粉尘螨变应原第2组分血清特异性IgE酶联免疫吸附测定法的建立及其检测效能的评价

Establishment of enzyme-linked immunosorbent assay for determining Dermatophagoides farinae mite group 2 allergen-specific IgE in serum and evaluation on its detection efficiency

目的建立粉尘螨变应原第2组分(Der f 2)血清特异性IgE的酶联免疫吸附测定(ELISA)法,并评价其检测效能。方法利用重组表达的Der f 2作为包被抗原,分别建立检测Der f 2血清特异性IgE的间接ELISA法和亲和素-生物素复合(ABC)-ELISA法,采用正交试验筛选两种方法的最优检测条件。分别采用上述两种ELISA法检测Der f 2特异性IgE阳性标准血清,绘制两种ELISA法检测Der f 2特异性IgE的标准曲线;同时以Pharmacia Uni Cap系统检测结果为金标准,计算两种ELISA法检测46例哮喘患者血清Der f 2特异性IgE的结果符合率。结果间接ELISA法检测Der f 2特异性IgE的最佳条件为:包被液浓度为10μg/ml,血清稀释倍数为1∶5,辣根过氧化物酶(HRP)标记抗人IgE抗体稀释倍数为1∶1 000。ABC-ELISA法检测Der f 2特异性IgE的最佳条件为:包被液浓度为15μg/ml,血清稀释倍数为1∶5,生物素标记抗人IgE抗体稀释倍数为1∶1 000,HRP标记的链霉和素稀释倍数为1∶2 000。间接ELISA和ABC-ELISA法的灵敏度值分别为0. 72 U/ml和0. 69 U/ml。间接ELISA和ABC-ELISA检测哮喘患者的符合率分别为93. 5%和95. 7%。结论成功建立基于重组表达Der f 2的ELISA法,其对Der f 2特异性IgE具有较好的检测效能。

Objective To develop the method of enzyme-linked immunosorbent assay(ELISA)for determining Dermatophagoides farinae mite group 2 allergen(Der f 2)-specific IgE in serum,and to evaluate its detection efficiency.Methods Indirect ELISA and avidin-biotin complex(ABC)-ELISA for the detection of Der f 2-specific IgE in serum were developed using the recombinant Der f 2 as the coating antigen.The orthogonal test was used to screen out the optimal detection conditions of the two methods.Standard serum with Der f 2-specific IgE positive was detected by the two methods of ELISA above separately,and the standard curves of the two methods of ELISA for detecting Der f 2-specific IgE were determined.Meanwhile,the coincidence rates of the two ELISA methods for detecting the serum Der f 2-specific IgE of the 46 asthmatic patients were calculated using the findings of the Pharmacia UniCap test as the golden standard.Results The optimal conditions of indirect ELISA for detecting Der f 2-specific IgE were defined as the concentration of coating buffer of 10μg/ml,the serum dilution rate of 1∶5,and the dilution rate of horseradish peroxidase(HRP)-labeled anti-human IgE antibodies of 1∶1 000.The optimal conditions of ABC-ELISA for detecting Der f 2-specific IgE were defined as the concentration of coating buffer of 15μg/ml,the serum dilution rate of 1∶5,and the dilution rates of biotin-labeled anti-human IgE antibodies and HRP-labeled streptavidin of 1∶1 000 and 1∶2 000 respectively.The sensitivities of indirect ELISA and ABC-ELISA were 0.72 U/ml and 0.69 U/ml respectively.The coincidence rates of indirect ELISA and ABC-ELISA in asthmatic patients were 93.5%and 95.7%respectively.Conclusion The ELISA method based on recombinant Der f 2 was established successfully,and it has a favorable detection efficiency for Der f 2-specific IgE.