An on-line purification and entrapment system was developed that could extract a protein from a sample such as serum and entrap this protein within a small column for use in high-performance affinity chromatography. Human serum albumin (HSA) was employed as a model protein for this work. Immunoextraction columns containing polyclonal anti-HSA antibodies were developed to capture and isolate HSA from applied samples. This was followed by the use of a strong cation-exchange column to recapture and focus HSA as it eluted from the immunoextraction columns. The recaptured HSA was entrapped within 1.0 cm × 2.1 mm I.D. columns containing hydrazide-activated silica and in the presence of oxidized glycogen as a capping agent. The binding and elution properties of HSA on the various components of this system were examined and optimized. The entrapped columns produced by this system were then evaluated for their use in binding studies with several sulfonylurea drugs. The HSA columns created by this approach typically contained 0.3-0.6 nmol HSA and were stable over several weeks and more than 50-60 sample injections. Drug binding constants could be determined with these columns in 8 min or less by zonal elution and gave good agreement with literature values. The same system could be used for the capture and entrapment of other proteins by utilizing antibodies against the given target for immunoextraction.

中文翻译：

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开发用于蛋白质捕获的在线免疫提取/诱捕系统，并通过高效亲和色谱法用于药物结合研究。

开发了一种在线纯化和截留系统，该系统可以从样品（例如血清）中提取蛋白质，并将该蛋白质截留在小柱中，以用于高性能亲和色谱。人类血清白蛋白（HSA）被用作该工作的模型蛋白。开发了包含多克隆抗HSA抗体的免疫提取柱，以从应用样品中捕获和分离HSA。随后，使用强阳离子交换柱重新捕获和聚焦从免疫萃取柱洗脱的HSA。重新捕获的HSA被截留在1.0 cm×2.1 mm ID的色谱柱中，该色谱柱包含酰肼活化的二氧化硅，并且存在氧化的糖原作为封端剂。检查并优化了HSA在该系统各个组成部分上的结合和洗脱特性。然后评估该系统产生的截留柱在几种磺酰脲类药物结合研究中的用途。通过这种方法创建的HSA色谱柱通常包含0.3-0.6 nmol HSA，并且在数周内稳定，并且进样量超过50-60。可以通过区域洗脱在8分钟或更短时间内用这些色谱柱确定药物结合常数，并且与文献值具有很好的一致性。通过利用针对给定靶标的抗体进行免疫提取，可以将同一系统用于其他蛋白质的捕获和包埋。可以通过区域洗脱在8分钟或更短时间内用这些色谱柱确定药物结合常数，并且与文献值具有很好的一致性。通过利用针对给定靶标的抗体进行免疫提取，可以将同一系统用于其他蛋白质的捕获和包埋。可以通过区域洗脱在8分钟或更短时间内用这些色谱柱确定药物结合常数，并且与文献值具有很好的一致性。通过利用针对给定靶标的抗体进行免疫提取，可以将同一系统用于其他蛋白质的捕获和包埋。