Purification of Emu IgY for Therapeutic and Diagnostic Use Based on Monoclonal Secondary Antibodies Specific to Emu IgY

Abstract

The emu is the second largest ratite; thus, their sera and egg yolks, obtained after immunization, could provide therapeutic and diagnostically important immunoglobulins with improved production efficiency. Reliable purification tools are required to establish a pipeline for supplying practical emu-derived antibodies, the majority of which belongs to the immunoglobulin Y (IgY) class. Therefore, we generated a monoclonal secondary antibody specific to emu IgY. Initially, we immunized an emu with bovine serum albumin multiply haptenized with 2,4-dinitrophenyl (DNP) groups. Polyclonal emu anti-DNP antibodies were partially purified using conventional precipitation method and used as antigen for immunizing a BALB/c mouse. Splenocytes were fused with myeloma cells and a hybridoma clone secreting a desirable secondary antibody (mAb#2-16) was established. The secondary antibody bound specifically to emu-derived IgY, distinguishing IgYs from chicken, duck, ostrich, quail, and turkey, as well as human IgGs. Affinity columns immobilizing the mAb#2-16 antibodies enabled purification of emu IgY fractions from sera and egg yolks via simple protocols, with which we succeeded in producing IgYs specific to the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) spike protein with a practical binding ability. We expect that the presented purification method, and the secondary antibody produced in this study, will facilitate the utilization of emus as a novel source of therapeutic and diagnostic antibodies.

INTRODUCTION

Immunoglobulin Y (IgY) is a class of immunoglobulins present in lungfish, amphibians, reptiles, and birds that are present in egg yolk and serum.^1,2) IgY shares a common ancestor with mammalian IgG and IgE, which is compatible with their antigen-binding properties and features of in vivo circulating as monomeric forms. Despite this, IgYs possess several advantages over mammalian IgGs that encourage their therapeutic and diagnostic applications^1,2): (1) long half-life (one month at 37 °C and 5 years at 4 °C); (2) potentially higher affinities toward mammalian proteins, which is ascribable to the phylogenetic distance between birds and mammals; and (3) the lack of complement and leukocyte activation and the binding ability to Fc receptors and rheumatoid factors, which may prevent immune activation and reduce nonspecific binding in diagnostic applications. In fact, chicken IgY against Pseudomonas effectively prevents infection with this pathogen,³⁾ and ostrich anti-pollen IgY has already been commercially available in a mask-conjugated form to prevent pollen allergy.⁴⁾ Another advantage is the strong resistance of IgY to degradation in the human digestive tract. Trials involving IgY as an oral drug to prevent infection and neutralize toxins are ongoing.⁵⁾

Although chicken and hen eggs have been used as sources of IgY for practical use, we have focused on the emu (Dromaius novaehollandiae) (Fig. 1A) as a promising species that can supply IgY with improved productivity. Emus are the second largest ratite after ostriches, reaching 1.5–1.8 m in height and weighing 38–55 kg.⁶⁾ They lay eggs (approx. 500 g) that are much larger than those of chickens (approx. 60 g)⁷⁾ (Fig. 1B). Furthermore, they are relatively easy to breed because of their ability to adapt to the environment. To establish a pipeline for supplying practical emu-derived antibodies, the majority of which belongs to IgY class, a reliable purification tool is required. Several protocols have been developed for purifying chicken IgY from egg yolk,^8,9) but only one of these methods including a time-consuming gel-filtration has been applied to emu IgY.⁹⁾ Therefore, here we generated a monoclonal secondary antibody specific to emu IgY, and constructed antibody-based affinity columns. A simple and efficient purification protocol was developed using these methods. Furthermore, we validated the feasibility of our protocol, by purifying IgY raised in sera and egg yolks against the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) spike protein (S protein, hereinafter),¹⁰⁾ which were obtained from emus immunized with a recombinant S protein.

Fig. 1. Emu (Dromaius novaehollandiae) (Male, 3.5 Years of Age) (A) and an Emu Egg (Left), in Comparison with a Chicken Egg (Right) (B)

MATERIALS AND METHODS

Animal studies with emus were carried out under the ethical guideline established between Japan Eco System Co., Ltd. and Research Institute for Production Development. Studies with mice were performed at Kobe Pharmaceutical University, with prerequisite permissions obtained from the Committee of Animal Experiments.

