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Research Communications |
a Institute of Physiology, University of Zürich-Irchel, CH-8057 Zürich, Switzerland
b Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107–5099, USA
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit of the human hypoxia-inducible factor 1 (HIF-1). Anti-HIF-1 IgY antibodies were affinity purified and tested for their performance in various applications. Abundant HIF-1 protein was detected by Western blot analysis in nuclear extracts derived from hypoxic cells of human, mouse, monkey, swine, and dog origin whereas in hypoxic quail and frog cells, the HIF-1 signal was weak or absent, respectively. In electrophoretic mobility shift assays, affinity-purified IgY antibody was shown to recognize the native HIF-1 (but not the related HIF-2) complex that specifically binds an oligonucleotide containing the HIF-1 DNA binding site. Furthermore, IgY antibody immunoprecipitated HIF-1 from hypoxic cell extracts. Immunofluorescence experiments using IgY antibody allowed the detection of HIF-1 in the nucleus of hypoxic COS-7 cells. For comparison, the application of a mouse monoclonal antibody raised against the identical HIF-1 fragment was more restricted. Because chicken housing is inexpensive, egg collection is noninvasive, isolation and affinity purification of IgY antibodies are fast and simple, and the applicability of IgY is widespread, immunization of hens represents an excellent alternative for the generation of polyclonal antibodies.—Camenisch, G., Tini, M., Chilov, D., Kvietikova, I., Srinivas, V., Caro, J., Spielmann, P., Wenger, R. H., Gassmann, M. General applicability of chicken egg yolk antibodies: the performance of IgY immunoglobulins raised against the hypoxia-inducible factor 1. FASEB J. 13, 81–88 (1999)
Key Words: monoclonal antibody • affinity purification • Western blotting • EMSA • immunoprecipitation • immunofluorescence
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hens and mice were immunized against the subunit of the human hypoxia-inducible factor 1 (HIF-1). The heterodimeric transcription factor HIF-1, consisting of the two subunits and ß, represents the master regulator of oxygen homeostasis (reviewed in ref 11). Null mutant mice lacking either the HIF-1 (12, 13) or the HIF-1ß subunit (14, 15) die shortly after midgestation. Unexpectedly, whereas HIF-1 represents a newly detected protein, HIF-1ß was found to be identical to the heterodimerization partner of the dioxin receptor/aryl hydrocarbon receptor (AhR), termed AhR nuclear translocator (ARNT) (16). Upon hypoxic exposure, HIF-1 induces mRNA expression of a variety of oxygen-dependent genes including erythropoietin, vascular endothelial growth factor, and transferrin (11). Transactivation occurs by binding to an HIF-1 DNA binding site (CGTG) present in the regulatory regions of such hypoxically regulated genes. In contrast to these genes, HIF-1 is not regulated at the mRNA but at the protein level (17).
Here we demonstrate that the affinity-purified anti-HIF-1 IgY antibody allowed detection of HIF-1 protein in Western blots, electrophoretic mobility shift assays (EMSAs), immunoprecipitation, and immunofluorescence experiments using hypoxic cells from different species. In contrast, the application of the mouse monoclonal antibody was more restricted.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cDNA spanning the carboxyl-terminal amino acids 530 to 825 of human HIF-1 (16) was cloned into the EcoRI-SmaI site of the bacterial overexpression vector pGEX4T1 (Pharmacia, Piscataway, N.J.). The resulting plasmid overexpressed HIF-1 as a fusion protein with glutathione S-transferase (GST) in the bacterial strain BL21(DE3)plysS (Novagen, Madison, Wis.) after induction with isopropyl-ß-D-1-thiogalactopyranoside. The bacteria were pelleted, resuspended in phosphate-buffered saline (PBS) containing 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride, and lysed in a French press. The GST-HIF-1 fusion protein was purified using glutathione Sepharose 4B (Pharmacia) according to the manufacturer's instructions. For further purification, the GST-HIF-1530–825 antigen was electrophoresed through a preparative sodium dodecyl sulfate (SDS) polyacrylamide gel and the corresponding band was excised, minced with a polytron (Kinematika, Kriens, Switzerland), and lyophilized.
