牛奶样品中志贺氏菌胶体金免疫试纸条检测方法的建立

Development of colloidal gold based immunochromatographic strip test for detection of Shigella spp. in milk

通过用超声波破碎的宋内氏志贺氏菌和福氏志贺氏菌的菌体碎片为免疫原免疫BALB/c小鼠,获得4株能稳定分泌抗志贺氏菌单克隆抗体的杂交瘤细胞株,选取其中一株制备单抗,并用胶体金标记。同时抗志贺氏菌的兔多抗和驴抗鼠抗体(二抗)分别喷涂于硝酸纤维素膜作为检测线(T线)和质控线(C线),研制了志贺氏菌胶体金快速检测试纸条。结果显示试纸条的灵敏度为105mL-1,并只与两株志贺氏菌有阳性反应外,而与其它23株常见的细菌无交叉反应。同时在检测添加有阪崎肠杆菌和长双歧杆菌的牛奶样品中,结果显示试纸条的灵敏度为106mL-1。志贺氏菌免疫胶体金试纸条的建立,为加强食品中志贺氏菌的检测工作,建立一种快速、简便的筛查方法具有重要的意义。

Monoclonal antibody against Shigella spp. was produced in Balb/c mice using ultrasonicated Shigella sonnei and S. flexneri as immunogen. Indirect ELISA was employed for screening of monoclonal antibodies from fusion cells （spleen cells from Balb/c mice fused with myeloma cells）. Four stable anti-Shigella spp. monoclonal antibody producing hybridoma cell lines were screened out and one of which was labeled with colloidal gold for following studies. Polyclonal antibody against Shigella spp. （rabbit source） and donkey-anti-mouse antibody were sprayed on the nitrocellulose membrane in line as test （T） and control （C） lines, respectively. The colloidal gold based immunochromatographic test strips for detection of Shigella spp. were fabricated and tested with bacteria pure culture for their sensitivity and specificity. Results showed that the sensitivity of the strip was 105 CFU/mL, and the test strip was specific to 2 Shigella strains with no cross reactivity with other 23 selected bacteria strains. The sensitivity of Shigella test strips was 106 CFU/mL in Enterobacter sakazakii and Bifidobacterium longum spiking milk samples. The established colloidal gold based immunochromatographic strip test method is important for rapid and simple screening of Shigella spp. in food samples.