Rapid assays for environmental and biological monitoring

Abstract

Rapid, inexpensive, sensitive, and selective enzyme‐linked immunosorbent assays (ELISAs) now are utilized in environmental science. In this laboratory, many ELISAs have been developed for pesticides and other toxic substances and also for their metabolites. Compounds for which ELISAs have recently been devised include insecticides (organophosphates, carbaryl, pyrethroids, and fenoxycarb), herbicides (s‐triazines, arylureas, triclopyr, and bromacil), fungicides (myclobutanil), TCDD, and metabolites of naphthalene and toluene. New rapid assays have been developed for mercury.     

褐菖鮋(Sebastiscusmarmoratus)金属硫蛋白的分离提纯及免疫测定

通过Ｓｅｐｈａｄｅｘ Ｇ－７５凝胶过滤和ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ ＣＬ－６Ｂ离子交换柱层析，从褐菖Ｙｏｕ肝脏分离金属硫蛋白。经氨基酸组成分析表明：褐菖Ｙｏｕ金属硫蛋白不含环状氨基酸，半胱氨酸含量达３５％，估算分子量为６７００道尔顿。等电聚焦显示两条区带，ＰＩ分别为４．０和４．２。金属硫蛋白经与牛血蛋白交联对兔进行免疫，兔抗血清用ＰｒｏｔｅｉｎＡ亲和纯化并标记上辣根过氧化物酶，利用酶联免疫吸附测     

Measurement of inhibin A: a modification to an enzyme-linked immunosorbent assay

Inhibin A is a useful prenatal marker of Down syndrome. Currently, the available enzyme-linked immunosorbent assays (ELISAs) for inhibin A are based upon the same paired monoclonal antibodies. In the present study we have confirmed for one of those ELISAs that short-term sample storage as whole blood leads to a significant decline in detectable inhibin A and that this is most likely due to erythrocyte catalase interference with a critical oxidation step in the assay. While this interference can be eliminated by heating the samples pre-assay, this process is labour intensive. In the present study we have demonstrated that the addition of 3-amino-1,2,4-triazole (AT), a catalase ‘suicide’ inhibitor, also prevents the decline of inhibin A levels in samples stored as whole blood. We suggest that the addition of AT to samples prior to assay is a simple modification to the inhibin A ELISA that affords optimum performance. Copyright © 2001 John Wiley & Sons, Ltd.     

水环境中大肠杆菌多特征抗原及抗体的制备研究

为了建立用于水环境中大肠杆菌的酶联免疫快速检测方法,针对水中大肠杆菌属多种血清型的特征,制备了水环境样品中大肠杆菌的多特征抗原,包括全菌体抗原、破碎全菌体抗原、菌体抗原、鞭毛抗原和菌毛抗原;采用这5种大肠杆菌抗原分别免疫新西兰大白兔,获得了5种大肠杆菌多克隆抗体,抗体效价高、纯度好,具有较强且稳定的特异性结合抗原的功能;间接ELISA方法表明,全菌体抗体和破碎全菌体抗体的效价均大于1×10⁵,性能优良.基于上述抗体,建立了水中大肠杆菌间接ELISA检测方法,实际水样的检测结果表明,检测限可达10⁴个/L.     

Cu/Zn superoxide dismutase quantification from fetal erythrocytes—an efficient confirmatory test for Down's syndrome after maternal serum screening and sonographic investigations

An enzyme immunoassay especially designed for the quantification of Cu/Zn superoxide dismutase (SOD) in erythrocytes has been applied to measure the SOD of outcomes with high risk for Down's syndrome. From 148 fetuses SOD was quantified from erythrocytes of umbilical vein blood and related to the number of cells, the content of haemoglobin (Hb), and to the haematocrit (Hc). Comparative studies between the SOD content of erythrocytes from the fetuses and their mothers resulted in similar SOD levels (14.09 ± 1.20 for fetal and 14.48 ± 1.63 for maternal cells) with a 1.84-fold smaller variance for fetal cells. The best differentiation between normal fetuses and fetuses with Down's syndrome resulted from the SOD/cell ratio followed by the SOD/Hb ratio. Fixing a cut-off value from the probability density functions that the method results in a specificity of 99.99 per cent, the sensitivity to detect cases of Down's syndrome was 99.71 per cent for the SOD/cell ratio, 70.92 per cent for the SOD/Hb ratio, and 60.21 per cent for the SOD/Hc ratio. Nine cases with Down's syndrome were correctly diagnosed by the SOD/cell ratio determination. Eight of these were confirmed as free trisomy 21 by karyotype analysis and one was found to be a triploidy. The latter was not detected by the SOD/Hb and SOD/Hc ratios because of the one-third higher content of haemoglobin and the larger volume of the erythrocytes which resulted in ratios within the normal range.     

