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目的探讨卵巢上皮性癌6B11抗独特型微抗体(6B11微抗体)的最佳免疫剂量和免疫间隔时间,为用于临床治疗卵巢上皮性癌打下基础。方法(1)采用基因工程方法构建6B11微抗体的重组表达载体,并将其转化到大肠杆菌BL21中进行表达。直接酶联免疫吸附试验(ELISA)法测定其免疫学活性。(2)将BALB/c小鼠随机分为A、B、C、D、E、F组,于第1、14、28天时均分别给予6B11微抗体150、100、50μg及小鼠IgG100μg、人IgG100μg、磷酸盐缓冲液100μl,其中A、B、C组为实验组,D、E、F组为对照组。间接ELISA法测定各组小鼠血清中抗卵巢上皮性癌6B11抗独特型抗体(6B11抗体)的抗体(即Ab3)的水平,并观察其变化规律。(3)将小鼠脾淋巴细胞作为效应细胞,人卵巢上皮性癌细胞株SKOV3细胞作为靶细胞,设定效靶比为250∶1、125∶1、60∶1、30∶1,51Cr释放实验检测各组小鼠脾淋巴细胞产生的抗体依赖的细胞毒效应(ADCC);设定血清稀释度为1∶2、1∶25、1∶50,51Cr释放实验检测各组小鼠脾淋巴细胞产生的补体依赖的细胞毒效应(CDC)。分别于紫外光波长490nm时测定其吸光度(A)值。结果(1)大肠杆菌BL21中成功表达6B11微抗体,并证实其保留了6B11抗体的免疫学活性。(2)用上述不同剂量6B11微抗体免疫BALB/c小鼠,在第14天注射后1周,血清中可检测到较低水平Ab3;第28天注射后1周,Ab3水平达到最高值,A、B、C组平均A值分别为1·16、1·11、1·06;此Ab3水平持续6周,6周内A、B、C组平均A值分别为1·05、1·06、0·94,B组略高于A、C两组,但3组间比较,差异无统计学意义(P>0·05);第7周Ab3水平明显下降。此时,给予加强免疫,1周后Ab3水平很快升高,并持续至少2周。(3)在4个不同的效靶比条件下,实验组小鼠脾淋巴细胞诱导产生的ADCC明显高于对照组(P<0·05);效靶比为1∶125时,A、B、C组51Cr自然释放率分别为23%、17%、12%,3组间比较,差异有统计学意义(P<0·05)。血清稀释度1∶50时,A、B、C组51Cr自然释放率分别为47%、39%、26%,A、B组BALB/c小鼠血清诱导产生的CDC明显高于C组(P<0·05),而A和B组相比,差异则无统计学意义(P>0·05)。结论6B11微抗体可作为卵巢上皮性癌疫苗,其较佳的动物免疫方案为:免疫剂量为100μg;基础免疫分别于第1、14、28天进行,共3次;间隔6周给予加强免疫。
Objective To investigate the optimal immunization dose and immunization interval of 6B11 anti-idiotypic antibody (6B11) in epithelial ovarian cancer and lay the foundation for the clinical treatment of epithelial ovarian cancer. Methods (1) The recombinant expression vector of 6B11 was constructed by genetic engineering and transformed into E. coli BL21 for expression. Direct enzyme-linked immunosorbent assay (ELISA) method to determine the immunological activity. (2) BALB / c mice were randomly divided into groups A, B, C, D, E and F, and 150,100 and 50μg of 6B11 and 100μg of mouse IgG were given on the 1st, 14th and 28th day, respectively IgG100μg, phosphate buffer 100μl, of which A, B, C group for the experimental group, D, E, F group as the control group. The indirect ELISA was used to detect the level of Ab3 in anti-idiotypic antibody 6B11 (anti-idiotypic antibody 6B11) in ovarian cancer of each group. (3) Using mouse spleen lymphocytes as effector cells and human ovarian epithelial cancer cell line SKOV3 as target cells, the target-to-target ratios of 250: 1, 125: 1, 60: 1, The antibody-dependent cytotoxicity (ADCC) of splenic lymphocytes was detected in each group. Serum dilutions were set at 1: 2, 1: 25, 1: 50, 51Cr release test to detect the splenic lymphocytes The resulting complement-dependent cytotoxicity (CDC). Absorbance (A) values were measured at UV light wavelength 490 nm. Results (1) The 6B11 mAb was successfully expressed in E. coli BL21 and confirmed that it retained the immunological activity of the 6B11 antibody. (2) BALB / c mice were immunized with the above-mentioned 6B11 minibody at different levels for 1 week after the 14th day, and Ab3 was detected in the serum at the 14th day. The Ab3 level reached the highest one week after the 28th day, The average A values of A, B and C groups were respectively 1.16, 1.11 and 1. 06. The Ab3 level lasted 6 weeks. The average A values of A, B and C groups within 6 weeks were 1.05 and 1.00 There was no significant difference between the three groups (P> 0.05), while the level of Ab3 in the seventh week decreased significantly. At this point, given booster immunization, Ab3 levels rose rapidly after 1 week and lasted at least 2 weeks. (3) ADCC induced by splenic lymphocytes in experimental group was significantly higher than that in control group (P <0.05) under the condition of 4 different effective target ratios. When the effective target ratio was 1:125, A, B The natural release rates of 51Cr in group C were 23%, 17% and 12%, respectively. The differences among the three groups were statistically significant (P <0.05). The serum 51Cr release rates of group A, B and C were 47%, 39% and 26% respectively when the serum dilution was 1:50. The CDC induced by serum of BALB / c mice in groups A and B was significantly higher than that of group C <0.05), while there was no significant difference between A and B groups (P> 0.05). Conclusions 6B11 micro-antibody can be used as ovarian epithelial cancer vaccine. The optimal animal immunization protocol is as follows: the immunization dose is 100μg; the basic immunization is carried out on the 1st, 14th and 28th days respectively for 3 times; and the boosting is given every 6 weeks.