Production of a monoclonal antibody specific for ovine immunoglobulin E and its application to monitor serum IgE responses to Haemonchus contortus infection

Abstract

Part of the C epsilon 3-C epsilon 4 region of the ovine immunoglobulin E (IgE) gene (nucleotides 1111-1575) was amplified by PCR. The recombinant protein (recIgE1-2) was expressed in E. coli and both monoclonal and polyclonal antibodies were produced. These antibodies recognized recIgE1-2 and native IgE on Western blots and in ELISA. The polyclonal serum showed cross-reactivity with other sheep immunoglobulin classes. The monoclonal antibody was specific for ovine IgE and goat IgE. Infection of sheep with the abomasal nematode Haemonchus contortus resulted in elevated IgE levels in serum 2-4 weeks after infection, as measured by sandwich ELISA using the rabbit polyclonal as capture antibody and the monoclonal antibody against ovine IgE as second antibody. A negative correlation between worm counts and total serum IgE levels at the end of the experiment was found in repeatedly infected sheep. Significant increased levels of excretory-secretory antigens specific IgE levels were found after H. contortus infection. In contrast, no significant changes in 3rd-stage larvae (L3) antigen-specific IgE titre in sera could be detected after infection.

Field study on nematode resistance in Nelore-breed cattle

“…Not only FEC but also total serum IgE level was associated with the resistance degree, corroborating the results obtained by Kooyman et al (1997) and Pettit et al (2005) in sheep. Therefore, the present study provides further evidence of a role of IgE in nematode immunity and suggests that total serum IgE level might serve as an additional marker for selecting Nelore cattle that are responsive to H. placei and C. punctata infections.…”

Serum antibody responses in Creole kids experimentally infected with Haemonchus contortus

“…Thus, in sheep increased total and nematode-specific serum IgE levels after gastrointestinal nematode infection have been reported in many studies (Huntley et al, 1998;Pernthaner et al, 2005). Furthermore, a negative correlation between worm counts and total serum IgE levels was found in sheep parasitized with H. contortus (Kooyman et al, 1997). Our results showed a significant rise in L3 crude extract and ESP specific serum IgE in both animal groups after H. contortus infection.…”

“…The plates were washed three times with PBST before addition of a horseradish peroxidase-conjugated Rabbit anti-goat IgA (Alpha Diagnostic) diluted 1:1000 in carbonate buffer (60 min of incubation at RT). For IgE ELISA, plates were sequentially incubated with mouse anti-ovine IgE monoclonal IE7 (Kooyman et al, 1997) diluted 1:1000 (kindly provided by Dr. F.N.J. Kooyman, Utrecht University, The Netherlands) and rabbit anti-mouse HRP conjugate (Serotec, Oxford, England).…”

“…Thus genetic resistance is immunogically mediated by proliferation of mucosal mast cells, globule leukocytes and eosinophils (Meeusen et al, 2005). The response against gastrointestinal nematodes also involves production of parasite-specific immunoglobulin A (IgA), IgG1 and IgE (Pfeffer et al, 1996;Kooyman et al, 1997;Shaw et al, 1998).…”

Generation and Characterization of a Clonotypic Antibody Specific for the T Cell Receptor of an Arthritogenic T Cell Clone—Studies in Adjuvant Arthritis

“…Subsequently, IgG2a molecules were purified by protein affinity chromatography (Amersham Pharmacia Biotech, Rosendaal, The Netherlands) and IgG1 molecules were purified by thiophilic affinity chromatography using AFFI-T agarose (Kem-En-Tec, Copenhagen, Denmark). As isotype controls IE7H11 (anti-IgE IgG2a, [25]) and IL-A51 (anti-CD8 IgG1, [26]) were used. …”