IgY antibodies are immunoglobulins derived from the egg yolks of birds, functioning similarly to IgG antibodies in mammals. Found in the serum and egg yolks of birds, reptiles, and amphibians, IgY has a distinct molecular structure, lacking the Fc fragment that binds to mammalian complement or Fc receptors. This feature reduces non-specific reactions, making IgY highly specific and advantageous in research. IgY antibodies hold significant value in biomedical research, particularly as an alternative to traditional mammalian antibodies. They are produced by immunizing hens with antigens, followed by antibody extraction from the egg yolk, offering a cost-effective, non-invasive, and sustainable production method.
Fig.1 Development of the IgY antibodies and their potential mechanisms of action.1
Isolation of IgY
The isolation of IgY antibodies primarily involves removing high levels of lipids and lipoproteins from egg yolks to obtain the water-soluble fraction. Several methods are commonly employed for this purpose:
- Organic Precipitation Method
This method utilizes substances like polyethylene glycol (PEG) or dextran sulfate (DS). The yolk is diluted fivefold with PBS or TBS buffer, followed by the addition of PEG6000 or DS at specific ratios. Lipids precipitate, leaving IgY in the supernatant. Dextran sulfate is particularly advantageous due to its simplicity, efficiency, and rapid processing.
- Organic Solvent Extraction Method
Lipids, being soluble in organic solvents, can be separated using agents like chloroform. A yolk treated with TBS produces 15 mL of yolk solution. After adding 40 mL of TBS and 40 mL of chloroform, the mixture undergoes freezing centrifugation, separating into three phases. The chloroform layer is discarded, the aqueous layer is retained, and the intermediate semi-solid phase is re-extracted with TBS. Combining the aqueous layers yields the IgY-rich water-soluble fraction. This method is effective but requires careful handling of organic solvents to ensure safety and minimize environmental impact.
- Natural Gum Method
Natural gums like carrageenan and xanthan gum can be used to remove lipids. A 0.1% (w/v) carrageenan solution (9x volume of yolk) is mixed with yolk and centrifuged to obtain the water-soluble fraction. Alternatively, diluting yolk 1:1 with distilled water and mixing it with xanthan gum (4x volume) achieves similar results. This method ensures minimal lipid content (<0.4%) in the aqueous solution, making it ideal for downstream applications requiring low lipid interference.
- Medium-Chain Fatty Acid Method
Medium-chain fatty acids, such as caprylic acid, are effective for IgY separation. The yolk is diluted 7.5x with deionized water and centrifuged. The supernatant is further diluted twofold with acetate buffer (pH 5.0), and 1% caprylic acid is added. Upon standing, the solution separates into layers, with IgY in the aqueous layer. This method is simple and efficient but may require pH adjustment for optimal separation.
- Detergent Separation
Surfactants like cetyltrimethylammonium bromide or Triton X-114 can efficiently separate lipids. Using Triton X-114 achieves a protein recovery rate of 85%-95%. Yolk is diluted 20x with TBS, and the supernatant is mixed with 6% TX-114. The mixture is heated to 37°C to form layers, with lipids in the detergent phase and IgY in the aqueous phase. Triton X-100 yields a protein recovery rate of 97%, combining simplicity, minimal antibody impact, and non-toxicity.
- Water Dilution Method
Yolk is diluted 10x with water, pH adjusted to 5.2, and incubated at 4°C for 6 hours. The supernatant constitutes the water-soluble fraction. Alternatively, a sevenfold water dilution is sufficient. While this method is straightforward and cost-effective, protein recovery may vary depending on pH and incubation conditions.
- Supercritical Fluid Extraction
This advanced method involves spray-drying yolks to produce powder, followed by lipid removal using supercritical CO₂ extraction. Under high pressure, CO₂ dissolves lipophilic substances, which are separated in a depressurized chamber. The defatted yolk powder is reconstituted in 20x phosphate buffer, stirred, and centrifuged to yield the water-soluble IgY fraction. This method offers high efficiency but requires specialized equipment.
Purification of IgY
After isolation, IgY is purified from the aqueous solution to achieve high purity. This is typically done using ultrafiltration or precipitation methods.
- Ultrafiltration
Ultrafiltration simultaneously concentrates, desalts, and purifies IgY. A molecular weight cut-off (MWCO) membrane of 100 kDa is commonly used to retain IgY (180 kDa) while removing impurities. This method is scalable and minimizes chemical use, but it requires specific equipment.
- Inorganic Precipitation
Inorganic salts like ammonium sulfate and sodium sulfate are used to precipitate IgY under high-salt conditions. For example, crude IgY can be obtained by freeze-drying the yolk supernatant, followed by sequential precipitation using 60% ammonium sulfate and 14% sodium sulfate. This approach achieves a recovery rate of 94% and a purity of 97%. Residual salts are removed through dialysis.
- Organic Solvent Precipitation
A two-step precipitation process using 12% PEG and -20°C 50% ethanol achieves IgY with a recovery concentration of 4.9 mg/mL and 89% purity. Variants of this method include combining PEG, ethanol, and other agents to enhance IgY yield and purity.
- Combined Precipitation
Combining different precipitants often yields higher purity IgY. For example, PEG8000 and ammonium sulfate, or sodium sulfate and caprylic acid, can be sequentially used. This multi-step process ensures efficient removal of impurities while maintaining high antibody activity.
Conclusion
IgY antibodies are widely applied in pathogen detection, drug development, disease research, and food safety due to their high specificity, low immunogenicity, and absence of cross-reactivity with mammalian systems. These characteristics make IgY an essential tool in various diagnostic and research applications. Creative Biolabs has extensive expertise in IgY production and purification. For more information on our IgY antibody services or other non-IgG antibodies, please feel free to contact us.
| Services | Features | Price |
| IgY Production and Purification | Creative Biolabs has established a comprehensive platform offering a variety of non-IgG related services to global clients. IgY production and purification is one of what we are good at. | Inquiry |
| IgM Production and Purification | Based on our comprehensive antibody platforms and professional experts, Creative Biolabs has absolute advantages in providing IgM production and purification services. | Inquiry |
| IgA Production and Purification | Our seasoned scientists can customize the most suitable IgA products and services according to the specific requirements of customers. | Inquiry |
| IgE Production and Purification | Our purification strategies utilize multiple methods, such as affinity chromatography, and size-exclusion chromatography, specifically designed to handle IgE's complex properties and ensure the highest purity. | Inquiry |
Reference
- El-Kafrawy, Sherif A., et al. "IgY antibodies: The promising potential to overcome antibiotic resistance." Frontiers in Immunology 14 (2023): 1065353. Distributed under Open Access license CC BY 4.0, without modification.
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