ELISA检测小鼠肉汁中旋毛虫抗体实验条件的研究

Studies on the experimental conditions of ELISA for detection of anti-Trichinella antibodies in muscle juice of the infected mice

目的建立检测小鼠肉汁中旋毛虫抗体的酶联免疫吸附试验(ELISA)方法。方法应用棋盘滴定方法,选择适宜的旋毛虫肌幼虫排泄-分泌(ES)抗原的包被浓度、酶结合物的种类及肉汁稀释液的种类。结果旋毛虫肌幼虫ES抗原的包被浓度为5.0μg/ml。辣根过氧化物酶(HRP)标记的羊抗小鼠IgG对感染旋毛虫小鼠血清及肉汁的抗体检出率均为100%(10/10),HRP标记的葡萄球菌A蛋白(HRP-SPA)的检出率均为0(0/10),两者相比其差异有显著性(P<0.05)。对感染旋毛虫的小鼠肉汁分别用磷酸盐缓冲液(PBS)、磷酸盐缓冲液-Tween20(PBS-T)、生理盐水及含3%脱脂奶粉的PBS-T稀释后进行检测,发现含3%脱脂奶粉的PBS-T在各稀释度的P/N值均明显高于其他3种稀释液(P<0.05)。结论建立了检测小鼠肉汁中旋毛虫抗体的ELISA方法,合适的酶结合物为HRP标记的羊抗小鼠IgG,肉样的最佳稀释液为含3%脱脂奶粉的PBS-T,而HRP-SPA不适合于ELISA检测小鼠抗体IgG。

Objective To establish the ELISA method for detection of anti-Trichinella antibodies in muscle juice of infected mice. Methods The chessboard titration was used for selection of the ELISA experimental conditions, such as the coating concentration of T. spiralis muscle larval excretory-secretory （ES） antigen, the kind of enzyme conjugates and the dilution of muscle juice samples. Results The coating concentration of T. spiralis muscle larval ES antigen was 5.0 μg/ml. The goat anti-mouse IgG conjugated with horseradish peroxidase （HRP） and staphylococal protein A（SPA） conjugated with HRP were used for detection of anti-Trichinella antibodies in serum and muscle juice samples of 10 mice infected experimentally with T. spiralis. The antibody positive rate in both of serum and muscle juice was 100% （10/10） by using HRP-goat antimouse IgG, comparing with the rate of 0 （0/10） by using HRP-SPA, being a significant difference （P 〈 0.05）. The muscle juice sample was diluted with PBS, PBS-T, 0.85% NaCI and PBS-T containing 3% skimmed milk, respectively. The results showed that the ratio of optical absorbance （A） value of positive juice/ negative juice diluted with PBS-T containing 3% skimmed milk was obviously higher than that with the other 3 kinds of dilution （P 〈 0.05）. Conclusion The ELISA method for detection of anti-Trichinella antibodies in muscle juice of infected mice was established. The optimal conjugate for detection of mouse IgG antibody is HRP-goat anti-mice IgG and the optimal solution for diluting muscle juice is PBS-T containing 3% skimmed milk. The results also suggest that HRP-SPA is not suitable for detection of mouse IgG antibody in ELISA.