Immunization of hens and isolation of IgY from egg yolks
Approximately 80 µg of GST-HIF-1 fusion protein was resuspended in 500 µl PBS and mixed with an equal volume of complete Freund's adjuvant (Behring Diagnostics, Sommerville, N.J.). Laying white leghorn hens obtained from a local breeder were immunized by injecting the antigen into the pectoral muscle. Two booster injections of 60 µg antigen mixed with incomplete Freund's adjuvant were given 2 and 4 wk later. The eggs were collected daily and stored at 4°C for up to 6 months until antibody extraction.
Isolation of polyclonal chicken immunoglobulins from individual eggs was performed by a simple two-step procedure modified from an earlier protocol (18). The egg yolk was separated from the egg white, washed with deionized water, and placed in a funnel. The skin of the egg yolk was removed with forceps and the yolk was collected in a 50 ml screw cap tube. The yolk volume was brought to 25 ml with sodium phosphate buffer (100 mM, pH 7.6) and mixed vigorously. Subsequently, 20 ml of chloroform was added and the mixture was shaken until a semisolid phase was obtained. After centrifugation at 1200 x g for 30 min, the supernatant was decanted into a centrifugation tube and solid polyethylene glycol 6000 (Fluka, St. Louis, Mo.) was added to a final concentration of 12% (w/v). After centrifugation at 15,700 x g for 10 min, the pellet was resuspended in 2 ml sodium phosphate buffer and stored at -80°C.
Affinity purification of IgY antibodies
One gram of freeze-dried cyanogen bromide-activated Sepharose 4B (Pharmacia) was reswollen according to the manufacturer's instructions, thereby yielding 3.5 ml of resin. Recombinant fusion protein (2.5 mg) containing the human HIF-1 carboxyl terminus was dialyzed against the coupling buffer (0.5 M NaCl, 0.1 M NaHCO3, pH 8.3) and added to the reswollen Sepharose. After end-to-end rotation for 2 h at room temperature and centrifugation at 40 x g for 5 min, the pellet was incubated with 10 ml of blocking agent (0.2 M glycine, pH 8.0) at 4°C for 16 h to inactivate the remaining active groups of the resin. The resin was poured into a column and unspecifically bound proteins were washed out by repeating five times an alternating washing procedure consisting of a washing step with 5 ml coupling buffer, followed by one with 5 ml acetate buffer (0.5 M NaCl, 0.1 M ammonium acetate, pH 4.0). After rinsing the column with 50 ml PBS, isolated IgY fractions from four eggs were pooled (8 ml), brought to 50 ml with PBS, and loaded overnight at 4°C at a flow rate of 30 µl/min. The column was washed with 50 ml PBS, and bound IgY antibodies were eluted with 8.5 ml of elution buffer (0.15 M NaCl, 0.2 M glycine, pH 2.2). For immediate neutralization, the eluted fractions (2 ml) were poured into 15 ml tubes containing 0.8 ml of 1 M Tris-HCl buffer (pH 8.0). The column was neutralized with Tris buffer, washed with PBS, stored in PBS containing 0.05% NaN3 at 4°C, and washed with PBS before re-use. Prior to storage of the affinity-purified IgY antibody (+4°C or -80°C), 0.1 mg/ml bovine serum albumin was routinely added as a carrier.
