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Poult Sci 2007. 86:1910-1914
© 2007 Poultry Science Association
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IMMUNOLOGY, HEALTH, AND DISEASE: Research Notes

Purification of Immunoglobulins from Chicken Sera by Thiophilic Gel Chromatography

C. C. Constantinoiu*,1, J. B. Molloy{dagger}, W. K. Jorgensen{dagger} and G. T. Coleman*

* School of Veterinary Science, University of Queensland, Brisbane, Queensland, 4072, Australia; and {dagger} Department of Primary Industries and Fisheries, Yeerongpilly, Australia

1 Corresponding author: c.constantinoiu{at}uq.edu.au


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Immunoglobulin Y is different from most of the other immunoglobulins because it does not bind protein A or protein G. Thiophilic gel chromatography has been successfully used to purify IgY from chicken egg yolk, but the technology has not previously been used to purify IgY from serum. In this research note, we describe the optimization of T-gel chromatography for purification of IgY from serum. Data are provided on the recovery and purity of IgY obtained using potassium sulfate buffers of different concentrations. Decreasing the strength of potassium sulfate buffer from 0.5 to 0.3 M did not alter the amount of IgY recovered but increased the purity. Using 0.3 M potassium sulphate, we recovered approximately 63.7% of the serum Ig as almost pure IgY.

Key Words: immunoglobulin Y • serum • purification • thiophilic chromatography


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The use of chicken IgY in various research fields has increased significantly in recent years, mainly because producing antibodies in eggs is both cost effective and minimizes animal welfare concerns. The phylogenetic distance between birds and mammals also makes them ideal for raising antibodies against mammalian proteins (Schade et al., 2005). There are many reports reviewing methods for purifying antibodies from yolk (Schwarzkopf and Thiele, 1996; Gee et al., 2003). However, some immunological techniques used in the research of avian infections, especially in broilers and cockerels, require the purification of IgY from serum. In these types of birds, studies on infected tissue aiming to identify and localize the pathogens and their antigens recognized by immune serum require purified IgY to avoid background interference caused by immunoglobulins present in the tissues. One option is to purify antibodies from sera and conjugate them with biotin or some other appropriate label and use a method of detection that avoids incubations of tissue sections with secondary antibodies directed against chicken immunoglobulins.

Little information about purifying IgY from sera is currently available (Bhanushali et al., 1994). Unlike IgG of mammalian species, IgY does not bind to protein A or G, making purification comparatively difficult (Ansari and Chang, 1983; Warr et al., 1995; Zhang, 2003). Hansen et al. (1998) described a method for purifying IgY from egg yolk using thiophilic gel (T-gel) chromatography. The same technology could potentially be used to purify IgY from sera.

The T-gel binds proteins at high concentrations of a lyotropic salt and releases them at low concentrations of a lyotropic salt or sodium chloride (Lihme and Heegaard, 1991). Its binding of IgG, IgM, and IgA from mammals (Porath et al., 1985; Belew et al., 1987) was thought to be via the Fc region (Oscarsson et al., 1991). However, the successful reports of purifying recombinant single chain antibody fragments (Schulze et al., 1994) as well as F(ab)2 and Fc fragments (Yurov et al., 1994) suggested a large contribution of the conserved framework sequences of the variable domains in the binding process (Schulze et al., 1994). The binding mechanism of IgY is probably similar to that of mammalian immunoglobulins despite its structure and biological activities being slightly different (Tini et al., 2002).

In this technical note we describe the optimization of T-gel chromatography for the purification of IgY from chicken sera by varying the concentration of the lyotropic salt (potassium sulfate) involved in immunoglobulin binding.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Inoculation of Chickens and Sera Collection
All experiments were conducted in accordance with Animal Ethics Committee guidelines (AEC Approved Application Number: ARI 01/02/2005). Rhode Island Red/Rhode Island White cross birds were housed in suspended wire cages in limited access, climate-controlled rooms. Food and water were supplied ad libitum. Beginning with the age of 4 wk, the birds were infected by oral gavage, on 3 occasions over a period of 6 wk, with 3,000 sporulated oocysts of Eimeria tenella Redland strain. Three weeks after the last inoculation the birds were anaesthetized with isoflurane (IsoFlo, Abbott Australasia, New South Wales, Australia), heart bled, and euthanized by cervical dislocation before recovery. The blood was allowed to clot for 1 h at room temperature and then overnight at 4° C, centrifuged at 800 x g for 10 min, and stored at – 20° C until usage. The IgY was purified from the sera of 4 individual birds in separate experiments.

