Signal enhancement of surface plasmon resonance-based immunoassays for the allergen detection

A surface plasmon resonance (SPR) biosensor was used to determine the recombinant group 1 house dust mite allergen (rDer f1) in both HBS-EP buffer and fetal bovine serum (FBS). The monoclonal antibody was immobilized onto the CM5 sensor chip surface using an amine coupling method. The procedures of antibody immobilization and the subsequent primary and enhanced immunoassay were monitored in real time. The sensitivity for rDer f1 detection was remarkably improved by using intact polyclonal antibody as signal amplifying agent. Using this signal enhanced SPR immunosensor, rDer f1 in HBS-EP buffer and FBS was detected at a concentration of 15.4 and 32.1 ng/ml, respectively. The result demonstrates that SPR biosensor is a simple and reliable method for allergen detection.     

Micro-piezoelectric immunoassay chip for simultaneous detection of Hepatitis B virus and α-fetoprotein

In this paper, a piezoelectric diaphragm-based immunoassay chip was developed to simultaneously detect anti-Hepatitis B virus (HBV) and anti-alpha-fetoprotein (AFP). The chip was fabricated by micro-machining technology and consists of eight individual circular sensors with a diameter of 800 μm. Hepatitis B surface antigen (HBsAg), Hepatitis C core antigen (HBcAg) and AFP as the probe molecules were immobilized on different sensing spots on the chip. A solution containing anti-HBsAg and anti-AFP was applied into the reaction chambers in all sensors of the chip, and significant frequency shifts were only observed in the sensors with HBsAg and AFP for immunoassay detection. The fluorescence image further confirmed the successful detection of anti-HBsAg and anti-AFP. The total assay time was less than 2 h. The frequency shift-based calibration curves show a detection limit of 0.1 ng/ml and a dynamic detection range of 0.1-10,000 ng/ml for both anti-HBsAg and anti-AFP, respectively, thus demonstrating that the developed piezoelectric immunoassay chip has potential applications for rapid, specific, sensitive, and multiple detections of HBV.     

贝类毒素大田软海绵酸的免疫分析检测技术研究进展

大田软海绵酸(okadaicacid,OA)是一种广泛存在于贝类等生物中的海洋生物毒素,可引起人或动物的急性中毒,对食品安全和海产养殖具有严重危害。因此建立快速、可靠、灵敏的OA检测技术具有重要意义。免疫分析检测技术基于抗原抗体的结合,特异性强、灵敏度高、应用范围广,是当前检测贝类毒素OA的主要手段。本文综述了近年来针对贝类毒素OA的免疫分析检测技术,其中包括酶联免疫吸附检测、免疫层析检测、时间分辨荧光免疫检测和基于免疫传感器的检测技术等。本文着重阐述了不同免疫分析技术的原理及其在OA检测中的实际应用,同时探讨了免疫分析技术在贝类毒素OA检测方面的挑战和发展趋势,以期为开发性能更加优异的OA免疫检测技术提供研究思路。     

Determination of IgG levels in bovine bulk milk samples from different regions of Spain

Colostrum addition to milk for consumption is an unlawful practice. Furthermore, the higher whey protein/casein ratio of colostrum compared with milk causes severe technological problems. A high concentration of immunoglobulins has been generally accepted as an index of colostrum presence in milk. In this work, the levels of IgG have been determined by a non-competitive ELISA in 3,248 bulk bovine milk samples from different regions of Spain throughout one year. The mean value for IgG concentration obtained from all the samples was 0.257±0.122 mg/ml, within the range generally considered as acceptable for mature milk. The mean concentration obtained for all the samples collected in the spring was 0.288±0.125 mg/ml, significantly higher than the values found at the other seasons. The finding of some samples with very high values of IgG would suggest that the determination of immunoglobulin levels periodically would be a good parameter to control milk quality.     

Clinical proteomics: Promises,challenges and limitations of affinity arrays

After the establishment of DNA/RNA sequencing as a means of clinical diagnosis, the analysis of the proteome is next in line. As a matter of fact, proteome-based diagnostics is bound to be even more informative, since proteins are directly involved in the actual cellular processes that are responsible for disease. However, the structural variation and the biochemical differences between proteins, the much wider range in concentration and their spatial distribution as well as the fact that protein activity frequently relies on interaction increase the methodological complexity enormously, particularly if an accuracy and robustness is required that is sufficient for clinical utility. Here, we discuss the contribution that protein microarray formats could play towards proteome-based diagnostics.     

