对硫磷酶联免疫吸附分析方法的建立和评价

通过制备对硫磷的多克隆抗体,建立了对硫磷的间接竞争酶联免疫吸附分析方法(icELISA)。通过活泼酯法将半抗原分别与牛血清蛋白(BSA)和卵清蛋白(OVA)偶联,获得免疫抗原和包被抗原。使用对硫磷抗血清,经优化条件参数建立了对硫磷的icELISA分析方法。该方法检测线性范围为3.39～533.05ng/mL,最低检测限为0.90ng/mL,对蔬菜样品和水样品的平均添加回收率为75.6%～127.4%,能用于实际样品中对硫磷的检测。     

Development of a direct competitive enzyme-linked immunoassay for carbofuran in vegetables

Three ELISA formats, antigen coated, antibody coated and the second antibody coated for the determination of carbofuran were investigated with conjugations including hapten–BSA, hapten–OVA, hapten–HRP and anticarbofuran IgG–HRP. Results showed that the second antibody-coated method of ELISA had a better performance in the establishment of standard curves and detection of carbofuran residue in vegetables samples. The sensitivity for detection, the I₅₀ value was 36.1 ng/ml at a practical working concentration range from 3.44 to 380.1 ng/ml and the limit of detection for carbofuran was 3.44 ng/ml. The average recoveries of determination for carbofuran spiked in cabbage, lettuce, carrot, winter fragrant-flowered garlic, bamboo shoot and green soy bean were 85.24%, 101.8%, 103.6%, 90.52%, 106.9% and 94.08%, respectively. Additional analyses confirmed that the results given by the ELISA method was in agreement with those of the gas chromatography (GC) method.     

Development of a McAb-based immunoassay for parathion and influence of the competitor structure

A specific monoclonal antibody (McAb) for parathion was produced. Based on this McAb, a battery of competitors as coating antigens were used to develop homologous and heterologous indirect competitive enzyme-linked immunosorbent assays (ELISAs) for parathion. The relationship between the heterology degree of the competitor and the sensitivity of the corresponding immunoassay was investigated. Results showed that, when the specific McAb was used in the ELISA experiment, competitors should have a certain degree of homology with the immunizing hapten for immunoassays, and the best performance occurred when the competitor hapten was highest heterologous to the target analyte. With the most suitable competitor, a sensitive and selective ELISA was developed. The IC₅₀ value of the ELISA was 2.94 ng/ml with a detection limit (IC₂₀) of 0.70 ng/ml. The average recoveries of parathion in spiked water, soil, cucumber and rice were 88.09%, 93.15%, 91.37% and 83.42%, respectively.     

白酒中邻苯二甲酸二乙酯的化学发光酶联免疫分析方法

以邻苯二甲酸二乙酯(diethyl phthalate,DEP)为研究目标,以4-氨基邻苯二甲酸二乙酯为半抗原,通过重氮法偶联载体蛋白并免疫动物,制备针对DEP的特异性兔多克隆抗体。通过棋盘滴定法和单因素实验确定最佳的实验参数,即包被抗原浓度为50 ng/m L,4℃环境下包被12 h;抗体用T液稀释;药物缓冲液选用p H 5.4、0.005 mol/L的PBS缓冲液;酶标二抗用P液稀释,稀释度为1/3000;反应时间是:一抗∶二抗=30 min:40 min。基于此建立了间接竞争化学发光酶联免疫法检测DEP。该方法对DEP的最低检测限(LOD)为3.09 ng/m L,检测范围(IC20~IC80)为5.93~42.03 ng/m L,半抑制浓度(IC50)值为16.57 ng/m L,与14种结构类似物及功能类似物交叉反应均远低于0.5%,通过对白酒样品添加回收率的测定,证明了该方法的准确性,适用于白酒中DEP的快速检测。     

直接竞争酶联免疫吸附法检测虾过敏蛋白

目的:建立酶标抗原的直接竞争酶联免疫吸附法(dcELISA)检测食品中虾过敏蛋白,为食品过敏诊断试剂的开发和应用提供理论基础。方法:提取虾主要过敏蛋白,免疫小鼠制备抗虾过敏蛋白多克隆抗体,辣根过氧化物酶(HRP)标记抗原,建立酶标抗原的dcELISA检测虾过敏蛋白。结果:所建立的dcELISA法最低检测限为3.94ng/mL,标准曲线在0.12～128.86ng/mL范围内线性良好,批内和批间变异系数分别为6.16%和2.73%,回收率为82%～98%。结论:该方法具有良好的特异性、敏感性和稳定性,为进一步研制检测虾过敏蛋白的ELISA试剂盒提供有效的方法。     

