标记免疫分析在微量蛋白检测中的应用概述

蛋白质定量检测涉及医学、环境和食品等众多领域,在疾病筛查、诊断和疗效评价等方面具有较大实用价值,如何快速、精确进行定量分析是现代检验科学面临的重要课题之一。本文综述了近年来国内外广泛使用的标记免疫分析方法,并结合实际应用重点介绍放射免疫分析、酶联免疫分析、荧光免疫分析、化学发光免疫分析、免疫传感器、免疫-PCR和蛋白质芯片等方法在微量蛋白定量检测中的应用特点和发展现状。     

兔抗金黄地鼠酶标抗体的制备及应用

目的 纯化金黄地鼠血清IgG,制备兔抗金黄地鼠酶标抗体(IgG-HRP),开展金黄地鼠仙台病毒的初步检测.方法 采用亲和层析纯化法纯化金黄地鼠IgG,用SDS- PAGE电泳测定IgG纯度并制备兔抗金黄地鼠IgG抗体(second antibody,Ab2);用免疫双扩散法检测抗血清效价后,再用亲和层析纯化抗血清IgG( Ab2);采用改良过碘酸钠标记法制备兔抗金黄地鼠酶标抗体( rabbit anti-hamster IgG-HRP);用直接ELISA和Western-blot法对兔抗金黄地鼠IgG酶标抗体进行工作浓度测定;应用金黄地鼠酶标抗体对金黄地鼠仙台病毒进行酶免检测(IEA).结果 金黄地鼠血清IgG纯度达95％;兔抗金黄地鼠IgG抗体(Ab2)免疫双扩散效价为1(:)64;兔抗金黄地鼠IgG -HRP经直接ELISA和Western-Blot测定工作浓度分别为1∶5000和1∶2000;酶免(IEA)效价为1:2000.结论 高效快速纯化了金黄地鼠IgG,制备了金黄地鼠IgG-HRP,为金黄地鼠病原微生物的血清学检测提供了条件.     

食品中农药残留的新型生物检测技术研究进展

食品安全是关系国计民生的重大战略问题。农药残留（简称农残）是造成食品质量与安全问题的重要因素。食品中农药残留不仅发生率高，而且超标严重，涉及范围广，长期接触或食用含有农药残留的食品会危害人体健康。传统的农残分析技术已经很难满足多样化食品安全检测的实际需求，现代新型生物技术因特异性强、灵敏度高、简单快速等优势，在食品的农残检测中得到广泛应用和持续发展。分析了食品中农药残留及检测技术现状，综述了酶联免疫吸附测定法、荧光免疫法和生物条形码免疫法等分析技术，及电化学、光学、压电传感器等生物传感技术在食品农残检测中的应用进展，讨论了各种技术的优势和局限，以期为相关研究提供参考，从而进一步提高我国食品农残的检测效率，保障食品安全，促进我国食品行业的可持续发展。     

大肠杆菌变种的两种分子生物学检测方法分析

目的:对大肠杆菌的一种重要的变种--肠出血性大肠杆菌O157-H7的几种检测方法进行比较研究.方法:以自动免疫磁珠收集系统(AIMS)、自动酶标免疫测试系统(VIDAS)与传统常规的分离方法进行对比分析.结果:运用自动免疫磁珠收集系统(AIMS)方法对80份可能含有肠出血性大肠杆菌O157-H7的实样进行检测,检出份数为6份,检出率为7.5％,而且在一周之内可以全部对上述检出实样进行鉴定.AIMS法能够检出浓度在10cfu/mL模拟实样之中的肠出血性大肠杆菌O157-H7,然后将此法与CHROMagar 0157琼脂板相结合,其效果则更为明显.而自动酶标免疫测试系统(VIDAS)与传统与的分离方法则检测的效果不佳,检出率为0.自动免疫磁珠收集系统(AIMS)检测方法与自动酶标免疫测试系统(VIDAS)、传统与的分离方法在检出率方面存在显著的统计学差异,P＜0.01.结论:运用自动免疫磁珠收集系统(AIMS)结合CHROMagar 0157琼脂板对出血性大肠杆菌O157-H7进行检测,检出率较高、灵敏度较高且快速便捷,可以用于O157-H7外环境检测与食品污染源的实际调查之中,应该对其加以广泛地推广并应用.     