Production and Partial Purification of Emu Polyclonal Immunoglobulins

4-(2,4-Dinitrophenylamino)butyric acid (CAS 10466-75-8) was coupled with bovine serum albumin (BSA) using the N-hydroxysuccinimide ester (NHS) method¹¹⁾ to prepare an immunogenic conjugate (DNP–BSA; coupling molar ratio 11 : 1) (Fig. 2). A male emu (4.5 years of age) raised in Japan Eco System (Chikushino, Fukuoka, Japan) was subcutaneously immunized with DNP–BSA (2.5 mg/head) with an emulsion of Freund’s complete adjuvant (FCA). Booster injections administered 14 and 28, and 42 d after the initial injection, using Freund’s incomplete adjuvant (FIA). The emu was sacrificed 10 d after the last injection to collect blood. Emu immunoglobulins (Igs) in the sera were partially purified using a protocol reported for chicken IgY, involving slightly modified caprylic acid and ammonium sulfate treatments⁸⁾ (Supplementary Fig. S1).

Fig. 2. Schematic Diagram of the Present Study

Generation of Monoclonal Secondary Antibody Specific to Emu IgY

A BALB/c mouse (female, 8 weeks of age) was immunized biweekly with the partially purified emu Igs obtained above, which were coupled with keyhole limpet hemocyanin (KLH) via the carbodiimide reaction.¹²⁾ The resulting emu-Igs-KLH conjugate (10 µg) was subcutaneously injected into the mouse with an emulsion of FCA (the primary and third immunizations) or FIA (the secondary immunization). After confirming an increase in the serum titer of antibodies against emu Igs using an enzyme-linked immunosorbent assay (ELISA) (Supplementary Fig. S2), the mouse received intrasplenic injections of the conjugate. After three days, splenocytes obtained from the mouse were fused with P3/NS1/1-Ag4-1 (NS1) myeloma cells.^11,13) The hybridomas producing antibodies that are estimated to be specific to emu IgYs (eIgYs) were selected, expanded, and then cloned. The established clones were cultured for 7–10 d, and the antibody (mAb#2-16) secreted in the supernatant was precipitated by adding 50%-saturated (NH₄)₂SO₄ and affinity-purified with an in-house-prepared emu-Ig-immobilized column to remove bovine immunoglobulins present in the hybridoma culture media (Fig. 2).

Purification of Emu IgYs Specific to S Protein from Emu Sera and Egg Yolks

The affinity-purified antibody mAb#2-16 was conjugated on NHS-activated HP column (1 mL) (Cytiva, Shinjuku, Tokyo, Japan) according to the appended instructions, to construct an affinity column containing immobilized mAb#2-16 (anti-eIgY column, hereinafter) (Fig. 2). Two female emus (4.25 years of age) were immunized with recombinant S protein (100 µg; ACRO Biosystems; Newark, DE, U.S.A.) emulsified with FCA or FIA, as described above. Sera (1.0 mL) were mixed with phosphate-buffered saline (PBS; 1.0 mL) and applied to an anti-eIgY column at room temperature. After washing with PBS, the IgY fraction adsorbed onto the column was eluted with glycine–HCl (pH 3.0, 6.0 mL) and neutralized with Tris–HCl (pH 8.0, 1.0 mL). The resulting mixture was applied to a Vivaspin20 (Sartorius; Göttingen, Germany) to concentrate and then to a PD10 column (GE Healthcare, Chicago, IL, U.S.A.) to exchange the buffer with PBS. To purify the IgY in the egg yolk (1 mL), water (9 mL) was added and mixed continuously for 1 min. After overnight incubation at 4 °C, the mixture was centrifuged at 1710 × g for 20 min and the supernatant was subjected similarly to the anti-eIgY column.

