Isolation and purification of immunoglobulins from ...

Journal of Immunological Methods 215 Ž1998. 1–7

Isolation and purification of immunoglobulins from chicken eggs using thiophilic interaction chromatography Per Hansen, Judith A. Scoble, Brendon Hanson, Nicholas J. Hoogenraad

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School of Biochemistry, La Trobe UniÕersity, Bundoora 3083, Victoria, Australia Received 16 May 1997; revised 25 November 1997; accepted 19 January 1998

Abstract We report on the use of thiophilic interaction chromatography for the purification of IgY from egg yolk. This procedure permits the purification to homogeneity of IgY in a single chromatographic step after ammonium sulfate fractication. This study also compares the use of an improved T-gel which has a higher capacity for immunoglobulin than the original T-gel, having a capacity in excess of 25 mg IgYrml resin. The recovery from this procedure is close to 100%, providing a simple and efficient means for purifying IgY from egg yolk. We also determined that the amount of specific antibody present in egg yolk from an immunised chicken is around 1% of total IgY. q 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Thiophilic chromatography; Immunoglobulin Y

1. Introduction Immunoglobulins from immunised chickens are a useful source of polyclonal antibodies. Due to the phylogenetic distance between birds and mammals, antibodies against conserved mammalian proteins are often produced more readily in chickens than in rabbits ŽCarroll and Stollar, 1983; Song et al., 1985; Gassmann et al., 1990; Goueli et al., 1990; Lee et al., 1991; Ntakarutimana et al., 1992; Hatta et al., 1993; Shimizu et al., 1995; Wooley and Landon, Abbreviations: ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; IgY, immunoglobulin Y; GST, glutathione S-transferase; DhTom20, soluble form of mitochondrial protein import receptor with residues 1–29 deleted; PBS, phosphate buffered saline; SAS, saturated ammonium sulfate ) Corresponding author. Fax: q61-3-9479-2467; e-mail: [email protected].

1995.. Birds, like mammals, pass on immunity to their offspring, and antibodies produced in immunised chickens are transferred from the serum to the egg yolk ŽJensenius et al., 1981.. The antibodies are thus produced in a conveniently packaged form and can be collected on a daily basis without invasive procedures such as bleeding, conforming better to today’s standards of animal-care. The antibodies can be stored in the eggs at 48C for at least 1 year ŽJensenius et al., 1981. and large amounts of antibody can be obtained at a relatively low cost. Several papers ŽRose et al., 1974; Jensenius et al., 1981; Gottstein and Hemmeler, 1985; Hatta et al., 1993. report that much higher amounts of specific immunoglobulin can be obtained in the eggs of immunised chickens than from the serum of rabbits. The antibody in egg yolk has been termed IgY ŽYolk Immunoglobulin. because it differs in molecu-

0022-1759r98r$19.00 q 1998 Published by Elsevier Science B.V. All rights reserved. PII S 0 0 2 2 - 1 7 5 9 Ž 9 8 . 0 0 0 5 0 - 7

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P. Hansen et al.r Journal of Immunological Methods 215 (1998) 1–7

lar weight and isoelectric point from mammalian IgG ŽBenedict and Berestecky, 1987.. In addition, IgY binds neither to mammalian complement or Fc-receptors ŽBenson et al., 1961. nor does it interfere with rheumatoid factors ŽLarsson et al., 1991., which has proven advantageous in many immunological tests. Furthermore, IgY from chickens is not recognised by Staphylococcus protein A or Streptococcus protein G ŽKronvall et al., 1970, 1974.. Several procedures for the isolation and purification of IgY from egg yolk have been described previously ŽJensenius et al., 1981; Song et al., 1985; Hassl and Aspock, 1988; Gassmann et al., 1990; ¨ Polson, 1990; Akita and Nakai, 1992, 1993.. As the yolk consists of almost 50% non-aqueous material the first step involves isolation of IgY in a water soluble fraction circumventing the tendency of IgY to fractionate with the yolk lipids. The next step generally involves the precipitation of IgY with sodium sulfate, ammonium sulfate or polyethylene glycol. The final step involves chromatographic procedures such as ion-exchange chromatography and gel filtration. Thiophilic interaction chromatography was first described by Porath et al. Ž1985.. The adsorbent, consisting of 2-mercaptoethanol reacted with a divinylsulfone activated support, was named ‘T-gel’ and showed selective binding of immunoglobulins in the presence of structure-forming salts. The T-gel has since been successfully used in the purification of antibodies from serum Žhuman, rat, rabbit and bovine ŽPorath et al., 1985, Porath and Belew, 1987; Lihme and Heegaard, 1991.. and bovine milk whey ŽKonecny et al., 1994. and in the isolation of monoclonal antibodies from hybridoma supernatants and ascitic fluid ŽBelew et al., 1987; Porath and Belew, 1987; Serres et al., 1995.. There have also been reports on the use of the T-gel in the purification of recombinant single chain antibody fragments ŽSchulze et al., 1994. and Fab fragments ŽYurov et al., 1994; Lutomski et al., 1995.. The advantages of producing specific antibodies in chicken and the difficulties in applying standard IgG purification procedures in the purification of IgY prompted us to investigate the use of thiophilic interaction chromatography for the purification of IgY from chicken eggs. We also report on the isolation and quantitation of specific antibodies from eggs

