零售药品；销售第三类医疗器械；销售生物检测试剂、医疗器械I类、Ⅱ类；机械设备、电子产品、汽车、化工产品（不含危险化学品及一类易制毒化学品）；技术服务、技术转让；代理进出口、货物进出口、技术进出口；生产快速检测试剂盒；销售北京明日达科技发展有限责任公司的兽用诊断制品（委托的生产企业名称和产品类别见有效委托代理或销售协议、不含强制免疫兽用生物制品）（有效期至2024年04月28日）。（市场主体依法自主选择经营项目，开展经营活动；零售药品、销售第三类医疗器械以及依法须经批准的项目，经相关部门批准后依批准的内容开展经营活动；不得从事国家和本市产业政策禁止和限制类项目的经营活动。）
This study aimed to establish a time-resolved fluorescence immunoassay method for the simultaneous quantitative detection of fumonisin B1, B2 and B3 in corn. The EDC/NHS active ester method was used to prepare time-resolved fluorescent microspheres and fumonisin antibody conjugates. Fumonisin antigen was immobilized on a nitrocellulose membrane as the test line. By optimizing the number of test line, a competitive immunoassay method for detecting fumonisin was eventually established. Subsequently, the sensitivity, specificity, accuracy, precision, and correlation with the national standard method were evaluated for the established method. The detection limits of the established method were 107.68~168.28 μg/kg, and the quantification limits were 283.46~444.63 μg/kg. The cross-reactivity with fumonisin B1, fumonisin B2 and fumonisin B3 were 100%, 85.59% and 72.72%, respectively, and no significant cross-reactivity was observed with five other common mycotoxins. The recovery rates ranged from 88.37% to 117.42%, and the coefficients of variation were lower than 10%. The conformity of the results obtained by the established method, as compared to the immunoaffinity column purification-high performance liquid chromatography (IAC-HPLC) method specified in the national standard GB 5009.240-2016, falls within the range of 92.17% to 107.21%. The established time-resolved fluorescence immunoassay method meets the requirements for rapid on-site quantitative detection of fumonisin B1, B2 and B3 in corn.
This article aimed to establish an amplified luminescent proximity homogeneous assay linked immune sorbent assay(AlphaLISA)for detecting quinolones(QNS)residues in milk.The EDC/NHS active ester method was used to connect 6-aminocaproic acid as the spacer arm on the norfloxacin molecule to prepare biotin-modified norfloxacin hapten(Biotin-6AS-NOR),and QNS monoclonal antibody was coupled on the receptor microsphere.Biotin-6AS-NOR could compete with QNS in the sample to bind quinolone antibodies.A competitive indirect AlphaLISA for QNS residues in milk was established by optimizing the parameters including QNS antibody coupling amount on receptor microspheres,Biotin-6AS-NOR,the dosage of donor/receptor microspheres,and dilution schemes for milk samples.By using the 1∶5 milk sample dilution scheme,the limit of detection and the limit of quantification of the established AlphaLISA for QNS in milk were 0.19 ng/mL and 0.46 ng/mL,respectively.The recovery values ranged from 85.97％and 110.11％,and the intraday and interday variation were both less than 10％.The cross-reactivity values for norfloxacin,enrofloxacin,ciprofloxacin,ofloxacin,pefloxacin,ofloxacin,and lomefloxacin were all higher than 70％.In conclusion,the established AlphaLISA showed high sensitivity,accuracy and good repeatability,without the need of tedious wash steps throughout the detection process;therefore,it is easy to achieve automated detection of chemical hazards residue in milk and has high application and promotion value.
Objective: This study aims to establish an indirect competitive enzyme-linked immunosorbent assay for the detection of amanitin (AMA) in mushroom. Methods: In this study, the hapten was obtained by introducing 6-aminocaproic acid into the carboxyl position of the α-amanotropin molecule through EDC/NHS, and further coupled with different carrier proteins to prepare immunogens and coating antigen. Afterwards, Balb/c mice were immunized with the immunogen to prepare monoclonal antibodies. Based on the obtained antibody, an indirect competitive ELISA method for the detection of amanitin in mushroom was established by optimizing the working concentrations of coating antigen and antibody, coating conditions, blocking conditions, working concentrations of enzyme labeled secondary antibody and incubation time. Finally, the sensitivity, recovery rate, intra batch and inter batch variation of the established method were evaluated. Results: The molecular weight of the synthesized hapten in this study was 1033.12, and the coupling ratio of the immunogen identified by MALDI-TOF was about 10.03. Based on hybridoma technology, the IC50 of the mouse monoclonal antibody 13H4 was 1.91 μg/L. Based on the obtained monoclonal antibody, the detection limit of the established ic-ELISA method for amanitin in mushroom was 0.88 μg/kg, the recovery rate was 85.66%~113.05%, the intra-assay coefficient of variation was 5.35%~9.54%, and the inter-assay coefficient of variation was less than 15%. Conclusion: The ic-ELISA method established in this study had high accuracy, precision, sensitivity, and stable performance. It provided a simple, reliable and rapid detection method for the analysis of the toxicogen of sudden mushroom poisoning.