酶标SPA免疫酶法检测宫颈癌患者血清中单纯疱疹病毒抗体

免疫酶法用于检测病人体液中病毒特异性抗体与其它方法相比。是一种简便易行、敏感性、特异性较好的方法,应用日广。用它检测病毒IgG抗体,通常以酶标抗人IgG(简称酶标抗体)作为第二抗体。本文将辣根过氧化物酶标记葡萄球菌A蛋白(简称酶标SPA)用于免疫酶法中检测宫颈癌患者血清中单纯疱疹病毒     

食品微生物快速检测技术研究进展

本研究概述了食品微生物引起的食源性疾病及对人类的危害;介绍了国内外9种食源性微生物快速检测技术的研究现状,指出电喷雾离子化多级质谱对氨基糖苷类抗生素的检测分析技术、分析即用型纸片技术、免疫分析检测技术、分子生物检测技术、阻抗技术以及其他新物理、生化技术检测方法,传统的检测的灵敏度和专一性有一定提高,但操作繁琐;现在发展的分子生物学方法为食品中快速、灵敏的病毒检测带来了更多的希望,各种快速检测方法还只是实践中的一个参考.其今后的发展方向主要是:克服现有分子生物学方法的缺点,绘制各种菌落图谱,开发在线检测软件,能定量测定微生物细胞特征和性能,简化采集、培养等程序,提高检测效率.     

食品微生物快速检测技术研究

在人们对食品安全重视程度日益加深的今天,要想保证食品卫生质量,就必须借助相应的检测手段,对食品的微生物情况进行监测。但是传统的检测手段存在众多的缺陷,不足以应付日益上涨的食品安全需求,所以一系列新型的微生物快速检测技术应运而生。目前用于食品微生物快速检测的技术主要包括免疫学技术、代谢学技术和分子生物学技术三个方面,这些检测技术以其快速、简便、高效等优点极大程度上保障了食品安全。本文主要对目前的食品微生物快速检测技术进行详细分析。     

食品微生物检测技术应用现状及展望

微生物引起的食源性污染正成为引起食品安全问题的主要原因,食品微生物检测技术的诞生为解决食品安全问题带来了福音,本文主要对食品微生物相关的检测技术进行阐述,同时对新型食品微生物快速检测技术的发展进行了展望。     

微生物的快速检验盒在日销售

日本味之素公司今春正式出售快速检查微生物的成套试剂“生物速测”系列产品。它是通过高灵敏度荧光反应法速检微生物的,可比过去为培养微生物所需时间缩短1/3以下。这种产品可用于冷藏食品厂为判断成品的检验结果后能当日出售以及食品产品、医药等工厂快速检查原料、工艺过程与成品中的微生物。系列产品中包括荧光分析仪、     

一种免疫生物传感器对单核细胞增生李斯特菌快速检测方法的初步研究

应用F0F1-ATP酶旋转分子马达和免疫技术相结合,建立免疫生物传感器快速检测技术。首先pH变化敏感荧光物质F1300标记到色素体(chrom atophore)的内表面,然后在F0F1-ATP酶上连接β亚基抗体-生物素-链亲和素-生物素-单核细胞增生李斯特菌多抗复合体,得到可以捕获单核细胞增生李斯特菌的免疫生物传感器。传感器上负载菌量不同,酶活性不同,酶活变化以pH敏感的荧光探针来感应,最后通过荧光扫描仪检测不同菌量负载下的荧光信号。结果表明,该方法对单核细胞增生李斯特菌标准菌株(ATCC 15313)的检测时间为4.5 h,检出浓度为100 CFU/孔。     

Impact of molecular biology on the detection of foodborne pathogens

Molecular biological methods that use antibodies and nucleic acids to detect specific foodborne bacterial pathogens were scarcely known a decade and a half ago. Few scientists could have predicted that these tools of basic research would come to dominate the field of food diagnostics. Today, a large number of cleverly designed assay formats using these technologies are available commercially for the detection in foods of practically all major established pathogens and toxins, as well as of many emerging pathogens. These tests range from very simple antibody-bound latex agglutination assays to very sophisticated DNA amplification methods. Although molecular biological assays are more specific, sensitive, and faster than conventional (often cultural) microbiological methods, the complexities of food matrices continue to offer unique challenges that may preclude the direct application of these molecular biological methods. Consequently, a short cultural enrichment period is still required for food samples prior to analysis with these assays. The greater detection sensitivity of molecular biological methods may also affect existing microbiological specifications for foods; this undoubtedly will have repercussions on the regulatory agencies, food manufacturers, and also consumers. The US government has the right to retain a nonexclusive royalty-free license in and to any copyright covering this article. Use of trade names is for identification only and does not imply an endorsement by the US FDA.     

