Purification and identification of polyclonal IgE antibodies from ragweed-sensitized dog sera

We have purified and characterized polyclonal dog IgE. Serum IgE was precipitated by (NH4)2SO4 and then purified by two different procedures. Ion exchange on DEAE-Sephacel, followed by HPLC using Tonen hydroxylapatite and then Protein G-Sepharose, produced a highly purified IgE fraction (No. 1) free of IgG, IgA and IgM as measured by ELISA, but recovery of IgE as measured by passive cutaneous anaphylaxis was low. Gel filtration on Sephacryl S-300, Con A-Sepharose and Protein G-Sepharose recovered 18% of initial IgE, 0.02% IgG, 0.4% IgM and 0.3% IgA. This IgE fraction (No. 2) was used to induce antibody production in rabbits. Western blot analysis was then performed for dog IgE fractions No. 1 and 2. Using the rabbit anti-dog IgE, a prominent IgE band with an apparent molecular mass of 226 kD was identified in fractions No. 1 and 2 subjected to nonreducing SDS-PAGE. This band also reacted with anti-human IgE, but not with anti-dog IgG or anti-dog IgA. Under reducing conditions the approximate molecular mass for the IgE & chain, estimated by Western blot using rabbit anti-dog IgE, was 73 kD, providing a molecular mass of 196 kD for dog IgE.