Isolation and Preparation of Antisera to Ovine IgE

Abstract

Elevated levels of ovine reaginic antibody were induced by immunization with Ascaris suum antigens. The PCA activity of this antibody persisted in the skin of sheep for 20 days and was abolished by heating to 56 °C, suggesting that it was immunoglobulin E. Attempts to isolate IgE from this hyperimmune serum by gel filtration, ion exchange and affinity chromatography resulted in the preparation of a PCA-positive fraction containing proteins with molecular weights of 70,56 and 22 kD on SDS-PAGE. This preparation was used to raise an anti-serum in a rabbit to IgE which was rendered specific by absorption. An anti-serum to the heavy chain of ovine IgE was also raised in a rabbit by immunization with a fraction prepared from the 68- to 80-kD region of SDS-PAGE by excision and electroelution. Both antisera were positive in reverse cutaneous anaphylaxis tests and recognized a single protein with a molecular weight of 70–72 kD on SDS-PAGE immunoblotting. The presence of this protein in reaginic sheep serum, the molecular weight of its heavy chain, its heat lability and long-term skin-sensitizing ability are characteristic of IgE.

IgE responses in the serum and gastric lymph of sheep infected with Teladorsagia circumcincta

“…Further evidence for the specificity of this monoclonal was provided by the demonstration of a single, but diffuse, reactive band on Western blots of serum or gastric lymph of about 80 kDa as described previously for 1E7 on lymphocyte extracts (Kooyman et al 1997). Similar broad bands on SDS-PAGE have been noted for ovine epsilon chain with MW 81-96Kd (Shaw et al 1996), although this is heavier than reported by Yilmaz et al (1993) of 70-72Kd, or by Clarke & Beh (1994) of 68Kd, but consistent with molecular weights reported for mouse (Ishizaka 1985), human myeloma (Ikeyama et al 1986 circumcincta L3, or for ten days during a secondary immune response in sheep previously repeatedly infected, treated with anthelmintic, and then given a challenge of 50 000 T. circumcincta L3. Reasons for the inability to adequately detect native ovine IgE in solution by ELISA was unclear since MoAbs recently produced by Shaw et al (1996) readily detect ovine IgE in a double-antibody capture ELISA.…”

“…Further evidence for the specificity of this monoclonal was provided by the demonstration of a single, but diffuse, reactive band on Western blots of serum or gastric lymph of about 80 kDa as described previously for 1E7 on lymphocyte extracts ( Kooyman et al 1997 ). Similar broad bands on SDS‐PAGE have been noted for ovine epsilon chain with MW 81–96Kd ( Shaw et al 1996 ), although this is heavier than reported by Yilmaz et al (1993 ) of 70–72Kd, or by Clarke & Beh (1994) of 68Kd, but consistent with molecular weights reported for mouse ( Ishizaka 1985), human myeloma ( Ikeyama et al 1986 ) and dog ( DeBoer et al 1993 ) epsilon chain.…”

“…The majority of studies on IgE in responses to nematode/ helminth infections have been in humans and rodents and have demonstrated increases in total and parasite specific IgE during infection (Jarrett & Miller 1982). In ruminants, specific antibodies to bovine (Thatcher & Gershwin 1988) and ovine (Yilmaz et al 1993, Shaw et al 1996, Kooyman et al 1997) IgE have only become available relatively recently and so the respone of these species has not been well documented. However, IgE levels increased in the serum of calves infected with Ostertagia ostertagi and Cooperia spp.…”

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

“…As listed in table 2, the molecular mass of the IgE 8 chains ranges from 69 to 73 kDa [21][22][23] in humans, 68 kDa in rats [3], and 75 kDa in mice [24], The polyclonal IgE e chains, with a similar molecular mass to those identified from monoclonal IgE antibodies, were recently purified and identified in sheep [6] and dogs [5] (table 2). Because of variations in the methods used, variations among laborato ries, and even variations among experiments, it is difficult to make comparisons between the molecular masses of the 8 chains from different studies.…”

“…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…”