Membrane filtration of food suspensions.

Factors affecting the membrane filtration of food suspensions were studied for 58 foods and 13 membrane filters. Lot number within a brand, pore size (0.45 or 0.8 micrometer), and time elapsed before filtration had little effect on filterability. Brand of membrane filter, flow direction, pressure differential, age (microbiological quality) of the food, duration of the blending process, temperature, and concentration of food in the suspension had significant and often predictable effects. Preparation of suspensions by Stomacher (relative to rotary blender) addition of surfactant (particularly at elevated temperature) and prior incubation with proteases sometimes had dramatic effects of filterability. In contrast to popular opinion, foods can be membrane filtered in quantities pertinent to the maximums used in conventional plating procedures. Removal of growth inhibitors and food debris is possible by using membrane filters. Lowering of the limits of detection of microorganisms by concentration on membrane filters can be considered feasible for many foods. The data are particularly relevant to the use of hydrophobic grid-membrane filters (which are capable of enumerating up to 9 X 10(4) organisms per filter) in instrumented methods of food microbiological analysis.     

The use of bacteriophage-based systems for the separation and concentration of Salmonella

Techniques for the separation/concentration of micro-organisms from background food matrices can be applied to increase the speed of analysis and ease of isolation and detection of target micro-organisms. One recent example of such a technique is the immunomagnetic separation (IMS) procedure that has been used for the separation of specific micro-organisms from foods. This paper describes the use of a novel biosorbent consisting of a Salmonella -specific bacteriophage (phage) immobilized to a solid phase that was used for the separation and concentration of Salmonella from food materials. This work has shown that a Salmonella -specific phage-based biosorbent could remove Salmonella from culture fluid and separate Salmonella from suspensions of other Enterobacteriaceae. The ease of production of phage, high affinity of phage–cell interaction and the ability of phage to infect host cells in heterogeneous environments indicates the potential of such a biosorbent as the basis for a reliable separation system in food microbiological analysis.     

Does microbiological testing of foods and the food environment have a role in the control of foodborne disease in England and Wales?

This review looks at the contribution of microbiological sampling to the safety of retail foods in England and Wales. It compares sampling methods available and assesses the value of testing as part of outbreaks of foodborne disease, as part of routine management by local authorities, as part of work done or commissioned by the food industry, and as part of research. It confirms that microbiological testing has a role during outbreaks as it makes a significant contribution to help identify foods and other areas of greatest risk for future study. The review suggests that routine testing by local authorities is often of limited use and could be improved by more targeted surveillance. Testing could be better used to validate primary control methods, such as Hazard Analysis and Critical Control Point (HACCP) system. Any public health benefit from testing in the food industry is often restricted by client confidentiality. Microbial research on foods is important as it can lead to significant improvements in safety. Current microbiological methods are slow and, in future, rapid molecular methods may make an even bigger contribution to the control of foodborne disease.     

Microbiological criteria for retail foods

This article proposes that microbiological criteria should only be drawn up and applied to foods if there is a full understanding and consideration of raw material selection, production process, packaging and finished product characteristics. The combined knowledge and expertise of public sector microbiologists and food industry microbiologists and technologists need to be harnessed in the establishment of such criteria. In this manner, microbiological criteria could be derived to the overall benefit of the consumer.
A meeting of the Society for Applied Bacteriology held in November 1994 considered the issue of microbiological standards for foods and, in this paper, the recently formed PFMG presents an opinion on the 'Provisional microbiological guidelines' published by the Food Surveillance Group of the Public Health Laboratory Service.     

The use of NASBA for the detection of microbial pathogens in food and environmental samples

The isothermal amplification method nucleic acid sequence-based amplification (NASBA), which amplifies RNA, has been reported as useful for the detection of microbial pathogens in food and environmental samples. Methods have been published for Campylobacter spp., Listeria monocytogenes and Salmonella enterica ser. Enteritidis in various foods and for Cryptosporidium parvum in water. Both 16S rRNA and various mRNAs have been used as target molecules for detection; the latter may have advantages in allowing specific detection of viable cells. Most of the methods to detect pathogens in foods have employed enrichment in nutrient medium prior to NASBA, as this can ensure sensitivity of detection and encourage the detection of only viable target cells. Although a relatively recent method, NASBA has the potential for adoption as a diagnostic tool for environmental pathogens.     