of chickens immunised with the human protein import receptor of mitochondria, hTom20 ŽHanson et al., 1996..

2. Materials and methods 2.1. Immunisation of chickens Chickens ŽWhite Leghorn, 20 weeks old. were immunised with 100 m g GST-DhTom20 ŽHanson et al., 1996. in complete Freund’s adjuvant followed by 100 m g GST-DhTom20 3 weeks later in incomplete adjuvant. The final boost was with 100 m g of cleaved and purified DhTom20. Immunization was subcutaneous in the breast region. Eggs were collected daily one week after the final boost and the titre of specific antibody was determined in an aqueous extract of yolk by ELISA Žsee below.. The eggs were stored at 48C for up to 12 months, before use. 2.2. Extraction The extraction of IgY and other water soluble proteins from egg yolk was adapted from the work by Akita and Nakai Ž1992.. The yolk from eight eggs Žtotal 80 ml. was separated from the white and carefully washed with deionised water. The yolk sac was disrupted by inserting a needle and the contents allowed to drip through a nylon mesh into a measuring cylinder. The egg yolk was diluted 10 times with cold 3 mM HCl to give the suspension a final pH of 5 Žadjusted with 10% acetic acid.. The suspension was incubated for at least 6 h at 48C before the supernatant containing the IgY was collected by centrifugation Ž10 000 = g for 15 min at 48C.. Solid ammonium sulfate was added to 60% saturation Ž390 grl. and the mixture stirred in the cold for 15 min. The precipitate was collected by centrifugation and washed once with 60% saturated ammonium sulfate ŽSAS.. 2.3. T-gel chromatography The precipitate was redissolved in 50 mM phosphate buffer pH 7.4 containing 0.5 M sodium sulfate Žapproximately 10 times the pellet volume.. Small amounts of undissolved material were removed by


centrifugation. Protein Ž200 mg. was applied at a linear flow rate of 15 cmrh Ž0.2 mlrmin. to a 2-ml T-gel column Ž2.6 cm = 1.0 cm I.D.. equilibrated with 50 mM phosphate buffer pH 7.4 containing 0.5 M sodium sulfate and the column effluent was monitored at 280 nm. The column was washed with equilibration buffer until base line absorbance was reached. Bound proteins were then eluted with 50 mM phosphate buffer pH 7.4. The purified IgY was stored as a 60% SAS fraction at 48C. 2.4. T-gel T-gel was prepared with improved binding capacity following the procedure of Scoble and Scopes Ž1997.. Sepharose CL-4B was activated for 6 h at pH 12 with 80 m l divinylsulfone ŽDVS. per ml of gel. The gel, activated to 48 m mol active groups per ml ŽScoble and Scopes, 1996., was reacted to completion with 0.1 M 2-mercaptoethanol for 1 h at pH 11. This T-gel ŽST-gel. was compared to the original T-gel ŽPT-gel. prepared according to the method of Porath et al. Ž1985. in which the DVS activated support has 20 m mol active groups per ml. 2.5. Enzyme-linked immunoassay Fifty microliters of a 20-m grml phosphate buffered solution ŽPBS; 137 mM NaCl, 2.7 mM KCl, 8.7 mM Na 2 HPO4 , 1.5 mM KH 2 PO4 , pH 7.4. of a mouse monoclonal anti-IgY antibody Žproduced against the Fc fragment of IgY. was coated onto each well of a 96-well microtitre plate Žpolyvinylchloride. for 4 h at room temperature. Wells were blocked with 3% bovine serum albumin ŽBSA. in PBS overnight at room temperature. Serially diluted fractions from the purification steps were added Ž50 m l in 0.1% BSA. for 4 h at room temperature. The plates were washed with three changes of PBS-T ŽPBS containing 0.05% Tween 20., and once with tap water. An excess of rabbit anti-chicken IgG coupled to horseradish peroxidase ŽHRP; Silenus. was added in 50 m l 0.1% BSA and the amount of second antibody bound was determined using tetramethylbenzidine ŽHarlow and Lane, 1988.. The amount of IgY present in each fraction was determined by reference to a standard curve for known quantities of purified IgY.

