Determnation of ochratoxin A in grain by monoclonal antibody-based enzyme-linked immunosorbent assay

ＤｅｔｅｒｍｎａｔｉｏｎｏｆｏｃｈｒａｔｏｘｉｎＡｉｎｇｒａｉｎｂｙｍｏｎｏｃｌｏｎａｌａｎｔｉｂｏｄｙ－ｂａｓｅｄｅｎｚｙｍｅ－ｌｉｎｋｅｄｉｍｍｕｎｏｓｏｒｂｅｎｔａｓｓａｙＹａｎｇＣｈｕａｎｈｅ；ＬｕｏＸｕｅｙｕｎ；ＬｉｕＣｈａｎｇ；ＬｉＷｅｎ...     

ELISA of polyclonal antibody to fish MT and study on heavy metal tolerance in fish

ＥＬＩＳＡｏｆｐｏｌｙｃｌｏｎａｌａｎｔｉｂｏｄｙｔｏｆｉｓｈＭＴａｎｄｓｔｕｄｙｏｎｈｅａｖｙｍｅｔａｌｔｏｌｅｒａｎｃｅｉｎｆｉｓｈＷａｎｇＹｉｎｇｙａｎ（ＮａｔｉｏｎＥｎｇｉｎｅｅｒｉｎｇＲｅｓｅａｒｃｈＣｅｎｔｅｒｆｏｒＵｒｂａｎＥｎｖｉｒｏ...     

对硫磷的人工抗原合成与鉴定

为防治病虫草害,确保农业丰收,每年有大量农药化学品施入环境中,从而使环境监测任务日趋繁重.气相色谱、液相色谱这些传统的农药残留检测手段,因其仪器昂贵,前处理过程复杂等难以满足快速监测需要.酶联免疫吸附分析(Enzyme Linked ImmunoSorbent Assay简称ELISA)技术,具有设备简单、成本低廉、专一性强、灵敏度高、样品预处理简化、适于同时检测大量样品等优点.近年来已发展成为一种简单、灵敏、快速的农药残留测试技术而应用于环境监测中.     

失去记忆性贝毒ASP酶联免疫检测方法的研究

建立了赤潮毒素失去记忆性贝毒中主要成分软骨藻酸(domoic acid,DA)的竞争酶联免疫检测方法.通过将DA偶联到蛋白载体上,免疫BALB/c小鼠,免疫数次后,得到抗DA的多克隆抗血清,以DA-OVA为包被抗原,利用抗原抗体反应,建立间接竞争酶联免疫吸附技术(Enzyme-linked immunosorbant assay,ELISA)分析检测海水样品和海洋贝类中的赤潮毒素失去记忆性贝毒DA的方法.结果表明,该方法最低检出限10μg/L,海水样品平均回收率102.%,批内变异系数12.%;贝类样品平均回收率为111.%,批内变异系数4.8%.     

在添加醋酸钾的营养富集培养基中培养杂交瘤细胞(英)

营养富集、添加醋酸钾和灌注培养都是改善杂交瘤细胞培养的策略在Cp9B细胞的静止分批培养中，添加氨基酸和维生素延长了培养时间，但对最大细胞密度影响不大，在反应器分批培养中，营养物明显促进细胞增殖，最大细胞密度比原来提高35倍．利用添加1g／L醋酸钾的反应器灌注培养实验说明，从普通培养基转到营养富集培养基，反应器中的平均细胞密度、抗体浓度和抗体滴度得到了进一步的提高，在培养上清中，乳酸、氨和丙氨酸大量积累，葡萄糖和某些氨基酸大量消耗，以致于可能造成对细胞生长和代谢的抑制。     

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.     

Homology modeling of anti-parathion antibody and its interaction with organophosphorous pesticides and analogues

The mechanism of specific recognition in pesticide immunochemistry was investigated by computer-based strategy, and a rapid method for the identification of antibody specificity was developed. Based on the previously produced anti-parathion monoclonal antibody (mAb), the DNA sequence was analyzed by polymerase chain reaction (PCR). From the translated amino acid sequences, a three-dimensional structure of the antibody was constructed by homology modeling method, and then it was coordinated by 1 ns molecular dynamics under the explicit solvent condition. The stereochemical property and folding quality were further assessed by Procheck and Profile-3D. The self-compatibility score for the antibody model was 98.7, which was greater than the low score 46.2 and close to the top score 102.6. In addition, parathion and several structural analogues were docked to the constructed antibody structure. The docking results showed that the interaction energy (-40.54 kcal/mol) of antibody-parathion complex was the lowest among all the tested pesticides, which accounted for the high specificity of the antibody to parathion and perfectly matched with the experimental data. Moreover, three residues, Phe165, Asp107 and Thr100 were recognized as the most important residues for antibody reacting with parathion. The interaction energy negatively correlated with the antibody specificity.     

