Generation and Characterization of Murine Monoclonal Antibodies Specific for Bovine Immunoglobulin E

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.

Section: Discussionmentioning

confidence: 99%

Isolation and Preparation of Antisera to Ovine IgE

Yılmaz

Roe²,

Morgan³

1993

“…These assays, including the use of positive and negative controls, have been previously de scribed by us [13,14]. Modification consisted of substitution of rab bit anti-bovine IgE with murine monoclonal anti-bovine IgE [16]. Individual serum samples were assayed in duplicate and the mean value determined for each day.…”

Section: Enzyme Immunoassay For Mf Specific Ige Igg and Brsv-specifimentioning

confidence: 99%

Immunoglobulin E Responses and Lung Pathology Resulting from Aerosol Exposure of Calves to Respiratory Syncytial Virus and <i>Micropolyspora faeni</i>

Gershwin

Dungworth

Himes

et al. 1990

Self Cite

Bovine respiratory syncytial virus (BRSV) infection of calves has been associated with a type-I hypersensitivity syndrome not unlike respiratory syncytial virus-associated pulmonary symptomatology in humans. To study the mechanism of pulmonary pathology in calves and define the relationship with both viral-specific IgE response and IgE titers to concurrent aerosol of Micropolyspora faeni (Mf) we subjected groups of calves to inhalation of Mf during acute BRSV infection. The calves were divided into 4 groups: exposed to virus only (group 1); exposed to aerosolized Mf over a 24-day period and then challenged with Mf during BRSV infection (group 2); similarly exposed to aerosolized Mf and then challenged with Mf without BRSV infection (group 3) and exposed to aerosolized Mf, infected with virus but not challenged with Mf (group 4). All calves were followed for appearance of IgE-specifïc responses to both BRSV and Mf, clinical disease expression, and pulmonary pathology including viral and allergen localization by immunohistology. Our data indicate an association of BRSV-specific IgE with increased development of lung pathology and clinical disease expression and an enhancing effect of aerosolized Mf on induction of virus-specific IgE. The presence of BRSV-specific IgE was solely in calves with the most significant macroscopic and microscopic pneumonic lesions.

“…sedimentation and diffusion data [24], electrophoresis [6], or SDS-PAGE under reducing condi tions [5,28]. Attempts to isolate polyclonal IgE antibodies have utilized sera from dogs [11], horses [29], cows [30], and guinea pigs [31], but the molecular mass has only been reported for horse IgE [29]. Estimation of its molecular mass by non reducing SDS-PAGE revealed a value of 6) and the IgE t; chain ( fig.…”

Section: Discussionmentioning

confidence: 99%

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

Peng

Arthur²,

Kierek-Jaszczuk

et al. 1993

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.