Microbes in food processing technology

Abstract: There is an increasing understanding that the microbial quality of a certain food is the result of a chain of events. It is clear that the microbial safety of food can only be guaranteed when the overall processing, including the production of raw materials, distribution and handling by the consumer are taken into consideration. Therefore, the microbiological quality assurance of foods is not only a matter of control, but also of a careful design of the total process chain. Food industry has now generally adapted quality assurance systems and is implementing the Hazard Analysis Critical Control Point (HACCP) concept. Rapid microbiological monitoring systems should be used in these cases. There is a need for rapid and simple microbiological tests which can be adapted to the technology and logistics of specific production processes. Traditional microbiological methods generally do not meet these high requirements. This paper discusses the tests, based on molecular biological principles, to detect and identify microbes in food-processing chains. Tests based on DNA technology are discussed, including in vitro DNA amplification like the polymerase chain reaction (PCR) method and identifications based on RFLP, RAPD and DNA fingerprinting analysis. PCR-haled methodology can be used for the rapid detection of microbes in food manufacturing environments. In addition, DNA fingerprinting methods are suitable for investigating sources and routes of microbial contamination in the food cycle.     

Clostridium botulinum and the safety of minimally heated, chilled foods: an emerging issue?

Foodborne botulism is caused by consumption of preformed botulinum neurotoxin, with as little as 30 ng of neurotoxin being potentially lethal. Consumption of minute quantities of neurotoxin-containing food can result in botulism. In view of the severity of foodborne botulism, it is essential that new foods be developed safely without an increase in incidence of this disease. Minimally heated, chilled foods are a relatively new type of food, sales of which are currently increasing by about 10% per annum. These products meet consumer demand for high-quality foods that require little preparation time. Their safety and quality depends on mild heat treatment, chilled storage, restricted shelf life and sometimes on intrinsic properties of the foods. The principal microbiological hazard is nonproteolytic Clostridium botulinum, and there is a concern that this may become an emerging issue. A considerable amount of research and development over the last 15 years has underpinned the safe production of commercial, minimally heated, chilled foods with respect to foodborne botulism, and it is essential that safe food continues to be developed. In particular, the desire to use lighter heat processes and a longer shelf life presents a challenge that will only be met by significant developments in quantitative microbiological food safety.     

Methods of identification and assessment of safety of genetically modified microorganisms in manufacture food production

Methods of identification of genetically modified microorganisms (GMM), used in manufacture food on control probes are presented. Results of microbiological and molecular and genetic analyses of food products and their components important in microbiological and genetic expert examination of GMM in foods are considered. Examination of biosafety of GMM are indicated.     

ASSESSMENT OF IMPEDANCE MICROBIOLOGICAL METHOD FOR THE DETECTION OF ESCHERICHIA COLI IN FOODS

This study was conducted to determine the sensitivity and specificity of the impedance-based microbiological method for the detection of Escherichia coli in foods within 24 h of testing. A Malthus Microbiological Analyzer system (Malthus System V, Malthus Instruments Ltd., Bury, United Kingdom), and a modified Malthus Coliform Broth Medium (MCBM), and an incubation temperature of 44C were used. The sensitivity of the impedance method was determined by testing E. coli-negative food samples spiked with different concentrations of E. coli. The specificity of the method was determined by testing E. coli -negative food samples spiked with Klebsiella pneumoniae, Enterobacter cloacae and Pseudomonas aeruginosa. The test results were compared with those obtained by the Most Probable Number (MPN) method. Milk, milk products, raw and ready-to-eat meats, and vegetables were tested for the presence of E. coli by both methods. The sensitivity of the impedance method and the MPN method for the detection of foods containing 10¹ CFU/g was 100% and 84.4%, respectively. Both methods had a specificity of 100% for food samples spiked with 10¹ CFU/g E. coli. The specificity of the impedance and the MPN methods for the detection of E. coli in naturally contaminated milk and meat samples was 100% and 95.7% respectively. E. coli was detected in foods by the impedance method within 4–24 h of testing at a detection limit of 1 CFU/mL. These results demonstrate that the impedance method can be used as a rapid and sensitive method for the detection of E. coli in foods.