A double-antibody sandwich ELISA for detection of Candida tropicalis in dairy product
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摘要: 本研究用于乳品中热带假丝酵母菌的快速检测。采用热带假丝酵母菌超声破碎后的上清蛋白作为免疫原分别免疫新西兰大耳白兔和Hartley豚鼠,获得热带假丝酵母菌的多克隆抗体。以兔抗体作为捕获抗体,豚鼠抗体作为检测抗体,通过矩阵法及正交分析建立乳品中热带假丝酵母菌快速、特异的双抗夹心ELISA检测方法。该方法检出限为98 ng/m L,板内变异系数小于2%,板间变异系数小于6%,特异性及重复性良好。实际样品检测中,通过对乳制品进行滤膜集菌并选择性增菌培养,超声后提取的蛋白上清应用本研究建立的双抗夹心ELISA检测方法,100 CFU/m L热带假丝酵母菌可在22 h内准确检测出阳性反应。Abstract: This study focused on developing an rapid detection of Candida tropicalis in dairy products.The soluble protein was expressed by Candida tropicalis as immunogen to obtain the polyclonal antibodies( PAbs) of rabbit and guinea pigs respectively.A sandwich ELISA detection method based on PAb pair targeting Candida tropicalis was then established,in which rabbit PAb was capture antibody and guinea pig PAb was detection antibody.There was no cross reaction with miscellaneous bacteria or microzymes and the detection limits for Candida tropicalis was 98 ng / m L,the intro- batch variation was less than 2%,and the inter- batch variation was less than 6%.During the actual sample tests,the Candida tropicalis in dairy were enriched by filter and selective cultural method,followed by the sandwich ELISA detection. The results showed that 100 CFU / m L Candida tropicalis could be detected accurately in 22 h.The test results of this improved method were validated by national standard method.
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