尘螨过敏原Der p 1、Der p 2特异性IgE化学发光免疫检测方法的建立及其性能评估

Establishment and performance evaluation of Der p 1/Der p 2 specific immunoglobulin E detection for the components of dust mite allergens based on chemiluminescence immunoassay

目的采用纳米磁微粒化学发光免疫技术建立尘螨过敏原第1、2组分(Der p 1、Der p 2)特异性免疫球蛋白E(sIgE)的定量检测方法。方法通过优化试剂组份和反应模式,评估纳米磁微粒化学发光免疫技术检测Der p 1、Der p 2 sIgE的各项性能指标。同时检测50例疑似尘螨过敏患者血清Der p 1、Der p 2 sIgE水平,与Phadia系统测定结果为标准进行比较,其一致性的比较采用Kappa检验。结果最佳实验体系为:磁微粒用量25μg,反应缓冲液(pH值7.4)含0.1 mol/L Tris-HCl、质量分数0.25%酪蛋白,包被液含20 mmol/L磷酸缓冲液(PB)、质量分数1%牛血清白蛋白(BSA),发光增强液含体积分数0.05%Triton X-100,采用两步法免疫反应,样本量为20μl,反应温度为37℃。检测体系最低检出限小于0.01 kU/L,线性范围0.2~100.0 kU/L,批内、批间精密度分别小于5%、7%,Der p 1和Der p 2交叉污染率分别为0.19%、0.21%。与Phadia系统测定结果比较,Der p 1阳性、阴性符合率分别为78.0%(32/41)、9/9,一致性较好(Kappa=0.65,P=0.008);Der p 2阳性、阴性符合率分别为93.3%(28/30)、85.0%(17/20),一致性也较好(Kappa=0.79,P=0.003)。结论成功建立了尘螨过敏原Der p 1、Der p 2 sIgE纳米磁微粒化学发光免疫检测方法,其各项性能指标良好,与Phadia系统有较好的一致性。

Objective To establish a quantitative detection method for the main components of dust mite allergens Der p 1,Der p 2 specific immunoglobulin E(sIgE)by using the nano-magnetic particle chemiluminescence immunoassay.Methods The performance indexes of the established method were evaluated after setting up and optimizing the chemiluminescence detection system and immune reaction conditions of sIgE for dust mite allergen.Serum sIgE levels of 50 suspected allergic patients with dust mite were determined by this chemiluminescence method.At the same time,this method was compared with the Phadia kit and the consistency was analyzed by Kappa test.Results The optimal amount of magnetic beads was 25μg,the optimal reaction buffer(pH=7.4)contained 0.1 mol/L Tris-HCl and 0.25%(W/W)casein,the optimal coating solution contatined 20 mmol/L phosphate buffer(PB)and 1%(W/W)bovine serum albumin(BSA),and the luminescence enhancement solution contained 0.05%(V/V)Triton X-100.The two-step immunoreaction was adopted,and the detection could be completed with 20μl sample at the optimal reaction temperature of 37℃.The limit of detection(LOD)of the established nano-magnetic particle chemiluminescence system in detecting Der p 1 and Der p 2 sIgE antibodies were both less than 0.01 kU/L,with the linear range of 0.2-100.0 kU/L,the precision of less than 7%,and the cross contamination rate of 0.19%and 0.21%.Compared with the Phadia system,the positive and negative coincidence rate of Der p 1 were 78.0%(32/41)and 9/9 with good consistency(Kappa=0.65,P=0.008),and the positive and negative coincidence rate of Der P 2 were 93.3%(28/30)and 85.0%(17/20)with good consistency(Kappa=0.79,P=0.003).Conclusion The nano-magnetic particle chemiluminescence immunoassay is successfully established for detecting dust mite allergen sIgE,which has good detection performance and good consistency with Phadia system.