The principle of the AW method is that mice sensitized with a protein antigen could exhibit an antigen specific anaphylactic reaction in abdominal wall on challenge with the same antigen. The method is applicable to the detection of protein allergens in food containing unknown components. It has been evident that the appearance of anaphylaxis on abdominal challenge with food samples in the mice sensitized previously with known protein antigen, could indicate the existence of the protein in the food. However, unless the single purified protein antigen as an object of the detection is available, raw food samples themselves have to be used to sensitize the experimental mice. In raw food samples a lot of protein antigens coexist and their concentration varies from food to food. In order to widen the range of application and to ascertain the reliability of the AW method, it is needed to investigate whether the coexisting protein antigens could interact at the both sensitization and elicitation phases, and to determine the dose of antigen required for induction of anaphylactic reaction. To answer these questions, in the present study, we performed three experiments as follows. First we examined the effects of coexisting proteins on the anaphylactic responses by the AW method, using two well-studied protein antigens, hen egg white lysozyme (HEL) and ovalbumin (OVA). Mice sensitized with a mixture of HEL and OVA were challenged with HEL or OVA on the abdominal wall 9 days after sensitization (Table 1). The other mice sensitized with OVA were challenged with a mixture of OVA and HEL 9 days after sensitization. The anaphylactic intensity was estimated by VPV (vascular permeability value) on abdominal wall. VPV of each experimental group was not significantly different from that of the control group. These results suggest that OVA and HEL does not influence each other on both the sensitization and challenge phases. Next, dose-dependent effects of OVA on the sensitization of anaphylactic reaction were studied. Mice were sensitized with various concentrations of OVA (0.0001〜10mg/mL, 50 μL/mouse) and challenged with the fixed concentration of OVA (5 μg/50 μL/site) 9 days later. The observed VPVs were significant at more than 0.01 mg/mL and reached plateau at more than 0.25 mg/mL. Finally we investigated the correlation between induction of anaphylactic reaction in mice and total protein concentration in food. A correlation coefficient between the total protein concentration and the anaphylactic intensity (VPV) was 0.6619. Table 1. Effect of coexisting protein-antigens on the sensitization phase [table]