晒干处理对花生过敏原蛋白潜在致敏性的影响

花生是八大食物过敏原之一,花生过敏通常是终身的。晒干是花生加工的重要环节,本研究通过对新鲜花 生进行去壳晒干和带壳晒干2 种不同的晒干处理,探索不同晒干方式对花生过敏原蛋白潜在致敏性的影响。采用凯 氏定氮法、二喹啉甲酸法和聚丙烯酰胺凝胶电泳法测定花生及蛋白提取液中的蛋白浓度和过敏原蛋白的组成,用圆 二色谱、紫外扫描光谱检测花生蛋白的结构变化,用血清免疫球蛋白E（immunoglobulin E,IgE）结合能力表征花 生蛋白潜在致敏性的变化。结果显示,晒干处理后,花生蛋白与血清IgE的结合能力显著增强（P＜0.05）,去壳晒 干的花生蛋白质二级结构比带壳晒干的花生更有序,三级结构更加紧凑,带壳晒干的花生蛋白可能因为其结构较为 松散,故与IgE结合能力更强。     

Dot-ELISA法同时检测多种食品过敏原的实验研究

通过花生、虾、蛋清过敏原浸液皮下免疫复制小鼠过敏模型,获特异性IgE的小鼠血清进行方法学研究。将不同的过敏原以适当浓度点状吸附于硝酸纤维素膜(NC膜)上,与待检血清作用,再依次与生物素化羊抗鼠IgE第二抗体,亲和素偶联的辣根过氧化物酶反应,DAB显色。结果表明:获得了小鼠抗花生、虾、蛋清过敏原高效价特异性IgE血清。Dot-ELISA法各种过敏原最适包被量为2μL(10μg蛋白),待检抗血清最佳稀释度为1∶80,通过肉眼观察NC膜上斑点的显色即可确定待检血清中相应过敏原特异性IgE,且3种过敏原之间无交叉反应性。不同时间同批试剂重复检测结果完全一致,包被过敏原的NC膜4℃可稳定保存3个月。实验初步建立了用含有过敏原特异性IgE的小鼠血清同时检测多种过敏原的Dot-ELISA实验方案,该法简便、特异、稳定、重复性好,可成为食品过敏原鉴定与评价的新方法。     

中国对虾肉中与虾过敏患者血清特异性IgE结合的组分分析

分析与血清特异性IgE结合的虾肉中的蛋白成分。方法 常规制备虾肉蛋白提取液,用SDS-PAGE技术分析提取液中的蛋白组分,以虾过敏患者血清(sIgE)作为探针,经免疫印记技术分析、鉴定与患者特异性IgE结合的蛋白成分。结果 虾肉蛋白提取液显示12种可辨认蛋白条带,其中65、50和36kD相对含量较高;不同过敏患者血清中与蛋白结合的强度和所识别的蛋白组分存在一定差别,但所识别的蛋白主要集中在分子量＞70kD的区域。结论 与阳性血清的反应强度与蛋白相对含量无关,提取有效组分有望提高血清特异性IgE检测的敏感度。     

花生致敏蛋白Ara h1双抗体夹心ELISA法的建立

为建立用于花生致敏蛋白Ara h 1快速、灵敏、特异检测的双抗体夹心ELISA法,以Ara h 1鼠源单抗(mAb)为捕获抗体,以Ara h 1兔源多抗(pAb)为检测抗体,通过棋盘法优化抗体工作浓度建立方法,并对方法的灵敏度、特异性、准确度、稳定性等检测特性进行鉴定。结果表明,双抗体夹心ELISA法的mAb和pAb最佳工作浓度分别为1∶10 000稀释和1∶8 000稀释;ELISA标准曲线线性范围为4～256ng/mL,检测限为4.16ng/mL;样品添加回收试验的平均回收率为85.9%～94.5%,且该方法与其他常见致敏蛋白无交叉反应;并能在4℃条件下避光密封保存90d内保持检测结果稳定。说明所建立的双抗体夹心ELISA法灵敏特异、准确稳定,能够用于花生致敏蛋白Ara h 1的快速筛查。     

