A flow cytometry-based strategy to identify and express IgM from VH1-69+ clonal peripheral B cells.
Pathologic rheumatoid factor (RF) levels are hallmarks of several human diseases. Production of monoclonal RF in vitro is essential for studies of the antigenic specificities of RF, as well as for a dissection of the mechanisms of aberrant RF+ B cell activation. We have expanded upon previous methods to develop a flow cytometry-based method to efficiently clone monoclonal antibodies (mAbs) from humans with expansions of RF-like, immunoglobulin heavy chain variable region (IgVH) 1-69 gene segment-containing B cells. The cloned variable regions are expressed as IgM and produced during culture at concentrations between 5 and 20 μg/ml. Using this system, we show that clonal Igs from patients with HCV-related mixed cryoglobulinemia, when expressed as IgM, have RF activity. We anticipate that this system will be useful for the cloning and expression of mAbs partially encoded by VH1-69 and for determination of the reactivity patterns of polyspecific, low-affinity IgMs of human pathogenic importance.
Charles
Edgar D
ED
Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA.
Orloff
Michael I M
MI
Dustin
Lynn B
LB
D000595:Amino Acid Sequence; D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular; D003449:Cryoglobulinemia / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry / Q000379:methods*; D016174:Hepacivirus / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007075:Immunoglobulin M / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012217:Rheumatoid Factor / Q000235:genetics / Q000276:immunology*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D007143:Immunoglobulin Heavy Chains; D007075:Immunoglobulin M; D007135:Immunoglobulin Variable Region; D012367:RNA, Viral; D012217:Rheumatoid Factor