Development of immunoaffinity restricted access media for rapid extractions of low-mass analytes.
Development of immunoaffinity restricted access media for rapid extractions of low-mass analytes.
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DOI：
10.1021/ac801233t
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发表时间：
2008-11-15
影响因子：
7.4
通讯作者：
Hage, David S.
中科院分区：
文献类型：
--
作者：
Wa, Chunling;Mallik, Rangan;Hage, David S.
使用抗体作为固定化配体的受限访问介质被开发用于通过免疫提取快速和选择性地捕获小分析物，从而产生了被称为免疫亲和力受限访问介质(IA-RAM)的材料。为了制造这种材料，首先将所需目标的完整抗体固定在多孔硅胶上，然后用木瓜酶(或相关试剂)处理载体外表面或附近的抗体，以释放和去除它们的结合域。其结果是，只有毛孔深处的抗体保持完好，并能够与靶标结合。在这种介质的开发中评估的项目包括用于抗体的固定方法、载体的孔大小、用于支持治疗的木瓜蛋白酶的量和时间。还建立了一个理论模型来描述结合结构域的去除程度，该模型基于木瓜酶处理前后IA-RAM载体的多肽含量。最后优化了制备IA-RAM载体的条件，并用于制备含有抗荧光素抗体的柱。将荧光素和荧光素标记的牛血清白蛋白注射到这些IA-RAM柱上，在1-2个S中实现了荧光素的选择性和定量提取。该方法可用于其他抗体和低质量靶标，对于从药物-蛋白质复合体中快速分离药物或从含有相同修饰或标记的完整蛋白中分离标记/修饰的多肽具有应用价值。
Restricted access media using antibodies as immobilized ligands were developed for the rapid and selective capture of small analytes by immunoextraction, giving rise to materials referred to as immunoaffinity restricted access media (IA-RAM). To make such a material, intact antibodies for the desired target were first immobilized onto porous silica, with antibodies at or near the outer surface of the support then being treated with papain (or a related agent) to release and remove their binding domains. The result was a support in which only antibodies deep within the pores remained intact and able to bind to the target. Items evaluated in the development of such media included the immobilization method used for the antibodies, the pore size of the support, and the amount of papain and time that were used for support treatment. A theoretical model was also developed to describe the extent of binding domain removal based on the measured polypeptide content of the IA-RAM support before and after treatment with papain. The final optimized conditions for making the IA-RAM supports were used to prepare columns that contained anti-fluorescein antibodies. Injections of fluorescein and fluorescein-labeled bovine serum albumin onto these IA-RAM columns gave selective and quantitative extraction of fluorescein in 1-2 s. This approach can be used with other antibodies and low mass targets and should be valuable for such applications as the rapid separation of drugs from drug-protein complexes or the isolation of labeled/modified peptides from intact proteins that contain the same modification or label.
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DOI：
10.1016/j.jchromb.2004.06.006
发表时间：
2004-09-25
影响因子：
3
作者：
Cheng, Y;Ho, E;Tseng, HL
通讯作者：
Tseng, HL
影响因子：
4.1
作者：
Clarke, W;Beckwith, JD;Hage, DS
通讯作者：
Hage, DS
影响因子：
2.2
作者：
HOFFMAN, WL;OSHANNESSY, DJ
通讯作者：
OSHANNESSY, DJ
影响因子：
2.9
作者：
Ikegawa, S;Isriyanthi, NMR;Goto, J
通讯作者：
Goto, J
DOI：
10.1016/j.jchromb.2004.06.012
发表时间：
2004-09-25
影响因子：
3
作者：
Chen, JZ;Ohnmacht, C;Hage, DS
通讯作者：
Hage, DS