论文部分内容阅读
目的制备抗脱氧雪腐镰刀菌烯醇(DON)单克隆抗体(McAb)并对其进行初步鉴定。方法采用小剂量长周期的免疫方案,以DON-牛血清白蛋白(BSA)偶联物免疫雌性BALB/c小鼠,采用细胞融合的方法制备杂交瘤细胞株,酶联免疫吸附试验(ELISA)检测上清中McAb,对抗体分泌阳性的细胞株进行克隆化,直至抗体阳性率100%,用竞争抑制ELISA法进一步检测McAb的特异性,腹水诱生法制备大量McAb,并用饱和硫酸铵对其进行纯化,用抗体亚类鉴定试剂盒对该McAb亚类进行鉴定,BCA蛋白测定试剂盒测定蛋白含量,ELISA检测McAb的滴度、参考工作稀释度和亲和常数。结果 DON-BSA免疫的BALB/c小鼠血清效价为1∶256000,与BSA有强烈的交叉反应。细胞融合后,ELISA筛选抗体分泌阳性的杂交瘤细胞株,经3轮克隆化,建立了1株能稳定分泌抗DON-BSAMcAb的杂交瘤细胞株3G5,腹水诱生法制备了大量的McAb。该McAb属IgG1,IgG含量为6.06g/L,抗体滴度为1∶500000,参考工作稀释度为1∶64000,抗体亲和常数为1.62×109。用间接竞争抑制ELISA测得校正曲线线性范围9.8～10000ng/mL,线性方程Y=-0.2726X+0.2259(r=0.9309)。结论获得了分泌抗DON-McAb的杂交瘤细胞株,该单克隆抗体灵敏度高,特异性强,可用于制备高质量国产DON-ELISA检测试剂盒。
Objective To prepare monoclonal antibodies against deoxynivalenol (DON) and preliminary identification of McAbs. Methods Female BALB / c mice were immunized with DON-bovine serum albumin (BSA) conjugate and the hybridoma cell lines were prepared by the cell fusion method with a small dose and long-term immunization protocol. The enzyme-linked immunosorbent assay (ELISA) The McAb in the supernatant was detected to clone the cell line positive to the antibody secretion until the positive rate of the antibody was 100%. The specificity of the McAb was further detected by competitive inhibition ELISA. A large number of McAbs were prepared by ascites induction and treated with saturated ammonium sulfate The subclass of McAb was identified by the subclass of antibody subclass. The BCA protein assay kit was used to determine the protein content. The titer of McAb was measured by ELISA. The working dilution and affinity constants were also determined. Results The serum titer of DON-BSA-immunized BALB / c mice was 1:256000, which was strongly cross-reactive with BSA. After the cells were fused, the hybridoma cell lines secreting the antibody secreted by ELISA were screened by ELISA, and one hybridoma cell line 3G5 capable of stably secreting anti-DON-BSAMcAb was established after cloning in 3 cycles. A large number of McAbs were prepared by ascites induction. The McAb belongs to IgG1, the IgG content is 6.06g / L, the antibody titer is 1: 500000, the reference working dilution is 1:64000, and the antibody affinity constant is 1.62 × 109. The linearity of the calibration curve was 9.8 ~ 10000ng / mL and the linear equation Y = -0.2726X + 0.2259 (r = 0.9309). Conclusions The hybridoma cell line secreting anti-DON-McAb was obtained. The monoclonal antibody is highly sensitive and specific and can be used to prepare high quality domestic DON-ELISA kit.