Purification and Identification of Polyclonal IgE Antibodies from Ragweed-Sensitized Dog Sera

Abstract

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 ε chain, estimated by Western blot using rabbit anti-dog IgE, was 73 kD, providing a molecular mass of 196 kD for dog IgE.

Binding Properties of Protein A and Protein G for Human IgE

“…Serum IgE was purified mainly according to previously described methods [12], Briefly, serum (2 ml) from an al lergic patient with a high IgE level was precipitated between 30 and 55% saturation of (NH4)2S 0 4, dissolved in 0.02 M phosphate-buffered saline (PBS) pH 7.2 and applied to a Sephadex G-25 column equilibrated with PBS. This IgE-containing fraction was then applied to a Con A-Sepharose co lumn (bed volume 12 ml).…”

Cross-Reactivity and Molecular Mass of the ε Chains of the IgE Antibodies in Dogs, Humans, Rats, and Mice

“…Dog IgE-enriched fractions were purified from ragweed-sensitized dos sera as previously described [5], Briefly, dog IgE-enriched frac tions were obtained by a combination o f salt precipitation, gel filtra tion, and affinity with concanavalin A-and Protein G-Sepharose. The fraction which contained other immunoglobulins and serum proteins had a PCA titer of 1,024 and its protein concentration was 2 mg/ml.…”

“…Polyclonal antidog IgE antibody was developed, purified and iden tified as previously reported [5], Rabbits were immunized by three injections at 2-week intervals with a dog IgE-enriched fraction in com plete Freund's adjuvant for the first injection and in incomplete Freund's adjuvant for the other injections. The rabbit antisera were obtained 2 weeks after the third injection.…”

“…The extremely low serum con centrations of IgE have limited the purification of polyclo nal IgE. To date, IgE e chains have been identified only from the polyclonal antibodies of dogs [5] and sheep [6], The cross-reactivity of IgE among different species is an impor tant aspect o f studies o f comparative immunology, IgE mo lecular structure, and identification of IgE in species in which IgE has yet to be identified. Previous studies have…”

What Can We Learn from the Allergic Zoo?

“…Spontaneously devel oping or experimentally induced IgE or IgE-like antibodies have also been identified in mice [2], rats [3], guinea pigs [4], rabbits [5], cows [6], horses [7], dogs [8,9], monkeys [ 10] and even kangaroos [11].…”