Bt蛋白免疫检测技术研究进展

在商用转基因大豆、玉米中,转Bt基因占绝大多数,由于目前越来越多的国家要求对转基因产品实行标签制度,因此转Bt基因成分的快速检测对进口转基因国显得非常重要,本文对Bt蛋白的一种快速检测技术-免疫检测技术的研究进展进行综述。      

转Bt基因水稻种子中Bt蛋白含量测定方法的研究

采用ELISA技术检测转Bt基因水稻中Bt蛋白的含量,判断水稻样品中是否含有转基因成分。利用研磨、酶标、孵育等技术对样品进行前处理。采用阳性质控物浓度等倍稀释方法,建立标准曲线,相关系数为0.997 4。用一系列不同转基因含量的标准基体材料,分析方法的最低检测限,灵敏度达0.1%。通过对我国进入生产性试验的转Bt基因水稻品系TT51-1和科丰6号的测试,表明该方法与普通PCR方法真实性和灵敏度一致,可以广泛应用于转Bt基因水稻及其粗加工产品的转基因成分检测。为转基因生物安全监管和安全性评价提供技术支撑。     

免疫检测技术的研究进展

综述了近几年来出现的免疫检测技术的新方法,简要介绍了它们的原理、特点及其应用,并做出相应的比较。免疫检测技术是基于抗体抗原反应的原理对待测物进行定量定性分析的检测方法,具有特异性强、灵敏度高、简便等优点,是现代生命科学的重要研究手段,在生物分析检测领域有着广泛的应用前景。此外,展望了免疫检测技术的发展方向,以期为有关的科研工作者提供参考。     

应用ELISA定量检测转基因玉米中Bt1蛋白的研究

应用纯化的Bt1杀虫晶体蛋白作为标准蛋白和免疫抗原,通过抗体-抗原-酶标抗体反应,建立了酶联免疫吸附测定法(ELISA),以定量检测转基因玉米中的Bt1表达蛋白。用建立的ELISA法对4种进口玉米产品进行了测定,实验结果得到了免疫印迹分析的验证,并与进口试剂盒方法的定量分析结果相一致,因而建立的ELISA法具有操作简便、快速特异、定量准确、经济实惠的优点,特别适合于大批量检测,有着良好的应用前景。     

拟除虫菊酯类农药及其代谢物免疫检测技术研究进展

拟除虫菊酯类农药是一类在世界范围内被广泛用于防治害虫的神经毒剂.由于用量大且存在一定毒性,因此,研究适合现场快速检测的筛查方法对于保障消费者身心健康具有重要作用.基于抗体特异性识别的免疫分析法具有简便、灵敏、快速及低成本等特点,是当前食品安全主流快速检测方法.该文重点介绍了拟除虫菊酯类农药及其代谢物半抗原设计策略、抗体...     

花生蛋白改性技术研究进展

花生是一年生草本植物,起源于南美洲热带、亚热带地区,是世界上主要的食用植物油料作物之一,在全国大部分地区都有种植。作为花生榨油之后的主要副产物,花生饼粕中约含有50%的蛋白质,丰富的花生资源为花生蛋白的研究与开发利用提供了充足的原料,由此也有力地推动了花生蛋白产品的迅速发展。花生蛋白不仅所含氨基酸种类比较齐全,而且所含人体必需氨基酸的比例较高,是植物蛋白中为数不多能替代动物蛋白的理想营养佳品,通过蛋白质改性技术可以修饰蛋白质的功能特性,提高其加工性能,拓宽花生蛋白在各领域中的应用范围。本文从物理改性、化学改性、酶法改性3个方面探讨花生蛋白改性技术对其功能特性所产生的影响,物理方法主要包括超高压均质、热处理、超声处理、低温等离子体、臭氧、反胶束和冻融循环等;化学方法包括糖基化、酰化、磷酸化、pH偏移处理和多酚化合物处理等;生物方法主要包括酶法水解和酶法交联处理两种。此外,本文总结了不同改性方法的作用机制及其对花生蛋白性质的影响,同时展望了花生蛋白改性技术的应用及发展趋势,旨在为花生蛋白的开发利用和未来发展奠定基础。     

拟除虫菊酯及其代谢物免疫检测的研究进展

作者主要介绍了检测拟除虫菊酯类农药及其代谢物的酶联免疫法和荧光免疫法。通过应用新型纳米材料(如磁纳米粒子,镧系发光纳米材料)以及噬菌体展示技术提高免疫检测的灵敏度,并对拟除虫菊酯类农药及其代谢物免疫检测的发展方向进行了展望。     

转Bt基因水稻的食用安全性评价研究进展

水稻（Oryza sativa）是我国重要的经济作物,其优化育种一直是研究的重点。苏云金芽孢杆菌（Bacillusthuringiensis,Bt）抗虫基因的出现,开辟了利用外源基因培育转基因抗虫作物的新领域。目前,转基因抗虫水稻的培育技术已日趋成熟,而其对人体的潜在安全性问题却备受争议。转Bt基因水稻能否实现商业化是人们关注的焦点。本文就转Bt基因水稻的食用安全性评价的研究进展进行综述,以期为我国转Bt基因水稻的风险评估和推广提供一些参考。     

