【摘要】 为建立快速检测布鲁氏菌抗原的双抗体类的胶体金免疫层析方法（CGIA），本实验采用杂交瘤细胞株4A12和6C12分别经腹腔接种小鼠，制备单克隆抗体（MAb）4A12和6C12，分别以4A12作为金标抗体，6C12作为检测线（T线）包被抗体，以羊抗鼠IgG-HRP作为质控线（C线），通过优化反应条件组装成双抗体夹心胶体金免疫层析试纸条。结果显示，最适金标抗体4A12的浓度为2μg/mL，且每毫升胶体金溶液中加入6μL 0.2 mol/L K₂CO₃以维持稳定性；布鲁氏菌MAb 6C12和羊抗鼠IgG-HRP的最佳包被浓度分别为2 mg/mL和1 mg/mL。利用该方法检测大肠杆菌、金黄色葡萄球菌、单增李斯特菌、沙门氏菌和布鲁氏菌灭活菌液，结果显示除布鲁氏菌灭活菌液检测为阳性外，其他均为阴性，该方法特异性强。将1×10⁹cfu/mL的布鲁氏菌灭活菌液10倍倍比稀释后利用方法检测，结果显示，该方法的检测限为1×10⁵cfu/mL。利用3个独立批次制备的试纸条对布鲁氏菌阳性和阴性样品进行检测，结果显示3次结果完全一致，重复性良好。采用本实验建立的双抗体夹心CGIA和试管凝集试验同时对42份临床羊血液样品检测，结果显示，二者阳性符合率为90.9%，阴性符合率为100%，总符合率为97.6%。本研究首次建立检测布鲁氏菌的双抗体夹心CGIA，该方法具有较好的特异性、敏感性与重复性，为临床布鲁氏菌感染的快速可视化检测提供了新的技术手段。更多还原

【Abstract】 To establish a rapid colloidal gold immunochromatographic assay（CGIA） for the detection of Brucella antigen, this study utilized monoclonal antibody（MAb） 4A12 as the gold-labeled antibody and MAb 6C12 as the test line（T line） coating antibody, respectively. Goat anti-mouse IgG-HRP was served as the control line（C line）. A double-antibody sandwich colloidal gold immunochromatographic test strip was assembled by optimizing the reaction conditions. The results showed that the optimal concentration of the gold-labeled antibody was 2??g/mL, meanwhile 6 ??L of 0.2 mol/L K₂ CO₃ was added per milliliter of colloidal gold solution to maintain stability. The optimal coating concentrations for Brucella MAb 6C12 and goat anti-mouse IgG-HRP were2 mg/mL and 1 mg/mL, respectively. When the method was used to detect inactivated suspensions of E. coli, Staphylococcus aureus,Listeria monocytogenes, and Salmonella, only the inactivated Brucella suspension tested positive, with no cross-reactions observed,indicating high specificity. After a 10-fold serial dilution of the inactivated Brucella suspension（1×10⁹ cfu/mL）, the CGIA showed the limit of detection of 1×10⁵ cfu/mL. Testing of Brucella-positive and negative samples with three independently produced batches of test strips yielded completely consistent results, demonstrating good reproducibility. Simultaneous testing of forty-two sheep blood clinical samples using the CGIA and the tube agglutination test showed a positive concordance rate of 90.9%, a negative concordance rate of 100%, and an overall concordance rate of 97.6% between two methods. This study for the first time reports development of a dual-antibody sandwich colloidal gold immunochromatographic test for Brucella detection based on the dualantibody sandwich mode. The CGIA demonstrates favorable specificity, sensitivity, and reproducibility, offering a novel strategy for discriminating natural Brucella infection from vaccine immunization and providing a method for rapid visual detection of clinical Brucella infection.更多还原