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Hapten-Branched Polyethylenimine as a New Antigen Affinity Ligand to Purify Antibodies with High Efficiency and Specificity
ACS Applied Materials & Interfaces ( IF 8.3 ) Pub Date : 2020-12-15 , DOI: 10.1021/acsami.0c15586 Xiangning Han 1 , Hong Lin 1 , Limin Cao 1 , Xiangfeng Chen 2 , Luefeng Wang 1 , Hongwei Zheng 1 , Ziang Zhang 1 , Tushar Ramesh Pavase 1 , Sai Wang 1 , Xun Sun 1 , Jianxin Sui 1
ACS Applied Materials & Interfaces ( IF 8.3 ) Pub Date : 2020-12-15 , DOI: 10.1021/acsami.0c15586 Xiangning Han 1 , Hong Lin 1 , Limin Cao 1 , Xiangfeng Chen 2 , Luefeng Wang 1 , Hongwei Zheng 1 , Ziang Zhang 1 , Tushar Ramesh Pavase 1 , Sai Wang 1 , Xun Sun 1 , Jianxin Sui 1
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Purification of antibodies has become a critical factor in antibody production. A high-purity specific antibody against antigens, especially small molecules, seems to be difficult to obtain, even with the help of a protein A affinity column, which is a conventional and broadly used ligand for the separation of antibody and non-antibody proteins. Therefore, it is urgent to develop a cheap, simple, efficient, and stable method to separate the specific antibody from other antibodies. In this study, to improve the sensitivity and accuracy of immunoassay results, enrofloxacin (ENR) was grafted onto polyethylenimine (PEI) by the abundant amino groups and then the whole ligand (ENR–PEI) was conjugated to CNBr–Sepharose 4B to prepare the affinity column for the purification of the specific antibody against ENR from polyclonal antibodies. Scanning electron microscopy and Fourier transform infrared spectroscopy verification showed that Sepharose 4B was successfully modified by ENR–PEI with excellent uniformity. The capacity of the prepared column could reach to 6.15 mg of specific antibody with high purity per milliliter resin due to the high coupling ratio (49.3:1) of ENR on PEI, and the IC50 value of the antibody after purification was 47.58 ng/mL with a lowest limit of detection (IC10) of 1.099 ng/mL—18 times lower than those of the antibody purified through the protein A column. All the results showed that this new kind of resin could be used as the potential ligand in the purification of the trace-specific antibody against antigens in complex mixtures with high efficiency and specificity.
中文翻译：
半抗原分支的聚乙烯亚胺作为一种新型抗原亲和配体，可高效高效地纯化抗体。
抗体的纯化已成为抗体生产的关键因素。即使借助蛋白质A亲和柱，也很难获得针对抗原（尤其是小分子）的高纯度特异性抗体，而A亲和柱是分离抗体和非抗体蛋白质的常规且广泛使用的配体。因此，迫切需要开发一种廉价，简单，有效和稳定的方法以将特异性抗体与其他抗体分离。在这项研究中，为了提高免疫测定结果的灵敏度和准确性，将恩诺沙星（ENR）通过丰富的氨基接枝到聚乙烯亚胺（PEI）上，然后将整个配体（ENR–PEI）与CNBr–Sepharose 4B偶联以制备亲和柱，用于从多克隆抗体中纯化抗ENR的特异性抗体。扫描电子显微镜和傅立叶变换红外光谱验证表明，ENR-PEI成功地改性了Sepharose 4B，具有出色的均匀性。由于ENR在PEI和IC上的高偶联比（49.3：1），制备的色谱柱的容量可以达到6.15 mg每毫升树脂高纯度的特异性抗体纯化后的抗体50值为47.58 ng / mL，最低检测限（IC 10）为1.099 ng / mL，比通过蛋白A柱纯化的抗体低18倍。所有结果表明，这种新型树脂可以作为潜在的配体，高效，高特异性地纯化复杂混合物中的痕量抗抗原抗体。
更新日期：2020-12-30
中文翻译：
半抗原分支的聚乙烯亚胺作为一种新型抗原亲和配体，可高效高效地纯化抗体。
抗体的纯化已成为抗体生产的关键因素。即使借助蛋白质A亲和柱，也很难获得针对抗原（尤其是小分子）的高纯度特异性抗体，而A亲和柱是分离抗体和非抗体蛋白质的常规且广泛使用的配体。因此，迫切需要开发一种廉价，简单，有效和稳定的方法以将特异性抗体与其他抗体分离。在这项研究中，为了提高免疫测定结果的灵敏度和准确性，将恩诺沙星（ENR）通过丰富的氨基接枝到聚乙烯亚胺（PEI）上，然后将整个配体（ENR–PEI）与CNBr–Sepharose 4B偶联以制备亲和柱，用于从多克隆抗体中纯化抗ENR的特异性抗体。扫描电子显微镜和傅立叶变换红外光谱验证表明，ENR-PEI成功地改性了Sepharose 4B，具有出色的均匀性。由于ENR在PEI和IC上的高偶联比（49.3：1），制备的色谱柱的容量可以达到6.15 mg每毫升树脂高纯度的特异性抗体纯化后的抗体50值为47.58 ng / mL，最低检测限（IC 10）为1.099 ng / mL，比通过蛋白A柱纯化的抗体低18倍。所有结果表明，这种新型树脂可以作为潜在的配体，高效，高特异性地纯化复杂混合物中的痕量抗抗原抗体。
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