牛乳酪蛋白源生物活性肽研究进展

乳酪蛋白源生物活性肽以其良好的消化性、低敏性及生物活性而日益受到重视。综述国内外在牛乳酪蛋白来源的阿片肽、阿片拮抗肽、ACE抑制肽、免疫调节肽和矿物元素结合肽等方面的研究,旨在为更好地利用牛乳酪蛋白源的生物活性肽提供参考。     

大米蛋白提取工艺的研究进展

综述国内外大米蛋白的提取工艺，从大米蛋白功能特性及产业化开发角度提出大米蛋白提取工艺的研究趋势。     

直接竞争酶联免疫吸附法检测虾过敏蛋白

目的:建立酶标抗原的直接竞争酶联免疫吸附法(dcELISA)检测食品中虾过敏蛋白,为食品过敏诊断试剂的开发和应用提供理论基础。方法:提取虾主要过敏蛋白,免疫小鼠制备抗虾过敏蛋白多克隆抗体,辣根过氧化物酶(HRP)标记抗原,建立酶标抗原的dcELISA检测虾过敏蛋白。结果:所建立的dcELISA法最低检测限为3.94ng/mL,标准曲线在0.12～128.86ng/mL范围内线性良好,批内和批间变异系数分别为6.16%和2.73%,回收率为82%～98%。结论:该方法具有良好的特异性、敏感性和稳定性,为进一步研制检测虾过敏蛋白的ELISA试剂盒提供有效的方法。     

An immunoassay based on bio-barcode method for quantitative detection of semicarbazide

A functionalized gold nanoparticles bio-barcode technology was developed to detect semicarbazide. In the assay, the hapten CPSEM (4-[[2-(aminocarbonyl) hydrazinylidene] methyl]-benzoic acid) was first prepared and conjugated to bovine serum albumin as the coating antigen. Subsequently, gold nanoparticles dual-labeled with anti-CPSEM monoclonal antibodies and DNA oligonucleotide were synthesized via a one-step preparation method. Enzymatic signal was converted to DNA signal by combining polymerase chain reaction with indirect competitive ELISA (icELISA). The limit of detection of this method could reach 8 pg mL⁻¹ for detecting SEM, about 25 times more sensitive than conventional ELISA. The immunosorbent bio-barcode assay is a rapid high-throughput screening tool adapted for ultra-high sensitive detection.     

Simple immunoassay for detection of PCBs in transformer oil

A rapid and inexpensive procedure to detect polychlorinated biphenyls (PCBs) in transformer oil is needed to facilitate identification and removal of PCB contaminated transformers. Here we describe a simple two-step liquid-liquid extraction using acidic dimethyl sulfoxide in conjunction with an immunoassay for detecting PCBs in transformer oil. The process described is faster and simpler than any previous immunoassay while maintaining comparable detection limit and false negative rate. Cross reactivity data, characterizing the immunoassay response to the four Kanechlor technical mixtures of PCBs in oil, are presented. Forty-five used transformer oil samples were analyzed by gas chromatography-high-resolution mass spectrometry and were also evaluated using the immunoassay protocol developed. Results presented show zero false negatives at a 1.4 ppm nominal cutoff for the transformer oils analyzed.     

Multi-allergen screening immunoassay for the detection of protein markers of peanut and four tree nuts in chocolate

A multiresidue enzyme immunoassay was developed to check for the presence of markers of peanut, hazelnut, almond, cashew and Brazil nuts in a single run. The assay was designed under the competitive indirect format and adapted for screening purposes applied to chocolate samples. The limit of detection for this assay was below 1 µg g^-1 protein for each allergenic food. In most cases, the high specificity of the antibodies used allowed the identification of each particular allergenic food with no possible confusion. This assay was proven to be useful as part of an analytical procedure involving the identification of the unknown allergenic food among peanut and other tree nuts in recalled samples before the application of a quantitative technique to determine the level of cross-contamination.     

色谱及免疫分祈在氰戊菊酯残留检测中的应用

介绍了气相色谱、毛细管气相色谱、气质联用、液相色谱、免疫分析等方法在氰戊菊酯残留检测中的应用.     