IgY Purification
The T-gel was initially used according to the manufacturer’s instructions for mammalian immunoglobulin purification. Prior to application to the column, the serum was diluted with potassium sulfate at a final concentration of 0.5 M (Belew et al., 1987), centrifuged (10,000 x g for 20 min), and filtered through a 0.45-µm filter. A 3-mL column of T-Gel Adsorbent (Pierce, Rockford, IL) was prepared and the flow rate adjusted to 0.5 mL/min. One milliliter of the processed serum was applied to the column, which was then washed with 12 column volumes (36 mL) of binding buffer (0.5 M potassium sulfate, 50 mM sodium phosphate buffer, pH 8) to remove unbound material. The immunoglobulins were then eluted with 12 column volumes of 50 mM sodium phosphate (pH 8). Three-milliliter fractions of the nonbound (NB) and bound (B) fractions were collected during the washing and elution steps, respectively.

To optimize the purification process, buffers containing decreasing concentrations of potassium sulfate were evaluated. Columns packed and loaded with sera as described above were washed sequentially with 10 column volumes of buffer containing 0.5, 0.4, 0.3, 0.2, and 0.1 M potassium sulfate and 50 mM sodium phosphate (pH 8) before IgY was eluted with 10 column volumes of 50 mM sodium phosphate buffer (pH 8). In a final optimization experiment dilution of serum with potassium sulfate at final concentrations of 0.3 or 0.2 M, and washing buffers containing 50 mM sodium phosphate and 0.3 or 0.2 M potassium sulfate were evaluated.

Purifications using 0.5 and 0.3 M potassium sulfate buffer were performed in triplicate, and purifications using 0.2 M potassium sulfate buffer were performed in duplicate. All purifications were carried out at room temperature.

Determination of Protein and IgY Concentration
The protein concentration of column fractions was determined by measuring the absorbance at 280 nm (Bio-Photometer, Eppendorf, Hamburg, Germany). The IgY, IgM, and IgA concentrations were determined using sandwich ELISA quantification kits (Bethyl Laboratories, Montgomery, TX) following the manufacturer’s instructions. The IgY recovery relative to the total IgY in the parent serum was calculated after summing the amounts of IgY found in all elution fractions.

Assessment of Purity
The purity and composition of all fractions were analyzed by SDS-PAGE under reducing conditions as described by Laemmli (1970). Undiluted NB and B fractions and parent serum diluted 1:30 in distilled water were mixed with an equal volume of sample buffer (61.7 mM Tris pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol), heated at 95° C for 4 min, and loaded onto a 4% stacking/12% resolving polyacrylamide gel. The proteins were electrophoresed at constant voltage (120 V) using a Mini Protean II cell (BioRad, Hercules, CA). The resolved proteins were stained by BioSafe Coomassie (BioRad) or electrotransferred onto Immobilon-P membrane (Millipore, Bedford, MA) for 1 h at 100 V of constant voltage using the Mini TransBlot cell (BioRad) in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) according to Towbin et al. (1979). The membranes were then blocked in 2% skim milk powder (SMP) in Tris-saline containing 0.05% Tween 20 (0.01 M Tris, 0.154 M NaCl, pH 7.4; TTS), washed once with TTS then incubated with biotin labeled goat antichicken IgY (1:200 in 2% SMP) (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Membranes were then washed 3 times in TTS and incubated with streptavidin-peroxidase (1:200 in 2% SMP; Kirkegaard & Perry Laboratories). After 3 final washes with TTS and 1 with Tris-saline (0.01 M Tris, 0.154 M NaCl, pH 7.4), the color reaction was developed with 4-chloro-1-naphtol (BioRad).

