Screen‐printed biosensor for allergens

Allergen levels in indoor environments, leading to many diseases, eg asthma, rhinitis and conjunctivitis, affect a large and increasing fraction of the population. A quite effective and inexpensive method of a rough but very rapid overall assessment of total allergen level in the environment has been developed. The method involved estimation of protein in allergen extracts by screen‐printed electrodes using two different techniques. The biosensor comprised a rhodinised carbon working electrode, a silver/silver chloride reference electrode and a carbon counter electrode. In the first method the enzyme protease reacted with allergen protein to release amino acid, which produced hydrogen peroxide in the presence of amino acid oxidase. This was detected amperometrically. The second method used potassium bromide as electrolyte and the electrode was subjected to dual potential. Bromine, released due to electrolysis at higher potential, was consumed by the allergen protein at lower potential. In the first method, a unique technique was used to microencapsulate the enzyme protease and immobilise it on the surface of the electrode by in‐situ polymerisation to avoid contact with the amino acid oxidase. A total of seven allergens were tested and the results gave a good correlation with the standard protein measurement method. Environmental specimens from indoors, schools and workplaces can be evaluated for the aeroallergens produced by dust mites, animal hairs, cockroach debris, pollens, etc as a means of determining the exposure risk. Copyright © 2005 Society of Chemical Industry     

腰果过敏原Ana o 3蛋白的分离纯化及热稳定性研究

Ana o 3蛋白是腰果主要的过敏原之一。以生腰果为原料,通过粉碎脱脂、低盐提取粗蛋白、冷冻干燥、阴离子交换层析及超滤浓缩等过程分离得到目的蛋白,利用小分子蛋白电泳、液相色谱-串联质谱和免疫印迹技术进行鉴定;此外,采用紫外光谱及圆二色光谱分析腰果过敏原Ana o 3蛋白在160℃持续加热10、15、20、25、30 min的结构变化,以评估分离纯化后腰果过敏原Ana o 3蛋白的稳定性。结果表明,成功建立了一种快速高效纯化腰果过敏原Ana o 3蛋白的方法,该方法获得的蛋白纯度高于95%。通过圆二色光谱发现腰果过敏原Ana o 3蛋白在160℃加热过程中α-螺旋构象逐步转变为β-折叠构象,β-转角含量变化较小,无规则卷曲含量稍有增加,表明该蛋白二级结构较为稳定;经紫外光谱发现加热后腰果过敏原Ana o 3蛋白的紫外特征吸收峰的吸光度升高,表明加热会使腰果过敏原Ana o 3蛋白变性,结构展开,更多的色氨酸和酪氨酸残基暴露,使得空间结构变得松散;经蛋白电泳和免疫印迹分析发现,热处理后腰果过敏原Ana o 3蛋白会发生降解,表位被破坏。希望研究可为高纯度腰果过敏原Ana o 3蛋白的纯化提...     

食品过敏原生物传感检测技术研究进展

食品过敏原已成为全球性的食品安全隐患。应对食品过敏反应最直接、有效的方法就是避免食用和接触各种含有致敏成分的食品。因此,快速和灵敏地检测食品中过敏原对预防和控制食品过敏反应是十分重要的。食品过敏原检测主要可以分为基于核酸和蛋白质的两大类检测技术。与基于聚合酶链式反应技术、液相色谱-串联质谱法的传统检测手段相比,生物传感技术检测平台因具有操作简单、高通量、灵敏度高、现场便携等优点,在食品过敏原快速检测领域备受关注。本文综述了近年来食品过敏原的生物传感检测技术研究进展,包括电化学传感器、光学传感器和其他新型传感器及其在食品过敏原检测中的应用,并展望了生物传感技术在食品过敏原分析中面临的挑战和发展趋势。     

Isolation of recombinant antibody fragments (scFv) by phage display technology for detection of almond allergens in food products

Tree nut allergies are considered an important health issue in developed countries. To comply with the regulations on food labeling, reliable allergen detection methods are required. In this work we isolated almond-specific recombinant antibody fragments (scFv) from a commercial phage display library bypassing the use of live animals, hence being consistent with the latest policies on animal welfare. To this end an iterative selection procedure employing the Tomlinson I phage display library and a crude almond protein extract was carried out. Two different almond-specific scFv (named PD1F6 and PD2C9) were isolated after two rounds of biopanning, and an indirect phage ELISA was implemented to detect the presence of almond protein in foodstuffs. The isolated scFvs demonstrated to be highly specific and allowed detection of 40 ng mL⁻¹ and 100 ng mL⁻¹ of raw and roasted almond protein, respectively. The practical detection limit of the assay in almond spiked food products was 0.1 mg g⁻¹ (110–120 ppm). The developed indirect phage ELISA was validated by analysis of 92 commercial food products, showing good correlation with the results obtained by a previously developed real-time PCR method for the detection of almond in foodstuffs. The selected phage clones can be affinity maturated to improve their sensitivity and genetically engineered to be employed in different assay formats.     

Apple Allergy: The cDNA Sequence of the Major Allergen of Apple,Determined by Performing PCR with a Primer Based on the N-Terminal Amino Acid Sequence,is Highly Homologous to the Sequence of the Major Birch Pollen Allergen

Considering the known N-terminal amino acid sequence of the major apple allergen, a polymerase chain reaction (PCR) primer was selected to amplify cDNA encoding this protein. A single PCR product was obtained, cloned into Escherichia coli and subsequently sequenced. The missing 5′-end of the apple cDNA sequence was obtained by a 5′-RACE method. The cDNA sequence showed 72% identity with the coding region of one of the known isoforms of Bet v 1, the major allergen of birch pollen. The deduced amino acid sequence resulted in a 158-residue protein with a calculated molecular mass of 17·5 kDa and 63% amino acid sequence identity to Bet v 1. In addition, further protein alignments showed a high degree of identity with allergens from other tree pollens and some ‘pathogenesis-related proteins’ from food plants. According to international regulations the allergen was termed Mal d 1 for this protein, it being the first major allergen discovered and characterised in fruits of apple (Malus domestica).     