RESULTS

Generation and Characterization of the Monoclonal Secondary Antibody Specific to Emu IgY

Secondary antibodies targeting IgY from chicken,¹⁴⁾ duck,¹⁴⁾ turkey,¹⁵⁾ macaw,¹⁴⁾ or pigeon¹⁶⁾ are now commercially available or reported, although the majority of which are the polyclonal products. However, to the best of our knowledge, emu-IgY-specific secondary antibodies are not commercially available. Therefore, we aimed to generate monoclonal antibodies specific to emu IgY. Initially, we planned to obtain emu IgY antibodies with defined specificity toward a xenobiotic compound as a reliable immunogen for eliciting IgY-specific secondary antibodies. Thus, an emu was repeatedly immunized with DNP–BSA (Fig. 2), and the increase in the serum titer of anti-DNP antibodies was confirmed using ELISA (Supplementary Fig. S3). Blood was collected, and Igs therein were partially purified using a conventional protocol⁸⁾ (Supplementary Fig. S1). We obtained 1.7 mg of protein from 16 mL of the serum, in which IgY antibodies were expected to be the major component. The purified proteins were conjugated with KLH to increase immunogenicity and used to immunize a mouse. A fusion experiment using splenocytes therefrom provided a hybridoma clone that secreted a desirable secondary antibody (mAb#2-16).

The mAb#2-16 antibody was composed of γ1-heavy and κ-light chains (approx. 50 kDa and approx. 25 kDa, respectively) (Fig. 3A), each having the V_H and V_L domains containing 119 and 107 amino acids, respectively (determined by nucleotide sequencing of the relevant genes).¹⁷⁾ The complementarity-determining regions (CDRs),¹⁸⁾ which may contribute to direct interaction with the antigen, were assigned as follows: V_H-CDR1, DYNMN; CDR2, LINPYNGGTTYNRKFKG; CDR3, GDGYDDALDY; V_L-CDR1, SVSSSVSFMH; CDR2, STSNLAS; and CDR3, HQWSSYRT. mAb#2-16 specifically bound to emu IgY, distinguishing it from IgYs derived from chicken, duck, ostrich, quail, and turkey, as well as human IgGs (Fig. 3B, Supplementary Fig. S4). We also confirmed by comparing the recognition profiles that, mAb#2-16 is more specific than some commercially-available secondary antibodies that are introduced as “reactive with emu IgY” in their product information (data not shown). Comparison of the reactivity to the anti-DNP emu antibodies before and after pepsin treatment allowed us to estimate that mAb#2-16 binds to the Fc region of emu IgY (Supplementary Fig. S5).

Fig. 3. Biochemical Characterization of the Secondary Antibody mAb#2-16

(A) SDS-PAGE profiles of mAb#2-16, before and after purification with the emu-Ig-immobilized column, in the presence (+) or absence (−) of 2-mercaptoethanol (2-ME). A 12.5% polyacrylamide gel was used and separated proteins were stained with Coomassie brilliant blue. M, M_r marker; fraction 1, unpurified culture supernatant; 2, precipitate obtained with 50%-saturated (NH₄)₂SO₄; 3, non-binding fraction passed through the emu-Ig-immobilized column; 4, adsorbed-and-eluted fraction to the emu-Ig-immobilized column. All fractions were loaded with approx. 10 µg protein. (B) Evaluation of the specificity of mAb#2-16. The assay procedure is detailed in Supplementary Fig. S6. (C) Comparison of protein A, protein G, and anti-eIgY columns for their ability to retain emu IgY. Protein A and G columns [HiTrap Protein A (or G) HP (1 mL) column; Cytiva, Tokyo, Japan] were operated according to the appended directions. The amount of emu IgY recovered in the pass-through and adsorbed-and-eluted fractions was determined using a protein assay kit (Bio-Rad Laboratories, Tokyo, Japan) (left). Purity of the recovered proteins in each fraction (1–7) was analyzed using SDS-PAGE in the same conditions as described above (right).

Purification of Emu IgY with Affinity Column Containing Immobilized mAb#2-16

Affinity columns with immobilized protein A and protein G have been widely used to purify mammalian IgGs, but are not available for IgY given the insufficient retention.^1,2) Therefore, we developed affinity columns specialized for purification of emu IgY (Fig. 2). The affinity column with immobilized mAb#2-16 (anti-eIgY column) successfully isolated IgY with high purity from the serum of the emu immunized with DNP–BSA (Fig. 3C). As previously reported,⁹⁾ the recovered IgY had a larger heavy chain (υ chain) than that of IgGs (γ chain) owing to an extra Cυ4 domain, which appeared as a doublet (probably due to differences in glycosylation) at the positions of approx. 60 and 70 kDa in sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE). In contrast, as expected, the majority of the applied IgY passed the protein A and G columns without being retained.