Fluorescent polarization immunoassay for sulphadiazine using a high specificity antibody

A rapid and sensitive fluorescent‐labelled polarization immunoassay for sulphadiazine (SDZ) is reported. Fluorescein‐labelled tracers were synthesized and a specific SDZ antibody has been used in the development of the method. The influence of the fluorescent label type on the immunoassay sensitivity was investigated. Homologous and non‐homologous tracer – antiserum combinations were varied to provide increased sensitivity of the immunoassay. A detection limit of 0.2 ng mL^?1 for SDZ in 50 μL aqueous sample has been achieved. The cross‐reactivity of other sulphonamide drugs has also been examined.     

Lateral flow immunoassay integrated with competitive and sandwich models for the detection of aflatoxin M1 and Escherichia coli O157:H7 in milk

Pathogens, mycotoxins, or antibiotics may exist in a food sample. Micro- and macromolecular substances must be detected quickly. A rapid and convenient lateral flow immunoassay (LFI) integrated with competitive and sandwich models was developed to detect micro- and macromolecular substances. In this study, aflatoxin M₁ (AFM₁) and Escherichia coli O157:H7 were selected as the micro- and macromolecular substances, respectively. Two test lines in the LFI test strip were evaluated to detect AFM1 and E. coli O157:H7 by competitive and sandwich models. Results showed that the limits of detection for detecting AFM1 and E. coli O157:H7 were 50 pg·mL^?1 and 1.58 × 10⁴ cfu·mL^?1, respectively. The whole assay time was 30 min. The recoveries of gold nanoparticle-LFI ranged from 78.0 to 111.6% with coefficients of variation in the range of 3.9 to 8.5% for the detection of AFM₁. For the detection of E. coli O157:H7, the range of recoveries was from 70.1 to 89.6% with coefficients of variation ranging from 4.9 to 13.0%. This study not only tested sensitivity and specificity, but also was a systematic study of location of 2 test lines of the LFI test strip integrated with competitive and sandwich models.     

Development and validation of an indirect competitive enzyme-linked immunosorbent assay for monitoring quinoxaline-2-carboxylic acid in the edible tissues of animals

The feed drug additive carbadox is a suspected carcinogen and mutagen. To monitor effectively residues of carbadox in the edible tissues of food-producing animals, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to detect quinoxaline-2-carboxylic acid, the marker residue of carbadox, was developed. Several haptens were synthesised and conjugated to the carrier protein. Nine female New Zealand white rabbits were immunised with the immunising conjugates to produce polyclonal antibodies according to the designed schemes of immunisation. The highly specific antibody that was very sensitive to N-butylquinoxaline-2-carboxamide with an IC₅₀ value of 7.75?µg?l^?1 was selected for the development of an ic-ELISA. The standard curves based on the N-butylquinoxaline-2-carboxamide matrix calibration ranged from 0.2 to 51.2?µg?l^?1. The decision limit and detection capability of the ic-ELISA were 0.60 and 0.83?µg?kg^?1 for liver and 0.68 and 0.79?µg?kg^?1 for muscle of swine, respectively. The recoveries were 57–108% with coefficients of variation of less than 20% when the quinoxaline-2-carboxylic acid was spiked into liver and muscle with the concentrations of 1.0–20.0?µg?kg^?1. Excellent correlations between the results of the ic-ELISA and an HPLC method (r?=?0.9956???0.9969) were observed for incurred tissues. These results suggest that the ic-ELISA is a sensitive, accurate and low-cost method that would be a useful tool for screening residues of carbadox in the edible tissues of food-producing animals.     

