96孔酶标板法检测家蝇血淋巴黑化反应的初步研究

目的 探讨用96孔酶标板法检测家蝇血淋巴黑化反应的可行性。方法 用大肠埃希菌、金黄色葡萄球菌、藤黄微球菌单菌和大肠埃希菌与金黄色葡萄球菌混合菌分别体外刺激家蝇3龄幼虫的血淋巴,通过96孔酶标板法分析菌刺引起的家蝇幼虫血淋巴酚氧化酶活性的变化,进一步探讨菌刺与家蝇黑化反应的关系。结果 相比正常对照的起始A490值,1~3号样品的起始A490值随加菌量的增多依次递增,其增幅分别为大肠埃希菌1.33~1.38倍,金黄色葡萄球菌1.30~2.40倍,藤黄微球菌2.60~3.00倍,大肠埃希菌与金黄色葡萄球菌混合菌1.40~3.80倍。显然,菌刺可使家蝇血淋巴酚氧化酶活性增高,菌刺浓度越大,黑化反应越强,单菌刺激的效果依次为藤黄微球菌〉金黄色葡萄球菌〉大肠埃希菌,大肠埃希菌与金黄色葡萄球菌混合菌刺激的效果要比单菌好。结论 用96孔酶标板法检测菌刺家蝇血淋巴黑化反应的变化可行。

A preliminary study on melanization of hemolymph in Musca domestica by 96-well microplate assay

Objective To investigate the feasibility of 96-well microplate assay in determination of melanization responses of Musca domestica to different bacterial challenges. Methods The hemolymph from third- instar larvae of M. domestica was stimulated in vitro with Escherichia coli, Staphylococcus aureus, Micrococcus luteus, or the mixture of E. coli and S. aureus. The phendoxidase activity of hemolymph was determined with a 96-well microplate reader, and the relationship between bacterial stimulation and housefly melanization was further analyzed. Results Compared with the initial A490 values of normal control sample, those from the stimulated samples （numbered 1-3） constantly increased along with the increase in bacterial quantity. The fold changes in initial A490 values with stimulation by different bacteria were as follows： 1.33-1.38 times with E. coli, 1.30-2.40 times with S. aureus, 2.60-3.00 times with M. luteus, and 1.40-3.80 times with the mixture of E. coli and S. aureus. Obviously, stimulation with bacteria enhanced the phenoloxidase activity of hemolymph of M. domestica in vitro; meanwhile, the melanization responses of hemolymph got stronger with increase in bacterial quantity. The stimulating effects of single bacteria were as follows： M. luteus〉S, aureus〉E, coli, while the mixture of S. aureus and E. coli showed a better effect than single bacteria. Conclusion The 96-well microplate assay is a reliable method to determine the melanization responses in hemolymph of M. domestica during bacterial challenge.