腰果主要过敏原Ana o 2原核表达及免疫学鉴定

目的 构建Ana o 2重组表达质粒,原核表达重组蛋白并评价其免疫活性。方法 将Ana o 2基因构建到pET-28a(+)载体中,测序正确的重组质粒转化Rosetta(DE3)感受态细胞,诱导表达目的蛋白并进行质谱鉴定。利用腰果过敏阳性血清,通过酶联免疫吸附试验(ELISA)和蛋白质免疫印记(Western blot)法评价重组Ana o 2的免疫活性。结果 重组蛋白进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)表明分子量为54 kD,与理论值相符,经质谱鉴定为Ana o 2。ELISA结果显示,用重组Ana o 2检测腰果过敏阳性血清与阴性血清特异性IgE抗体(sIgE)水平差异有统计学意义(t=2.44,P<0.05)。Western blot结果表明,重组Ana o 2与腰果过敏患者血清反应性良好。结论 利用原核系统表达了Ana o 2,并且重组Ana o 2与腰果过敏患者血清具有良好的反应性。     

花生过敏患者血清中抗Ara h1的IgG与IgE分离纯化研究

Ara h1是花生的一种主要致敏原蛋白,能够使花生过敏患者产生特异的IgG与IgE,从而导致花生过敏症状。Ara h1与其特异IgG和IgE的结合是导致花生致敏的一个重要原因。本研究利用Protein A亲和层析柱与Ara h1作为配体的亲和层析柱纯化抗Ara h1的IgG与IgE。高纯度的抗Ara h1特异IgG与IgE对探讨研究其与Ara h1之间相互作用具有重要意义。     

山羊乳与牛乳配方乳粉的致敏性比较

山羊乳是一类营养丰富且易于消化的乳品,因其营养成分与牛乳相近而受到广泛关注。本研究通过蛋白质潜在致敏性树状评估策略（生物信息学、消化稳定性、血清学研究、动物模型）对山羊乳配方乳粉的致敏性进行评价,并研究山羊乳配方乳粉与牛乳过敏患者血清中免疫球蛋白（immunoglobulin,Ig）E之间存在的交叉反应性。结果表明,山羊乳配方乳粉的167 种蛋白质中,β-乳球蛋白和α-乳清蛋白含量显著低于牛乳配方乳粉（P＜0.05）,山羊乳配方乳粉在模拟胃液中更易消化,且与过敏患者血清的结合能力弱于牛乳配方乳粉,动物模型结果表明山羊乳配方乳粉致敏小鼠体温下降幅度、临床过敏症状评分、血清中特异性IgE和IgG1抗体水平、血浆中组胺水平、血管渗透性及组织炎症程度等均显著低于牛乳配方乳粉组致敏小鼠（P＜0.05）,交叉反应性结果表明山羊乳配方乳粉能与牛乳过敏患者血清结合,但结合能力明显低于牛乳配方乳粉。结论：与牛乳配方乳粉相比,山羊乳配方乳粉的致敏性较弱,可作为牛乳的替代品,降低牛乳过敏性疾病的发病风险。     

荞麦过敏原特异性抗体酶联免疫吸附法检测试剂制备

以天然苦荞过敏蛋白包被聚苯乙烯微孔板,以鼠抗人IgE抗体标记辣根过氧化物酶,以间接酶联免疫吸附法(indirect enzyme-linked immunosorbent assay,IELISA)检测病人血清中的特异性抗体,分析并建立关键操作步骤及反应条件。结果表明:ELISA反应的最适工作条件为包被的抗原质量浓度100μg/mL、阳性血清稀释度1:50、酶标二抗稀释度1:1000,37℃时封闭的最佳时间为40min,包被抗原与阳性血清中抗体的最佳作用时间为40min,阳性血清与酶标二抗的最佳作用时间为40min。本方法具有良好的特异性、重复性和精密度。     

