论文部分内容阅读
目的制备促肾上腺皮质激素 ( ACTH)单克隆抗体 ,拟建立高灵敏度 ACTH- IRMA或 EL ISA。方法 ACTH1 - 1 3、ACTH1 - 2 8、ACTH 1 - 39和 ACTH1 8- 39分别与牛血清白蛋白偶联制备免疫原 ,在背部皮下和腹腔注射免疫 BAL B/ c鼠。按常规方法进行细胞融合 ,以 RIA法检测培养上清液 ,阳性孔经亚克隆 ,获得 2株分泌特异性抗体的杂交瘤细胞株 ;瘤细胞种于预致敏的BAL B/ c鼠腹腔 ,批量生产单抗性腹水 ;琼脂扩散法鉴定抗体类型 ;利用标准曲线的参数计算单抗的亲合常数 ;亲合纯化单抗并进行抗体固化试验。结果从 ACTH1 - 2 8免疫鼠获得单抗株为 5 G2 ,从 ACTH1 - 1 3免疫鼠获得单抗株为 D3 ;单抗腹水 5 0 %结合率时腹水最终稀释度分别为 5× 10 3和 2× 10 3;抗体类型均为 Ig G1/λ;5 G2和 D3亲合常数分别为 5 .1× 10 9L M- 1 和 4 .9× 10 7L M- 1 ;抗体包被实验显示 5 G2和 D3的固化抗体量分别为 1.4μg和 8.8μg时结合率达到饱和。结论 5 G2和 D3可以用于构建灵敏的 ACTH- IRMA或 EL ISA。
Objective To prepare monoclonal antibodies to adrenocorticotropic hormone (ACTH) and to establish a high sensitivity ACTH-IRMA or EL ISA. Methods BALB / c mice were immunized subcutaneously and intraperitoneally with ACTH1 - 13, ACTH1 - 2 8, ACTH 1 - 39 and ACTH1 8-39 conjugated with bovine serum albumin respectively. According to the conventional method, the cells were fused and the culture supernatant was detected by RIA method. The positive wells were subcloned to obtain 2 hybridoma cell lines secreting specific antibodies. The tumor cells were seeded in the pre-sensitized BALB / c mice abdominal cavity, Mass production of monoclonal antibody ascites; agar diffusion method to identify the type of antibody; the use of standard curve parameters to calculate the affinity of monoclonal antibodies; affinity purification of monoclonal antibodies and antibody curing test. Results The McAbs obtained from ACTH1 - 28 immunized mice were 5 G2, and the McAbs obtained from ACTH1 - 13 mice were D3. The final dilutions of ascites were 5 × 10 3 and 50%, respectively 2 × 10 3, respectively. The antibody types were both Ig G1 / λ. The affinity constants of 5 G2 and D3 were 5.1 × 10 9 L M -1 and 4.9 × 10 7 L M -1 respectively. The binding rate reached saturation when the amount of immobilized antibody of D3 and that of D3 were 1.4 μg and 8.8 μg, respectively. Conclusions 5 G2 and D3 can be used to construct sensitive ACTH-IRMA or EL ISA.