Generation of the monoclonal antibody mgc3
HIF-1 (100 µg) antigen was resuspended in 0.5 ml complete Freund's adjuvant and injected subcutaneously into mice. Additional aliquots of 100 µg of antigen were resuspended in incomplete adjuvant and administered 21 and 42 days later. After decapitation, isolated splenocytes were fused to myeloma X65Ag8 cells, as described (19), and supernatants from the resulting hybridoma cell lines were screened for the presence of specific antibodies against HIF-1 by enzyme-linked immunosorbant assay (ELISA) using standard methodology. The positive clone was isotyped (using IsoStrip from Boehringer Mannheim, Mannheim, Germany), revealing a monoclonal IgG1 antibody (named mgc3), and affinity purified using a protein A Sepharose column (Pharmacia).
Cell culture and hypoxic exposure
The human epitheloid carcinoma cell line HeLa and the African green monkey cell line COS-7 were obtained from American Type Culture Collection in Rockville, Md. (ATCC numbers CCL-2 and CRL-1651, respectively). The mouse hepatoma cell line Hepa1 (also termed Hepa1c1c7) was a kind gift of L. Poellinger (Stockholm), the porcine kidney cells LLC-PK1 (ATCC CL-101) and the canine kidney cells MDCK (ATCC CCL-34) were kindly provided by J. Forgo (Zürich), and the quail sarcoma cell line QT6 (ATCC CRL-1708) and the Xenopus laevis kidney cell line A6C1 (ATCC CCL-102) were obtained from L. Vogt and B. Spindler (Zürich), respectively. All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM, high glucose, Life Technologies, Paisley, U.K.) supplemented with 10% heat-inactivated fetal calf serum (Boehringer-Mannheim), 1 x minimal essential medium nonessential amino acids, 2 mM L-glutamine, 1 mM natrium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin (all Gibco-BRL). Except for frog A6C1, the cell lines were cultured in a humidified atmosphere containing 5% CO2 at 37°C. Oxygen tensions in the incubators (Forma Scientific, model 3319) were either 140 mm Hg (20% O2 v/v, normoxia) or 7 mm Hg (1% O2 v/v, hypoxia). Routinely, cells were subjected to hypoxic induction for 4 h at a density of approximately 1–2 x 105 cells/cm2.
The kidney cell line A6C1 derived from X. laevis was cultured at 28°C in 5% CO2 in air. To mimic hypoxia, cells were exposed to 130 µM deferoxamine mesylate (Sigma, St. Louis, Mo.) for 6 h.
HeLaS3 cells (ATCC CCL-2.2), a subline of the HeLa cell line that was adapted to grow in suspension, were cultured in Ham's F-12 medium supplemented as described above. Hypoxic induction of HeLaS3 cells was performed as previously described (20). In brief, approximately 107 cells/ml were discontinuously stirred in a tonometer (Instrumentation Laboratory, model IL 237) at 37°C using gas mixtures of either 20% O2, 5% CO2, and 75% N2 (normoxia) or 0.5% O2, 5% CO2, and 94.5% N2 (hypoxia) for 4 h at a flow rate of 500 ml/min.
Cellular fractionation, in vitro translation, Western blot analysis, and EMSA
Nuclear extracts from hypoxic and normoxic cells were isolated as previously described (20). For total protein lysates, the culture medium was removed and the cells were washed twice with ice-cold PBS. Ice-cold lysis buffer consisting of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1% sodiumdeoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 2 µg/ml pepstatin, and 1 mM Na3VO3 (all from Sigma) was added and the cells were kept on ice for 10 min. The lysates were collected, sonicated, and centrifuged with 700 x g at 4°C for 10 min. The supernatant was frozen at -80°C.
In vitro transcription and translation of the recombinant HIF-1 and ß subunits were performed using the plasmids pBSKhHIF1T7 (16), pBSK-HLF (21), and pBSArntKS+ (22) and the TNT-coupled transcription-translation rabbit reticulocyte lysate system (Promega, Southampton, U.K.), following the manufacturer's instructions.