乳制品中牛IgG含量间接竞争性ELISA检测方法的研究

建立了牛初乳制品和添加了牛初乳成分的乳制品中牛IgG含量间接竞争性酶联免疫吸附测定方法,主要研究步骤包括:制备牛IgG保守区(Fc段),以Fc段为免疫原免疫Balb/C小鼠,取其脾细胞进行细胞融合,制备特异性单克隆抗体及合成适合实际测定用途的ELISA试剂盒。应用本研究建立的方法对标准品和实际样品进行牛IgG含量检测的结果表明,该方法回收率在78.9%～117.5%,批内变异系数小于10%,批间变异系数小于15%,检测结果稳定可靠,可满足目前国内牛初乳制品和添加了牛初乳成分的乳制品品质监控的需要。     

新型高灵敏赭曲霉毒素A间接竞争化学发光免疫分析法

建立了一种测定赭曲霉毒素A的新型高灵敏化学发光间接竞争酶联免疫分析方法。以酶标赭曲霉毒素A二抗上的辣根过氧化物酶催化过氧化脲氧化3-(4-羟苯基)丙酸,生成具有荧光的3-(4-羟苯基)丙酸二聚体。并利用乙腈介质中双[2,4,6-三氯苯基]草酸酯和过氧化脲在增强剂咪唑的作用下反应产生强化学发光,以发光强度确定待检物中赭曲霉毒素A含量。结果表明,在最佳条件下IC50为0. 55 ng/m L,在0. 05～6. 08 ng/m L范围内有良好的线性关系,最低检出限为0. 01 ng/m L。样品加标回收实验显示葡萄干和葡萄汁样品的平均回收率分别为84. 55%～91. 36%和73. 32%～87. 64%,批内与批间变异系数均小于10%,精密度良好。该新型化学发光方法检测赭曲霉毒素A时发光强度更大、发光时间更长,可用于食品中赭曲霉毒素A的高灵敏度痕量检测。     

Intra- and inter-laboratory validation of a dipstick immunoassay for the detection of tropane alkaloids hyoscyamine and scopolamine in animal feed

Tropane alkaloids (TAs) are toxic secondary metabolites produced by plants of, inter alia, the genera Datura (thorn apple) and Atropa (deadly nightshade). The most relevant TAs are (–)-L-hyoscyamine and (–)-L-scopolamine, which act as antagonists of acetylcholine muscarinic receptors and can induce a variety of distinct toxic syndromes in mammals (anti-cholinergic poisoning). The European Union has regulated the presence of seeds of Datura sp. in animal feeds, specifying that the content should not exceed 1000 mg kg^–1 (Directive 2002/32/EC). For materials that have not been ground, visual screening methods are often used to comply with these regulations, but these cannot be used for ground materials and compound feeds. Immunological assays, preferably in dipstick format, can be a simple and cost-effective approach to monitor feedstuffs in an HACCP setting in control laboratories. So far no reports have been published on immunoassays that are capable of detecting both hyoscyamine and scopolamine with equal sensitivity and that can be used, preferably in dipstick format, for application as a fast screening tool in feed analysis. This study presents the results obtained for the in-house and inter-laboratory validation of a dipstick immunoassay for the detection of hyoscyamine and scopolamine in animal feed. The target level was set at 800 µg kg^–1 for the sum of both alkaloids. By using a representative set of compound feeds during validation and a robust study design, a reliable impression of the relevant characteristics of the assay could be obtained. The dipstick test displayed similar sensitivity towards the two alkaloids and it could be concluded that the test has a very low probability of producing a false-positive result at blank level or a false-negative result at target level. The assay can be used for monitoring of TAs in feedstuffs, but has also potential as a quick screening tool in food- or feed-related poisonings.     

Development of a lateral flow immunoassay strip for screening of sulfamonomethoxine residues

A rapid lateral flow immunoassay screening method has been developed for the determination of sulfamonomethoxine (SMM) residues in swine urine. For this purpose, a specific monoclonal antibody (mAb), SMM4B9 for SMM, was generated and characterized. The mAb showed low cross-reactivity (not larger than 0.3%) to other sulfonamides and other potentially occurring analytes. Based on the competitive immunoassay principle, the strip was developed with the mAb SMM4B9 and applied to the screening of SMM residues. The test strip is made up of a sample pad, a gold-conjugate SMM4B9 reagent pad, a blotted test membrane containing a test line, a control line (a nitrocellulose membrane spotted with SMM-BSA and goat anti-mouse antibody, respectively), and an absorbent pad. The test could be accomplished within 8-10 min. It was shown that the sensitivity of the test strip was as low as 5 ng ml^-1 of SMM and the half of maximal inhibition concentration (IC₅₀) was calculated to be 10.78 ± 0.22 ng ml^-1 by relative optical density. In unaided visual assessment the detection limit of the strip was 15 ng ml^-1. For samples spiked at 20 and 30 ng ml^-1 the coefficient of variation (CV (%)) was between 2.3 and 7.1%. When the test strip was compared with high-performance liquid chromatography (HPLC) analysis for naturally contaminated swine urine samples, the difference in results was less than 6.1%. The data suggest that the method has advantages of high sensitivity, specificity, simplicity and speed of performance, as well as the characteristics of repeatability, reproducibility or accuracy and assurance. Therefore, the test strip is suitable to determine SMM residues in swine urine rapidly and reliably by quantitative, semi-quantitative or qualitative detection.     

