Schofield Peter, Vazquez-Lombardi Rodrigo, Abdelatti Mahmoud, Nevoltris Damien, Goodnow Christopher C, Christ Daniel, Reed Joanne H
Garvan Institute of Medical Research, Sydney, NSW, Australia.
Faculty of Medicine, St. Vincent's Clinical School, University of New South Wales, Sydney, NSW, Australia.
Methods Mol Biol. 2018;1827:287-309. doi: 10.1007/978-1-4939-8648-4_15.
Here we describe methods for screening human blood to isolate peripheral blood mononuclear cells (PBMCs) capable of binding fluorescently labeled antigen, as well as methods for the amplification and sequencing of B cell receptor (BCR) heavy and light chain genes. Detailed protocols are provided for transient mammalian expression in a hexahistidine-tagged Fab format, purification by immobilized metal affinity chromatography (IMAC), and affinity determination by BioLayer interferometry (BLI).
在此，我们描述了筛选人血以分离能够结合荧光标记抗原的外周血单核细胞（PBMC）的方法，以及B细胞受体（BCR）重链和轻链基因的扩增和测序方法。还提供了详细的方案，用于以六组氨酸标签的Fab形式进行瞬时哺乳动物表达、通过固定金属亲和色谱（IMAC）进行纯化以及通过生物层干涉术（BLI）进行亲和力测定。
