新型食物不耐受sIgG定量检测方法的构建

【摘要】目的以检测鸡蛋特异性免疫球蛋白G（sIgG）为例，探讨新型食物不耐受的定量检测方法。方法选择行食物过敏原sIgG检测的患者173例，另择健康体检者78例为阴性对照。制备生物素化牛血清白蛋白-链酶亲和素酶标反应板，同时加入生物素化鸡蛋抗原和待检血清，洗涤后再加入酶标记抗人IgG，建立一种抗原间接包被-液相反应模式的酶联免疫方法；优化生物素化过敏原和酶标记抗体的浓度，确定反应条件。应用此方法检测血清标本中的sIgG。结果选用生物素化蛋清作为抗原，其最适稀释比例是1∶2000，酶标抗体的最适稀释比例是1∶12000。此方法批内及批间变异系数分别为4.83%~8.55%,4.88%~7.93%；与蟹、牛奶、羊奶等的交叉反应率均<10%；与临床现用食物不耐受sIgG检测法有较好的相关性（=0.977X+8.45，r=0.961, P<0.05）。结论本检测方法可用于检测鸡蛋不耐受患者血清sIgG，操作简便，具有较高的准确性和特异性，重复性好，并显示较好灵活性潜能，为今后研制“个性化”随机组合食物品种的检测试剂盒奠定基础。

The New Establishment of Quantitative Determination Method for Food Intolerance Specific IgG

[Abstract]ObjectiveTo explore a novel quantitative detection method for the concentration of specific IgG (sIgG)in food intolerance, taking egg sIgG detection for the example.MethodsA total of173patients underwent food allergen sIgG detection were included in this study, and 78healthy subjects were used as negative controls. The microtiter plates were coated with biotinylated bovine serum albumin (BSA) and linked with streptavidin. Then, the biotinylated egg antigen and specimen were successively added into the wells of plate．After washing, enzyme labeled anti-human IgG was added to establish an antigen indirectly coated liquid-phase reaction patterns of ELISA method. The concentration of the biotinylated allergens and enzyme labeled antibody were optimized and the reaction conditions were determined. This method was used to detect the sIgG in serum samples.ResultsThe biotinylated egg white was selected as antigen, the optimal dilution rate was 1∶2000，and the most suitable enzyme labeled antibody dilution ratio was1∶12000in this method. The within-run and the between-run coefficients of variation were4.83%-8.55% and4.88%-7.93% respectively. The specificity is preferable, the species-crossed reaction rates with crab, cow milk and goat milk were <10%. There was a good correlation between the assay developed in this study and the food intolerance detection kit provided by United States BIOMERICA Inc ( =0.977X+8.45,r=0.961, P<0.05).Conclusion The detection method can be used to detect serum sIgG for egg intolerance patients with easy operation, highly accuracy and specificity. Furthermore, it showed the capacity of excellent repeatability and flexibility potential, which provided a good foundation for developing a kind of“personalized”random combination of food varieties ELISA kit.