME in the 2x Laemmli solution, or was simply a contaminant, we conducted a densitometry with two of the wells containing our protein along with a higher concentration of BME. 

DENSITOMETRY

Figure 4. The results of our Coomassie stained and de-stained IgY and BSA Densitometry [left-most well: NEB marker. Well 2-3: 10% BME + IgY. Well 4-5: IgY + normal Coomassie. Wells 6-10: decreasing concentrations of BSA] The red arrow points to the heavy IgY chain, which is a band we did not observe in the previous (SDS PAGE) experiment. The blue arrow points to the light IgY chain. The yellow arrow points to the BSA chain. 

SDS-PAGE was run, this time with more BME and varied BSA amounts to both analyze the light/heavy chains and to possibly get a reading of the bands that would help us determine the concentration of protein in each band. Figure 4 shows there is clearly a large amount of BSA in wells 6-10, which would have been difficult to analyze our IgY density with, so we were unable to carry out analysis of the IgY. 

We found that the gel with the higher percentage of BME had a more prominent band around 72 kDa, along with bands around 27 kDa and 130 kDa. The finding of a band around 72 kDa and 27 kDa was consistent with current literature on IgY protein chains. The bands around 27 kDa and 130 kDa were consistent with our previous SDS PAGE (Figure 4). We still assume that the band around 130 kDa could be the IgY protein’s heavy chain that remains a dimer, however, there is a possibility that this could be a contaminant.  

This experiment does not tell us the identity of each of the protein bands found in the densitometry. If we had more time, we would have run the experiment again, using lower BSA concentrations and using the BSA band densities to make a standard curve in order to determine the total integrated density of the protein. From there, we would have calculated the density and concentration of the IgY, which would have told us the purity of the protein in relation to BSA. However, since we were unable to conduct this experiment, we suggest that further experiments should be performed to determine whether the IgY was effectively purified. 

CONCLUSION

Based on preliminary results, we can reasonably conclude that our IgY purification was successful and we were able to extract around 24.3 mg of pure IgY protein from the 8 mL of store-bought chicken eggs. Current literature indicates that the expected concentration of protein from each egg yolk is 2-4 mg/mL, which was what we observed in our results. Other studies using similar methods to our experiment have also found that it is possible to purify around 40-80 mg of IgY per egg using similar methods [7]. The reason we predict 40-80 mg per egg yolk is because we only extracted 8 mL of yolk, whereas the average egg yolk is about 15 mL, [8] so the total protein amount would be around 40 mg for 15 mL of yolk. The 24.3 mg of IgY extracted from one egg shows that eggs are a reliable source of IgY protein. Since each hen produces hundreds of eggs annually, and each egg contains such a large portion of IgY, it is reasonable to conclude that mass purification of IgY is feasible. 

The extraction of IgY has important implications for further application. In the future, we hope to see multiple pathways for immunization built from IgY. One is the immunization of humans through digestion of IgY antibodies [9]. Another is epitope mapping, [10] identification and characterization of antibody binding sites on cells, which could help us understand the structures of antigen binding sites to combat diseases. The results of our purification of IgY seem promising for the future of IgY extraction and execution of further research. 

ACKNOWLEDGEMENTS 

We thank Dr. Thomas and Dr. Catanese for all their support and mentorship throughout the course. OURI, CUR, and the Biosciences Department for funding BIOS 211.  Anika Sonig and Aaron Lin for their partnership through this project and aid with the creation of the figures. BIOS 211 TAs for their guidance. 

Works Cited

(1) Pereira, E P V et al. International immunopharmacology 2019 vol. 73, 293-303. doi:10.1016/j.intimp.2019.05.015  

(2) Aymn Talat Abbas, Sherif Aly El-Kafrawy, Sayed Sartaj Sohrab & Esam Ibraheem Ahmed Azhar, Human Vaccines & Immunotherapeutics. 2019, 15:1, 264-275, doi: 10.1080/21645515.2018.1514224  

(3) Nagaraj et al. International Journal of Food Microbiology. 2016, 237, 136-141.

(4) da Silva M.C, Schaefer R, Gava D, Journal of Immunological Methods. 2018, 461, 100-105. https://doi.org/10.1016/j.jim.2018.06.023. 

(5) ThermoScientific PierceChicken IgY Purification Kit manual https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011404_Pierce_Chicken_IgY_Purifi_UG.pdf

(6) Sudjarwo, S. A., Eraiko, K., Sudjarwo, G. W., & Koerniasari, Journal of advanced pharmaceutical technology & research. 2017, 8(3), 91-96. https://doi.org/10.4103/japtr.JAPTR_167_16 

(7) Pauly, Diana, et al. Journal of Visualized Experiments. 2011, www.ncbi.nlm.nih.gov/pmc/articles/PMC3197133/.    

(8) Egg Cooking Tips: Cooking With Eggs: Egg Farmers Of Alberta. eggs.ab.ca/eggs/egg-cooking-tips/. (Accessed November 24, 2021)

(9) Constantin, C.; Neagu, M.; Diana Supeanu, T.; Chiurciu, V.; A. Spandidos, D, Exp Ther Med, (2020), 20, 151–158. https://doi.org/10.3892/etm.2020.8704. 
(10) Lu, Y.; Wang, Y.; Zhang, Z.; Huang, J.; Yao, M.; Huang, G.; Ge, Y.; Zhang, P.; Huang, H.; Wang, Y.; Li, H.; Wang, W, Journal of Immunology Research (2020). https://doi.org/10.1155/2020/9465398.