Detection Of Immunoglobulin Research Articles

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

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.

Enhanced sensitivity of immunoblotting with peroxidase-conjugated antibodies using an adsorbed substrate method

Immunoblots probed with immunoglobulin E (IgE)-containing sera from allergic patients are frequently used in allergy research. Current techniques for detection of specific IgE include radiolabeled and enzyme-linked methods. Although radiolabeled methods are very sensitive, many research groups prefer non-radioactive procedures with equal or greater sensitivity. Alkaline phosphatase (AP) and horse radish peroxidase (HRP) are the most frequently used conjugating enzymes for immunoblotting with the former generally recognized as more sensitive. We describe a method of immunoblot detection using HRP-conjugated immunochemicals with sensitivity equal to and for some systems greater than that of AP conjugates. An adsorbed substrate method for developing immunoblots probed with HRP immunochemical conjugates is compared with traditional AP and HRP methods. The adsorbed substrate system, when used to detect IgE binding to allergenic proteins, gives high resolution and delineates bands not otherwise seen. The system has advantages of high sensitivity, rapid development and conservation of immunochemicals. Problems of fading, sensitivity to heat and light, and high background can be solved with increased washing, prompt photography and computer scanning.

Structure and Function of Human Immunoglobulin E

Publisher Summary This chapter discusses the isolation, and the physicochemical characteristics of immunoglobulin E (IgE) properties of antigenically and biologically active structural regions of IgE, levels of IgE in disease, and detection of antibody activity in the IgE Class. IgE represents a minor, but a distinct class of proteins present in the serum of man and higher primates, and possibly also in the serum of other species. The detection and quantitation of IgE require very sensitive methods. Immunoglobulin E is elevated 4-30 times normally in various diseases, among which atopic disorders and parasitic infestations appear to be the most prominent. Pathological amounts of IgE have also been found in the serum of patients with γE myeloma. The best way to determine IgE is to use the immunochemical methods based upon the reaction between IgE and the antibodies specific for the class-related antigenic determinants of IgE. Gel diffusion methods, such as single radial diffusion in gel are handicapped by low sensitivity; radioimmune assays have proven to be superior.