家蚕成品卵微粒子病McAb-ELISA检测方法的研究

Studies on the Method of McAb-ELISA Detection of Pebrine of Bombyx mori Egg

用纯化的微孢子虫抗兔多抗包被,做为捕获抗原,加入检测样品,然后用辣根过氧化物酶标记的单克隆抗体作为检测抗原,并对各步实验条件进行优化,建立检测家蚕微孢子虫的双抗体夹心ELISA检测方法。试验结果表明,多抗最适包被浓度为10ng/mL,最适包被条件为4℃过夜,酶标二抗工作浓度为1∶2000,待检样品和酶标二抗的反应时间分别为37℃1.5h和1h,底物37℃显色15min。此方法对纯化孢子的检测量为3.125×105spores/mL,对体外"添毒"蚕卵的检测量为5×106spores/mL。

The purified rabbit polyclone antibody of Nosema bombycis was coated use as capturing antibody. The horseradish peroxidase conjugated monoelonal antibodies was used as antigen to detect test samples. Each of steps were optimized to establish a double-antibody sandwich ELISA method for Nosema bombycis detection. It was showed that the optimal concentration of recombinant polyclonal antibody for coating was 10 ng / ml, the optimal coating condition for ELISA was 4℃ overnight, the work concentration of HRP-labeled monoclonal antibody was 1 ： 2000, the sample for detecting and HRP- labeled monoclonal antibody should be incubated at 37℃ for 1.5 h. and 1 h. respectively, the substrate for ELISA was incubated at 37℃ forl5 rain before terminated with the stopping solution. The detection density of Nb was 3.125 × 10^5 spore / ml for purified spore and was 5 × 10^6 spore / ml for Nosema bombycis infected silkworm egg.