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PF IgY (A) and yield (B, %) obtained for each system with different amounts of WSPF and 20 wt% of Triton X-114, upon phase separation at 35.0 ± 0.1 • C.
Source publication
Immunoglobulin Y (IgY) represents an important class of antibodies, being present in egg yolk, with relevant medical applications and involving non-invasive methods of extraction. However, due to the complexity of egg yolk, the purification levels required for use in most medical applications demands the application of multi-step and cost-intensive...
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... may contribute to the IgY purification factor increase in the surfactant-poor phase. As a second optimization step, the WSPF concentration was studied, keeping constant the surfactant concentration at 20 wt% and varying the WSPF content (10.0, 17.5 and 25.0 wt%) in the AMTPS. The respective PF IgY and extraction yield results are presented in Fig. 4. Overall, the increment in the WSPF concentration leads to an increase in the IgY purification factor (Fig. 4.A), meaning that more IgY is being retained in the surfactant-poor phase when compared to the remaining proteins. These results also suggest that none of the AMTPS phase has reached saturation. This set of studies allowed a PF ...
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... step, the WSPF concentration was studied, keeping constant the surfactant concentration at 20 wt% and varying the WSPF content (10.0, 17.5 and 25.0 wt%) in the AMTPS. The respective PF IgY and extraction yield results are presented in Fig. 4. Overall, the increment in the WSPF concentration leads to an increase in the IgY purification factor (Fig. 4.A), meaning that more IgY is being retained in the surfactant-poor phase when compared to the remaining proteins. These results also suggest that none of the AMTPS phase has reached saturation. This set of studies allowed a PF IgY increase from 1.28 ± 0.01 to 1.48 ± 0.02, with 25 wt% of WSPF, without any IgY loss as can be seen through ...
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... that more IgY is being retained in the surfactant-poor phase when compared to the remaining proteins. These results also suggest that none of the AMTPS phase has reached saturation. This set of studies allowed a PF IgY increase from 1.28 ± 0.01 to 1.48 ± 0.02, with 25 wt% of WSPF, without any IgY loss as can be seen through the IgY yield given in Fig. ...
Citations
... Madera- Contreras et al. (2022) optimized the PEG 6000 extraction method developed by Pauly et al. (2011), adding an extra step to enhance IgY purity. In a different approach, Vicente et al. (2022) employed thermoresponsive aqueous micellar two-phase systems with ionic liquids, achieving a promising one-step (54 % purity) or two-step (69 % purity) IgY purification method. Similarly, Almeida et al. (2022) used aqueous biphasic systems of PEG 1000 and phosphate buffer combined with centrifugal partition chromatography, reaching up to 56 % IgY purity. ...
This study presents a novel and efficient method for extracting immunoglobulin Y (IgY) antibodies from egg yolk based on the principle of liquid-liquid phase separation (LLPS) induced by polyethylene glycol 8000 (PEG 8000). Initial delipidation of egg yolk samples with varying PEG 8000 concentrations demonstrated optimal delipidation efficiency and protein recovery at 2.5% PEG 8000 concentration. Subsequent IgY extraction involved inducing LLPS by raising PEG 8000 concentration to 6.5%, resulting in turbid solutions and the formation of globular droplet-like condensates observed under a microscope. Unlike the PEG 6000 method that induced aggregation, the method developed here using PEG 8000 does not lead to the appearance of aggregates of IgY. SDS-PAGE analysis confirmed that IgY extracted was no different from the conventional PEG 6000 method, with similar purity levels (77% vs 79%). Enzyme-linked immunosorbent assay and western blot analysis confirmed the antigen recognition properties of the isolated IgY. This method significantly reduces the amount of PEG used, leading to substantial cost savings compared to PEG 6000. The method can be completed within one hour. Despite a slightly lower IgY yield by the method, the time- and cost-saving advantages of this method make it a promising alternative for IgY extraction in research. This proposed IgY extraction technique utilizing protein LLPS has the potential to improve the study of the physicochemical properties of IgY and optimized production, while offering a quicker and cost-effective solution for various applications in biomedical research.
