General applicability of chicken egg yolk antibodies: the performance of IgY immunoglobulins raised against the hypoxia‐inducible factor 1α

Abstract

Avian embryos and neonates acquire passive immunity by transferring maternal immunoglobulins from serum to egg yolk. Despite being a convenient source of antibodies, egg yolk immunoglobulins (IgY) from immunized hens have so far received scant attention in research. Here we report the generation and rapid isolation of IgY from the egg yolk of hens immunized against the alpha subunit of the human hypoxia-inducible factor 1 (HIF-1alpha). Anti-HIF-1alpha IgY antibodies were affinity purified and tested for their performance in various applications. Abundant HIF-1alpha 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-1alpha 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-1alpha from hypoxic cell extracts. Immunofluorescence experiments using IgY antibody allowed the detection of HIF-1alpha in the nucleus of hypoxic COS-7 cells. For comparison, the application of a mouse monoclonal antibody raised against the identical HIF-1alpha 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.

Prolyl-4-hydroxylase PHD2- and hypoxia-inducible factor 2-dependent regulation of amphiregulin contributes to breast tumorigenesis

“…Human MCF-7 breast carcinoma cells were cultured in highglucose Dulbecco's modified Eagle's medium (Sigma, Buchs, Switzerland) as described previously (Camenisch et al, 1999). HUVEC cells were kindly provided by TF Lu¨scher (Zu¨rich, Switzerland) and cultured in complete endothelial basal medium-2 containing 2% fetal calf serum (Lonza, Basel, Switzerland).…”

Constitutive upregulation of hypoxia-inducible factor-1α mRNA occurring in highly metastatic lung carcinoma cells leads to vascular endothelial growth factor overexpression upon hypoxic exposure

“…The resolved proteins were electrophoretically transferred to nitrocellulose membrane. After blocking with 5% dry milk in TBS-T (150 mm NaCl, 50 mm Tris-HCl, pH 7.4, 0.05% Tween 20), the membrane was incubated with chicken anti-HIF-1a antibodies (Camenisch et al, 1999) or rabbit anti-c-Jun antibodies (Oncogene Science Inc., Manhasset, NY, USA). The membrane was washed extensively with TBS-T, and then incubated with horse raddish peroxidaseconjugated rabbit anti-chicken IgY (Promega) or goat antirabbit IgG, respectively.…”

Onconeuronal cerebellar degeneration-related antigen, Cdr2, is strongly expressed in papillary renal cell carcinoma and leads to attenuated hypoxic response

“…Cell culture and transient transfections Human HeLa cervical carcinoma, MCF-7 and BT474 breast carcinoma, SKOV3 and OVCAR3 ovary adenocarcinoma, HCT116 colorectal carcinoma, HepG2 hepatoma, U2-OS osteosarcoma, HEK293 embryonic kidney carcinoma, MEFs lacking HIF-1a (MEFs Hif1a À/À ) or PHD1 (MEFs Phd1 À/À ) cells were cultured in high-glucose Dulbecco's modified Eagle's medium (Sigma, Buchs, Switzerland), as described previously (Camenisch et al, 1999). For long-term hypoxia, cells were grown in a gas-controlled glove box (InvivO 2 400, Ruskinn Technologies, Leeds, UK).…”