文章摘要
李小燕,李振兴,林洪,SAMEE Haider,张轶群.菲律宾蛤仔过敏原可视化抗体微阵列玻片的检测方法[J].水产学报,2010,34(3):422~427
菲律宾蛤仔过敏原可视化抗体微阵列玻片的检测方法
Development of visible antibody microarray chips for detecting mottled calm (Ruditapes philippinarum) allergen
投稿时间:2009-07-14  修订日期:2009-12-09
DOI:10.3724/SP.J.1231.2010.06520
中文关键词: 菲律宾蛤仔  过敏原  夹心法  抗体微阵列  可视化
英文关键词: Ruditapes philippinarum  allergen  sandwich  antibody microarray  visualization
基金项目:国家自然科学基金项目(30800859);国家“八六三”高技术研究发展计划(2006AA09Z427)
作者单位E-mail
李小燕 中国海洋大学 yanqunfeng10@163.com 
李振兴 中国海洋大学  
林洪 中国海洋大学  
SAMEE Haider 中国海洋大学  
张轶群 中国海洋大学  
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中文摘要:
      采用辣根过氧化物酶(HRP)与3,3′,5,5′四甲基联苯胺(TMB)H2O2作为信号示踪系统,建立了一种可视化抗体微阵列检测菲律宾蛤仔过敏原的方法。对孵育时间、抗体浓度等免疫条件进行了优化。采用改良双抗体夹心法,将菲律宾蛤仔过敏原的兔多克隆抗体固定于琼脂糖三维芯片上,依次加入待检样品、菲律宾蛤仔鼠多克隆抗体和HRP-羊抗鼠IgG,孵育后加TMB显色,肉眼观察后,用平板扫描仪获取扫描图象,采用GenePixPro 6.0软件分析灰度值。试验结果表明,该方法最低可检出10 ng/mL的菲律宾蛤仔过敏原,片内平均变异系数(CV)为5.99%,片间为10.3%;香肠及蟹棒中3个不同浓度的加标回收率为73.54%~95.44%;4 ℃存放4个月内抗体微阵列玻片活性保持稳定。该方法不需要大型精密仪器,结果直观可见,可以发展为对多种过敏原进行同时检测,具有良好的实用和推广价值。
英文摘要:
      Ruditapes philippinarum has a widespread consumption in China. It is enjoyed for the uniqueness of its flavour and has a wide variety of applications in foods, such as soup and flavouring. Associated with the diversiform consumption of Ruditapes philippinarum is the general increase of allergic disorder. To provide guarantee for the allergy sufferers, it is important to establish a more convenient method for the detection of food allergen. A visible antibody microarray for detecting Ruditapes philippinarum allergen was developed in this study, which was based on the 3, 3′, 5, 5′ Tetramethylbenzidine (TMB)H2O2 reaction catalyzed by horseradish peroxidase (HRP). Several physicochemical parameters such as the incubation time and the dilution ratio of antibodies were optimized. A modified sandwich reaction was performed with immobilization of rabbit anti-Ruditapes philippinarum antibody onto the three dimensional slide, followed by adding antigen,mouse anti-Ruditapes philippinarum,HRP goat anti-mouse IgG. After incubating, TMB was employed to give signals, the slides were observed by naked eyes and recorded with flatbed scanner, analysed with GenePix Pro 6.0. The results showed that allergen from Ruditapes philippinarum can be detected sensitively with a sensitivity of 10 ng/mL. The method has good reproducibility with average intra assay CV of 5.9% and average inter assay CV of 10.3%. The recoveries of three different spiked concentrations in sausage and surimi stick ranged from 73.54% to 95.44%. During storage at 4 ℃, the activity of the chips kept stable for 4 months. Moreover, some cross reactivity with Penaeus vannamei was found. With visible and stable results, this method has shown great potential in the massive parallel analysis of various food allergens simultaneously.
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