Purification of Antibodies from Industrially Separated Egg Yolk

Abstract

Industrially separated egg yolk was diluted and water soluble proteins separated by sedimentation. The supematant was filtered and applied to a column packed with cation exchanger within an automated liquid chromatography system. This was scaled-up from a 50 mL to a 1500 mL column. Two cation exchangers were investigated and immunoglobulin recoveries of 60-65% were obtained with 6069% purities. Batch separation resulted in lower recovery (55%) and purity (57%). Further purification was investigated using anion exchange chromatography and salt precipitation. Results were improved with one step salt precipitation where purity was increased. The process is simple, economical and should prove useful for large production of IgY.

Avian egg antibodies: basic and potential applications

“…Chang et al (2000) recently reported the precipitation of over 90% of lipoproteins from yolk using lcarrageenan, sodium alginate, carboxymethyl cellulose, and pectin. Ion exchange chromatography has also been reported as a ®nal step in IgY puri®cation (McCannel and Nakai, 1990;Akita and Nakai, 1992;Fichtali et al, 1992Fichtali et al, , 1993, as well as hydrophobic interaction chromatography (Hassl and Aspock, 1988). Because of the failure of IgY to bind proteins A and G, and its sensitivity to traditional af®nity puri®cation conditions, several other methods of af®nity chroma-tography have been examined for the puri®cation of IgY, including immobilized metal ion af®nity chromatography (McCannel and Nakai, 1989;Greene and Holt, 1997), thiophilic interaction chromatography (Hansen et al, 1998), af®nity chromatography using alkaline conditions (Kuronen et al, 1997), and synthetic peptide ligands, designed speci®cally for immobilizing antibodies (Fassina et al, 1998;Verdoliva et al, 2000).…”

“…Similar to IgG puri®cation, ammonium sulfate precipitation has also been reported for the puri®cation of IgY from WSF, following lipoprotein precipitation (Akita and Nakai, 1992;Svendsen et al, 1995). Other methods of IgY separation include: lipoprotein separation by ultracentrifugation (McBee and Cotterill, 1979), delipidation by organic solvents (Bade and Stegemann, 1984;Polson et al, 1985;Hatta et al, 1988;Polson, 1990;Kwan et al, 1991;Horikoshi et al, 1993;McLaren et al, 1994;Svendsen et al, 1995), lipoprotein precipitation by polyethylene glycol (Polson et al, 1980b, Akita andNakai, 1993;Svendsen et al, 1995), sodium dextran sulfate (Jensenius et al, 1981;Akita and Nakai, 1993), and dextran blue (Bizhanov and Vyshniausskis, 2000), and natural gums such as xanthan gum (Akita and Nakai, 1993a) and sodium alginate (Hatta et al, 1990). Chang et al (2000) recently reported the precipitation of over 90% of lipoproteins from yolk using lcarrageenan, sodium alginate, carboxymethyl cellulose, and pectin.…”

The potential application of avian egg antibodies with emphasis on immunotherapeutic and immunodiagnostic purpose

“…Chang et al (2000) recently reported the precipitation of over 90% of lipoproteins from yolk using lcarrageenan, sodium alginate, carboxymethyl cellulose, and pectin. Ion exchange chromatography has also been reported as a final step in IgY purification (Fichtali et al, 1993), as well as hydrophobic interaction chromatography (Hassl and Aspock, 1988).immobilized metal ion affinity chromatography (Greene and Holt, 1997), thiophilic interaction chromatography (Hansen et al, 1998), affinity chromatography using alkaline conditions (Kuronen et al, 1997), and synthetic peptide ligands, designed specifically for immobilizing antibodies (Verdoliva et al, 2000). As well, Erhard et al (1996) described a method for the purification of mouse IgG subclass specific IgY using indirect affinity chromatography with protein G Sepharose (Deignan et al, 2000).…”

Development of simple and rapid procedures for purification of egg yolk immuno-globulin and ovotransferrin from chicken egg and antimicrobial activity of ovo-transferrin against Escherichia coli O157:H7 and Listeria monocytogenes

“…After removal of lipids and lipoproteins from egg yolk, egg yolk antibodies were precipitated by ammonium sulfate Nakai, 1992, 1993;Hansen et al, 1998), sodium sulfate or caprylic acid-ammounium sulfate McLaren et al, 1994). From the precipitated egg yolk proteins, antibodies were further purified by affinity column chromatography (Greene and Holt, 1997;Yokoyama et al, 1993), gel filtration (Devi et al, 2002), cation exchange chromatography (Fichtali et al, 1992(Fichtali et al, , 1993 or DEAE-sephacel anion exchange chromatography . suggested that a protocol using ammonium sulfate precipitation followed by ultrafiltration and gel filtration was the most efficient for IgY purification.…”