虾制品致敏性诱发豚鼠相关细胞因子及Th1/Th2细胞平衡的变化

以消减致敏性南美白对虾的虾仁、虾肉和虾蛋白为样品,以pH 7.5的磷酸盐缓冲液为阴性对照,未处理的虾蛋白为阳性对照,建立豚鼠过敏模型,研究过敏豚鼠血清中相关细胞因子与食物过敏的相关性,并推断食物过敏对辅助性T（type 1/type 2 T-helper, Th1/Th2）细胞平衡的影响。收集过敏豚鼠的血清,采用酶联免疫吸附测定法检测血清中免疫球蛋白E（immunoglobulin E,IgE）、组胺（histamine,HIS）、相关细胞因子（白细胞介素（interleukin,IL）-1、IL-2、IL-3、IL-4、IL-6、IL-10、肿瘤坏死因子-α（tumor necrosis factor α,TNF-α）、干扰素-γ（interferon-γ,IFN-γ）质量浓度。结果表明,用未处理的虾蛋白致敏豚鼠,用致敏性消减程度不同的虾制品（虾蛋白、虾肉、虾仁）提取的蛋白激发,消减致敏性的虾蛋白、虾肉和虾仁提取蛋白激发后豚鼠血清中IgE含量分别为（3.905±0.120）、（4.813±0.188）、（5.199±0.327）U/mL,HIS质量浓度分别为（16.437±1.120）、（19.656±1.080）、（21.071±1.732）μg/mL,激发后血清中IgE和HIS质量浓度变化与致敏性程度呈正相关,致敏性越低,血清中IgE和HIS质量浓度越低。过敏豚鼠血清中IL-1、IL-2、IL-3、IL-4、IL-6、TNF-α质量浓度的变化与虾制品致敏程度呈正相关;同时,与IgE和HIS质量浓度变化规律具有一致性;血清中IL-10质量浓度变化与致敏性程度呈负相关;因此,这些细胞因子与食物过敏具有很好的相关性。过敏血清中IFN-γ没有呈现规律性变化,但IFN-γ/IL-4随着致敏性的增强而减小。因此,推测虾制品激发过敏豚鼠的Th1/Th2细胞平衡向Th2细胞偏移。     

新抑制剂有望防止花生过敏

正美媒称,花生是最常见的过敏诱因,美国的科学团队开发出一种首创的针对特定过敏原的抑制剂,可以让病人的免疫系统无法辨识出花生过敏原。据美国《新闻周刊》网站4月8日报道,当人体的免疫系统误把食物蛋白当做威胁时,会发生过敏反应。人体制造出一种名为免疫球蛋白E(IgE)的蛋白,用来袭击过敏原。身体分泌这种抗体会导致可能致命的过敏反应症状,包括瘙痒、呼吸困难和风疹。     

加工方式和蛋白提取方法对花生蛋白致敏性的影响

加工方式影响花生蛋白的致敏性。本实验系统的研究了中国传统花生制品(水煮和油炸花生)和美国常食用的烘烤花生致敏性的差异,并比较了不同蛋白提取缓冲溶液对花生致敏蛋白提取效率的影响。结果表明:不同花生制品在不同的缓冲溶液中蛋白提取效率不同,水煮和油炸并没有较烘烤方式降低致敏蛋白的数量,而降低花生蛋白的致敏性。因此加工方式并不是导致中国花生过敏发病率低的主要原因。     

Peanut polyamines may be non‐allergenic

Polyamines such as putrescine, spermidine and spermine have been implicated in preventing food allergies in early life, but they have also been reported to be able to bind to immunoglobulin E (IgE) antibodies in vitro (ie they are possibly allergenic). The objective of this study was to determine if polyamines bind in vitro to IgE antibodies from a pooled serum of five peanut‐allergic individuals. Levels of polyamines were also determined by ion‐exchange chromatography. Indirect and inhibition enzyme immunosorbent assays (ELISAs) were used to determine the IgE binding or allergenic properties of polyamines. Results showed that, of the three polyamines, spermidine was predominant in peanuts. In both indirect and inhibition ELISAs, IgE antibodies did not bind to the polyamines. It was concluded that polyamines from peanuts, unlike peanut proteins, are not allergenic or an additional threat to patients who are allergic to peanuts. Published in 2005 for SCI by John Wiley & Sons, Ltd.     

Peanut varieties with reduced Ara h 1 content indicating no reduced allergenicity

Peanut allergy is a major cause of food‐induced severe anaphylactic reactions. To date, no medical care is available to prevent and treat peanut allergy and therefore hypoallergenic peanut varieties are of considerable health political and economic interest. Major allergens that induce IgE‐responses in peanut‐sensitive patients are Ara h 1, Ara h 2 and Ara h 3/4. In order to identify hypoallergenic peanuts, commercially locally available peanut varieties were screened for their allergen content. Ara h 1‐deficient peanuts from Southeast Asia were identified by SDS‐PAGE, immunoblotting, inhibition assays and ELISA. 2‐D PAGE analyses demonstrated the different compositions of the tested extracts and revealed a number of variations of the allergen patterns of peanuts from different varieties. Mediator release experiments of these peanut extracts demonstrated similar allergenicities as compared with standard peanut extract. These results indicate that the allergenicity of peanuts with reduced Ara h 1 content might be compensated by the other allergens, and thus do not necessarily cause a reduction of allergenicity.     

