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 metho...

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Bibliographic Details
Published in:Journal of immunological methods 2011-01, Vol.363 (2), p.210-220
Main Authors: Charles, Edgar D., Orloff, Michael I.M., Dustin, Lynn B.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
B-Lymphocytes - immunology
Base Sequence
Cloning
Cloning, Molecular
Cryoglobulinemia - immunology
Enzyme-Linked Immunosorbent Assay
Expression
Flow cytometry
Flow Cytometry - methods
genes
Hepacivirus - genetics
Hepacivirus - immunology
human diseases
Humans
IgM
immunoglobulin heavy chains
Immunoglobulin Heavy Chains - genetics
Immunoglobulin Heavy Chains - immunology
immunoglobulin M
Immunoglobulin M - biosynthesis
Immunoglobulin M - genetics
Immunoglobulin M - immunology
Immunoglobulin Variable Region - genetics
Immunoglobulin Variable Region - immunology
Mixed cryoglobulinemia
Molecular Sequence Data
monoclonal antibodies
patients
Reverse Transcriptase Polymerase Chain Reaction
Rheumatoid factor
Rheumatoid Factor - genetics
Rheumatoid Factor - immunology
RNA, Viral - chemistry
RNA, Viral - genetics
Sequence Alignment
Sequence Analysis, DNA
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Summary: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.
ISSN:0022-1759
1872-7905
DOI:10.1016/j.jim.2010.09.022