Anastase-Ravion S, Ding Z, Pellé A, Hoffman A S, Letourneur D
INVIMAT, Université Paris 13, Villetaneuse, France.
J Chromatogr B Biomed Sci Appl. 2001 Sep 25;761(2):247-54. doi: 10.1016/s0378-4347(01)00336-x.
Through their specificity and affinity, antibodies are useful tools in research and medicine. In this study, we investigated a new type of chromatographic method using a thermosensitive polymer for the purification of antibodies against a dextran derivative (DD), as a model. The thermally reversible soluble-insoluble poly(N-isopropylacrylamide)-dextran derivative conjugate, named poly(NIPAAm)-DD, has been synthesized by conjugating amino-terminated poly(N-isopropylacrylamide) to a DD via ethyl-3-(3-dimethylaminopropyl)-carbodiimide. On one hand, this report describes the two steps of poly(NIPAAm)-DD conjugation and characterization. On the other hand, the poly(NIPAAm)-DD conjugate was used as a tool to purify polyclonal antibodies in serum samples from rabbits subcutaneously immunized with the derivatized dextran. Antibodies were purified and quantified by immunoenzymatic assays. Our results indicate that antibodies recognized both DD and poly(NIPAAm)-DD. In contrast, they did not bind to native poly(NIPAAm) or poly(NIPAAm) conjugated with another anionic dextran. We conclude that the conjugation of a polysaccharide to poly(NIPAAm) leads to an original and efficient chromatographic method to purify antibodies. Moreover, this novel method of purification is rapid, sensitive, inexpensive and could be used to purify various types of antibodies.
凭借其特异性和亲和力，抗体是研究和医学领域的有用工具。在本研究中，我们以一种新型的基于热敏聚合物的色谱方法作为模型，用于纯化抗葡聚糖衍生物（DD）的抗体。通过使用N-乙基-3-(3-二甲基氨基丙基)碳二亚胺将氨基端基化的聚N-异丙基丙烯酰胺与DD共轭，合成了热可逆的可溶性-不溶性聚N-异丙基丙烯酰胺-葡聚糖衍生物共轭物，即聚（NIPAAm）-DD。一方面，本报告描述了聚（NIPAAm）-DD共轭的两个步骤及其表征。另一方面，聚（NIPAAm）-DD共轭物被用作一种工具，用于纯化皮下注射衍生化葡聚糖的兔血清样本中的多克隆抗体。通过免疫酶测定法对抗体进行纯化和定量。我们的结果表明，抗体既能识别DD，也能识别聚（NIPAAm）-DD。相比之下，它们不与天然聚（NIPAAm）或与另一种阴离子葡聚糖共轭的聚（NIPAAm）结合。我们得出结论，多糖与聚（NIPAAm）的共轭产生了一种新颖且高效的纯化抗体的色谱方法。此外，这种新型纯化方法快速、灵敏、廉价，可用于纯化各种类型的抗体。