... That is why, recently, the utilization of laying hens as immunoglobulin producers has received wider attention. Methods to isolate IgY from chicken eggs have recently been intensified [10]. On the other hand, methods to isolate immunoglobulin from chicken serum have not been widely explored. ...
This study aimed to isolate IgG from chicken serum using a combination of coconut caprylic triglyceride and ammonium sulfate precipitation techniques. This study used serum from chickens that had previously been vaccinated repeatedly using a commercial rabies vaccine. Initially, the serum was treated with 2.5% coconut caprylic triglyceride, followed by serum IgG precipitation using 40% ammonium sulfate. The 10% SDS-PAGE analysis showed that a protein of about 190 kDa with a purity above 95% was successfully isolated. Whether the isolate protein is IgG and specific to rabies antigen still needs to be investigated further.
... [C16 MIM][Cl]) and the non-ionic surfactant Triton X-114 was used as the extraction phase in both works. The SAIL-CPE technique was also used for the purification of immunoglobulin Y from egg yolk using a Triton X-114/1-tetradecyl-3-methylimidazolium chloride ([C 14 MIM][Cl]) mix[66] . ...
Cloud-point extraction (CPE) is a pre-treatment technique for the extraction and preconcentration of different chemical compounds, such as metal ions, pesticides, drugs, phenols, vitamins etc., from various samples. CPE is based on the phenomenon of two phases (micellar and aqueous) forming after the heating of an aqueous isotropic solution of a non-ionic or zwitterionic surfactant above the cloud-point temperature. If analytes are added to the surfactant solution under suitable conditions, they should be extracted into the micellar phase, also called the surfactant-rich phase. Recently, the traditional CPE procedure is being increasingly replaced by improved CPE procedures. In this study, recent advances in CPE over the last three years (2020 - 2022), including the application of various innovative approaches, are reviewed. In addition to the basic principle of CPE, alternative extraction media in CPE, CPE supported by various auxiliary energies, a different modified CPE procedure and the use nanomaterials and solid-phase extraction in combination with CPE are presented and discussed. Finally, some future trends for improved CPE are presented.
... IL's outstanding properties make them attractive for catalysis, electrochemistry, separation, and extraction, among others (Fontanals et al., 2009;Kianfar and Mafi, 2020). Although less investigated, within the pharmaceutical field, ILs have already shown to be important enhancers of the solubility, activity, permeability, and stability of several synthetic drugs and biopharmaceuticals (Vijayaraghavan et al., 2010;Byrne et al., 2012;Foureau et al., 2012;Kumar and Venkatesu, 2013;Mukesh et al., 2013;Kumar and Venkatesu, 2014;Mazid et al., 2015;Todinova et al., 2016;Banerjee et al., 2018;Jagannath et al., 2018;Pedro et al., 2018;Reslan et al., 2018;Guncheva et al., 2019;Lin et al., 2019;Quental et al., 2019;Mandal et al., 2020;Lin et al., 2021;Dhiman et al., 2022;Vicente et al., 2022), and also in the extraction and purification of these biomolecules (Shu et al., 2010;Qu et al., 2012;Yuan et al., 2012;Chen et al., 2015;Ding et al., 2015;Ren et al., 2015;Taha et al., 2015;Ferreira et al., 2016;Mondal et al., 2016;Ramalho et al., 2018;Santos et al., 2018;Song et al., 2018;Xu et al., 2018;Capela et al., 2019;Magri et al., 2019;Marchel et al., 2019;Quental et al., 2019;Vicente et al., 2019;Castro et al., 2020;Neves et al., 2020;Vicente et al., 2022;Capela et al., 2023). therapeutic proteins, including monoclonal antibodies (mAbs) (Rosa et al., 2010;Foldvari et al., 2016;Chen et al., 2018;Tavares et al., 2019;O'Flaherty et al., 2020). ...
