Tomoaki Inoue1 and Ikuo Horii2
Original paper :AATEX 9(1):17-28
To develop an antigenicity study with exposure to test compounds in in vitro systems, we investigated suitable experimental conditions for the in vitro exposure. In the antigenicity evaluation system, mouse peritoneal exudate cells (PECs) were exposed to test compounds in in vitro priming culture, the primed PECs were intraperitoneally injected into nontreat mice, and the sera were isolated after an immunizing period. The antigen-specific IgE in the sera was measured by in vitro anaphylaxis test which rat mast cells were exposed to the mouse sera with following exposure to the test compounds (antigens). Histamine levels in the supernatant was measured to evaluate the degranulation of the mast cells. Optimum conditions for the in vitro anaphylaxis test were as follows. The mast cell number: 1-3x104cells/test mast cells, dilution of the test sera: 10:1, incubation of the mast cells with the test sera: 4 hr, concentrations of antigens: 100ug/ml for macromolecular antigens or maximum non-cytotoxic concentrations for low molecular weight antigens, incubation with the antigens: 45 min. By using low molecular weight reference antigens such as penicillin G and nafamostat mesilate, low but positive reactions were observed in the antigenicity evaluation system consisting of in vitro priming culture and in vitro anaphylaxis test. From these results, this antigenicity testing system would help reductions of the number of animals, required amount of test compounds, and the experimental period, and make it possible to evaluate antigenicity in early stages of drug discovery.
Keywords: antigenicity, in vitro, priming culture, anaphylaxis, peritoneal cells