Development of a lateral flow immunoassay strip for screening of sulfamonomethoxine residues

A rapid lateral flow immunoassay screening method has been developed for the determination of sulfamonomethoxine (SMM) residues in swine urine. For this purpose, a specific monoclonal antibody (mAb), SMM4B9 for SMM, was generated and characterized. The mAb showed low cross-reactivity (not larger than 0.3%) to other sulfonamides and other potentially occurring analytes. Based on the competitive immunoassay principle, the strip was developed with the mAb SMM4B9 and applied to the screening of SMM residues. The test strip is made up of a sample pad, a gold-conjugate SMM4B9 reagent pad, a blotted test membrane containing a test line, a control line (a nitrocellulose membrane spotted with SMM-BSA and goat anti-mouse antibody, respectively), and an absorbent pad. The test could be accomplished within 8–10?min. It was shown that the sensitivity of the test strip was as low as 5?ng?ml^?1 of SMM and the half of maximal inhibition concentration (IC₅₀) was calculated to be 10.78?±?0.22?ng?ml^?1 by relative optical density. In unaided visual assessment the detection limit of the strip was 15?ng?ml^?1. For samples spiked at 20 and 30?ng?ml^?1 the coefficient of variation (CV (%)) was between 2.3 and 7.1%. When the test strip was compared with high-performance liquid chromatography (HPLC) analysis for naturally contaminated swine urine samples, the difference in results was less than 6.1%. The data suggest that the method has advantages of high sensitivity, specificity, simplicity and speed of performance, as well as the characteristics of repeatability, reproducibility or accuracy and assurance. Therefore, the test strip is suitable to determine SMM residues in swine urine rapidly and reliably by quantitative, semi-quantitative or qualitative detection.     

Application of competitive indirect enzyme-linked immunosorbent assay (Ci-ELISA) for monitoring the degree of frozen denaturation of bovine myosin

The denaturation of myosin on freezing and frozen storage was monitored using competitive indirect enzyme-linked immunosorbent assay (Ci-ELISA) formatted with polyclonal antibodies anti-MWM IgG, anti-S-1 IgG and anti-LMM IgG raised against the antigens (Ags) bovine myosin whole molecules (MWMs), heavy meromyosin S-1 (myosin head part, S-1) and light meromyosin (myosin tail part, LMM) respectively. Beef slices and cuts stored at −20 °C or −50 °C lost immune affinity with all antibodies, in particular anti-LMM IgG. Repeated thawing–refreezing treatment caused more myosin denaturation than simple freezing. Myosin from beef stored at −20 °C was denatured more than that stored at −50 °C. The immune affinities between anti-LMM IgG and thawed samples were similar to those from anti-MWM IgG. We were unable to differentiate reliably between fresh and thawed beef using anti-S-1 IgG. Myosin was denatured by freezing, in particular its tail part (LMM).     

间接酶联免疫吸附检测克伦特罗条件优化

优化间接酶联免疫吸附法的测定条件。采用间接测定克伦特罗抗血清效价和血清阻断率。采用0.5μg/mL的包被浓度最佳,2%卵清蛋白的封闭效果最好,抗血清工作浓度可选择为1∶1000,抑制曲线表明,小鼠抗血清中抗体特异性很好,呈现线性关系。相同条件下邻苯二胺(OPD)显色值高于四甲基联苯胺(TMB),OPD的浓度以及过氧化氢的添加量对保质期影响很大,抗体稀释液选用0.5%明胶较好。经过实验,建立了实用的检测条件。     

