不同热加工处理方式对中华绒螯蟹原肌球蛋白的消化稳定性和致敏性的影响

目的 探究不同热加工处理方式对中华绒螯蟹原肌球蛋白的消化稳定性和致敏性的影响。方法 以中华绒螯蟹（Eriocheir sinensis）为原料，采用4种不同热加工方式（蒸煮、超声波结合蒸煮、反压蒸煮、高温高压）对蟹肉进行处理，参照美国药典中模拟胃、肠液消化实验方法，对处理后蟹肉的TM粗蛋白进行体外消化，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（Sodium dodecyl sulfate-polyacrylamide gel electrophoresis，SDS–PAGE）、免疫印迹（Western blotting）和抑制性酶联免疫吸附测定（Inhibition enzyme linked immunosorbent assay，Inhibition ELISA）等技术分析其消化稳定性及消化产物的致敏性。结果 结果显示，与未处理的样品相比，普通蒸煮处理后TM的消化稳定性及致敏性没有明显变化（P>0.05）；超声波结合蒸煮、反压蒸煮以及高温高压处理对TM降解具有明显的促进作用，TM及其消化产物的致敏性也显著降低（P<0.05）。结论 实验中反压蒸煮与高温高压处理对致敏性消减的效果较好。本实验为过敏原消减机制的研究和低致敏性水产品的开发提供了理论依据。     

加工食品中虾蟹类过敏原ELISA检测前处理方法的研究

目的建立了一种可应用于加工食品中虾蟹类过敏原检测的前处理方法。方法针对提取液和提取步骤2个部分选取条件先后进行优化,通过对相应样品提取后的蛋白溶出量等进行评估,选取最适合本ELISA检测的条件。结果采用含有0.5%SDS和0.5%DTT的磷酸盐缓冲液(pH 8.5),按照食物样品与提取液的1:9 (m/V)进行混合,在室温下均质30 min,3020×g离心15 min,取上清液进行检测效果最佳,此方法与PBS(pH7.4)溶液提取方法相比,蛋白质溶出量增加了约5倍。结论在已建立的检测虾蟹类过敏原夹心ELISA的基础上,建立优化前处理方法进一步提高了加工食品中过敏原的提取率。     

Cyclic Peptide Mimotopes for the Detection of Serum Anti–ATIC Autoantibody Biomarker in Hepato-Cellular Carcinoma

Tumor-associated (TA) autoantibodies have been identified at the early tumor stage before developing clinical symptoms, which holds hope for early cancer diagnosis. We identified a TA autoantibody from HBx-transgenic (HBx-tg) hepatocellular carcinoma (HCC) model mouse, characterized its target antigen, and examined its relationship to human HCC. The mimotopes corresponding to the antigenic epitope of TA autoantibody were screened from a random cyclic peptide library and used for the detection of serum TA autoantibody. The target antigen of the TA autoantibody was identified as an oncogenic bi-functional purine biosynthesis protein, ATIC. It was upregulated in liver cancer tissues of HBx-tg mouse as well as human HCC tissues. Over-expressed ATIC was also secreted extracellularly via the cancer-derived exosomes, which might cause auto-immune responses. The cyclic peptide mimotope with a high affinity to anti-ATIC autoantibody, CLPSWFHRC, distinguishes between serum samples from HCC patients and healthy subjects with 70.83% sensitivity, 90.68% specificity (AUC = 0.87). However, the recombinant human ATIC protein showed a low affinity to anti-ATIC autoantibody, which may be incompatible as a capture antigen for serum TA autoantibody. This study indicates that anti-ATIC autoantibody can be a potential HCC-associated serum biomarker and suggests that autoantibody biomarker’s efficiency can be improved by using antigenic mimicry to native antigens present in vivo.     

Comparison of two Analytical Methods (ELISA and LC-MS/MS) for Determination of Aflatoxin B1 in Corn and Aflatoxin M1 in Milk

The aim of this paper is to assess the closeness of agreement between results of ELISA and LC-MS/MS methods for determination of aflatoxin B₁ in corn and aflatoxin M₁ in milk. Samples of corn (n=100) and milk (n=250) were simultaneously analyzed using ELISA and LC-MS/MS methods, after the severe drought that affected Serbia in summer 2012 resulting in occurrence of aflatoxin B1 in corn and aflatoxin M₁ in milk. Regression analysis showed higher level of agreement between aflatoxin B₁ samples (R²=0.994), compared to aflatoxin M₁ samples (R²=0.920). However, both techniques were satisfactory in meeting the requirements for official control purposes.     

土霉素完全抗原的制备及ELISA检测方法的建立

为制备土霉素(Oxytetracycline,OTC)完全抗原,获得免疫学特性良好的土霉素鼠源多抗血清并建立OTC免疫学检测方法,采用重氮化法在OTC分子上引入羧基,进而利用改进碳二亚胺法将其与载体蛋白偶联制备完全抗原,通过红外扫描、紫外扫描和凝胶电泳鉴定其偶联效果,然后将完全抗原免疫BLAB/c小鼠获得多抗血清(pAb)并对其免疫学特性进行鉴定,通过优化条件建立OTC间接竞争ELISA检测方法。结果表明,偶联效果良好,免疫的4只小鼠多抗血清效价均达到1∶12 800;4只小鼠多抗血清均有抑制效果,其中2号小鼠产生的多抗血清效果最佳,基于该多抗血清建立的OTC间接竞争ELISA检测方法半数抑制浓度(IC_(50))为32.92ng/mL,检测限(LOD)为1.3ng/mL,牛奶样品中添加回收率在84.70%～87.45%,与金霉素、四环素的交叉反应率分别为5.27%,4.10%,与其他竞争物交叉反应率0.4%。该试验成功合成了OTC完全抗原,获得了免疫学特性良好的OTC多抗血清,并建立了OTC免疫学检测方法。     

