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

Avian embryos and neonates acquire passive immunity by transferring materna l 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 a s ubunit of the human hypoxia-inducible factor 1 (HIF-1 alpha). Anti-HIF-1 al pha IgY antibodies were affinity purified and tested for their performance in various applications. Abundant HIF-1 alpha protein was detected by Weste rn blot analysis in nuclear extracts derived from hypoxic cells of human, m ouse, monkey, swine, and dog origin whereas in hypoxic quail and frog cells , the HIF-1 alpha signal was weak or absent, respectively. In electrophoret ic mobility shift assays, affinity-purified IgY antibody was shown to recog nize the native :HIF-1 (but not the related HIF-2) complex that specificall y binds an oligonucleotide containing the HIF-1 DNA binding site. Furthermo re, IgY antibody immunoprecipitated HIF-1 alpha from hypoxic cell extracts. Immunofluorescence experiments using IgY antibody allowed the detection of HIF-1 alpha in the nucleus of hypoxic COS-7 cells. For comparison, the app lication of a mouse monoclonal antibody raised against the identical HIF-1 alpha 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 generatio n of polyclonal antibodies.