The effect of ensiling on PCR-based detection of genetically modified Bt maize

 The detection of the genetic modification in silage obtained from insect-resistant Bt maize by means of the polymerase chain reaction (PCR) is described. The detectability of the transgene was shown to be dependent on the length of the genomic target sequence chosen for amplication by the PCR. By amplifying a Bt-maize-specific DNA sequence of 211 bp the genetic modification was detected up to 7 months after ensilage. The effect of maize DNA degradation in the course of the ensilage on the detectability of target sequences was demonstrated in model experiments. Received: 17 December 1998     

A sensitive monoclonal-antibody-based test for gluten detection: Quantitative immunoassay

An enzyme-coupled monoclonal antibody has been used to quantify “gliadin-like immunoreactivity” in a variety of foods. Small discs of nitrocellulose are soaked in food extract or a series of standard gliadin solutions, and incubated with antibody and an enzyme substrate yielding a soluble product. By use of a photometer, standard curves for gliadin may be constructed and the apparent gliadin content of samples calculated. The reproducibility and reliability of the procedure were examined using a variety of common foods and food proteins. The limit of detection for wheat gliadin was approximately 20μg/ml extract; gliadin levels in excess of this value were found in some “gluten-free bread mixes” and starch sources. The overall time for analysis is 5–6 h, although for large numbers of samples, overnight blocking of non-specific antibody binding may be used. It is possible that a “library” of enzyme-linked monoclonal antibodies could be developed as useful tools for specific food analysis.     

Development of an innovative immunoassay for CP4EPSPS and Cry1AB genetically modified protein detection and quantification

An innovative immunoassay, called enzyme-linked immunoabsorbant assay (ELISA) Reverse, based on a new conformation of the solid phase, was developed. The solid support was expressly designed to be immersed directly in liquid samples to detect the presence of protein targets. Its application is proposed in those cases where a large number of samples have to be screened simultaneously or when the simultaneous detection of different proteins is required. As a first application, a quantitative immunoassay for Cry1AB protein in genetically modified maize was optimized. The method was tested using genetically modified organism concentrations from 0.1 to 2.0%. The limit of detection and limit of quantitation of the method were determined as 0.0056 and 0.0168 (expressed as the percentage of genetically modified organisms content), respectively. A qualitative multiplex assay to assess the presence of two genetically modified proteins simultaneously was also established for the case of the Cry1AB and the CP4EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) present in genetically modified maize and soy, respectively.     

Fluorescence polarization immunoassay based on a monoclonal antibody for the detection of ochratoxin A

A fluorescence polarization immunoassay (FPIA) based on a monoclonal antibody for the determination of ochratoxin A (OTA) was developed. Fluorescein‐labelled OTA derivative (tracer) was synthesized and purified by thin‐layer chromatography. The optimized OTA FPIA had a dynamic range from 5 to 200 ng mL^?1 with IC₅₀ value of 30 ng mL^?1 and a detection limit of 3 ng mL^?1. The method developed was characterized by high specificity and reproducibility. Cross‐reactivity with other mycotoxins (zearalenone, aflatoxins, patulin and T‐2 toxin) was negligible (<0.1%). Methanol extracts of barley samples were used for the analysis. The results of OTA determination in barley were compared with those determined by indirect competitive enzyme‐linked immunosorbent assay (ELISA). Recoveries for the samples spiked at 50, 100 and 500 ng g^?1 levels were 91, 90 and 97%, respectively, for FPIA, and 98, 98 and 102%, for ELISA. Naturally contaminated barley samples were analysed by these methods but some disagreement was observed between the results. The FPIA method can be applied for screening of food samples for OTA residues without a complicated clean‐up.     

Evaluation of culture enrichment procedures for use with Salmonella detection immunoassay

To design efficient culture strategies for use with immunoassays to detect Salmonella in food, the growth of these organisms was investigated according to the Bacteriological Analytical Manual (BAM) and enrichment-immunoassay (EI) culture procedures. The cultures were further evaluated using a commercial enzyme-linked immunosorbent assay (ELISA) kit. The BAM procedure includes pre-enrichment in nutrient broth (NB) for 16 h followed by selective enrichment in either Rappaport-Vassiliadis (RV) or tetrathionate brilliant green (TBG) broth for 16 h. The EI procedure includes pre-enrichment in NB for 4 h, selective enrichment in RV for 16 h and post-enrichment in NB for 4 h. The effects of different incubation times for pre- and post-enrichment, and different culture media for selective enrichment (TBG and RV) and post-enrichment in NB and Brain Heart Infusion broth (BHI) on the growth of the bacteria and ELISA titers in the EI procedure were also investigated. Salmonella enteritidis and S. typhimurium inoculated at different initial concentrations between 0.1 and 35 CFU/ml grew to similar concentrations of 10(7) to 10(8) colony forming unit (CFU)/ml in pure culture and generally 2 to 4 fold lower concentrations (P<0.05) in mixed culture using spiked chicken rinse. In the BAM procedure, the concentration of Salmonella cultured in RV was higher (P<0.01) than that in TBG. The cultures in TBG showed positive results for ELISA, but those in RV were generally negative. In the EI procedure, the ELISA titers from cultures post-enriched in NB or BHI were higher (P<0.01) when TBG, as compared to RV, was used for selective enrichment. Post-enrichment in BHI yielded higher numbers of Salmonella and higher ELISA titers than those in NB (P<0.05) for post-enrichment. This study demonstrated that in both culture procedures small numbers of Salmonella could be increased to at least 10(7) CFU/ml which is detectable by most ELISAs, and that the type of the culture media used may have a significant impact on ELISA results.