Quantification of total sheep IgE concentration using anti-ovine IgE monoclonal antibodies in an enzyme immunoassay

Abstract

Monoclonal antibodies (mAbs) which recognize separate epitopes on ovine immunoglobulin E (IgE) have been used to develop a non-competitive antibody sandwich enzyme immunoassay (EIA) for quantitating ovine IgE. Purified anti-IgE mAb (YD3) coated onto polystyrene microtitre plates was used to capture IgE in serum samples. Biotinylated anti-IgE mAb (XB6) followed by streptavidin conjugated with horseradish peroxidase were used to detect captured IgE. Tetramethylbenzidine and H 2O 2 were used as enzyme substrate. A reference serum was prepared by pooling sheep sera containing elevated IgE levels. This reference serum was assigned a value of 100 units ml −1 and used to prepare standard curves for the EIA. The linear region of log-log transformed standard curve data covered a range of 0.05-0.8 units ml −. The equation of a linear regression line fitted to this curve was used to determine sample concentrations. Using purified IgE, 1 unit of reference serum was equivalent to 0.86 μg ml − IgE. Maximum intra- and inter-assay coefficients of variation for the EIA were 4.6% and 9.7%, respectively. Subjecting serum samples to 15 freeze/thaw cycles, storage at room temperature for 16 days or incubation at 37°C for 8 h resulted in minimal loss of IgE detection. Incubation of serum at 56°C resulted in rapid reduction in detection of IgE by the EIA. The assay was used to determine IgE levels in adult sheep monospecifically infected with weekly doses of the nematode Trichostrongylus axei. Serum IgE levels increased from 9 to 16 days following first infection and reached maximum levels by days 35–58. Serum IgE responses closely followed IgE positive cell responses in the abomasal mucosa.