Immunoassay of red dyes based on the monoclonal antibody of β-naphthol

The objective of the present study was to develop a multi-analyte immunoassay for the determination of eight red dyes in food samples. Two dye intermediates (2-hydroxy-1-naphthoic acid and 1-amino-2-naphthol) were used as the haptens to produce the monoclonal antibodies. The obtained monoclonal antibodies recognized Sudan 1–4, Para red, Sudan red G, Sudan red B and Acid orange II simultaneously. After evaluation of different antibody/coating antigen combinations, a heterologous indirect competitive enzyme linked immunosorbent assay was developed to determine the eight red dyes in food samples (chili oil, chili powder, tomato sauce, hotpot seasoning). The crossreactivities to the eight analytes were in the range of 61%–79% (with β-naphthol as 100%), and the limits of detection were in the range of 1.3–1.9 ng/mL. The recoveries of the eight analytes from the fortified blank samples were in the range of 84.2%–115% with coefficients of variation lower than 18.3%. Therefore, this method could be used as a rapid and simple tool to detect the residues of the eight red dyes in foods.     

High specificity detection of Pb2+ ions by p-SCN-Bz-DTPA immunogen and p-NH2-Bn-DTPA coating antigen

Only one bifunctional metal-chelator was used to prepare immunogen and coating antigen in all of the previous researches. However, the antibody-specific recognition to the spacer arm of the bifunctional metal- chelator might lower the specificity of heavy metal ions immunoassay. Two different bifunctional metal-chelators were adopted to prepare the immunogen and coating antigen respectively in our study to avoid this problem. The conjugates of keyhole limpet hemocyanin （KLH） and p-SCN-Bz-DTPA-Pb were used as immunogen, whereas the conjugates of bovine sentrn albumin （BSA） and p- NH2-Bn-DTPA-Pb were used as coating antigen. Poly- clonal antibodies specific to DTPA-Pb chelates were obtained from rabbits. Indirect competitive enzyme-linked immunosorbent assay （ELISA） was adopted to detect Pb^2＋ ion solutions prepared by Pb^2＋ standard solution and ultrapure water. In the mixing microplate, DTPA and Pb2＋ ions formed chelates and combined with specific anti- bodies. After incubation, the DTPA-Pb and the antibodies complex were added into the wells of the reaction microplate. The reaction microplate was coated by the conjugates ofBSA andp-NH2-DTPA-Pb, which competed for the specific antibodies. The result signals presented a good sigmoid curve when the Pb^2＋ concentration ranges from 0.01 to 100mg·L^-1 The IC50 of the indirect competitive ELISA is 0.23±0.04mg·L^-1 Pb2＋ ion. The cross-reaction with Cd^2＋, Cu^2＋, Fe^2＋, Mn^2＋, Zn^2＋, and other divalent ions were less than 5%.     

Determination of 3,4-dichlorinated biphenyl in soil samples by real-time immuno-PCR assay

A real-time fluorescent quantitative immuno-polymerase chain reaction (RT-IPCR) assay was developed for the detection of non-dioxin-like polychlorinated biphenyl (PCB) congener in soil samples. Based on the construction of 3,4-dichlorinated biphenyl (IUPAC PCB12) hapten and its immunogen, the specific polyclonal antibodies (pAbs) to PCB12 was obtained and used to develop a direct competitive RT-IPCR assay. Using the optimized assay, a standard curve for PCB12 was prepared. The linear range for the determination of PCB12 was from 10.0 to 1.0 × 10⁶ fg/mL with a correlation coefficient of 0.98 and a detection limit of 1.53 fg/mL. The RT-IPCR assays were tested for their cross-reactivity profiles using four selected congeners and four Aroclor products. The results for the soil samples correlated with the concentrations of PCBs obtained by gas chromatography/mass spectrometry. This highly specific, sensitive, and robust assay can be applied to on-site tests of PCBs and serve as a model for other pollutant immunoassays.