... IL's outstanding properties make them attractive for catalysis, electrochemistry, separation, and extraction, among others (Fontanals et al., 2009;Kianfar and Mafi, 2020). Although less investigated, within the pharmaceutical field, ILs have already shown to be important enhancers of the solubility, activity, permeability, and stability of several synthetic drugs and biopharmaceuticals (Vijayaraghavan et al., 2010;Byrne et al., 2012;Foureau et al., 2012;Kumar and Venkatesu, 2013;Mukesh et al., 2013;Kumar and Venkatesu, 2014;Mazid et al., 2015;Todinova et al., 2016;Banerjee et al., 2018;Jagannath et al., 2018;Pedro et al., 2018;Reslan et al., 2018;Guncheva et al., 2019;Lin et al., 2019;Quental et al., 2019;Mandal et al., 2020;Lin et al., 2021;Dhiman et al., 2022;Vicente et al., 2022), and also in the extraction and purification of these biomolecules (Shu et al., 2010;Qu et al., 2012;Yuan et al., 2012;Chen et al., 2015;Ding et al., 2015;Ren et al., 2015;Taha et al., 2015;Ferreira et al., 2016;Mondal et al., 2016;Ramalho et al., 2018;Santos et al., 2018;Song et al., 2018;Xu et al., 2018;Capela et al., 2019;Magri et al., 2019;Marchel et al., 2019;Quental et al., 2019;Vicente et al., 2019;Castro et al., 2020;Neves et al., 2020;Vicente et al., 2022;Capela et al., 2023). therapeutic proteins, including monoclonal antibodies (mAbs) (Rosa et al., 2010;Foldvari et al., 2016;Chen et al., 2018;Tavares et al., 2019;O'Flaherty et al., 2020). ...
... Several IL-based approaches, namely liquid-liquid and solid-liquid extractions, have been proposed for the efficient recovery and purification of a myriad of compounds, such as therapeutic enzymes , antibodies (Taha et al., 2015;Ferreira et al., 2016;Mondal et al., 2016;Ramalho et al., 2018;Capela et al., 2019;Vicente et al., 2022), nucleic acids (Pereira et al., 2021), viruses (Marchel et al., 2019), and interferons (Castro et al., 2020). Liquidliquid extraction based on ILs has been proposed for the recovery and purification of a variety of biopharmaceuticals, most of them relying on the use of IL-based Aqueous Biphasic Systems (IL-ABS). ...
The emergence of biopharmaceuticals, including proteins, nucleic acids, peptides, and vaccines, revolutionized the medical field, contributing to significant advances in the prophylaxis and treatment of chronic and life-threatening diseases. However, biopharmaceuticals manufacturing involves a set of complex upstream and downstream processes, which considerably impact their cost. In particular, despite the efforts made in the last decades to improve the existing technologies, downstream processing still accounts for more than 80% of the total biopharmaceutical production cost. On the other hand, the formulation of biological products must ensure they maintain their therapeutic performance and long-term stability, while preserving their physical and chemical structure. Ionic-liquid (IL)-based approaches arose as a promise alternative, showing the potential to be used in downstream processing to provide increased purity and recovery yield, as well as excipients for the development of stable biopharmaceutical formulations. This manuscript reviews the most important progress achieved in both fields. The work developed is critically discussed and complemented with a SWOT analysis.
... In fact, one of the main challenges for the broader use of IgY technology is to obtain highly purified compositions. Currently, most innovative methods for the depletion of protein contaminants from the water-soluble protein fraction of the egg yolk include anion-exchange chromatography (Zhou et al. 2020;González Viacava et al. 2022) and aqueous biphasic systems (Vicente et al. 2022). ...
The use of egg yolk antibodies—IgY technology—represents an alternative to the production of mammalian immunoglobulins and has several advantages regarding animal welfare and lower costs of production. The use of adjuvants to achieve the hyperimmunization of laying hens plays a key role in the success of the production of high levels of the antibodies. In the present work, two different adjuvant systems (Freund’s adjuvants and MontanideTM ISA 71 VG) were compared to produce IgY anti-Bothrops alternatus. For the first immunization, formalin-inactivated Salmonella was added to MontanideTM ISA 71 VG to emulate Freund’s complete adjuvant which includes a mycobacteria antigen. After eight immunizations, IgY produced by using either adjuvant was able to neutralize the lethal activity of the venom in a mouse model, but differences were found regarding the recognition of components of the venom between the two adjuvants tested. Overall, MontanideTM adjuvant used in this work could be a good alternative choice to produce antibodies capable of neutralizing the lethality of complex antigens. This adjuvant is commercially available and used in the formulation of several poultry vaccines and could be used for the IgY technology instead of traditional immunomodulators such as Freund’s adjuvants.
