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An easy and simple separation method for Fc and Fab fragments from chicken immunoglobulin Y (IgY).
Journal of Chromatography B ( IF 2.8 ) Pub Date : 2020-01-31 , DOI: 10.1016/j.jchromb.2020.122011
Xin Zhou 1 , Yanru Wang 1 , Dong Uk Ahn 2 , Zhaoxia Cai 1
Affiliation  

Antigen-binding (Fab) and crystallizable (Fc) fragments are the active components of yolk immunoglobulin (IgY), which have been widely used in the pharmaceutical field. However, the common purification methods for the Fab and Fc fragments use combinations of multi-columns are complex and time-consuming. The objective of this study was to improve the separation efficiency of the Fab and Fc fragments from the hydrolyzed IgY and increase the purity of the isolated Fab and Fc fragments. Natural IgY was hydrolyzed using papain for 6 hr and then treated with 45% saturated ammonium sulfate to remove small molecular-weight-peptides. The fraction containing Fab and Fc fragments was loaded on a DEAE-Sepharose ion exchange column and the Fab fraction was washed out first with 10 mM Tris-HCl buffer (pH 7.6). Then, the Fc fraction bound to the DEAE Sepharose was eluted with 10 mM Tris-HCl buffer (pH 7.6) containing 0.21 M NaCl. The purity of the two fragments was 88.7% and 90.1%, respectively. The results of Western blotting and MS analyses indicated that this method purified Fab and Fc fractions with high purity. This method is easy and simple compared with other methods, and the active fragments separated can be easily used.

中文翻译:

从鸡免疫球蛋白Y(IgY)中分离Fc和Fab片段的简便方法。

抗原结合(Fab)和可结晶(Fc)片段是蛋黄免疫球蛋白(IgY)的活性成分,已被广泛用于制药领域。然而,用于Fab和Fc片段的常用纯化方法使用多柱的组合是复杂且费时的。这项研究的目的是提高Fab和Fc片段与水解IgY的分离效率,并提高分离的Fab和Fc片段的纯度。使用木瓜蛋白酶将天然IgY水解6小时,然后用45%饱和硫酸铵处理以去除小分子量肽。将含有Fab和Fc片段的馏分上样到DEAE-Sepharose离子交换柱上,并首先用10 mM Tris-HCl缓冲液(pH 7.6)洗涤Fab馏分。然后,用含有0.21 M NaCl的10 mM Tris-HCl缓冲液(pH 7.6)洗脱与DEAE Sepharose结合的Fc部分。两个片段的纯度分别为88.7%和90.1%。Western印迹和MS分析的结果表明该方法以高纯度纯化了Fab和Fc级分。与其他方法相比,该方法简便易行,并且可以轻松使用分离出的活性片段。
更新日期:2020-01-31
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