Manil L, Motté P, Pernas P, Troalen F, Bohuon C, Bellet D
J Immunol Methods. 1986 Jun 10;90(1):25-37. doi: 10.1016/0022-1759(86)90379-0.
Protocols for purification of mouse monoclonal antibodies (MAbs) from nude mice ascites were investigated in order to assess the yield and to compare the purified products in two-dimensional gel electrophoresis (2DGE). Three MAbs (one IgG2 and two IgG1), selected for their differing behaviours towards protein A, were purified by ammonium sulphate precipitation and/or gel filtration, anion exchange (DEAE), hydroxylapatite and affinity (protein A) chromatography, or by a combination of these methods. Protein A constantly provided the highest purity whatever the IgG subclass. The best results in terms of yields and purity were a function of the optimization of the protein A protocol. In our study, they were obtained in a 3 h protocol (IgG2), a 16 h protocol with discontinuous pH gradient method (IgG1 with sufficiently high affinity for protein A) or a multi-step protocol involving DEAE and protein A (IgG1 with low affinity for protein A). DEAE chromatography alone provided a slightly better yield, but only moderate purity. Hydroxylapatite chromatography appeared to be less potent in terms of yield, purity and day-to-day reproducibility. Salt precipitation and gel filtration enabled only relative enrichment of the MAb solution. Some degradation products of both heavy and light chains clearly appeared in the 2DGE patterns of antibodies purified by different protocols, and seem to be partly related to the elution pH and to the duration of the purification procedure. Finally, this work highlights considerable heterogeneity not only between two different MAbs of the IgG1 subclass but also within a monoclonal population of immunoglobulins.
为了评估产量并在二维凝胶电泳（2DGE）中比较纯化产物，对从小鼠腹水纯化小鼠单克隆抗体（MAb）的方案进行了研究。选择了三种对蛋白A表现出不同行为的单克隆抗体（一种IgG2和两种IgG1），通过硫酸铵沉淀和/或凝胶过滤、阴离子交换（DEAE）、羟基磷灰石和亲和（蛋白A）色谱法，或这些方法的组合进行纯化。无论IgG亚类如何，蛋白A始终能提供最高的纯度。就产量和纯度而言，最佳结果取决于蛋白A方案的优化。在我们的研究中，在3小时的方案（IgG2）、采用不连续pH梯度法的16小时方案（对蛋白A具有足够高亲和力的IgG1）或涉及DEAE和蛋白A的多步方案（对蛋白A具有低亲和力的IgG1）中获得了最佳结果。单独的DEAE色谱法产量略高，但纯度一般。羟基磷灰石色谱法在产量、纯度和日常重现性方面似乎效果较差。盐沉淀和凝胶过滤仅能使单克隆抗体溶液相对富集。在通过不同方案纯化的抗体的二维凝胶电泳图谱中，重链和轻链的一些降解产物清晰可见，并且似乎部分与洗脱pH值和纯化过程的持续时间有关。最后，这项工作不仅突出了IgG1亚类的两种不同单克隆抗体之间存在相当大的异质性，而且在单克隆免疫球蛋白群体中也存在这种异质性。
