Centro de Investigação de Engenharia Quimica e Biotecnologia, Instituto Superior de Engenharia de Lisboa Rua Conselheiro Emidio Navarro, 1,1959-007 Lisboa, Portugal.
Mol Biotechnol. 2006 Jun;33(2):103-14. doi: 10.1385/MB:33:2:103.
The chromatographic behavior of monoclonal antibodies (MAbs) of immunoglobulin (Ig) M class against mutant (T103I) amidase from Pseudomonas aeruginosa was investigated on immobilized metal chelates. The effect of ligand concentration, the length of spacer arm, and the nature of metal ion were investigated in immobilized metal affinity chromatography (IMAC). The MAbs against mutant amidase adsorbed to Cu(II), Ni(II), Zn(II), Co(II), and Ca(II)-iminodiacetic acid (IDA) agarose columns. The increase in ligand concentration (epichlorohydrin: 30-60 and 1,4-butanediol-diglycidyl ether: 16-36) resulted in higher adsorption to IgM into immobilized metal chelates. The length of spacer arm was found to affect protein adsorption, as longer spacer arm (i.e., 1,4-butanediol-diglycidyl ether) increased protein adsorption of immobilized metal chelates. The adsorption of IgM onto immobilized metal chelates was pH dependent because an increase in the binding of IgM was observed as the pH varied from 6.0 to 8.0. The adsorption of IgM to immobilized metal chelates was the result of coordination of histidine residues to metal chelates that are available in the third constant domain of heavy chain (CH3) of immunoglobulins, as the presence of imidazole (5 mM) in the equilibration buffer abolished the adsorption of IgM to the column. The combination of tailor-made stationary phases for IMAC and a correct design of the adsorption parameters permitted to devise a one-step purification procedure for IgM. Culture supernatants containing IgM against mutant amidase (T103I) were purified either by IMAC on EPI-60-IDA-Co (II) column or by gel filtration chromatography on Sephacryl S-300HR. The specific content of IgM and final recovery of antibody activity exhibited similar values for both purification schemes. The purified preparations of IgM obtained by both schemes were apparently homogeneous on native polyacrylamide gel electrophoresis with a M(r) of 851,000 Da. The results presented in this work strongly suggest that one-step purification of IgM by IMAC is a cost-effective and processcompatible alternative to other types of chromatography.
研究了针对铜绿假单胞菌突变（T103I）酰胺酶的 IgM 类单克隆抗体（MAb）在固定化金属螯合物上的色谱行为。考察了配体浓度、间隔臂长度和金属离子性质对固定化金属亲和色谱（IMAC）的影响。抗突变酰胺酶的 MAb 吸附到 Cu(II)、Ni(II)、Zn(II)、Co(II)和 Ca(II)-亚氨基二乙酸（IDA）琼脂糖柱上。增加配体浓度（表氯醇：30-60 和 1,4-丁二醇二缩水甘油醚：16-36）导致对固定化金属螯合物中 IgM 的吸附增加。间隔臂的长度被发现影响蛋白质的吸附，因为较长的间隔臂（即 1,4-丁二醇二缩水甘油醚）增加了固定化金属螯合物对蛋白质的吸附。IgM 对固定化金属螯合物的吸附取决于 pH，因为当 pH 值从 6.0 到 8.0 变化时，观察到 IgM 的结合增加。IgM 对固定化金属螯合物的吸附是由于组氨酸残基与金属螯合物的配位，而金属螯合物存在于免疫球蛋白重链（CH3）的第三个恒定域中，因为在平衡缓冲液中存在咪唑（5 mM）会消除 IgM 对柱子的吸附。定制的 IMAC 固定相和吸附参数的正确设计相结合，允许设计一种一步法纯化 IgM 的方法。含有针对突变型酰胺酶（T103I）的 IgM 的培养上清液通过 EPI-60-IDA-Co(II)柱上的 IMAC 或 Sephacryl S-300HR 上的凝胶过滤色谱法进行纯化。两种纯化方案的 IgM 的特异性含量和抗体活性的最终回收率具有相似的值。两种方案获得的 IgM 纯化制剂在天然聚丙烯酰胺凝胶电泳中均表现出明显的均一性，分子量（Mr）为 851,000 Da。本工作的结果强烈表明，通过 IMAC 一步法纯化 IgM 是一种具有成本效益且与其他类型色谱法兼容的替代方法。
