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摘要：本实验以人重组IL6R为免疫原，采用动物免疫、杂交瘤技术、BCA、ELISA、SDS-PAGE等方法，进行了抗IL6R抗体的制备、浓度、灵敏度、特异性及中和活性的检测，并通过双抗体夹心的ELISA检测方法制备了人IL6R检测试剂盒。通过筛选获得两株稳定分泌抗IL6R抗体的杂交瘤细胞株，其细胞上清分泌单抗均为IgG1亚类，其中一株单抗具有较好的中和活性，制备了高纯度抗IL6R兔多抗，通过双抗体夹心的ELISA检测方法制备了人IL6R检测试剂盒，检测灵敏度能够达到3.125pg/ml，利用单抗和多抗成功建立了灵敏度高、稳定性好的ELISA双抗体夹心检测方法。本研究制备的高亲和力抗IL6R抗体和人IL6R检测试剂盒为IL6R相关基础和临床研究奠定了良好基础。50335
毕业论文关键词：人白介素6受体；单克隆抗体；多克隆抗体
Preparation and Application of Antibodies Against IL6R
Abstract: The preparation, concentration, sensitivity, specificity and neutralizing activity of against IL6R antibody were detected by the methods of animal immunization, hybridization technique, BCA, ELISA, SDS-PAGE with Human recombinant IL6R as the primary immune.By double antibody sandwich ELISA method for detection of prepared people IL6R detection kit. Two cell strain secreting McAb against IL6R was screened. The McAbs secreted by both strains were IgG1 and the one was neutralizing antibody. Polyclonal antibody specifically binding to human IL6R was developed. With the method of sandwich ELISA, the minimum detectable dose of human IL6R was determined to be approximately 3.125 pg/mL. Successfully established using monoclonal and polyclonal antibody of high sensitivity, good stability of the detection method of double antibody sandwich ELISA. The high affinity against IL6R antibody and human IL6R ELISA kit prepared in this study laid a good foundation of construction of correlative basic and clinical researches on IL6R.
Key words: IL6R; Monoclonal antibody; Polyclonal antibody
目 录
摘要 1
引言 2
1材料与方法 2
1.1实验材料 3
1.1.1实验动物、细胞及重组蛋白 3
1.1.2主要试剂 3
1.2实验方法 3
1.2.1人IL6R蛋白小鼠单克隆抗体的制备 3
1.2.2人IL6R蛋白兔多克隆抗体的制备 3
1.2.3抗体基础鉴定 4
1.2.4 IL6中和抗体的筛选 4
1.2.5检测人IL6R的酶联免疫试剂盒的制备 4
2结果与分析 5
2.1抗体基础鉴定结果 5
2.1.1抗体浓度及纯度结果 5
2.1.2单抗灵敏度 6
2.1.3单抗的特异性 7
2.1.4抗体亲和力测定 7
2.1.5单抗亚型鉴定 7
2.2IL6R中和抗体的筛选 8
2.3IL6R试剂盒鉴定 8
3讨论 10
参考文献 11
致谢 14
抗人IL6R抗体的制备及应用引言
人白细胞介素-6（interleukin-6，IL-6）是一种细胞因子，它具有多重免疫调节功能及广泛的生物学功能。研究显示许多疾病例如炎症性疾病、自身免疫性疾病及肿瘤的发生、发展和预后都与IL6有关。某些急性炎症、自身免疫性疾病及淋巴细胞恶性肿瘤等疾病与体内IL6水平的异常增高有关系[1-4]。IL-6能够由T淋巴细胞、B淋巴细胞、成纤维细胞、内皮细胞、巨噬细胞、心肌细胞、成骨细胞、系膜细胞、肿瘤细胞等分泌[5-6]产生。IL-6传导其生物活性是通过细胞上的两种蛋白，分别是人白细胞介素-6受体（interleukin-6 receptor，IL6R）和分子为130kd的gp130蛋白。IL-6和IL6R结合能够形成二元复合物，此复合物能与细胞膜结合的gp130相互作用，然后诱导其进行信号传导[7-10]。