Extracts were electrophoresed through SDS polyacrylamide gel and electrotransferred to nitrocellulose membranes (Schleicher & Schuell, Keene, N.H.) using standard procedures. The Western blot was blocked with blocking buffer (4% w/v instant nonfat milk powder in PBS) and incubated for 2 h with the affinity-purified IgY antibody or the hybridoma mgc3 supernatant in blocking buffer. The membranes were washed and subsequently incubated with horseradish peroxidase-coupled rabbit anti-chicken antibodies (Promega, G1351) for IgY detection or goat anti-mouse antibodies (Pierce, 31430) for mgc3 detection for 1 h. HIF-1 protein was detected using enhanced chemiluminescence (Pierce, 34080). To test their acidic resistance, affinity-purified IgY containing bovine serum albumin (final concentration of 2%) were adjusted to pH 2.35 and neutralized again after the indicated periods of time. Immediately after neutralization, the antibodies were diluted in PBS containing 4% milk powder.
For Western blot analysis of bacterially expressed fusion protein, cDNA fragments of human HIF-1 corresponding to amino acids 530–653 and 530–825, as well as a 575–793 mouse HIF-2 fragment, were cloned in frame to the carboxyl terminus of GST (pGEX4T1, Pharmacia). Alternatively, HIF-1530–652, HIF-1530–825, or HIF-2576–874 fragments were cloned in frame to the carboxyl terminus of a (His)6 tag (pQE32, Qiagen, Chatsworth, Calif.). Bacterial expression and fusion protein purification were performed as described by the manufacturers (Pharmacia and Qiagen, respectively).
EMSA analysis was performed exactly as described, using an erythropoietin gene-derived oligonucleotide containing the HIF-1 DNA binding site (20). For supershifts analysis, each 1 µl of affinity-purified IgY antibody or mgc3 supernatant was added to the binding reaction.
Immunoprecipitation
Hypoxic COS-7 cells were washed three times with PBS and lysed by exposure for 10 min to ice-cold lysis buffer consisting of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1% sodiumdeoxycholate, 1% SDS, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 µg/ml pepstatin (all from Sigma). The lysate was sonicated and centrifuged at 10,000 x g for 20 min at 4°C. Total cell lysate (1 mg) was incubated with 30 ng of affinity-purified IgY antibody overnight on ice. A rabbit anti-chicken antibody (Promega, C2691) was added to the mixture for 2 h on ice. Protein A Sepharose (Pharmacia) was added and incubation was continued by rotation for 30 min at 4°C. The precipitate was centrifuged at 15,000 x g; the pellet was washed three times with total cellular lysis buffer (see above) and twice with 10 mM Tris-HCl (pH 7.5). Finally, the precipitates were analzed by Western blotting using IgY antibody.
Similarly, 350 µg of total cellular lysate from HeLa cells was incubated with 50 µl hybridoma mgc3 supernatant overnight on ice. Protein A/G Sepharose (30 µl) (Santa Cruz) was allowed to bind for 3 h at 4°C and the precipitates were analzed by Western blot, as described above, using the monoclonal mgc3 antibody for HIF-1 detection.
Indirect immunofluorescence microscopy
Hypoxic COS-7 cells were fixed with 4% formaldehyde (pH 8.0) for 10 min at room temperature, washed three times with PBS, permeabilized with 0.5% Triton X-100 (Fluka) in PBS for 5 min, and rinsed three times with PBS. To block nonspecific binding, PBS containing 10% fetal calf serum was added for 30 min. Cells were then incubated with or without the affinity-purified IgY antibody (diluted 1:10 in PBS) at 37°C for 1 h. HeLa cells were fixed as described above and then incubated with or without affinity-purified mgc3 antibody diluted 1:10 in PBS.
