花椒（Zanthoxylum bungeanum Maxim.）是一种常见的调味品和中药材，也是一种潜在的食物过敏原。2005 年花椒过敏及其临床交叉过敏特点被我们的研究团队首次报道。然而其过敏原成分和交叉致敏情况尚不清楚。因此，本研究旨在明确并鉴定花椒主要致敏组分的致敏性，评估致敏风险，并深入剖析花椒与柑橘、腰果和开心果的交叉反应性。

首先，我们收集了15例花椒过敏/致敏患者的临床资料和血清；采集了10位健康受试者的血清作为阴性对照；通过SDS-PAGE、Western blot（WB）对主要致敏蛋白组分进行分析；通过WB、ELISA和竞争性ELISA评估蛋白致敏性和交叉反应性。

       通过分析临床资料发现：（1）约76.9%（10/13）的花椒过敏患者曾因进食花椒发作过严重过敏反应，表明花椒致敏性强；（2）100% （n=8）的花椒过敏患者花椒籽皮肤点刺试验（skin prick test，SPT）阳性，25%（2/8）的患者花椒皮SPT阳性。

ELISA和WB结果显示：（1）100%（n=8）花椒过敏患者的血清与花椒籽粗蛋白具有IgE反应性，而均不与花椒皮粗蛋白反应，结合SPT结果，表明花椒主要致敏组分蛋白来源于花椒籽，而不是花椒皮；（2）花椒籽WB实验中，有一半以上的阳性血清识别了10 kDa蛋白（84.6%）和55 kDa蛋白（53.8%），表明花椒籽主要致敏组分蛋白分子量约为10 kDa和55 kDa（在对花椒10 kDa和55 kDa蛋白进行纯化和生物化学性质鉴定后，确定了两者分别属于2S和11S蛋白）；（3）花椒纯化蛋白WB试验中，分别有92.3 %和100 % 的阳性血清识别了花椒2S和11S蛋白；（5）ELISA结果显示花椒过敏患者血清与2S和11S纯化蛋白的IgE反应性，分别为92.3%（12/13）和 69.2%（9/13）。表明: 纯化的花椒2S和11S蛋白是花椒主要致敏组分蛋白。

致敏风险评估方面的试验结果为：（1）在花椒籽WB试验中，识别2S蛋白的阳性血清比例（84.6%）高于识别11S 蛋白的比例（53.8%）；（2）ELISA试验中，2S蛋白与花椒过敏患者血清的IgE阳性反应率（92.3%）高于11S蛋白（69.2%），且2S蛋白组的OD均值要显著高于11S蛋白组（p = 0.0034）；（3）在5个不同抑制浓度梯度下（0.01-10ug/ml），2S蛋白对花椒籽粗提蛋白的抑制率均高于11S蛋白。说明花椒2S蛋白的致敏性强于11S蛋白。因此在花椒过敏的临床诊疗工作中2S蛋白应得到更多重视。

交叉反应性分析结果显示：（1）花椒过敏患者合并橘子、腰果、开心果过敏的比例很高，分别为69.23 %（9/13）、84.6 %（11/13）和92.3 %（12/13）；（2）83.3%（10/12）以上的花椒过敏患者 f33、f202、f203＞0.35kUA/L，且ELISA试验中，花椒过敏/致敏血清与橘子、腰果、开心果蛋白均具有IgE反应性；（3）ELISA抑制试验结果显示，在7个不同抑制浓度梯度下（0.1-300ug/ml），橘子对花椒致敏蛋白（花椒籽、2S蛋白、11S蛋白）的抑制率最高，然后是开心果和腰果；（4）橘子、腰果、开心果对11S蛋白的抑制率最高，其次是花椒粗提蛋白和2S蛋白。这些数据表明，橘子与花椒的交叉致敏性最强，然后是开心果和腰果；且橘子、腰果、开心果与11S蛋白的交叉致敏性要强于花椒籽粗提蛋白和2S蛋白，因此在交叉致敏的诊疗中11S蛋白需要得到更多的关注。

综上，本研究验证了花椒主要致敏组分蛋白主要来源于花椒籽，而不是花椒皮；花椒致敏性极强，容易引发严重过敏反应；本研究在血清学水平上对花椒主要致敏组分蛋白进行了致敏性鉴定；发现了2S蛋白的致敏性强于11S蛋白；确定了橘子、腰果、开心果与花椒致敏蛋白之间具有高度交叉致敏性，且11S蛋白与三者之间的交叉致敏性最强。另外，我们和中国科学技术大学合作获得了花椒主要致敏组分的部分编码序列。最终，2S和11S蛋白被过敏原命名小组委员会（Allergen Nomenclature Subcommittee）分别命名为Zan b 1和Zan b 2。本研究为将来花椒、橘子、腰果、开心果过敏患者的个体化诊断、免疫治疗和患者管理提供了科学依据；也对将来发展食品过敏原安全评价技术方法发挥了积极作用。

Sichuan pepper (Zanthoxylum bungeanum Maxim.), huajiao in Chinese, which is widely used as a spice around the world, is also a potentially life-threatening food allergen, and we first published a report on the allergenic situation of Sichuan pepper in 2005. However, so far, its allergenic composition is unknown. Therefore, this study aimed to clarify and characterize the allergenicity of the main allergenic components of Sichuan peppers (SP), to assess the risk of allergenicity and to provide insight into the cross-reactivity of SP with citrus, cashews and pistachios. First, we collected clinical data and sera from 15 patients with SP allergy/sensitization; sera from 10 healthy subjects were collected as negative controls; the main allergenic protein fractions were analyzed by SDS-PAGE and Western blot (WB); protein allergenicity and cross-reactivity were assessed by WB, ELISA and competitive ELISA.

