鼠IgG定量ELISA实验参比品的制备及其应用

Development and application of experimental standards of quantitive ELISA for mice IgG.

目的 制备一组用于小鼠IgG定量ELISA检测的实验参比品。方法 将三株杂交瘤细胞制备的小鼠腹水等体积混合，以葡萄球菌A蛋白（SPA）亲和层析纯化IgG，凯氏定氮法测定IgG含量，SDS-聚丙烯酰胺凝胶（SDS-PAGE）电泳作纯度鉴定并据此校正其IgG浓度，以特定保存液将其稀释成既定浓度系列，根据其在ELISA中的反应特征，制备浓度（自然对数）-A450值标准曲线，并将其用于对一组杂交瘤细胞培养上清中鼠单克隆抗体的IgG定量测定。结果提纯的鼠IgG经鉴定纯度达97．1％，且反应特性良好，其IgG含量与ELISA检测信号间有高度的相关性（r=0．990）。不同浓度的稀释系列经8次反复冻融，检测信号无显著变化，变异系数（CV）为1．87％～6．47％。将标准曲线计算所得浓度对设定浓度做回收实验分析，回收率89．1％～108．3％。并用其测得一组杂交瘤细胞培养上清的鼠IgG含量为8．0～51．5mg／L。结论 鉴定结果显示制备物定量准确，浓度范围适中，稳定性好，对于早期杂交瘤细胞分泌能力以及抗体稳定性的评价、单抗制剂的鉴定具有一定的应用价值。

Objective To develop a series of experimental standards of quantitive ELISA for mice IgG. Methods Three strains of mice hybridoma cells-produced ascitic fluid was mixed in equal volume. Mice IgG was purified by SPA affinity chromatography. The content of mice IgG was detected by using Kjeldah methed, the purity identified by SDS-PAGE and the concentration of IgG adjusted. The mice IgG was diluted into the fixed series of concentrations by a special preservative fluid, and a standard curve for concentration （ln）-A450 was drawn according to the characters of reaction in ELISA. These standards were used for the quantitive detection of a series of hybridoma cell culture supernatant. Results The purity of mice IgG extracted was 97. 1% with a good reactive characters. The content of IgG had a high correlation to the signal in ELISA （r = 0. 990）. After freezing the standards of different concentrations repeatedly for 8 times, the results of detection for them had no significant change, and the coefficient of variation was between 1.87% - 6. 47%. The recovery rate of the experimental standards in ELISA was between 89. 1% - 108.3%, which were well controlled. These experimental standards were used in the quantitive detection for IgG of a series of hybridoma cell culture supernatant, and the mice IgG concentration was between 8. 0 - 51.5mg/L. Conclusion These experimental standards had an accurate quantitive detection, high stability and moderate concentration, and also had a good value in evaluation for the secretory ability of hybridoma cell, stability of monoclonal antibody and assessment of related praeparatum.