褐菖鮋（Sebastiscusmarmoratus）金属硫蛋白的分离提纯及免疫测定

通过ＳｅｐｈａｄｅｘＧ－７５凝胶过滤和ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ离子交换柱层析，从褐菖的肝脏分离金属硫蛋白，经氨基区组成分析表明：褐菖的金属硫蛋白不含环状氨基酸，半胱氨酸含量达３５％，估算分子量为６７００道尔顿。等电聚焦显示两条区带，ＰＩ分别为４．０和４．２。金属硫蛋白经与牛血清蛋白交联对兔进行免疫，兔抗血清用ＰｒｏｔｅｉｎＡ亲和纯化并标记上辣根过氧化物酶，利用酶联免疫吸附测定法（ＥＬＩＳＡ）对褐菖金属硫蛋白进行分析，测定范围为５—１００ｎｇ／ｍＬ，经诱导和没诱导的肝提取液金属硫蛋白含量分别为４５０μｇ／ｍＬ和２０μｇ／ｍＬ，两者之间差别为２０多倍。     

Enzyme-immunoassay techniques for the detection of atrazine in water samples: Evaluation of a commercial tube based assay

Environmental immunoassays can help lower the operating costs and improve the effectiveness of residue laboratories. The present study assesses the ability of a commercially available enzyme immunoassay (EIA) to detect triazine herbicides in water. The tube based EIA could detect atrazine in lake and river water with detection limits of 62 pg/mL and 180 pg/mL respectively. The assay's ability to quantify atrazine in a set of 124 water samples taken from many parts of Canada was compared with a reference method that used gas chromatographic separation combined with a nitrogen phosphorous detector (GC-NPD) (R=0.919). A 71 % reduction in analytical load was achieved at a threshold concentration of 1 ng/mL. There were 2.4 % false negative and 0.8 % false positive results associated with that load reduction. The variability of the assay control parameters was generally within two standard deviations of the mean response for 65 assays. The EIA for atrazine is recommended for use as a screening technique and as an inexpensive way to monitor triazine levels in waters that are known to be contaminated with those herbicides.     

Optimization and validation of enzyme-linked immunosorbent assay for the determination of endosulfan residues in food samples

In this study, an enzyme-linked immunosorbent assay (ELISA) was optimized and applied to the determination of endosulfan residues in 20 different kinds of food commodities including vegetables, dry fruits, tea and meat. The limit of detection (IC₁₅) was 0.8 μg kg^?1 and the sensitivity (IC₅₀) was 5.3 μg kg^?1. Three simple extraction methods were developed, including shaking on the rotary shaker at 250 r min^?1 overnight, shaking on the rotary shaker for 1 h and thoroughly mixing for 2 min. Methanol was used as the extraction solvent in this study. The extracts were diluted in 0.5% fish skin gelatin (FG) in phosphate-buffered saline (PBS) at various dilutions in order to remove the matrix interference. For cabbage (purple and green), asparagus, Japanese green, Chinese cabbage, scallion, garland chrysanthemum, spinach and garlic, the extracts were diluted 10-fold; for carrots and tea, the extracts were diluted 15-fold and 900-fold, respectively. The extracts of celery, adzuki beans and chestnuts, were diluted 20-fold to avoid the matrix interference; ginger, vegetable soybean and peanut extracts were diluted 100-fold; mutton and chicken extracts were diluted 10-fold and for eel, the dilution was 40-fold. Average recoveries were 63.13–125.61%. Validation was conducted by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The results of this study will be useful to the wide application of an ELISA for the rapid determination of pesticides in food samples.     

Synthesis of novel hapten and production of generic monoclonal antibody for immunoassay of penicillins residues in milk

The objective of this study was to produce a generic monoclonal antibody for determination of penicillins residues in milk. The compound 6-aminopenicillanic acid was used as the template to synthesize two novel generic haptens that were used to produce the monoclonal antibodies. The obtained monoclonal antibodies simultaneously recognized 11 penicillin drugs (amoxicillin, ampicillin, penicillin G, penicillin V, sulbenicillin, carbencillin, methicillin, cloxacillin, dicloxacillin, oxacillin, and nafcillin). After evaluation of different reagent combinations, a heterologous indirect competitive enzyme immunoassay was developed to multi-determine the 11 drugs in milk. The crossreactivities to the 11 drugs were in a range of 16%–117% and the limits of detection were in a range of 0.7–9.3 ng/mL depending on the drug. The recoveries from the fortified blank milk were in a range of 77.6%–99.4% with coefficients of variation lower than 13.5%. This method could be used as a rapid screen tool for routine monitoring the residues of the 11 penicillin drugs in animal derived foods.     

环境样品免疫检测基质效应分析与控制

免疫检测技术以其特异性强、灵敏度高、分析容量大、反应速度快、检测费用低廉等优点,逐渐成为环境样品大规模快速筛查和预警监测的首选技术.由于免疫检测无需样品预处理、能实现对样品的在线检测,因此环境样品中复杂的基质可能对免疫检测产生非常大的干扰和影响.以水环境中微囊藻毒素-LR的酶联免疫吸附试验（ELISA）检测为基础,系统地考察了环境样品中各种背景基质对ELISA检测的影响,评价了各个基质的影响规律和程度,并研究提出了相应的控制和消除各不利影响的方法,最后统一提出水环境样品免疫检测抗基质效应干扰的控制方案,即采用10×PBS＋1%BSA＋0.5%EDTA作为免疫检测反应的缓冲体系,能有效消除各基质效应对免疫检测的干扰.该方案同样适应于其它不需要对环境样品进行预处理的免疫检测技术.