2种用于检测市售核桃露(乳)饮品中花生、大豆成分方法的比较分析

摘 要: 目的 比较实时荧光定量PCR法与酶联免疫吸附法在核桃露(乳)饮品中检测花生和大豆成分的灵敏性的差异。方法 应用实时荧光定量PCR方法检测样本DNA的核桃、花生和大豆源性成分, 应用酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA)检测样本的花生和大豆特征蛋白成分。结果 加热处理对实时荧光PCR和ELISA法检测花生和大豆的结果均有显著性影响。实时荧光定量PCR方法检出2个品牌4批次样本含有花生源性, 3个品牌的6批次样本含有大豆源性; ELISA方法除检出这些批次含有花生和大豆源性成分外, 另检出3个品牌5批次样本含有大豆源性成分。ELISA方法与实时荧光定量PCR方法的大豆检测结果差异主要集中在低蛋白浓度样品中, 这与2类方法的检出限不同有关。结论 鉴于2类方法检测结果在高浓度水平的高度一致性, 说明实时荧光PCR和ELISA方法对花生和大豆源性成分检测结果均具有很高的可靠性。     

A longitudinal study of gastrointestinal parasites in English dairy farms. Practices and factors associated with first lactation heifer exposure to Ostertagia ostertagi on pasture

The gastrointestinal nematode Ostertagia ostertagi is an important cause of lost production, health, and welfare in cattle. Detailed records were obtained over a 5-yr period (2010–2015) by questionnaires and qualitative interviews to investigate the practices adopted by dairy farmers to control cattle helminth infections and the factors associated with heifer exposure to O. ostertagi on pasture. In total, 1,454 heifers' individual milk samples were collected over a 1-yr period (2014–2015) in 43 dairy farms in England and tested for O. ostertagi antibody by ELISA. Multilevel linear regression models were used to investigate the association between individual milk optical density ratio (ODR) against O. ostertagi and heifer management from birth to time of sampling. Farm and heifer median ODR against O. ostertagi were 0.98 (interquartile range = 0.76–1.02) and 0.64 (interquartile range = 0.42–0.84), respectively. The majority of heifers (88%) received an anthelmintic treatment before sampling in this study. After controlling for the effect of anthelmintic treatments, heifer individual milk ODR against O. ostertagi significantly increased with high stocking rate at first grazing and co-grazing with adult cows before calving. Conversely, heifer individual milk ODR against O. ostertagi significantly decreased when heifers had co-grazed with sheep and pasture grass had frequently been mowed. Overall, these results provide evidence to support targeting grazing management toward limiting the use of anthelmintics in dairy young stock to enable sustainable control of cattle helminth infections in England. However, to be accepted and adopted by farmers, these best practices would need to take into account farmers' perspectives and contextual challenges.     

乳铁蛋白纯化和检测方法研究进展

乳铁蛋白是一种具有抗微生物活性的铁离子结合糖蛋白,属于转铁蛋白家族的一员,具有调节炎症反应和激活免疫系统等功能,因此乳铁蛋白被广泛应用于医学和食品领域。本综述汇集了乳铁蛋白纯化和检测方法的研究进展,并比较了各方法之间的优缺点。     

龙血素A和龙血素B的酶联免疫检测方法的建立

建立了龙血素A和龙血素B间接竞争酶联免疫吸附分析方法,并用于测定龙血素A和龙血素B的含量。分别合成龙血素A和龙血素B人工抗原制备其多克隆抗体,建立二者间接竞争酶联免疫吸附分析方法并评估其分析性能。结果表明:抗龙血素A血清和抗龙血素B血清的效价分别达到8 000和30 000;龙血素A和龙血素B的最低检测限分别为3.9ng·mL-1和26.1ng·mL-1,批内精密度分别为CV≤10.7%和CV≤9.9%,批间精密度分别为CV≤11.5%和CV≤12.3%,回收率分别为87.4%~110.8%和109.8%~113.5%,与各自的6种类似物的交叉反应率最大分别为9.20%和3.64%,在4种龙血竭药物中的最大含量和最小含量分别相差4.05倍和3.57倍。龙血素A和龙血素B间接竞争酶联免疫吸附分析方法的检测精密度、准确度和特异性均较好,可快捷有效地用于药物检测和分析。     

酶免分析仪孵育过程的分段温控方法研究

针对酶免分析仪孵育过程耗时过长、温度稳定性差导致试剂反应差异性较大的问题,提出了一种基于改进型数字PID的分段温度控制方法。首先对孵育过程中试剂温度及孵育位温度变化曲线特性进行分析,确定预升温段及恒温段的分段控制温度参数;然后根据温度参数及各段控制指标,分别对孵育过程的各温度变化段采用不同算法分阶段进行控制;最后基于酶免分析仪框架搭建了温控实验系统,对反应液温度上升时间及温度稳定性进行验证。实验结果表明,试剂由21℃升至37.5℃的温度上升时间为239±5 s,恒温阶段的温度偏差小于±0.2℃。该方法可实现反应液迅速升温并且稳定恒温,保证了孵育过程中生物酶反应的快速及稳定。