Distribution of, and immune response to, chicken anti-αGal immunoglobulin Y antibodies in wild-type and αGal knockout mice

Chicken antibodies (immunoglobulin Y; IgY) to the αGal epitope (galactose α-1,3-galactose) bind to αGal antigens of mouse and porcine tissues and endothelial cells in vitro and block human anti-αGal antibody binding, complement activation and antibody-dependent cell-mediated lysis mechanisms. The activities and toxicity of anti-αGal IgY have not been tested in vivo. In this study, we tested the effects of multiple injections of affinity-purified anti-αGal IgY (AP-IgY) in both wild-type (WT) and α-1,3-galactosyltransferase knockout (Gal KO) mice. WT and Gal KO mice were injected once, twice, three, or four times intravenously (i.v.) with AP-IgY and killed at 1 hr or 24 hr. Mice displayed no toxicity to four injections of AP-IgY. Heart, lung, liver, kidney, spleen and pancreatic tissue were evaluated using immunohistochemical techniques for the presence of the αGal epitope using the GSI-B4 lectin, and for bound IgY, as well as mouse IgM and IgG. The binding of AP-IgY antibodies to the endothelium of WT mouse tissues was essentially identical to the pattern of binding of the GSI-B4 lectin after injection of WT mice and death at 1 hr. WT mice killed 24 hr after i.v. injection of AP-IgY showed little remaining bound IgY in their endothelia, indicating that IgY is cleared over that time period. We also evaluated the blood drawn at the time of death for the presence of anti-αGal IgY, anti-IgY IgM and anti-IgY IgG by enzyme-linked immunosorbent assay. Anti-αGal IgY was almost undetectable in WT mouse sera at all injection and killing times. In contrast, Gal KO mouse sera showed increasing anti-αGal IgY levels until 24 hr after the fourth injection, when anti-αGal IgY levels were almost undetectable. Anti-IgY IgM and IgG levels in WT and Gal KO mouse sera showed a typical increase in anti-IgY IgM 24 hr after the second injection (3 days after the first injection) and an increase in anti-IgY IgG 24 hr after the third injection (5 days after the first injection). These results show that IgY binds to αGal epitopes in the WT mice and is cleared sometime over a 24-hr time period and that IgY is an expected immunogen in mice eliciting a rather typical anti-IgY IgM and IgG response.