尘螨变应原治疗性产品鉴别试验解吸附方法的建立

Development of identification test on dust mite therapeutic products after desorption

目的建立尘螨变应原治疗性产品鉴别试验的解吸附方法。方法选取两个厂家的尘螨变应原治疗性产品,分别用1 ml 20%柠檬酸溶液、0.4 mol/L磷酸盐溶液和0.5 mol/L EDTA-Na溶液解吸附后,进行SDS-PAGE和Western blot鉴别试验。将两个厂家各3批尘螨变应原治疗性产品于常温及37℃下分别解吸附16和24 h,进行Western blot鉴别试验。结果两个厂家的制品经3种溶液解吸附后,SDS-PAGE结果均可见相对分子质量约28 000及15 000的组分Ⅰ丝氨酸蛋白酶和组分ⅡNPC家族蛋白目的条带,Western blot结果可见同样大小的与鼠抗螨变应原组分Ⅰ、Ⅱ单克隆抗体特异性结合的蛋白条带,而未解吸附的制品未检测到以上目的条带。3批制品的Western blot鉴定结果相近,表明两种解吸附温度及时间对鉴别试验的结果影响不大。结论尘螨变应原治疗性产品需经解吸附后才能进行鉴别试验,本研究建立的解吸附方法可有效稳定地对制品进行解吸附。

Objective To develop the identification test on dust mite therapeutic products after desorption. Methods Dust mite therapeutic products from two manufacturers were selected and desorbed with 1 ml 20% citrate solution,0. 4 mol / L phosphate solution and 0. 5 mol / L EDTA-Na solution separately, and identified by SDS-PAGE and Western blot. The dust mite therapeutic products from two manufacturers, three batches for each, were desorbed at room temperature and 37 ℃ for 16 and 24 h separately, and identified by Western blot. Results After desorption with three solutions, the products from two manufacturers showed the target bands of components Ⅰ（serine proteinase） and Ⅱ（NPC family protein）, with relative molecular masses of about 28 000 and 15 000 respectively, on SDS-PAGE profile, and the bands with the same molecular masses, which showed specific bindings to mouse monoclonal antibodies against componentsⅠandⅡ, on Western blot profile. However, the products without desorption showed no above-mentioned target bands. The identification results of three batches of products by Western blot were similar, indicating no significant effect of temperature and time for desorption on identification test. Conclusion Therapeutic products of dust mites needs to be desorbed before identification test, which may be desorbed effectively and stably by the developed method.