Primary antibodies were detected using either an FITC-conjugated rabbit anti-chicken antibody (Promega, G2691) or Cy3-conjugated donkey anti-mouse antibody (Jackson Immunoresearch Laboratory, West Grove, Pa.). Secondary antibodies were incubated at room temperature for 30 min, washed extensively in PBS and mounted in DABCO solution (Sigma). Immunofluorescent staining was detected by confocal laser scanning microscopy.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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fusion protein. Isolation of IgY antibodies from individual eggs was performed by a simple two-step procedure that consisted of a chloroform extraction of the egg yolk, followed by a single polyethylene glycol precipitation step (see Materials and Methods). Using this procedure, IgY antibodies were purified to better than 90% homogeneity (data not shown). Western blot analysis revealed the presence of specific IgY antibodies in eggs collected 10 days after the last boost injection. Because strong unspecific signals were obtained with nuclear fractions derived from HeLaS3 cells, the IgY antibodies were affinity purified. The same fusion protein used for immunization was bound to a resin and specific IgY antibody was eluted using an acidic buffer (pH 2.2). Pooled polyethylene glycol fractions from four egg yolks yielded 14–16 ml affinity-purified IgY antibody, which detected HIF-1 in nuclear extracts from hypoxic HeLaS3 cells at a dilution as high as 1:1000 (
Fig. 1).
In addition to nuclear fractions, the affinity-purified IgY antibody also allowed detection of HIF-1 protein in total cellular extracts from hypoxic HeLaS3 cells (data not shown). We also generated a mouse monoclonal antibody termed mgc3, which was raised against the same human HIF-1 fragment as that used for hen immunization. Like the IgY antibody, mgc3 recognized HIF-1 protein in nuclear and total cellular extracts from human HeLaS3 cells previously exposed to hypoxia (data not shown).
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The stability of IgY antibodies to acidic denaturation has been described to be lower than that of rabbit IgG (10, 23), implying that acidic elution from the affinity column (see above) might be harmful to the IgY antibodies. To test their tenacity toward low pH conditions, affinity-purified IgY were exposed to pH 2.35. Western blot analysis using nuclear extracts from hypoxic HeLaS3 cells showed only slight reduction of HIF-1 signals when IgY antibodies that underwent acidic exposure for up to 3 h were used (
Fig. 1).
Next, we analyzed whether the affinity-purified IgY antibody was capable of recognizing native HIF-1 protein in electrophoretic mobility shift assays (EMSAs), using as probe an HIF-1 DNA binding oligonucleotide derived from the erythropoietin gene, as described (20). After incubation of the probe with nuclear extracts from normoxic or hypoxic HeLa cells, the expected nonspecific, constitutive, and hypoxia-inducible factors (20) were detected ( Fig. 2A).
Addition of the chicken polyclonal antibody partially abolished binding of the HIF-1 heterodimer to the HIF-1 DNA binding site and partially supershifted the HIF-1 complex, whereas the constitutive and the nonspecific factors were not affected ( Fig. 2A). On the other hand, addition of the monoclonal antibody mgc3 recognized HIF-1 protein in the same hypoxic HeLa extract and resulted solely in a supershifted HIF-1 complex. Analogous results were obtained when adding IgY antibody or mgc3 to hypoxic nuclear extracts from the mouse hepatoma cell line Hepa1 (data not shown). Thus, the carboxyl-terminal HIF-1 fragment contains residues that are necessary for functional formation of the HIF-1 complex.
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Recently, four groups independently isolated a novel endothel-specific transcription factor that is similar to HIF-1 and was hence termed HIF-2 (reviewed in ref 11). HIF-2, like HIF-1, forms a functional heterodimer with ARNT (termed HIF-2), is activated by hypoxia, and transactives oxygen-dependent genes by utilizing the same DNA binding site as HIF-1 (21). To rule out cross-reactivity of our IgY and IgG antibodies between both hypoxia-inducible transcription factors, we performed EMSAs using in vitro synthesized HIF-1, HIF-2, and ARNT. As shown in Fig. 2B, the monoclonal mgc3 and the polyclonal IgY antibody were able to recognize in vitro translated HIF-1 but not HIF-2 protein. These results were confirmed by Western blot analysis with deletion constructs of bacterially expressed fusion proteins. As shown in Fig. 3,
both antibodies were specific for HIF-1 and did not cross-react with HIF-2. Even though the polyclonal IgY antibody was affinity purified using the GST-HIF-1 fusion protein, its cross-reactivity with GST alone was very weak, indicating that the GST protein per se displays a low immunogenicity in chicken. In contrast to the IgY antibody, mgc3 failed to react with the 530–653 HIF-1 fragment, indicating that the mgc3 epitope is localized between amino acids 654 and 825.