Key points
• IgY extracts recognized major components of the venom.
• Avidity indexes of the IgY extracts increased after the successive immunizations.
• IgY obtained by two adjuvant systems neutralized the lethal activity of the venom.
... IgY purification from a chicken egg. IgY was purified using the Pierce® Chicken IgY purification kit (Thermo Scientific, US) according to the manufacturer's instructions [13]. Eggs from the same hen were pooled together for IgY purification. ...
Coronavirus disease (COVID)-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has become a global pandemic disease that has social and economic chaos. An alternative mitigation strategy may involve the use of specific immunoglobulin (Ig)-Y derived from chicken eggs. Our study aimed to evaluate the neutralizing potential of specific IgY targeting S1, receptor-binding-domain (RBD) of spike glycoprotein and nucleocapsid (N) of SARS-CoV-2 to inhibit RBD and angiotensin-converting-enzyme-2 (ACE2) binding interaction. Hy-Line Brown laying hens were immunized with recombinant S1, RBD spike glycoprotein, and nucleocapsid (N) of SARS-CoV-2. The presence of specific S1,RBD,N-IgY in serum and egg yolk was verified by indirect enzyme-linked immunosorbent assay (ELISA). Specific S1,RBD,N-IgY was purified and characterized from egg yolk using sodium-dodecyl-sulfate-polyacrylamide-gel-electrophoresis (SDS-PAGE), and was subsequently evaluated for inhibition of the RBD-ACE2 binding interaction in vitro. Specific IgY was present in serum at 1 week post–initial immunization (p.i.i), whereas its present in egg yolk was confirmed at 4 weeks p.i.i. Specific S1,RBD,N-IgY in serum was able to inhibit RBD-ACE2 binding interaction between 4 and 15 weeks p.i.i. The results of the SDS-PAGE revealed the presence of bands with molecular weights of 180 kDa, indicating the presence of whole IgY. Our results demonstrated that S1,RBD,N-IgY was able to inhibit RBD-ACE2 binding interaction in vitro, suggesting its potential use in blocking virus entry. Our study also demonstrated proof-of-concept that laying hens were able to produce this specific IgY, which could block the viral binding and large production of this specific IgY is feasible.
The cloud point extraction (CPE) approach has received a lot of interest, and many studies and reviews have already been written on this topic. The CPE is a good separation technique even at ultra-trace analyte levels. The researchers proposed numerous adjustments to make it environmentally friendly, affordable, and precise. Several reviews were handled in conventional CPE, but no analysis was offered that evaluated new CPE procedures based on green solvent-assisted surfactants and evaluated greenness scores. The present study tries to address the observed issue. This overview looks at the clouding phenomena and the applicability of CPE techniques that use green solvents. These approaches involve mixed-micellar media CPE (MM-CPE), ionic liquids CPE (IL-CPE), rapid synergistic CPE based on deep eutectic solvents (RS-DES-CPE), and others. They are simple to apply, efficient, fast, and safe. Also, they are reducing the heating step, chemical amounts, and extraction time compared with the conventional CPE methods. Additionally, the mechanisms and interactions of green solvents and surfactants were discussed.
The green solvent selection website and three recent greenness tools have been described, such as Sample Preparation Metric of Sustainability (SPMS), Complex Modified GAPI (ComplexMoGAPI), and Analytical Greenness Metric of Sample Preparation (AGREEprep). Also, a critical comparison was made between the greenness ratings of CPE approaches. In this overview, we present readers with classified descriptions and comparisons of CPE forms that have green solvent-assisted surfactants to help them acquire the knowledge they need for future CPE design. In addition, multiple limitations and potential patterns were introduced.