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2.6. Affinity purification of antibodies To affinity purify the anti-hTom20 and anti-GST antibodies, IgY from one egg was passed through a GST–Sepharose column and then through a DhTom20-Sepharose column Žboth equilibrated with PBS.. Non-specific antibodies were washed off the column with 20 ml PBS. The specific antibodies were eluted with 100 mM glycine pH 3.0 and immediately neutralised with 0.1 vol. of 10 = PBS. The affinity purified antibodies were concentrated to 150 m grml and assayed on immunoblots. The affinity resins were prepared by covalent attachment of the appropriate protein to cyanogen bromide-activated Sepharose ŽPharmacia. in PBS at 1.5 mg proteinrml resin according to the manufacturer’s recommendations. 2.7. Electrophoresis The fractions from the T-gel chromatography or Escherichia coli expressing GST-DhTom20 were analysed by SDS-PAGE on 12% slab-gels. Gels were stained with Coomassie Blue R-250 and destained according to standard procedures ŽHarlow and Lane, 1988.. 2.8. Immunoblotting Proteins separated on SDS-PAGE slab-gels were transferred to nitrocellulose filters using a semi-dry blotting apparatus ŽPharmacia. as described by Harlow and Lane Ž1988.. The filters were blocked with 3% BSA in PBS for 1 h at room temperature, then washed three times in PBS-T. The blots were incubated for 1 h at room temperature with affinity purified anti-hTom20 or GST antibody or the rabbit anti-chicken IgG-HRP conjugate and then washed again three times in PBS-T. The blots were analysed by enhanced chemiluminescence ŽECL; Amersham Life Science.. 2.9. Protein determination Protein was measured using the modified Bradford method ŽSedmak and Grossberg, 1977. with BSA as standard. The concentration of purified IgY was measured at 280 nm using an extinction coeffi-

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Table 1 Isolation and purification of IgY from chicken eggs a Fraction

Total IgY Žmg.

Total protein Žmg.

Specific activity Žmg IgYrmg protein.

Purification Žfold.

Recovery Ž%.

Soluble yolk extract Pellet after 60% ammonium sulfate

60.0 51.6

310 200

0.19 0.26

1.0 1.4

100 86

52.2 19.6 26.8

200 187 27.3

0.26 0.98

5.2

47

52.2 0.6 51.0

200 175 50.7

1.0

5.3

85

P T-gel

chromatography Applied Unbound Eluate

S T-gel

chromatography Applied Unbound Eluate

a

The data represent the average from eight eggs. The equivalent of two egg yolks was applied to each 2-ml T-gel column. IgY and protein were measured as described in the text.

Fig. 1. Purification of IgY by T-gel chromatography. ŽA. Coomassie stained 12% SDS-PAGE slab-gel of fractions from the purification of immunoglobulins derived from chicken eggs. In lanes 7 and 10, 10 m g of protein were loaded, in all other lanes, 25 m g. Lane 1: supernatant of egg yolk extract. Lane 2: supernatant after 60% SAS. Lane 3: wash with 60% SAS. Lane 4: Pellet after 60% SAS. Lane 5: material applied to ST-gel. Lane 6: unbound material, ST-gel. Lane 7: eluted material, ST-gel. Lane 8: material applied to P T-gel. Lane 9: unbound material, PT-gel. Lane 10: eluted material, P T-gel. ŽB. Immunoblot of slab-gel in A Žlanes 5–10.. Lanes are the same as in A except that only 1r10 of the material was loaded. The blot was probed with polyclonal rabbit anti-chicken HRP-conjugate.


cient of ´ s 1.51 cmy1 mgy1 ml ŽBenedict and Berestecky, 1987. because IgY, particularly the light chain, was found to bind Coomassie Brilliant Blue G-250 poorly.

