作　　者： (张远利）; (王红勋）; (李春梅）; (张玥）; (兰燕宇）; (孙祥明）;

机构地区： 贵州医科大学药学院,贵州贵阳550004

出　　处： 《生物技术》 2017年第4期378-382,共5页

摘　　要： [目的]制备鼠抗人PCNA Mab与人IgG的交联物作为临床分析阳性参考品。[方法]利用杂交瘤技术制备鼠抗人PCNA Mab,在SPDP的作用下与人IgG交联,建立ELISA方法评估其作为参考品的可行性。[结果]获得2株鼠抗人PCNA杂交瘤细胞;免疫印迹表明鼠抗人PCNA Mab与人IgG交联后对人PCNA和抗人IgG抗体均具有反应性;以该交联物作为参考品建立的ELISA中,300μg/L^4.69μg/L范围内,R2达0.994、准确度为88.26%~99.66%、相对标准偏差4.66%~17.95%;稀释400倍后,血清基质对检测结果没有明显干扰;SLE病人样本(n=41)的ELISA均值和高值均大于健康人(n=22);临床免疫膜条确认的阴阳性样本的ELISA结果分别16.44±1.30μg/m L和6.05±0.50μg/m L,与判别结果相关。[结论]鼠抗人PCNA Mab-人IgG交联物可作为阳性参考品应用于PCNA自抗体的免疫分析。 [Objective]To develop the coupled complex of mouse anti-human PCNA Mab and human Ig G as a reference substance in immunoassay of human serum PCNA autoantibody. [Methods]Hybridoma technique was used to prepare the mouse anti-human PCNA Mab,which was then coupled with human Ig G by SPDP. The coupled complex was evaluated as a reference substance in ELISA which was established to detect the PCNA autoantibody. [Results]Two hybridoma cell lines secreting mouse anti-human PCNA Mab were obtained. The coupled complex of mouse PCNA Mab and human Ig G,well responsive to human PCNA and anti-human Ig G antibody in Western Blot assay,was used as the calibrator at 300 μg/L-4. 69 μg/L in the established ELISA. The ELISA assay had a good linear coefficient with R2 reaching 0. 994,and good accuracy of 88. 26%-99. 66% with the relative standard error of 4. 66%-17. 95%. The serum substrate had no obvious effect on the assay after the 400-fold dilution. The PCNA autoantibody titers detected by ELISA in SLE patient serum is higher than that in health people with respect to both the average and the high values. The positive and negative specimen by the clinical licensed immunological membrane strip kit were re-determined by ELISA with a comparable result at 16. 44 ± 1. 30 μg/m L and 6. 05 ± 0. 50μg/m L,respectively. [Conclusion]The coupled complex of mouse anti-human PCNA Mab and human Ig G can be used as the positive reference substance in the PCNA autoantibody assay of human serum.

关 键 词： 单克隆抗体 免疫分析 杂交瘤 自身免疫 增殖细胞核抗原