       The analysis of the clinical data revealed that: (1) about 76.9% (10/13) of the SP allergic patients had anaphylaxis to SP, indicating that SP is highly allergenic; (2) 100% (n=8) of the SP allergic patients had a positive skin prick test (SPT) for Sichuan pepper seeds (ScPS), and 25% (2/8) of the patients had a positive SPT for Sichuan pepper peels (ScPP).

The ELISA and WB results showed that: (1) 100% (n=8) of the sera from pepper-allergic patients were IgE-reactive with pepper seed crude protein, whereas none of them reacted with pepper skin crude protein, which, in combination with the SPT results, suggests that the main allergenic component proteins of SP originate from ScPS, not from ScPP; (2) More than half of the positive sera in the ScPS WB assay recognized the 10 kDa protein ( 84.6 %) and 55 kDa protein (53.8 %), indicating that the molecular weights of the main allergenic component proteins of ScPS were about 10 kDa and 55 kDa (after purification and biochemical characterization of SP 10 kDa and 55 kDa proteins, it was determined that they belonged to the 2S and 11S proteins family, respectively); (3) in the peppercorn purified proteins WB assay, 92.3% and 100 % of the positive sera identified the 10 kDa and 55 kDa proteins, respectively; (5) ELISA results showed that sera from SP allergic patients showed IgE reactivity with 2S and 11S purified proteins of 92.3% (12/13) and 69.2% (9/13), respectively. The purified 2S and 11S proteins are the main allergenic component proteins of SP.

The results of the allergenic risk assessment were as follows: (1) the proportion of positive sera identifying 2S protein (84.6%) was higher than that identifying 11S protein (53.8%) in the ScPS WB test; (2) in the ELISA test, the IgE positive reaction rate between 2S protein and ScPS allergic patients' sera was 92.3% (92.3%) higher than that of 11S protein (69.2%) and the mean OD value of the 2S protein group was significantly higher than that of the 11S protein group (69.2%). The mean OD value of the 2S protein group was significantly higher than that of the 11S protein group (p = 0.0034); (3) under five different inhibitory concentration gradients (0.01-10ug/ml), the inhibition rate of 2S protein against the crude extracted protein from ScPS was higher than that of 11S protein. It indicates that the allergenicity of peppercorn 2S protein is stronger than 11S protein. Therefore, 2S protein should be paid more attention in the clinical diagnosis and treatment of pepper allergy.

The results of cross-reactivity analysis showed that: (1) the proportion of SP allergy patients with combined citrus, cashew, and pistachio allergy was high, 69.23 % (9/13), 84.6 % (11/13), and 92.3 % (12/13), respectively; (2) more than 83.3 % (10/12) of pepper allergy patients with f33, f202, and f203 > 0.35 kUA/L. and pepper allergy/sensitisation sera were IgE-reactive with citrus, cashew and pistachio proteins in the ELISA test; (3) the results of the ELISA inhibition test showed that citrus had the highest inhibition rate of pepper allergenic proteins (ScPS, 2S protein, 11S protein) followed by pistachio and cashew at seven different inhibitory concentration gradients (0.1-300ug/ml); (4) the results of the Tangerine, cashew and pistachio had the highest inhibition of 11S protein, followed by pepper crude extract protein and 2S protein. These data suggest that citrus has the strongest cross-sensitization with ScPS, followed by pistachio and cashew; and that citrus, cashew, and pistachio have stronger cross-sensitization with 11S proteins than with ScPS crude extract proteins and 2S proteins, so 11S proteins need to be paid more attention in cross-sensitisation clinics.

In this study, we verified that the main allergenic component proteins of SP mainly originated from ScPS rather than ScPP. ScPS are extremely allergenic and are prone to cause anaphylaxis. The allergenicity of the main allergenic component proteins of ScPS was identified at the serological level. The 2S protein was found to be more sensitizing than the 11S protein. Identified high cross-sensitization between citrus, cashew, pistachio, and pepper sensitizing proteins and the strongest cross-sensitization between the 11S protein and all three. In addition, we collaborated with the University of Science and Technology of China (USTC) to obtain the partial coding sequences of the major allergenic components. Finally, the 2S and 11S proteins were named Zan b 1 and Zan b 2, respectively, by the Allergen Nomenclature Subcommittee. These two new protein allergens may play an active role in individualized precision diagnosis and better prevention of food allergy.