氮、磷、铁、锌对铜绿微囊藻生长及产毒的影响

为探讨氮、磷、铁、锌对铜绿微囊藻生长及产毒的影响，以HGZ为基础培养基，调整氮、磷、铁、锌的浓度，于无菌条件下对两种藻株进行培养，定期取培养液进行细胞计数及用ELISA方法测定微囊藻毒素的含量。结果表明，浓度的改变没有引起无毒株产生毒素，也没有改变产毒株的产毒特性。高浓度的氮(1.6～245.1mg/L)有利于两藻株的生长及产毒株的毒素合成，而对磷、铁、锌的需求量较低。当磷的浓度低于1.4mg/L时，无毒株随其浓度增加而生长加快，但浓度超过此范围，则变化不大。产毒株的生长和产毒对磷盐的浓度变化不敏感，过高浓度的磷(62.1mg/L)可抑制其生长。两藻株的生长对铁、锌浓度的变化也不敏感，但产毒株的毒素合成却随铁离子浓度升高而缓慢上升。在氮、磷、锌3个单因素研究中，培养液中藻细胞的数量与总毒素含量均呈正相关(P＜0.05)，而铁未呈相关关系。     

甲萘威ELISA方法的建立及初步应用

以方阵法筛选甲萘威ELISA的抗原和抗体浓度 ,并建立了甲萘威的标准抑制曲线 ;以直接竞争性ELISA为基础 ,考察了单克隆抗体的特异性和ELISA法的各种影响因素 ;在黄瓜样品中添加定量甲萘威 ,用ELISA法和HPLC法检测其中甲萘威的含量 .结果显示 ,ELISA法的最低检测限为 4 .1ng/mL ,单克隆抗体对甲萘威的特异性较强 ;甲萘威的ELISA法受分析液中离子强度、pH值、有机溶剂和样品基质浓度的影响 ;ELISA回收率在 89.4 %～ 10 3.2 %之间 ,变异系数为 7.8%～ 10 .7% ;ELISA和HPLC两种方法分析结果无显著性差异 (P >0 .0 5 ) .本研究成功建立了甲萘威的直接竞争ELISA法 ,并将该方法初步应用于黄瓜样品中甲萘威的快速检测 .图 5表 2参 6     

农产品中黄曲霉素B1的酶联免疫吸附分析法

使用牛血清白蛋白(BSA)与黄曲霉素B_1(AFB_1)的结合物免疫兔子获得了高特异性、高效价的抗AFB_1的抗体,用此抗体建立了测定AFB_1的酶联免疫吸附分析法(ELISA),最低检出限为1.0pg/m。应用于花生、花生酱、玉米和大豆等农产品中AFB_1分析,其回收率在81—116％范围,变异系数在1.51—10.65％之间。此法具有简单、快速和适于大批量样品分析等优点。     

Expression and Immunoreactivities of Hepatitis E Virus Genotype 3 Open Reading Frame-2 (ORF-2) Recombinant Proteins Expressed in Insect Cells

Hepatitis E virus (HEV) is a fecal-orally transmitted virus that is endemic in many geographical areas with poor sanitary conditions and inadequate water supplies. In Europe, a low-endemic area, an increased number of autochthonous sporadic human cases of patients infected with HEV strains of genotype 3, have been reported lately. The relatively high prevalence of HEV genotype 3 infections in European pigs has raised concerns about a potential zoonotic transmission to humans. Determination of HEV seroprevalence in pigs would help to clarify its incidence and possible zoonotic implications. To this purpose, we have expressed and partially characterized swine genotype 3 HEV open reading frame-2 proteins upon infection of Sf21 insect cells with recombinant baculoviruses. The use of the expressed proteins as diagnostic antigens for the detection of antibodies to HEV has been further assayed with human and swine sera.     

