摘要
食物过敏反应是当前世界性的卫生学问题和主要的食品安全问题之一，且随着人们生活水平的提高，食物过敏呈逐年上升的趋势。根据流行病学调查，世界有8%的儿童和2%的成年人对食物过敏，一些发达国家受食物过敏性疾病困扰的人群超过20%。花生和鸡蛋分别被列入8种主要食物过敏原，且其营养丰富而广受消费者的喜爱，而致敏性却严重影响着部分人群的日常生活。因此，研究快速高效的致敏蛋白检测技术和纯化方法显得极其重要。
本课题从鸡蛋致敏蛋白检测方法和花生致敏蛋白纯化方法两方面进行研究，研制高效快速的鸡蛋致敏蛋白ELISA检测试剂盒，实现鸡蛋致敏蛋白的体外快速检测；用免疫亲和柱法纯化花生致敏蛋白Arah1，实现对致敏蛋白快速高效的分离纯化。
本文的主要研究内容和结果如下：（1）研究并制备了鸡蛋致敏蛋白检测试剂盒。首先通过硫酸铵沉淀法提取鸡蛋中致敏蛋白，用免疫印迹进行活性鉴定，接下来免疫新西兰大白兔制备抗鸡蛋致敏蛋白多克隆抗体。通过对ELISA条件优化，得出碳酸盐缓冲液（pH9.6）为最佳包被缓冲液，先4℃过夜，再37℃放置1h为最佳包被时间，5%的脱脂奶粉为最佳封闭液，酶标二抗的最佳稀释度为2000倍，抗原的最佳反应浓度为1μg/mL，抗体的最佳稀释倍数为50000倍，优化后方法的最低检测限IC10=1.1ng/mL，所建立试剂盒回收率在90%-105%范围之内，特异性良好，试剂盒批内平均变异系数低于5%，批间平均变异系数低于10%，且再现性差异不显著，经37℃加速破坏实验，试剂盒可在4℃可保存6个月，冻融次数不应超过三次，用所建立的方法对样品进行检测分析，检测结果正确率较高。（2）研究了免疫亲合法纯化花生致敏蛋白Arah1。首先通过硫酸铵沉淀法和分子筛层析法提取纯化花生致敏蛋白Arah1，纯度达90%，用所提取的Arah1致敏蛋白免疫新西兰大白兔制备特异性多克隆抗体，效价高达5×105。制备免疫亲和柱：以溴化氰活化的琼脂糖凝胶4B（CBr-activatedSepharose4B）为偶联介质，与抗Arah1特异性多克隆抗体进行偶联（偶联率达到95.9%），制备免疫亲和柱，通过条件优化，筛选出glycine-HCI（pH2.4)为最佳洗脱液，洗脱流速可在0.5～2ml/min内进行选择，具体流速大小可依具体实验而定，选用5min为最佳上样孵育时间，所制备的免疫亲和柱每毫升亲和介质的最高吸附容量在11mg左右，回收率在73.6%-89.2%之间，符合技术要求。综上分析，本研究所制备的鸡蛋致敏蛋白检测试剂盒检测限及稳定性等指标均符合技术要
求，为鸡蛋致敏蛋白的检测分析提供技术支持；对Arah1免疫亲和层析纯化方法的研究表明，此法高效、快速、特异性强，是一种实用性较强的纯化致敏蛋白的方法，为致敏蛋白的纯化及深入研究奠定基础。
Allergy reaction is regarded as global hygienic and food safety problem, and is becoming seriously from year to year. According to epidemiological investigation,Food allergy affects up to8%of enfants and infants and2%of adults,and exceeding20%of total population in developed countries. Peanuts and eggs are respectively enrolled in8main foods allergy,and they are also favorite foods because they are rich in nutrients, but some people are affected by their allergenicity.so it is necessary to research the rapid testing and purification of allergic proteins.
The main content and result were as follows:
(1) Indirect ELISA kit for detecting egg allergy proteins had been developed. Firstly, egg allergy proteins were extracted by ammonium sulfate decantation,and verified by Western Blotting,and then got polyclonal antibody was collected by getting New Zealand white rabbit a shot with extracted proteins. The best condition for ELISA was optimized influencing factors, which shown as:oating with CBS(0.01mol/L,pH9.6) at4℃for12h and then37℃for1h, blocking with5%skim milk powder, the best react content for antigen1μg.mL-1,for antibody50000times'dilution and for HRP-IgG2000times'dilution, Based on those conditions, the kit was with lowest detectable limit at1.1ng.mL-1.Secondly,kit was evaluated:the sample recovery was at90%-105%, specificity was good, the average coefficient of variation of infra and inner plate were less than5%and10%, repeatability discrepancy was Non-significant. The ELISA kit was stable at37℃for at least4days,which could be accumulate more than6months at4℃. Kit could tolerate three times frozen.
(2) The method of Immunoaffinityfor peanut allergy proteins Ara h1purification was developed.Firstly, Ara h1allergy protein were gained by ammonium sulfate decantation and molecular sieve chromatography,and the purity of Ara h1was above90%,then we got polyclonal antibody by getting New Zealand white rabbit a shot with Ara hl,whose titer was5×105.The next step was to couple the CBr-activated Sepharose4B with antibody(the coupling rate was up to95.9%), then chromatographic column was prepared.The best condition for chromatographic column was measured by optimizing influencing factors, which showed as, elution with glycine-HCI (pH2.4), elution velocity at0.5-2mL/min, the best incubation time was5min, loading capacity was11mg. The sample recovery was at73.6%-89.2%.
Above results showed that the kit's parameter,for example detectable limit stability and so on,fulfilled with technical specification, which supported help for egg allergic proteins detecting. From this study,we found that Immunoaffinity for proteins purification timesaving,highly efficient and strong specificit, which was a good support for proteins deep-going study.
引文
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