虾过敏症特异性IgE的ELISA检测方法的研究

Research of ELISA reagent of specific IgE for Metapenaeu ensis anaphylaxis

目的研制虾过敏症特异性IgE的ELISA检测试剂。方法以纯化的刀额新对虾的2种主要变应原30 kD和33 kD蛋白为抗原分别包被酶标反应板,对抗原的包被浓度和时间、封闭液的选择与封闭时间、酶标二抗稀释度等ELISA条件进行优化。结果 30 kD和33 kD变应原的最适包被浓度分别为200 ng/孔和12.5ng/孔,最适包被条件为37℃孵育3 h,4℃过夜;酶标反应板的封闭以1.0%BSA、4℃封闭过夜为最适条件;HRP标记的羊抗人IgE抗体以1∶1 500为最适稀释度。结论以刀额新对虾的33 kD主要变应原为诊断抗原研制特异性IgE的ELISA检测试剂,适用于临床虾过敏症的实验诊断。

Objective To develop ELISA reagent of specific IgE for Metapenaeu ensis anaphylaxis.Methods Micro titer plates were coated with two major allergens of 33 kD and 30 kD proteins purified from Metapenaeu ensis,respectively.The ELISA conditions including the allergens＇ coating concentration,coating time and temperature,the blocking conditions for the coated micro titer plate,and the dilution of HRP labeled goat anti-human IgE antibody were detected with different conditions and optimized.Results The optimal coating conditions of the 33 kD and 30 kD allergens were 12.5 ng per well and 200 ng per well respectively with coating for 3 h at 37 ℃ then for one night at 4 ℃.The optimal blocking agent for the micro titer plate was 1.0% BSA for one night at 4 ℃,and the optimal dilution of HRP labeled goat anti-human IgE antibody was 1∶1 500.Conclusion The ELISA reagent developed with the 33 kD allergen of Metapenaeu ensis was applicable for laboratorial diagnoses of Metapenaeu ensis anaphylaxis in clinic.