Hapten-Branched Polyethylenimine as a New Antigen Affinity Ligand to Purify Antibodies with High Efficiency and Specificity
2020
Han, Xiangning | Lin, Hong | Cao, Limin | Chen, Xiangfeng | Wang, Luefeng | Zheng, Hongwei | Zhang, Ziang | Pavase, Tushar Ramesh | Wang, Sai | Sun, Xun | Sui, Jianxin
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.
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