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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.

Original publication

DOI

10.1016/j.jim.2010.09.022

Type

Journal article

Journal

Journal of immunological methods

Publication Date

01/2011

Volume

363

Pages

210 - 220

Addresses

Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA.

Keywords

B-Lymphocytes, Humans, Hepacivirus, Cryoglobulinemia, Immunoglobulin M, Immunoglobulin Variable Region, RNA, Viral, Rheumatoid Factor, Enzyme-Linked Immunosorbent Assay, Flow Cytometry, Cloning, Molecular, Reverse Transcriptase Polymerase Chain Reaction, Sequence Alignment, Sequence Analysis, DNA, Amino Acid Sequence, Base Sequence, Molecular Sequence Data, Immunoglobulin Heavy Chains