Nisin的生产、提纯和检测

Nisin是一种由乳酸乳球菌产生的羊毛硫氨酸类细菌素 ,在许多国家被许可作为生物防腐剂。Nisin的产量受许多因素的制约 ,如产生菌性能、培养基组成 (碳源、氮源、磷源和阳离子 )、发酵条件 (pH、温度、搅拌、通风 )、发酵类型 (分批发酵、连续发酵、自由细胞、固定化细胞 )等 ;大规模回收和纯化Nisin主要采用一些基于吸附-解析或者相分配原理的方法 ;最常用的定量检测Nisin的方法主要有生物分析法和免疫检测法 ,采用各种特定nisin抗体的免疫检测方法具有迅速、灵敏、准确等特性并能实现Nisin的在线检测。     

An immunoassay to assess lamb and kid rennets authenticity

The potential adulteration of kid or lamb rennet with calf rennet is of interest for some Protected Designation of Origin cheeses producers and those looking for a specific cheese typicality. The approach proposed here for the authentication of kid or lamb rennet is based on the immuno-detection of bovine pepsin possibly present in calf rennet in varying quantities. The developed immunoassay (indirect ELISA) used a monoclonal antibody (mAb) raised against bovine pepsin. This mAb was found to be specific as it didn't cross-react with the pepsin of animal species other than bovine (kid, lamb, pig) and with other milk-clotting enzymes (chymosin and microbial enzymes). Adulteration tests were conducted with kid and lamb rennets spiked with a wide range of calf rennet (from 0 to 100% v/v). The presence of bovine pepsin was detected at low levels down to 6 mg/L in kid and lamb rennets. Good linear relationships were obtained between added bovine pepsin concentration and the absorbance values over the range 1.25–120 mg/L. Results showed that indirect ELISA proved to be an interesting tool for testing rennets authenticity targeting bovine pepsin as an indicator of the bovine adulteration of kid and lamb rennets.     

Development of a novel electrochemical immuno-assay using a screen printed electrode for the determination of secretory immunoglobulin A in human sweat

This paper reports on the steps involved in the development of a novel electrochemical immunoassay for secretory immunoglobulin A (sIgA) determinations in human sweat. This novel immunoassay involves an initial step whereby sweat sIgA is adsorbed onto a polyvinylidene fluoride (PVDF) membrane (sweat patch). Following a wash step, a solution containing bovine serum albumin (BSA) is used to block any sites at which non-specific binding might occur. After a second wash step, the PVDF sweat patch is transferred to a second tube to which is added diluent and anti-sIgA antibody conjugated to horseradish peroxidase (HRP). Following an incubation step, an aliquot of supernatant containing unbound antibody is transferred to a well in a 96 well plate format which had been previously coated with sIgA. After a second incubation step and wash step, 3,3′,5,5′-tetramethylbenzidine (TMB) is added to the same well and left to undergo enzymatic oxidation. Finally a screen-printed carbon electrode (SPCE) is inserted into the well and any oxidised TMB is detected by chronoamperometry using an applied potential of +50 mV. The resulting reduction current is then referred to a calibration plot to deduce the unknown sIgA concentration. The optimisation of the steps involved in this assay is described in detail. Some preliminary data is presented on sweat sIgA levels collected from human volunteers who had undergone a controlled exercise regime on a bicycle (Ergociser). To our knowledge this is the first report where a sweat patch and a SPCE have been successfully incorporated into an immunoassay for sIgA.     

Development of monoclonal antibodies and a competitive ELISA detection method for glycinin,an allergen in soybean

Soybean glycinin is a major food allergen causing anaphylaxis. A sensitive detection method for glycinin is needed to evaluate soybean allergies in food and feed products. In the present study, monoclonal antibodies (Mabs) against glycinin were prepared using purified glycinin as the immunogen. The generated Mabs, named 3B2 and 4B2, were identified as being IgG_2b and IgG_2a iso-types respectively, and exhibited high specificity to glycinin. Then we developed a competitive ELISA based on Mab 4B2 to measure glycinin which showed an IC₅₀ value of 1.7 ng/mL with a detection limit of 0.3 ng/mL, and the linear portion of the curve was 0.3–11.2 ng/mL. Recovery tests indicated that the competitive ELISA based on Mab 4B2 gave reliable reproducibility. The produced Mab 4B2 and the developed ELISA could provide a valuable tool for sensitive determination of glycinin and for future studies conducted to reveal the mechanism of how glycinin functions in anaphylaxis.     

虾类过敏原的识别、纯化和检测技术研究

虾类是人类优质的食用蛋白资源之一,也是联合国粮农组织公布的八大类过敏食物之一。虾类过敏反应严重影响着过敏人群的身体健康和生活质量,为此开展虾类过敏原的识别、纯化和检测技术研究非常必要。通过问卷调查初步了解食物过敏现状,获取自诉虾类过敏患者血清和正常人阴性对照血清,采用特异性IgE检测试剂盒筛选虾类过敏血清。提取南美白对虾蛋白,进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳及免疫印迹识别虾类过敏蛋白,并对患者识别率最高的虾类的主要过敏原进行分离纯化。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、免疫印迹和酶联免疫吸附测定等方法对纯化虾蛋白进行检测分析。患者识别的南美白对虾致敏原的分子质量依次约为200、175、116、85和36kDa,通过硫酸铵盐析及等电点沉淀等方法可以得到电泳纯的虾主要过敏蛋白。免疫印迹结果证实纯化的虾蛋白是具有过敏原性的原肌球蛋白,在此基础上,建立了虾原肌球蛋白的酶联免疫吸附测定方法。