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As mentioned above, both antibodies recognized native HIF-1 protein present in hypoxic extracts derived from human HeLa or mouse Hepa1 cell lines. Unexpectedly, and in contrast to the EMSA results described in Fig. 2A, the monoclonal antibody mgc3 repeatedly failed to react with denatured HIF-1 protein in Western blot analysis when using extracts from hypoxic mouse Hepa1 cells ( Fig. 4A).
Probably the specific mouse HIF-1 epitope was altered upon protein denaturation by SDS. Thus, the different mammalian, avian and amphibian cell lines were examined by the affinity-purified chicken IgY antibody only. To this end, human (HeLaS3), monkey (COS-7), swine (LLCPK), dog (MDCK), and quail (QT6) cells were exposed to hypoxia (1% oxygen) for 4 h. In the case of the X. laevis cell line A6C1, which requires a culture temperature of 28°C, hypoxic conditions were mimicked by addition of deferoxamine (130 µM for 6 h), an iron chelator known to induce hypoxically regulated genes including HIF-1 (24). Western blot analysis using affinity-purified IgY and nuclear extracts derived from induced cells detected abundant HIF-1 protein in all mammalian cells tested. In contrast, the HIF-1 protein signal in induced quail and frog cells was very weak or absent, respectively ( Fig. 4B). That HIF-1-like DNA binding activity has been observed in induced Drosophila melanogaster SL2 cells (25), however, suggests that an HIF-1-like mechanism is widespread throughout the animal kingdom. Assuming that this notion is correct, our data imply that the HIF-1 carboxyl-terminal part (used as antigen) is highly conserved within the mammalian species.
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Chicken IgY antibodies do not bind protein A or G. To circumvent this disadvantage in immunoprecipitation experiments, we made use of a commercially available rabbit anti-chicken IgY antibody. Total cellular lysates from hypoxic COS-7 cells were incubated with IgY antibody (30 ng) overnight, followed by a 2 h incubation with rabbit anti-chicken antibodies. Immunoprecipitation was performed by adding protein A Sepharose to the mixture and the precipitates were analyzed by Western blotting. As shown in Fig. 5,
the affinity-purified IgY antibody was able to immunoprecipitate HIF-1. We also demonstrated that, despite some background signals, the monoclonal antibody mgc3 also immunoprecipitated the HIF-1 protein present in total cellular lysates from human HeLa cells.
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Finally, the affinity-purified IgY antibody was tested for its ability to detect HIF-1 in the African green monkey cell line COS-7 by immunofluorescence. As shown in Fig. 6,
HIF-1 protein was localized in the nucleus of COS-7 cells that were previously exposed to hypoxia (1% oxygen) for 4 h. No signal was obtained when omitting the primary antibody. Similar results were obtained when immunofluorescence was performed with hypoxic HeLa cells using the monoclonal antibody mgc3. This data demonstrates that, upon hypoxic exposure, HIF-1 is translocated to the nucleus.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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affinity column. Apart from GST-based fusion protein, a synthetic peptide (26 amino acids) has also been used successfully to affinity purify IgY antibodies, which in turn allowed purification of human 2-antiplasmin (30).