Assessment of Binding Activity
The binding activity of the purified IgY was compared with that of the parent serum by Western blotting. Briefly, E. tenella gametocyte proteins were resolved by SDS-PAGE on a 12% gel and transferred to Immobilon-P membrane as described above. After blocking, the membranes were incubated with chicken sera diluted 1:20 in SMP or selected IgY fractions using a miniblotter system (Immunetics, Boston, MA) followed by washing with TTS and incubation with peroxidase labeled goat anti-chicken IgY (H+L) conjugate (Kirkegaard & Perry Laboratories) diluted 1:200 in SMP. After washing the color reaction was developed as described above. The serum dilution (1:20) was calculated so that the concentrations of IgY in serum and column fractions were roughly equivalent.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
The T-gel proved to be extremely efficient in binding IgY from serum when 0.3 to 0.5 M potassium sulfate buffers were used. However, the binding was not efficient with 0.2 M potassium sulfate buffers. The IgY recoveries obtained using buffers of different concentrations of potassium sulfate are presented in Table 1Go.


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  		Table 1. Recovery of IgY and purity of fractions for the potassium sulfate buffers tested
 
When serum was diluted with potassium sulfate at 0.5 M final concentration and columns washed with a buffer containing 0.5 M potassium sulfate and 50 mM sodium phosphate (pH 8), some IgY was detected in NB fractions by ELISA, but the amount (12 ng/mL or less) was too small to be visible on the gels stained by Coomassie blue (Figure 1AGo). The IgY recovered represented 62.9% of the amount loaded on the column, and the largest amounts were found in the first few elution fractions. However, Coomassie blue stained gels and immunoblots with anti-chicken IgY revealed that the initial fractions contained a large amount of contaminating protein (Figures 1AGo and 2Go). There was therefore a considerable discrepancy between the total protein concentration in the fractions and the IgY concentration. A relatively pure IgY could be obtained by discarding those initial, impure fractions, but the process was then very inefficient with IgY recoveries ranging between 25.5 to 27.8%. This led us to evaluate the effect of sequential washing of the columns with decreasing concentrations (0.4, 0.3, 0.2, and 0.1 M) of potassium sulfate buffer. It was found that 0.3 and 0.2 M buffers removed some of the impurities but with some loss of IgY. In contrast, 0.4 M buffer did not remove significant amounts of contaminants, whereas the 0.1 M buffer removed the contaminant proteins but with substantial loss of IgY (data not shown). Subsequently, the 0.3 and 0.2 M potassium sulfate buffers were further assessed. When serum was diluted with potassium sulfate at 0.3 M final concentration, and the columns washed with 0.3 M potassium sulfate, 50 mM sodium phosphate buffer before elution of IgY with 50 mM sodium phosphate, the IgY recovery was 63.7%, the fractions were purer than those obtained with the 0.5 M buffer (Figure 1BGo), and the IgY losses in the NB fractions were insignificant (2.3% of the total amount recovered). The amounts of IgA and IgM copurified with the IgY were very low (less than 8.4 µg/mL for IgA and less than 19 µg/mL for IgM). This indicates that purification of IgA and IgM, at least under the conditions described here, is relatively inefficient and that IgA and IgM could not account for the contaminants observed in stained gels. Weak bands were noticed on the Western blots of purified fractions probed with anti-chicken IgM and IgA antibodies, but they did not match any of the contaminant bands identified on the gels or membranes stained by Coomassie blue. This observation confirmed the conclusion that the contaminants were not IgA or IgM (data not shown).


Figure 1
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  		Figure 1. The elution profile obtained after thiophilic gel chromatography of 1-mL serum diluted with 0.5 (A), 0.3 (B), or 0.2 M potassium sulfate (C). The purity of fractions is showed on gels stained by Coomassie blue. The superimposed graph shows protein and IgY concentrations for each fraction as determined by spectrophotometry and ELISA, respectively. Lanes labeled PS contain protein size standards, whereas B and NB stand for bound and nonbound fractions. Arrows show the heavy chain of IgY.

 

Figure 2
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  		Figure 2. The identification of the heavy chain of chicken IgY in the fractions purified with 0.5 M potassium sulfate buffer. Adjacent wells were loaded with antigens from the same fraction and, after transfer on polyvinylidene fluoride membranes, were stained by Coomassie blue (left lane) or incubated successively with biotin labeled anti-chicken IgY and streptavidinperoxidase followed by color reaction development with 4-chloro-1-naphtol (right lane). Lanes labeled PS contain protein size standards, whereas B2’B4 stand for bound fractions. Arrow shows the heavy chain of IgY.