Mitigation of Major Peanut Allergens by Pulsed Ultraviolet Light

Peanut allergy represents one of the most severe IgE-mediated reactions with food, but to date, the only effective way to prevent peanut allergy is total avoidance. If allergens could be mitigated during food processing before a product reaches the consumer, this would substantially lessen the food allergy problem. The efficacy of pulsed ultraviolet light (PUV), a novel food processing technology, on reducing peanut allergens, was examined. This study revealed for the first time that PUV was also capable of deactivating Ara h 2, the most potent allergenic protein of peanut. Protein extracts from raw and roasted peanuts were treated for 2, 4, and 6?min and peanut butter slurry was treated for 1, 2, and 3?min in a Xenon Steripulse XL 3000? PUV system. The distance from the central axis of the lamp was varied at 10.8, 14.6, and 18.2?cm. The SDS?CPAGE showed a reduction in the protein band intensity for Ara h 1, Ara h 2, and Ara h 3 at the energy levels ranging from 111.6 to 223.2?J/cm². Reduction of the protein band intensity for peanut allergens increased with treatment time but decreased with increased distance from the PUV lamp. The ELISA for peanut extracts and peanut butter slurry showed a reduction in IgE binding of up to 12.9- and 6.7-folds, respectively, compared to control.     

Reduction of IgE Immunoreactivity of Whole Peanut (Arachis hypogaea L.) After Pulsed Light Illumination

Pulsed light (PL), a novel food processing and preservation technology, has been shown in literature to reduce allergen levels on peanut, soybean, almond and shrimp protein extracts. This study investigated how PL affected the immunoreactivity of whole peanut kernels at two sample-to-lamp distances (7 and 10 cm) and a PL frequency of 3 pulses/s. A combination of different illumination durations and distances were tested to explore the effective conditions for PL roasting of whole peanuts. After the PL treatments, crude protein of the whole peanuts was extracted and compared to that of the raw peanuts (control) in vitro for allergen reactivity using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, dot blot, and enzyme-linked immunosorbent assay (ELISA) with pooled plasma of allergenic patients. The SDS-PAGE, Western blot, dot blot results all showed that PL was capable of mitigating all major allergens of whole peanut kernels to an undetectable level. Indirect ELISA results showed 80 % signal reduction for the PL-treated peanut as compared to raw peanut. A closer distance (7 cm) between the PL lamp and the sample resulted in stronger reduction of IgE immunoreactivity than 10 cm.     

Serum testing of genetically modified soybeans with special emphasis on potential allergenicity of the heterologous protein CP4 EPSPS

Roundup Ready soy contains the CP4-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein. Serum IgE from two distinct populations of soy-allergic patients were recruited to determine their IgE-binding specificity. One population consisted of 10 adult patients from Europe, whose primary diagnosis was soy food allergy with some also having mite allergy. In addition, 6 primarily mite-allergic, 6 food-allergic (celery, carrot, milk, shrimp, walnut, and apple), and 5 non-allergic patients were tested. Another population consisted of 13 children from Korea, whose primary diagnosis was atopic dermatitis and secondarily soy and egg sensitization. In addition, 11 non-allergic patients were tested. Each patient population was extensively characterized with respect to clinical symptoms, specific IgE (CAP) scores, and total IgE. Immunoblots and ELISA assays were developed using serum IgE from these patients and soy extracts, CP4 EPSPS, rice extract, ovalbumin, rubisco, purified major peanut allergen Ara h 2, the putative soy allergen Gly m Bd 30k and mite allergen Der f 2 proteins as the intended targets. Immunoblot results indicated that soy-allergic patients bound soy extracts but did not specifically bind rubisco or CP4 EPSPS. ELISA results were in general agreement with the immunoblot results except that rubisco bound significant quantities of serum IgE from some patients. These results indicate that the CP4 EPSPS protein does not bind significant quantities of IgE from two geographically distinct sensitive populations and there is no evidence for an increased allergenic potential of this biotech protein.     