Preparation of hapten-specific monoclonal antibody for cadmium and its ELISA application to aqueous samples

High-affinity and specific monoclonal antibodies against cadmium-ethylene diamine tetraacetic acid (EDTA) complex have been produced using the hybridoma technique. A hapten was synthesized and characterized by Fourier Transform Infrared Spectroscopy (FT-IR) and UV-Vis. Competitive enzyme-linked immunosorbent assay (ELISA) for quantitative detection of cadmium in aqueous sample was developed. The monoclonal antibody with high level of binding affinity for Cd-IEDTA-BSA and high specificity for soluble Cd-EDTA complex showed less than 0.99% cross-reactivity with other 11 metals. The limit of detection was 0.10 μg·L^-1, and the effective linear range was 10^-1–10³ μg·L^-1. The intra- and inter-assay coefficient variations were 1.5%–6.3% and 3.2%–7.4%, respectively. The spike recovery in different water samples were between 98.5% and 110.3%. The detection limit of this assay was well below the allowable concentration of cadmium (3 μg·L^-1), and the working range was wider than that of other methods which showed the range of 2.19–86.38 and 0–10³ μg·L^-1. The competitive ELISA established in this paper was sensitive and accurate in the screening of cadmium in aqueous samples. The results will lay a solid foundation for construction of an immunoassay kit for cadmium.     

Enzyme-linked immunosorbent assay for bisphenol A: Assay optimization and its application for surface water analysis

A range of ELISAs in indirect and direct formats for the determination of bisphenol A (BPA) was developed. Bisphenol A carboxymethyl ether (BPA-CME), (BPA-CPE) and 4,4-bis(4-hydroxyphenyl)valeric acid were coupled with bovine serum albumin (BSA) (immunogens for the production of polyclonal antibodies), ovalbumin (OVA) and horseradish peroxidase. In general, the indirect assay was more sensitive and specific than the direct one. Using heterologous combinations of immunoreagents in the indirect assay allowed to increase assay sensitivity and specificity. The highest sensitivity was obtained for the antibodies produced against a conjugate of BPA-CPE with BSA and a conjugate of 4,4-bis(4-hydroxyphenyl)valeric acid with OVA. The detection limit of BPA in phosphate buffer was 0.03?ng?mL^?1. The assay developed was also the most specific towards BPA. Maximum cross-reactivity values did not exceed 11% for 4-cumylphenol, 5% for bisphenol E and 2% for bisphenol S. Finally, the developed assay was used to analyze surface water samples spiked with known amount of BPA. The assay showed good recovery values (85–109%) for surface water with mineralization level lower than 500?mg?L^?1.     

EVALUATION OF SELECTED PESTICIDES IN NORTH CAROLINA SURFACE WATER SUPPLIES: INTAKE STUDY1

ABSTRACT: Studies were conducted to analyze the presence of 11 selected pesticides in 12 surface water supply intakes in the Piedmont and coastal plain regions of North Carolina. Samples were assayed using enzyme linked immunosorbent assays (ELISAs). Samples with pesticide detection of 1 μg/L or greater were extracted and confirmed using gas chromatography/mass spectrometry (GC/MS). Detection limits of the immunosorbent assays for pesticide residues were generally an order of magnitude higher than GC/MS. Atrazine was detected in approximately 45 percent of the samples, and on two occasions was at or above the lifetime Maximum Contaminant Level of 3.0 μg/L set by the Environmental Protection Agency for an annual average in finished drinking water. Metolachlor was detected in 58 percent of the samples. Of the remaining nine pesticides, including carbaryl, aldicarb, 2,4‐D, chiorpyrifos, acetochlor, methomyl, carbofuran, alachlor, and chlorothalonil, only aldicarb, 2,4‐D, and chlorpyrifos were detected in less than 9 percent of the samples for each pesticide.     