 
Purer IgY was obtained when 0.2 M potassium sulfate buffer was used both for serum and column washing, but the recovery was much lower (36.8%), a large amount of IgY being lost in the NB fractions (Figure 1CGo).

The activity of the antibodies was largely unaffected by purification, purified IgY reacting with the same antigens as the parent serum. Apart from 1 weak band approximately midway down the gel that was present in the blot with parent serum but not in the blots with purified IgY, the reaction patterns were identical (Figure 3Go).


Figure 3
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  		Figure 3. Binding of antibodies in column fractions and in parent serum with Eimeria antigens. Equal amounts of Eimeria tenella gametocyte proteins were separated by SDS-PAGE on a 12% gel and transferred on polyvinylidene fluoride membrane. The lanes were incubated with column fractions (B2-5) or the parent serum (S) followed by peroxidase labeled goat anti-chicken IgY. The color reaction was developed using 4-chloro-1-naphtol.

 
In our experiment, the recovery of IgY was lower and the fractions less pure than those obtained by others when purifying mammalian serum antibodies (Lihme and Heegaard, 1991) or yolk IgY antibodies (Hansen et al., 1998) but still adequate for most applications. Bhanushali et al. (1994) used sequential precipitation of chicken serum with caprylic acid and ammonium sulfate and got pure IgY but did not report IgY recoveries. Furthermore, the purity of IgY extracted from yolk by ammonium sulfate precipitation did not exceed 65%. Recoveries were lower using other methods such as precipitation by ammonium sulphate, dextran sulphate, or polyethylene glycol (Schwarzkopf and Thiele, 1996).

Under the conditions described we demonstrated that IgY purity could be increased without compromising recovery by reducing the concentration of the lyotropic salt (potassium sulfate) from 0.5 to 0.3 M. We do not know whether the difference between our optimum potassium sulfate concentration and that recommended by the manufacturer for mammalian immunoglobulin is due to the slightly different properties of IgY or to differences in other parameters (lyotropic salt used, sample to support ratio, elution speed). Hardoiun et al. (2007) found that T-gel is not as selective as thought in binding immunoglobulins and that immunoglobulins actually compete with the other serum proteins for the binding sites. This implies that increasing the volume of the affinity support to maximize immunoglobulin recovery might compromise purity. In choosing the sample/support ratio we followed the manufacturer’s instructions for mammalian immunoglobulins, but those ratios may not be optimal for purifying IgY from sera. It would of interest to check the effect of different sample to support ratios in purifying sera IgY.


   ACKNOWLEDGMENTS
 
The authors thank Jan-Maree Hewitson and Anthea Bruyeres (Department of Primary Industries and Fisheries, Yeerongpilly, Australia) and Lyn Knott (School of Veterinary Science, University of Queensland, Brisbane, Queensland, Australia) for their expert technical assistance.

Received for publication February 17, 2007. Accepted for publication May 11, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Ansari, A. A., and T. S. Chang. 1983. Immunochemical studies to purify rabbit and chicken immunoglobulin G antibody by protein A-Sepharose chromatography. Am. J. Vet. Res. 44:901–906.[ISI][Medline]

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Bhanushali, J. K., J. M. Gilbert, and L. R. McDougald. 1994. Simple method to purify chicken immunoglobulin G. Poult. Sci. 73:1158–1161.[ISI][Medline]

Gee, S. C., I. M. Bate, T. M. Thomas, and D. B. Rylatt. 2003. The purification of IgY from chicken egg yolk by preparative electrophoresis. Protein Expr. Purif. 30:151–155.[ISI][Medline]

Hansen, P., J. A. Scoble, B. Hanson, and N. J. Hoogenraad. 1998. Isolation and purification of immunoglobulins from chicken eggs using thiophilic interaction chromatography. J. Immunol. Methods 215:1–7.[ISI][Medline]

Hardoiun, J., M. Duchateau, L. Canlle, C. Vileghe, J. Joubert-Caron, and M. Caron. 2007. Thiophilic adsorption revisited. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 845:226–231.[ISI][Medline]

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Schade, R., E. G. Calzado, R. Sarmiento, P. A. Chacana, J. Porankiewicz-Asplund, and H. R. Terzolo. 2005. Chicken egg yolk antibodies (IgY-technology): A review of progress in production and use in research and human and veterinary medicine. Altern. Lab. Anim. 33:1–26.[Medline]

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