基于Arah6的花生问接竞争ELISA检测方法的建立

建立了基于花生主要过敏原蛋白Arah6的间接竞争酶联免疫检测法,实现了对食物中花生的定量检测。利用花生过敏原Arah6纯品免疫新西兰大白兔,制得兔抗Arah6的多克隆抗体,以Arah6纯品作为包被抗原、自制抗体为一抗、辣根过氧化物酶标记的羊抗兔IgG为二抗,确定了包被抗原质量浓度为1μg／mL,一抗最佳稀释度为1：50000,酶标二抗稀释度为1：5000。此检测方法对Arah6的定量检测范围为16．5～10000ng/mL（折合成舍花生的含量,约为165ng／mL～100μg／mL）,抑制方程为抑制率I=21．4181gC-6．0633（c为Amh6的质量浓度,单位ng／mL）,IC50为414．6ng/mL,相关系数R2=0．9989。该检测方法灵敏度高,检测范围广,适用于食品中花生现场快速检测。     

免疫检测新技术在食品检测中的应用

免疫分析技术具有特异性强、灵敏度高、方便快捷、检测成本低、安全可靠等优点,已成为现代生命科学的重要研究手段,在食品安全检测中有着良好的应用前景。本文综述了近几年来出现的免疫分析新方法,简单介绍了它们的特点及在食品安全检测中的应用。     

Development of a Luminex based competitive immunoassay for 2,4,6-trinitrotoluene (TNT)

Previously, a displacement immunoassay for 2,4,6-trinitrotoluene (TNT) was demonstrated using the Luminex 100. The work presented utilized this same specialized flow cytometer to demonstrate a highly sensitive and rapid competitive immunoassay for TNT. This required a TNT analog to be attached to the microsphere surface. Various linkers were evaluated; bovine serum albumin provided over 3 times more binding sites in comparison to various shorter diamine linkers. For this assay TNB-coated microspheres were added to samples; then biotinylated anti-TNT antibody and the reporter molecule, Streptavidin-R-Phycoerythrin, were added. In the absence of TNT, a highly fluorescent complex was formed on the surface of the microsphere. The presence of TNT resulted in dose-dependent decreased fluorescence. Various anti-TNT antibodies were evaluated; Mab 30-1 gave the strongest response, yielding the lowest limit of detection (<1.0 ng/mL) and a dynamic range up to 1 microg/mL. Other factors such as reaction time, cross reactivity to other nitro-compounds, evaluation of acetone extracts of TNT contaminated soils, testing in environmental matrices such as fresh water and seawater were all completed. Finally, a multiplex assay for TNT and three protein toxins was successfully conducted using the competitive format.     

Detection of irradiated food by immunoassay – development and optimization of an ELISA for dihydrothymidine in irradiated prawns

This paper describes the development and use of a competitive enzyme‐linked immunosorbent assay (ELISA) to detect prawns which have been irradiated. The ELISA utilizes a monoclonal antibody against a modified DNA base, dihydrothymidine. A comparison of extraction procedures demonstrated that DNA purification was not required and that crude prawn homogenate could be used in the ELISA. The ELISA was applied successfully to two prawn species, North Atlantic prawn (Pandalus borealis) and Tiger prawn (Penaeus monodon). The ELISA has a working range of 0.5–2 kGy with CVs typically below 10%. Storage of irradiated prawns for up to 12 months at ?20 °C had no effect on ELISA performance. As most food contains DNA the assay has potential to be applied in a wide range of foodstuffs.     

Monitoring of organophosphorus pesticides in vegetables using monoclonal antibody-based direct competitive ELISA followed by HPLC-MS/MS

A multi-analytes method for monitoring of organophosphorus pesticides (OPs) using a combination of broad-specificity direct competitive enzyme-linked immunosorbent assay (dcELISA) and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed. The reaction formats of dcELISA as well as the matrix effects of vegetable samples by different treatments were studied. The dcELSIA based on horseradish peroxidase-labelled monoclonal antibody and solid-phase extraction can analyse 42 samples in duplicate simultaneously for 12 OPs with a limit of detection at 20 μg L⁻¹ within 40 min, with good accuracy and reproducibility. For screening purpose, the dcELISA can distinguish positive samples from hundreds of negative samples at a rapid, high-throughput and low cost manner. The positive samples can be following confirmed by HPLC-MS/MS for the kinds and the relative amounts of OPs. The method is suitable for monitoring of OP contamination in vegetables samples with high-efficiency and low cost.