Ohlson S, Lundblad A, Zopf D
Perstorp Biolytica AB, Lund, Sweden.
Anal Biochem. 1988 Feb 15;169(1):204-8. doi: 10.1016/0003-2697(88)90275-8.
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 recognized with low affinities (dissociation constant greater than 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 10-micron microparticulate silica particles. Using a temperature-sensitive antibody, we observed that when the ligand-ligate dissociation constant is greater than 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.
溶解在流动相中的一种分子物种（被连接物）被选择性地吸附到与固体支持物偶联的另一种物种（配体）上。被连接物的解吸通常需要苛刻的条件，这会降低纯化产物的生物活性。作为这种常规方法的替代方法，我们研究了弱亲和力模式下的亲和色谱，其中配体 - 被连接物相互作用处于动态平衡。以低亲和力识别的被连接物（解离常数大于10^(-4) M）在温和的等度条件下作为延迟峰从亲和柱上洗脱，与在空体积中洗脱的非相互作用溶质分离。为了说明该方法，我们报道了在一个2毫升柱上对寡糖进行色谱分析，该柱含有86毫克与10微米微粒硅胶颗粒偶联的单克隆抗体。使用一种对温度敏感的抗体，我们观察到当配体 - 被连接物解离常数大于10^(-3) M时，该系统的性能超过300理论塔板数/10厘米柱长，并接近通常与高效液相色谱相关的效率。
