TY  - JOUR
TI  - A Refined Protocol for Identifying Citrulline-specific Monoclonal Antibodies from Single Human B Cells from Rheumatoid Arthritis Patient Material
AU  - Amara , khaled  
AU  - Israelsson, Lena 
AU  - Stålesen, Ragnhild 
AU  - Sahlström, Peter 
AU  -  Steen, Johanna 
AU  - Malmström, Vivianne 
AU  - Grönwall, Caroline 
VL  - 9
IS  - 17
PY  - 2019
DA  - 2019/09/05
SP  - e3347
C1  - Bio-protocol 2019;9:e3347
DO  - 10.21769/BioProtoc.3347
UR  - https://doi.org/10.21769/BioProtoc.3347
AB  - We describe here a detailed, refined protocol for the generation of citrulline-specific monoclonal antibodies from single human B cells from rheumatoid arthritis (RA) patients. This protocol provides a detailed guide of the procedure starting from single B cells of your choice and followed by amplification of the variable region of immunoglobulin genes by RT-PCR, subsequent immunoglobulin gene cloning, recombinant IgG1 monoclonal antibody (mAb) production and quality controls. The produced mAbs can be used for further studies including reactivity towards candidate antigens and functionality both in vitro and in vivo. This protocol can be used to generate antigen-specific mAbs from B cells derived from different tissues and compartments, including peripheral blood, synovial fluid, digested biopsies, bone marrow aspirations, and bronchoalveolar lavage fluid. Notably, although examples are given on how to identify citrulline-specific autoantibodies the general methods can also be applied to other reactivities.
KW  - Autoantibodies
KW  - B cells
KW  - Antigen-specific monoclonal antibodies
KW  - Citrulline
KW  - Rheumatoid arthritis
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False