Unlike mammals, the Fc fragment of hens and most birds does not bind protein A or G (31). Therefore, after incubation of the extracts with IgY antibodies, we added commercially available rabbit anti-chicken antibodies, which allowed immunoprecipitation of HIF-1 with protein A beads. Similarly, the use of a crude IgY fraction and protein G-Sepharose beads preabsorbed with goat anti-chicken antibodies allowed immunoprecipitation of neurofibromatosis type 3 protein (32). It is noteworthy that duck immunoglobulins have been reported to bind to protein A or G; however, this high affinity was not attributed to the Fc fragment but to unique histidine residues occuring predominantly in the CH1 domain (31). Nonetheless, immunization of ducks is not recommended since they lay eggs during a short period of the year and since duck-specific housing is demanding.
Apart from the methods used in this work, IgY antibodies have been successfully applied in a variety of assays such as radioimmunoassay (RIA), ELISA, indirect hemagglutination assay, and immunodiffusion (7, 33–36), as well as in immunogold labeling experiments (37). From a therapeutic point of view, egg yolk from immunized hens has been proposed to be a convenient vehicle for oral passive immunization against enteral germs (10, 38, 39) or even against dental plaque formation in humans (40). Egg yolk antibodies from hens immunized with rattlesnake or scorpion venom have been used to generate antivenoms that neutralize the lethal effects of these venoms in vivo (41).
When comparing different adjuvants for chicken immunization, the use of complete Freund's adjuvants was reported to yield the highest IgY titers (42). In view of its tissue-damaging property, which is not necessarily observed macroscopically, the use of alternative adjuvants has been proposed recently (42). Considering animal welfare, it should be pointed out that chicken can be kept under conventional conditions without special veterinary support. If required, chicken housing and immunization can be outsourced. Despite their general applicability, IgY antibodies are scarcely used. We hope that the protocols presented in this work will help to increase the acceptance of the IgY technology.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: AhR, aryl hydrocarbon receptor; ARNT, AhR nuclear translocator; ATCC, American Type Culture Collection; ELISA, enzyme-linked immunosorbant assay; EMSAs, electrophoretic mobility shift assays; GST, glutathione S-transferase; HIF-1, hypoxia-inducible factor 1; Ig, immunoglobulin; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate.
Received for publication July 6, 1998.
Revision received September 9, 1998.
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 H. H. Marti, D. M. Katschinski, K. F. Wagner, L. Schaffer, B. Stier, and R. H. Wenger Isoform-Specific Expression of Hypoxia-Inducible Factor-1{alpha} During the Late Stages of Mouse Spermiogenesis Mol. Endocrinol., February 1, 2002; 16(2): 234 - 243. [Abstract] [Full Text] [PDF]
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 D. CHILOV, T. HOFER, C. BAUER, R. H. WENGER, and M. GASSMANN Hypoxia affects expression of circadian genes PER1 and CLOCK in mouse brain FASEB J, December 1, 2001; 15(14): 2613 - 2622. [Abstract] [Full Text] [PDF]
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D. M. STROKA, T. BURKHARDT, I. DESBAILLETS, R. H. WENGER, D. A. H. NEIL, C. BAUER, M. GASSMANN, and D. CANDINAS HIF-1 is expressed in normoxic tissue and displays an organ-specific regulation under systemic hypoxia FASEB J, November 1, 2001; 15(13): 2445 - 2453. [Abstract] [Full Text] [PDF]
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R. M. Wanner, P. Spielmann, D. M. Stroka, G. Camenisch, I. Camenisch, A. Scheid, D. R. Houck, C. Bauer, M. Gassmann, and R. H. Wenger Epolones induce erythropoietin expression via hypoxia-inducible factor-1alpha activation Blood, August 15, 2000; 96(4): 1558 - 1565. [Abstract] [Full Text] [PDF]
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H. E. Ryan, M. Poloni, W. McNulty, D. Elson, M. Gassmann, J. M. Arbeit, and R. S. Johnson Hypoxia-inducible Factor-1{{alpha}} Is a Positive Factor in Solid Tumor Growth Cancer Res., August 1, 2000; 60(15): 4010 - 4015. [Abstract] [Full Text]
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