嗜酸性粒细胞阳离子蛋白检测试剂盒的制备及其临床应用

发布时间：2018-06-01 01:01

  本文选题：嗜酸性粒细胞阳离子蛋白 + 酶联免疫吸附试验　； 参考：《天津医科大学》2008年硕士论文

【摘要】： 目的1.制备双抗夹心ELISA(enzyme linked immunosorbent assay,酶联免疫吸附试验)嗜酸性粒细胞阳离子蛋白(eosinophilia cationic protein,ECP)检测试剂盒。2.对不同的包被方法进行比较。3.评估ECP水平检测在过敏性疾病诊疗中的意义。 方法1.双抗夹心ELISA ECP检测试剂盒的制备:(1)葡萄球菌A蛋白亲和层析法纯化抗-ECP单克隆抗体(monoclonal antibody,mAb),(2)过碘酸钠法HRP标记抗-ECP多克隆抗体(polyclonal antibody,pAb),(3)ELISA试剂盒的制备与条件优化,(4)实验效能评价。2.ECP检测试剂盒的方法学比较:(1)抗-ECPmAb的生物素化,(2)BSA的生物素化,(3)先包被生物素的ELISA试剂盒的制备与条件优化,(4)先包被亲和素的ELISA试剂盒的制备与条件优化,(5)比较三种检测方法。3.评估ECP水平检测对过敏性疾病诊疗的意义:(1)评估采血方式对ECP结果的影响,(2)评估ECP检测对过敏性皮肤病诊疗的意义:检测过敏性皮肤病患者血清ECP水平:检测患者总IgE水平,并评估其与ECP的相关性;评估荨麻疹患者治疗前后ECP水平的变化,以及其ECP水平与症状评分、总IgE、特异性IgE相关性;评估异位性皮炎患者疾病症状评分与ECP水平、总IgE、嗜酸性粒细胞(EOS)计数的相关性。4.评估ECP水平检测在过敏性鼻炎诊疗中的意义。 结果1.双抗夹心ELISA ECP检测试剂盒的制备:(1)通过对该方法中各种反应条件的摸索和优化,确定了最适工作条件:单抗最适包被浓度1:500稀释,酶标多抗最适浓度为1:8000稀释,最佳封闭液为1%的BSA,最适血清稀释液为稀释液Ⅱ,最佳反应时间为60min,反应温度为37℃。(2)本研究制备的试剂盒线性范围为2.5～200ng/ml,灵敏度为2.75ng/ml,相对偏差为11.5%,批内变异系数＜10%,批间变异系数＜15%,稳定性较好,与ADL试剂盒相比较相关系数为0.97。2.三种方法的比较:(1)生物素亲和素包被法线性范围为2.5～500ng/ml,灵敏度为2.61ng/ml,相对偏差为10.9%;(2)亲和素包被法线性范围为2.5～500ng/ml,灵敏度为2.68ng/ml,相对偏差为11.9%。(3)三种方法的重复性、精确性没有显著性差异,稳定性亲和素包被法最为稳定,抗体包被法次之,生物素包被法最不稳定,抗体直接包被法操作最为简易,但抗体用量最多。3.ECP水平检测对过敏性疾病诊疗的意义:(1)标本采集后37℃放置1h的结果显著高于室温与0℃组,抗凝剂对ECP水平无影响,而标本溶血使ECP水平显著升高。(2)过敏性皮肤
[Abstract]:Objective 1. Preparation of double antibody sandwich ELISA(enzyme linked immunosorbent assay, enzyme linked immunosorbent assay) eosinophilia cationic protein detection kit. 2. Compare different envelope methods. 3. 3. To evaluate the significance of ECP level in the diagnosis and treatment of allergic diseases. Method 1. Preparation of double Antibody Sandwich ELISA ECP Assay Kit: 1) purification of Monoclonal body Monoclonal Antibody of Staphylococcus Staphylococcus by Affinity Chromatography Comparison of methods for evaluating .2. ECP Detection Kit; (1) Biotinylation of anti-ECPmAb and biotinylation of BSA; preparation and condition optimization of ELISA kit coated with biotin; preparation and condition optimization of ELISA kit coated with avidin; preparation and condition optimization of ELISA kit precoated with avidin (5) Compare three detection methods. To evaluate the significance of ECP level in the diagnosis and treatment of allergic dermatosis; to evaluate the effect of blood collection on the results of ECP; to evaluate the significance of ECP detection in the diagnosis and treatment of allergic dermatosis; to detect the serum ECP level of allergic dermatosis patients; and to detect the total IgE level in patients with allergic dermatosis. To evaluate the correlation between ECP and ECP, to evaluate the changes of ECP level in patients with urticaria before and after treatment, and to evaluate the correlation between ECP level and symptom score, total IgE and specific IgE, and to evaluate the relationship between ECP and ECP level in patients with ectopic dermatitis. Correlation of total IgE, eosinophil EOS count. 4. To evaluate the significance of ECP level in the diagnosis and treatment of allergic rhinitis. Result 1. Preparation of double Antibody Sandwich ELISA ECP Detection Kit: by exploring and optimizing various reaction conditions in this method, the optimum working conditions were determined: the optimal concentration of McAb was diluted by 1: 500, the optimal concentration of enzyme labeled polyantibody was diluted by 1: 8000. The best blocking solution is 1% BSA, and the optimal serum dilution solution is diluent 鈪，

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