Production of the broad specific monoclonal antibody against sarafloxacin for rapid immunoscreening of 12 fluoroquinolones in meat

The objective of this study was to produce a generic antibody for immunoassay of fluoroquinolone drugs in meat. Two novel haptens of sarafloxacin were synthesized that were used to produce the monoclonal antibodies. The obtained monoclonal antibodies simultaneously recognized 12 fluoroquinolone drugs (sarafloxacin, diflocaxin, marbofloxacin, ofloxacin, ciprofloxacin, enrofloxacin, norfloxacin, pefloxacin, lomefloxacin, amifloxacin, enofloxacin and danofloxacin). After evaluation of different coating antigen/antibody combinations, a heterologous competitive indirect enzyme linked immunosorbent assay (ELISA) was developed to determine the 12 drugs. The crossreactivities to these analytes were in the range of 18%–113% and the limits of detection were in the range of 0.8–6.5 ng/mL depending on the compound. Eight fluoroquinolones licensed as veterinary drugs in China were fortified into blank chicken for ELISA analysis. The recoveries were in the range of 67.6%–94.6% with coefficients of variation lower than 12.4%. Therefore, this method could be used as a screen tool for routine monitoring of the residues of these fluoroquinolone drugs in animal derived foods.     

Monitoring of atrazine in milk using a rapid tube-based ELISA and validation with HPLC

Although atrazine has been banned in the European Union since 2007 it still persists in soil from where it can enter the food chain. Milk-producing animals accumulate atrazine from contaminated feed and water and since large quantities of milk and milk products are consumed its quality should be constantly monitored. The objective of this investigation was to develop a simple tube ELISA procedure suitable for use in non-specialised laboratories and in the field. A polyclonal antibody raised in sheep and the hapten-gelatine conjugate was immobilised onto polystyrene tubes. This enables the colour produced to be read on a basic spectrophotometer. Milk samples were collected from three farms in different regions of Poland and diluted before immunoassay was performed. Samples were extracted with hexane-acetone for HPLC analysis. The amount of fat in the milk samples interferes with the dose response so it essential that the standards are prepared in the same samples matrix. A good correlation between 1% and 2% was found between the two methods in the analysis of real samples. However the ELISA procedure was more sensitive that the HPLC method since atrazine was detected in some samples by the ELISA but was not confirmed by the HPLC method. The study demonstrated that the simple antigen-coated tube assay provides a cost effective and valuable screening test that can be easily modified for direct use as a screening tool in the field.     

Immunoassays as high-throughput tools: Monitoring spatial and temporal variations of carbamazepine, caffeine and cetirizine in surface and wastewaters

Carbamazepine (CBZ), caffeine and cetirizine were monitored by enzyme-linked immunosorbent assays (ELISAs) in surface and wastewaters from Berlin, Germany. This fast and cost-efficient method enabled to assess the spatial and temporal variation of these anthropogenic markers in a high-throughput screening. CBZ and cetirizine were detected by the same antibody, which selectively discriminates between both compounds depending on the pH value used in the incubation step. To our best knowledge, this is the first dual-analyte immunoassay working with a single antibody.The frequent sampling with 487 samples being processed allowed for the repeated detection of unusually high concentrations of CBZ and caffeine. ELISA results correlate well with the ones obtained by liquid chromatography tandem mass spectrometry (LC-MS/MS). Caffeine concentrations found in surface waters were elevated by combined sewer overflows after stormwater events. During the hay fever season, the concentrations of the antihistamine drug cetirizine increased in both surface and wastewaters.Caffeine was almost completely removed during wastewater treatment, while CBZ and cetirizine were found to be more persistent. The maximum concentrations of caffeine, CBZ and cetirizine found in influent wastewater by LC-MS/MS were 470, 5.0 and 0.49 μg L⁻¹, while in effluent wastewater the concentrations were 0.22, 4.5 and 0.51 μg L⁻¹, respectively. For surface waters, concentrations up to 3.3, 4.5 and 0.72 μg L⁻¹ were found, respectively.