Allergenicity of roasted peanuts treated with a non-human digestive protease

Peanut allergy is a severe and lifelong type of food allergy triggered by allergenic proteins and peptides in peanuts. This study investigated the effects of ultrasound-assisted alcalase treatment on the concentrations of major allergenic proteins (Ara h 1 and Ara h 2) in roasted peanut kernels and the allergenicity of treated peanut extracts. Peanut kernels were sonicated for 1 h in buffer solution, incubated with different amount of alcalase for various time, then vacuum dried. The variations of Ara h 1 and Ara h 2 contents in soluble and insoluble portions of peanuts treatments were evaluated by sandwich ELISA and SDS-PAGE, respectively. The in vitro IgE-binding capacity of treated peanut extracts was determined by a competitive inhibition ELISA using pooled plasma of 10 peanut allergic patients. Samples with lower in vitro IgE-binding were used for human skin prick tests (SPTs) in peanut allergic individuals. Results indicate that alcalase digestion of sonicated peanuts significantly increased protein solubility while decreasing Ara h 1 and Ara h 2 concentrations in both soluble and insoluble portions of peanuts relative to untreated peanuts. The maximum reductions of Ara h 1 and Ara h 2 levels were obtained following 3 hour digestion with alcalase at concentrations of 4.54 and 6.05 U/100 g. Samples obtained under these conditions showed the lowest in vitro IgE-binding and caused the least allergic response in human SPTs. The current study suggests that the allergenic potential of peanuts could be reduced by postharvest processing such as ultrasound-assisted enzymatic treatment of peanuts kernels.     

花生过敏原检测方法研究进展

花生是重要食物过敏原之一,能引起严重的过敏反应。目前尚没有花生过敏的特效疗法,严格避免食入含花生的食物是花生过敏患者的最佳选择,所以食物中花生的检测就显得尤为重要。本文对花生主要过敏原及其过敏反应研究进展进行简要介绍,重点介绍花生过敏原的主要检测方法的新发展,包括传统的酶联免疫吸附实验(ELISA)、免疫印迹法、聚合酶链式反应(PCR)等,ELISA和免疫印迹法都是应用较为普遍的检验方法,PCR方法应用较少,但它能从DNA/RNA方面检测花生过敏原的存在;新兴检测方法主要包括生物传感器和质谱法,这两种方法在花生检测方面具有可观的前景。未来的这些检测方法将朝着快速、灵敏、简便的方向发展。     

Digestibility of peanut and hazelnut allergens investigated by a simple in vitro procedure

 Stability under digestion is thought to be an important prerequisite determining the allergenicity of food proteins. To test this hypothesis, two important allergenic plant-derived foods were selected for this investigation. Protein extracts from roasted peanuts and unprocessed (native) hazelnuts were digested by a static, two-step in vitro procedure with commercial enzyme tablets containing peptic and pancreatic enzymes, respectively. The food extracts were subjected to gastric digestion for 2 h followed by a 45-min treatment under duodenal conditions. Undigested control samples and the two digests were investigated by analytical sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), by SDS-PAGE immunoblotting, by an enzyme allergosorbent test (EAST) with human IgE, and by a rat basophil leukaemia (RBL) cell mediator release assay that depends on specific IgE raised in mice. Peanut proteins appeared to be more stable under digestion than hazelnut proteins, as shown by electrophoresis. These results were also underlined by immunblot data. The gastric digest from peanut contained various protein fragments that were detected by antibodies from a peanut-specific rabbit antiserum and by IgE from patients allergic to peanuts. These immunoblot reactivities decreased strongly after subsequent pancreatic digestion. In the gastric digest from hazelnuts, a rabbit antiserum with a broad reactivity against native hazelnut proteins exclusively recognized small protein fragments of <15 kDa. This serum showed no binding to blots of the pancreatic digest. Sera from hazelnut-allergic patients presented IgE reactivities to an 18-kDa major allergen with homology to major tree-pollen allergens, to a minor allergen of 12 kDa, and to multiple bands  1 30 kDa in native hazelnut extract. No binding was observed with these sera on blot strips of the two digests prepared from hazelnut extract. Under the native conditions of EAST, both digests from peanuts strongly reacted with human IgE. Their IgE binding capacity persisted at a level of about 50% when compared to undigested peanut. In the case of hazelnuts the IgE reactivity of the untreated samples was reduced to <10% by both gastric and combined gastric/duodenal digestion for a serum pool prepared from four patients and sera from three additional participants. By contrast, a constantly high immunoreactivity of the hazelnut digests was detected with serum from one patient. The results of the EAST were confirmed by dose-related mediator release experiments performed with RBL cells passively sensitized with allergen-specific murine IgE. As a whole, our results indicated that the EAST and the RBL cell assay are superior to immunoblotting for the immunologic testing of digests. In accordance with clinical observations, the allergenicity of peanut proteins was very persistent during digestion, whereas the native birch-pollen-related hazelnut allergens appeared to be relatively labile under identical conditions. Received: 6 July 1998