NOVEL-APPROACH TO AFFINITY-CHROMATOGRAPHY USING WEAK MONOCLONAL-ANTIBODIES
NOVEL-APPROACH TO AFFINITY-CHROMATOGRAPHY USING WEAK MONOCLONAL-ANTIBODIES
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DOI：
10.1016/0003-2697(88)90275-8
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发表时间：
1988-02-15
影响因子：
2.9
通讯作者：
ZOPF, D
中科院分区：
文献类型：
--
作者：
OHLSON, S;LUNDBLAD, A;ZOPF, D
亲和纯化通常依赖于两种生物分子之间的特异性高亲和性识别：溶解在移动的相中的一种分子种类（连接物）被选择性地吸附到与固体支持物偶联的另一种种类（配体）。连接物的解吸通常需要降低纯化产物的生物活性的苛刻条件。作为一种替代方案，这个一般的程序，我们研究了亲和色谱在弱亲和模式，其中配体-配体相互作用是在动态平衡。以低亲和力（解离常数> 10-4 M）从亲和柱在温和的等度条件下重新识别的连接体作为延迟峰，与在空隙体积中分离的非相互作用溶质分离。为了说明该方法，我们报道了寡糖在含有86 mg与10 μ m微粒二氧化硅颗粒偶联的单克隆抗体的2 ml柱上的层析。使用温度敏感性抗体，我们观察到当配体-连接物解离常数> 10-3 M时，系统的性能超过300个理论塔板/10 cm柱长，接近通常与高效液相色谱相关的效率。
Affinity purification generally relies on specific high-affinity recognition between two species of biological molecules: one molecular species (the ligate) dissolved in a mobile phase is selectively adsorbed to the other species (the ligand) coupled to a solid support. Desorption of the ligate often requires harsh conditions that degrade biological activity of the purified product. As an alternative to this general procedure, we have studied affinity chromatography in a weak affinity mode, where ligand-ligate interactions are in dynamic equilibrium. Ligates reocognized with low affinities (dissociation constant > 10-4 M) elute from affinity columns under mild, isocratic conditions as retarded peaks, separated from noninteracting solutes that elute in the void volume. To illustrate the procedure we report chromatography of an oligosaccharide on a 2-ml column containing 86 mg of a monoclonal antibody coupled to a 10-.mu.m microparticulate silica particles. Using a temperature-sensitive antibody, we observed that when the ligand-ligate dissociation constant is > 10-3 M, performance of the system exceeds 300 theoretical plates/10 cm column length and approaches the efficiencies generally associated with high-performance liquid chromatography.