Codina R, Oehling A G, Lockey R F
Division of Allergy and Immunology, Department of Medicine, University of South Florida and James A. Haley V.A. Hospital, Tampa, Fla., USA.
Int Arch Allergy Immunol. 1998 Oct;117(2):120-5. doi: 10.1159/000023998.
During the process of harvest, transport and storage, microbial and mold contamination can raise the temperature of soybeans to 75 degreesC or higher. The purposes of this study were (1) to evaluate the allergenicity of fresh and stored soybean hulls and (2) to ascertain whether heat alters the allergenicity of stored soybean hulls.
Allergen extracts were prepared from (1) stored soybean hulls, (2) fresh soybean hulls and (3) stored soybean hulls heated to 37 degreesC (E1), 55 degreesC (E2) and 80 degreesC (E3) or kept at room temperature (E4) for 16 h. Individual serum from 68 soybean asthmatic (SA) subjects, 30 nonallergic subjects and two serum pools made from 4 SA sera and 4 sera from asthmatics not sensitive to soybean were studied. All sera and serum pools were assayed for content of specific IgE (radioallergosorbent test) and IgG4 (ELISA). The following additional studies were done for extracts E1-E4: (1) SDS-PAGE, (2) SDS-PAGE/Western blot for specific IgE and IgG4 using both serum pools, and (3) study of the effects of heat on inhibiting activity of the extracts prepared from stored soybean hulls using the pool of SA sera.
Test results demonstrated a reduced binding of specific IgE and IgG4 to fresh soybean hull extract compared to stored soybean hull extract, and an increased binding for heated extracts (E1-E3) compared to unheated ones (E4). Moreover, there was an increase in potency for IgE and IgG4 bindings for the heated (E1-E3) compared to unheated (E4) extract, as measured by the amount of protein to produce 50% inhibition. Several protein bands with a molecular weight (MW) higher than 20 kD were absent from the SDS-PAGE for E3 but were present in E1, E2 and E4, and a new protein band (MW 15.3 kD) appeared for E3 only. Two new protein bands, with MWs of 15.3 and 10 kD, which bind specific IgE, were present on Western blot and one of the 3 main soybean hull allergens, probably Gly m 2, disappeared in E3. IgG4 Western blot showed similar results, but only the 10 kD protein band was present.
The results demonstrate that soybean hull allergenicity is affected by heat, and suggest that the heat generated during storage and transport of soybeans could generate 2 new allergen determinants or increases in epitope exposure as a result of conformational changes. The significance of these new IgE and IgG4 binding proteins has yet to be determined.
在收获、运输和储存过程中，微生物和霉菌污染可使大豆温度升至75摄氏度或更高。本研究的目的是：（1）评估新鲜和储存大豆皮的致敏性；（2）确定加热是否会改变储存大豆皮的致敏性。
制备过敏原提取物，分别来自：（1）储存大豆皮；（2）新鲜大豆皮；（3）加热至37摄氏度（提取物E1）、55摄氏度（提取物E2）和80摄氏度（提取物E3）或在室温下保存16小时（提取物E4）的储存大豆皮。研究了68名大豆哮喘（SA）患者、30名非过敏受试者的个体血清以及由4份SA血清和4份对大豆不敏感的哮喘患者血清制成的两个混合血清。对所有血清和混合血清进行特异性IgE（放射变应原吸附试验）和IgG4（酶联免疫吸附测定）含量检测。对提取物E1 - E4进行了以下额外研究：（1）十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）；（2）使用两个混合血清对特异性IgE和IgG4进行SDS - PAGE/蛋白质印迹分析；（3）使用SA患者混合血清研究加热对储存大豆皮提取物抑制活性的影响。
测试结果表明，与储存大豆皮提取物相比，特异性IgE和IgG4与新鲜大豆皮提取物的结合减少，与未加热提取物（E4）相比，加热提取物（E1 - E3）的结合增加。此外，通过产生50%抑制所需的蛋白量测定，与未加热提取物（E4）相比，加热提取物（E1 - E3）的IgE和IgG4结合效力增加。提取物E3的SDS - PAGE中不存在分子量（MW）高于20 kD的几条蛋白带，但在E1、E2和E4中存在，并且仅提取物E3出现了一条新的蛋白带（MW 15.3 kD）。蛋白质印迹分析显示存在两条与特异性IgE结合的新蛋白带，分子量分别为15.3 kD和10 kD，并且三种主要大豆皮过敏原之一（可能是Gly m 2）在提取物E3中消失。IgG4蛋白质印迹分析显示类似结果，但仅存在10 kD的蛋白带。
结果表明大豆皮致敏性受加热影响，提示大豆在储存和运输过程中产生的热量可能产生2种新的过敏原决定簇，或因构象变化导致表位暴露增加。这些新的IgE和IgG4结合蛋白的意义尚待确定。