Therefore, the applicability of the anti-eIgY column was examined via purification of emu IgY antibodies specific to the S protein. Two emus were repeatedly immunized with the S protein and an increase in serum levels of S protein-specific IgY was confirmed using ELISA (Supplementary Fig. S6). The serum was diluted with PBS and applied to the anti-eIgY column. We obtained S-protein-specific IgY antibodies with satisfactory purity (Fig. 4A), as no additional purification step was required. For egg yolk, only a water extraction step was necessary prior to column purification. We confirmed that these affinity-purified IgY antibodies, obtained from both sera and egg yolk, exhibited high binding activity against the S protein coated on ELISA plates (Fig. 4B, Supplementary Fig. S6A). The anti-eIgY column was operated at room temperature and recovery was completed within one hour. The column was stable at 4 °C for more than 2 months and was effectively reusable as shown with the reproducible recovery of the emu IgY specific to DNP residues: i.e., 2.4 and 2.5 mg emu IgY were recovered from 2.0 mL serum on a day and two months later, respectively, using the same anti-eIgY column stored under 4 °C.

Fig. 4. Purification of Emu IgY against SARS-CoV-2 Spike Protein (S Protein) Using Anti-eIgY Column

(A) SDS-PAGE profiles of emu IgY before and after purification, performed as described in Fig. 3(A). M, M_r marker; fraction 1, serum of immunized emu No. 1; 2, pass-through fraction obtained with serum from emu No. 1; 3, adsorbed-and-eluted fraction obtained with serum from emu No. 1; 4, adsorbed-and-eluted fraction obtained with serum from emu No. 2; 5, water-extracted fraction of egg yolk from immunized emu No. 2; 6, pass-through fraction obtained with the water-extracted fraction of egg yolk from emu No. 2; 7, adsorbed-and-eluted fraction obtained with the water-extracted fraction of egg yolk from emu No. 2. All lanes were loaded with a fixed amount of protein (approx. 8 µg) to visualize the components of each fraction clearly. It should be noted that we unfortunately failed to obtain eggs from emu No. 1. (B) Binding of purified IgY obtained from sera and egg yolk in the ELISA using microplates coated with S protein. ELISA procedure employed in this study is described in Supplementary Fig. S5.

DISCUSSION

In this study, we first established a hybridoma clone producing mAb#2-16, a reliable “secondary antibody” specific to emu IgY. A few polyclonal anti-bird (or anti-chicken) IgY antibodies, which have been reported to widely react with IgY from several birds including emu, are commercially available¹⁹⁾; however, the mAb#2-16 antibody is a monoclonal product and, thus, exhibited improved specificity and suited for mass-production. This feature facilitates a constant supply and use of affinity columns, requiring a large amount of antibodies. This antibody was also highly specific to emu-derived IgY antibodies, as described, possibly enabling low-background probing of emu-derived IgY antibodies specific to various biomarkers in diagnostic applications. The high specificity of mAb#2-16 could be attributed to the presence of highly emu-specific epitopes in emu IgY, which is plausible considering the relatively low homology between the emu Cυ (constant regions of IgY heavy chain) sequences and those of chicken, ostrich, and duck²⁰⁾ (approximately 40–70%).

The current anti-eIgY column, containing immobilized mAb#2-16, showed satisfactory performance in purifying IgYs from sera and eggs obtained from immunized emus, as demonstrated by isolation of biochemically active IgY against the S protein. As mentioned previously, emu could serve as a more efficient source of therapeutic and diagnostic antibodies than conventional mammalian- and chicken-based systems. Utilizing emu eggs, which are much larger than chicken eggs (Fig. 1B) removes the requirement for bleeding and sacrificing emus and thus allows for more desirable antibody production to be performed from an ethical perspective.

In conclusion, our newly established purification method is simple and efficient for purifying emu IgY from serum and egg yolk. This approach can accelerate the application of emu IgY antibodies for therapeutic and diagnostic purposes, as well as various biochemical studies.

Acknowledgments

This work was financially supported by Japan Eco System Co., Ltd., which also provided the research material and advice during the course of the study.

Conflict of Interest

Hirotaka Fujisawa and Yoshiki Kawata are employees of Japan Eco System Co., Ltd.

Supplementary Materials

This article contains supplementary materials.

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