腹壁法(AW法)を用いるアレルゲン食品試験、及び食品中のタンパク質性アレルゲンの検出に関する研究  [in Japanese] Studies on the Allergenic Food Tests and Detection of the Protein Allergens in Foods Using the Abdominal Wall Method (AW Method)  [in Japanese]

Food allergy, which is a serious health problem in the world, can be controlled by avoidance of the causative allergens. For the purpose of avoidance of the allergen, it is essential to recognize the information of not only allergenic foods but also the allergens contained in the processed foods. In Japan, like several foreign countries, the duty to label the raw materials containing well known allergenic substances in the processed food will be bound in the year 2001. Previously, we reported an in vivo assay system on the basis of the mouse anaphylactic reaction using the abdominal wall (AW method). This system has widely been applicable to the detection of the food allergens. When the mice were sensitized with a purified protein allergen, the induction of the specific anaphylactic reaction by the allergenic protein contained in the processed food was attained comfortably, but it isn't always easy to obtain respective purified protein allergens for the foods to be tested. In order to normalize the practical detection of the protein allergen in the processed food, it is important to establish the conditions for the sensitization of mouse with not only the purified protein allergen but also the food itself containing the numerous ingredient. In the present paper, the screening tests of the allergenicity under the same condition [sensitization, FIA emulsion of 10 (W/V) % sample (50μL/mouse); challenge, 10 (W/V) % sample (50μL/site)] were performed and several cases with unclear results of estimation for allergenicity were studied further in detail. Foods known as the causes of food allergy, not only egg, milk, and soybean, but also peanut, chicken, oyster, calamary, kiwi, banana, and strawberry gave clear results in the usual screening for allergenic food tests. On the other hand, buckwheat, rice, wheat, shrimp, mackerel, codfish, and tomato did not show significant differences as compared to normal control group in the screening tests. For these foods, the screening conditions were modified as follows: (1) In the case of the poor sensitization; (a) improvement of the preparation method of the sensitizing solution (ultrafiltration treatment, elongation of the extraction time, or concentration of extraction, etc. of the food-extracted samples), (b) elongation of the sensitization term; (2) In the case of the valid sensitization; (a) removal of the contaminant in the challenging solution which causes a positive error in the challenging solution (elimination of the low molecule contaminant by the ultrafiltration or dialysis etc.), (b) improvement of the preparation method of the challenging solution (consideration of this modified conditions is similar to that described above (1-a)). The results of inducted anaphylactic reactions for the several allergenic foods by the most appropriate condition tested are shown in Table 1. Table 1. Induction of the Anaphylactic Reaction of several allergenic foods by AW (Abdominal Wall) Method [table] Mice were not sensitized (cont.) or sensitized intraperitoneally with several foods in Freund's incomplete adjuvant (FIA) (expl.) on day 0 (50μL/mouse). On day 14, mice of both groups were challenged on the abdominal wall with the food which was used for the sensitized group (50μL/site). Statistical analysis of the each VPV (Vascular Permeability Value) was performed by Mann-Whitney's U test (n=8-12). Asterisks indicate significant differences from the control group (***, p<0.001; **, p<0.005; *, p<0.05). The conditions of the food sampls were as follows: F-10, 10 (W/V) %; F-10α, 10 (W/V) % (24h-extraction, samples prepared after 24h-extraction were expressed with symbol α); F-41.7 α, 41.7 (W/V) % (24h-extraction); F-50, 50 (W/V) %; F-J, food juice. The conditions of the sensitizing samples were as follows: S1, FIA emulsion of F-10 (screening condition); S2 and S3, preparation methods of the samples were described previously (Jpn. J. Toxicol. Environ. Health, 44, 277-288 (1998).); S4, FIA emulsion of F-10α; S5, FIA emulsion of F-41.7α; S6, FIA emulsion of F-50; S7, FIA emulsion of F-J. The conditions of the challenging samples were as follows: Ch1, it equals to F-10 (screening condition); Ch2 and Ch3, preparation methods of the samples were also described previously in the above literature; Ch4, 1.5 fold diluted solution of Ch7 described below; Ch5, it equals to F-10α; Ch6, 150 fold diluted solution of pre-Ch6 which was a 1.5 fold concentrated sample of F-41.7α; Ch7, 1.5 fold concentrated sample of Ch1; Ch8, it equals to F-50; Ch9, 15 fold diluted solution of Ch7; Ch10, 15 fold diluted solution of pre-Ch10 which was a 1.5 fold concentrated sample of F-J. Each concentrated sample was a preparation of high molecular weight fraction after respective ultrafiltration at MW 10kD. a) Jpn. J. Toxicol. Environ. Health, 44, 277-288 (1998). b) Mice were challenged on day 9. C=Chicken, Q=Quail.