Wistar Strain Rats as the Model for IgE Antibody Experiments.

Abstract

In recent years, an increase has been reported globally in the number of cases of allergic disease. Japan is no exception, and allergic diseases including cedar pollenosis, atopic diseases, and extrinsic asthma have raised grave social concern.2) These allergic symptoms are generally classified as the type I allergy and well-known immediate-type reactions. They are also known to be caused by the release of mediator from target cells such as mast cells and basophils following exposure to an antigen. When an antigen enters the body, it is phagocytosed by macrophages, which then present it to T cells. Differentiated (proliferated) T cells present the antigen to B cells. After differentiation to antibody-forming cells by the action of cytokines such as interleukin (IL)-4, B cells produce IgE antibody.3) An increase in plasma IgE level is observed in about 80% of patients with atopic dermatitis and this is closely related to their long-term serious symptoms. 4,5) We attempted to prepare rats with high IgE as a model of patients with allergic diseases. Rats were activated using 2,4-dinitrophenylated ascaris extract (DNP-As) as antigen and were immunized with killed Bordetella pertussis as adjuvant, which is reported to enhance IgE antibody formation in rats and mice. [6][7][8] The amount of IgE antibody formed and its change over time were then studied in the rats. MATERIALS AND METHODSAnimals Six-week-old male and female, Sprague-Dawley (SD), Donryu, and Wistar strain rats were kindly supplied by Japan SLC Inc. The animals were used in the experiments after preliminary feeding for one week.Reagents DNP-As and killed B. pertussis were purchased from LSL Co., Ltd., and Chiba Serum Research Laboratory, respectively. Morinaga Institute of Biological Sciences kindly donated enzyme-linked immunosorbent assay (ELISA) kits (Morinaga Rat IgE ELISA Kit).Immunization Immunization of rats was performed with reference to the method of Matsuura et al.9) DNP-As (1 mg per animal) was dissolved in physiological saline 0.1 ml and the solution was mixed with killed B. pertussis 0.5 ml of containing 10 10 cells. The mixture was subcutaneously injected in the paws of each rat for early immunization. Five days after early immunization, a solution of DNPAs 0.5 mg in physiological saline 0.25 ml was injected into the back muscle of each rat to produce additional immunization.Blood Sampling and Measurement Blood was drawn from the jugular vein of each rat 0, 5, 10, 20, and 25 d after additional immunization. The day of additional immunization was taken as day 0. The blood was centrifuged at 4°C for 10 min at 700ϫg and the plasma obtained was used as the sample. The plasma was frozen at Ϫ20°C and stored until the time of IgE determination. The amount of IgE was determined by the EIA sandwich method using ELISA. The sample was placed in a plate coated with solid phase-bound anti-rat IgE monoclonal antibody and the plate was allowed to stand at room temperature for 4 h, during which the antibody formed a complex with rat IgE. After washing each...

Allergen-sensitization in vivo enhances mast cell-induced inflammatory responses and supports innate immunity

“…This is consistent with the fact that cellular signaling induced in mast cells by monomeric IgE persists as long as free IgE is present [9,19]. In standard sensitization protocols, it has been demonstrated that the peak in the level of total IgE is exhibited 15 days after first immunization with antigen [20].…”