Sequencing and Affinity Determination of Antigen-Specific B Lymphocytes from Peripheral Blood
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).
|ISBN||1940-6029 (Electronic) 1064-3745 (Linking)|
|Authors||Schofield, P.; Vazquez-Lombardi, R.; Abdelatti, M.; Nevoltris, D.; Goodnow, C. C.; Christ, D.; Reed, J. H.|
|Responsible Garvan Author|
|Publisher Name||Methods in Molecular Biology|
|URL link to publisher's version||https://www.ncbi.nlm.nih.gov/pubmed/30196503|