食物过敏原检测与调控研究进展

食品过敏已成为一个重要的食品质量和安全问题,对食品加工行业构成挑战,并影响消费者的健康。一方面,从食品加工行业的角度来看,食品原料成分、外源添加剂和加工形式的多样性使得现代食品加工中过敏原的存在更加复杂。此外,由于缺乏过敏原识别和有效的检测与评价系统,导致目前食品过敏原筛选与检测、跟踪与预测、干预与控制的理论和技术存在严重不足;另一方面,从公共卫生的角度来看,满足消费者对包括食物过敏原在内的不同类型原料来源的知情权,提高政府的公信力和人民的满意度也成为当务之急;此外,随着人们接触的食物种类越来越多,食物过敏的发生概率也越来越高,日趋复杂化、广域化和严重化的食物过敏所带来的食品安全健康问题已很难避免。鉴于此,针对大健康背景下日益严重的食品过敏安全问题,本综述介绍了食物过敏原的检测方法,总结了食物过敏原消减与控制技术,阐述了低致敏性食品以及抗过敏活性物质,综述了目前食物过敏原口服免疫治疗进展,旨在为预防和控制食物过敏,保障食物过敏患者的身体健康提供依据。     

Food allergy,a summary of eight cases in the UK criminal and civil courts: effective last resort for vulnerable consumers?

Food allergy has a forensic context. The authors describe eight cases in the UK courts involving fatalities, personal injury or criminal non‐compliance with food law from mainly ‘grey’ literature sources. The potentially severe consequences for people with food allergy of contraventions of labelling law have led to enforcement action up to criminal prosecution for what might otherwise be regarded as ‘trivial’ non‐compliance. The authors suggest there should be central collation of such cases. Non‐compliances should be followed up in a more rapid and robust manner. Evidence of fraud in the catering supply chain supports recent calls for zero tolerance of food fraud. Businesses must guard against gaps in allergen management, for which there are readily available sources of training and guidance, but also against fraudulent substitution in the supply chain, about which training and guidance should be developed. New allergen labelling legislation and case law appear to place responsibility on food businesses even for the forensically problematic area of allergen cross‐contamination. The courts can be an effective last resort for vulnerable consumers; however, there is evidence of knowledge and skill gaps in both the investigation and prosecution of potentially serious incidents of food allergen mismanagement and mislabelling. Thorough investigation of food allergy deaths is required with a tenacious and skilled approach, including early realisation that samples of the food and/or stomach contents from a post mortem examination should be retained and analysed. The supply chain must be rigorously examined to find out where adulteration or contamination with the fatal allergen occurred. © 2014 Society of Chemical Industry     

表面等离子体共振生物芯片无标记实时检测虾血蓝蛋白

利用自行研制的便携式表面等离子体共振生物芯片检测系统,在生物芯片表面分别固定虾血蓝蛋白、虾血蓝蛋白的单克隆抗体、虾血蓝蛋白的单克隆抗体腹水作为生物探针,利用免疫反应的特异性,研究虾血蓝蛋白与其单克隆抗体的相互作用,分析动力学反应过程,建立标准曲线。用同一个生物芯片检测了8个抗体样品、7个未纯化的抗体腹水,为现场检测大量食品中过敏原、检测临床血清中过敏原特异性抗体进行基础研究。表面等离子体共振生物芯片检测过敏原,仪器便携、操作简便、无需标记、无污染、成本低,可进行现场大量样品的实时连续检测和快速筛选,适用于超市、集市、工厂等需要实时快速检测和质量监控的场所,也可以应用于临床上患者血清样品的过敏原抗体检测。     

牛奶过敏原制备方法的研究和免疫活性鉴别

目的探索牛奶主要过敏原的制备工艺。方法采用等电点沉淀、凝胶层析和分子筛等技术纯化牛奶中主要过敏原组分;采用SDS-PAGE鉴定蛋白纯度,采用双抗原夹心-ELISA鉴定免疫活性。结果成功获得牛奶中的四种主要过敏原组分:酪蛋白、β乳球蛋白、α乳白蛋白和分子量较高的P1组分,并经过免疫实验证实这四种组分均能与过敏血清产生反应,而其中β乳球蛋白和P1组分反应性较高。结论本研究探索出一种简单、实用的牛奶主要过敏原制备工艺,并证实牛奶中的β乳球蛋白和高分子量蛋白质为主要过敏原。     

酶解对乳清蛋白抗原性影响的研究

研究了酶解对乳清蛋白抗原性的影响。选择了7种常见蛋白酶在同一水解模式下水解乳清蛋白，用竞争ELISA法测定水解物的残留抗原性，从而间接测定其过敏性变化。结果表明，酶解能有效降低乳蛋白抗原性，但水解物仍能与特异抗体反应，保留一部分抗原性。不同酶对乳清蛋白过敏原的影响不同，酶的特异性对乳清蛋白水解物的抗原性有较大的影响，碱性蛋白酶降低乳蛋白抗原性的效果最佳，对抗β-乳球蛋白（β-LG）和抗α-乳白蛋白（α-LA）抗体的抗原性分别降低了50．02％和99．72％。