3. Results and discussion The first step in the isolation of IgY from egg yolk involves the extraction of soluble proteins from non-aqueous material such as lipids and lipoproteins. Methods for this include the precipitation of the non-aqueous material by diluting the yolk in buffer or water and with the addition of a precipitant ŽJensenius et al., 1981; Song et al., 1985; Hassl and Aspock, 1988; Gassmann et al., 1990; Akita and ¨ Nakai, 1992, 1993.. Extraction with chloroform has also been reported ŽPolson, 1990.. We found that including a chloroform extraction step resulted in a decrease in the amount of protein eluted from the T-gel. This could be due to denaturation of the IgY or the presence of chloroform affecting binding in a salt-promoted mode. To avoid the chloroform step we decided to use the ‘water dilution’ method which had been thoroughly optimised to give high yields of functionally active IgY in a simple and rapid procedure ŽAkita and Nakai, 1992.. The results from the extraction are summarised in Table 1. We introduced an ammonium sulfate precipitation step in order to concentrate the IgY extract and facilitate binding to T-gel. Simultaneously, the extract was partially purified without a substantial loss of IgY ŽFig. 1A, lanes 1–4.. The extract could then be stored at 48C as an ammonium sulfate precipitate awaiting further purification. For the purification of IgY by T-gel chromatography, a T-gel ŽPT-gel. synthesized according to the original procedure of Porath et al. Ž1985. and having 20 m molrml coupled mercaptoethanol was compared with a gel having a mercaptoethanol concentration of 48 m molrml ŽST-gel.. The comparison of IgY purification on ST-gel and PT-gel is summarised in Table 1. The IgY eluted from both the ST-gel and the PT-gel was essentially pure ŽFig. 1A, lanes 7 and 10.. The ST-gel adsorbent has a higher capacity Ž; 25 mg IgYrml gel. than the original PT-gel Ž; 13 mg IgYrml gel.. The ST-gel bound almost all

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the IgY of the applied sample as can be seen in Fig. 1A and B Žlane 6.. Based on the ELISA analysis ŽTable 1. and the immunoblot analysis ŽFig. 1B, lane 6., only about 0.6 mg IgY was not bound. This represents approximately 1% of the protein not binding to the ST-gel, providing a recovery of IgY of close to 99%. IgY did not bind to the PT-gel as well ŽTable 1 and Fig. 1A and B, lane 9., although the recovery was still more than 50%. Based on the results obtained in these experiments, IgY represents about 17% of the total extractable protein of egg yolk. The use of the more highly-substituted T-gel would thus be better for large scale purification of IgY than the original T-gel. The effect of ligand density on the capacity of T-gels is discussed elsewhere ŽScoble and Scopes, 1997.. We noticed that IgY seemed to stain poorly with the Coomassie dye. This can be seen in Fig. 1A Žlanes 7 and 10. where IgY and particularly the light chain show up only faintly. Thus, measurement of protein concentration by the modified Bradford’s dye binding assay ŽSedmak and Grossberg, 1977. results in a substantial underestimation of IgY. By comparison with UV absorption at 280 nm Ž ´ 280 s 1.51 cmy1 mgy1 ml. the protein concentrations for the pure IgY fractions are underestimated by a factor of ; 1.8. The total IgY eluted from the T-gel columns given in Table 1 is therefore based on the extinction coefficient rather than the dye binding assay. In order to determine the fraction of total IgY specifically directed against the GST-DhTom20 used to immunise the chicken, anti-hTom20 antibodies and anti-GST antibodies were affinity purified on immobilised DhTom20-Sepharose and GST–Sepharose, respectively. Following elution with glycine, the specificity of the eluted antibodies Žconcentrated to 150 m grml. was tested on immunoblots at a 1r1000 dilution. The total amount of affinity purified antibody isolated from one egg was 225 m g for hTom20 and 326 m g for GST. This represents around 1% of IgY for these particular antigens.

4. Conclusions The experiments reported demonstrate the isolation of immunoglobulins from chicken eggs to high purity using thiophilic interaction chromatography.

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The procedure is simple, rapid and cheap and large amounts of IgY were recovered.

Acknowledgements We wish to thank Joan Hoogenraad and Graeme Garth for the production of the monoclonal antibody against IgY. This work was supported by a grant from the Australian Research Council. Judith Scoble and Brendan Hanson are recipients of Australian Post Graduate Awards.

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