Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 97, Issue 16, Pages 9234-9239Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.97.16.9234
Keywords
antibody phage display; cDNA expression libraries; autoimmune neutropenia; human mAb; elongation factor 1A
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Funding
- NIAID NIH HHS [R01 R37 AI29549] Funding Source: Medline
- NIDDK NIH HHS [R01DK51406] Funding Source: Medline
- NIGMS NIH HHS [R01GM55722] Funding Source: Medline
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An increasing number of studies suggest the importance of antibodies in the pathogenesis of most systemic and organ-specific autoimmune diseases, although there is considerable controversy over the precise role of the autoantibodies involved. In humans, a major obstacle to progress is the identification and cloning of the relevant autoantibodies and autoantigens. Here, an approach based on the sequential use of antibody phage display and antigen expression libraries is developed and applied to a donor suffering from rheumatoid arthritis (RA), splenomegaly, and peripheral destruction of neutrophils leading to neutropenia (Felty's syndrome). An antibody phage display library was constructed from bone marrow from the donor and a high-affinity human mAb, ANA15, selected by panning against fresh neutrophils and independently by panning against a fixed cell line. The antibody showed strong staining of neutrophils and a number of cell lines. Probing of a lambda gt11 expression library from an induced myelomonocytic cell line with the mAb ANA15 identified the eukaryotic elongation factor 1A-1 (eEF1A-1) as a novel autoantigen, The specificity of ANA15 was confirmed by reactivity with both purified and recombinant eEF1A-1, Screening of a large panel of sera revealed that 66% of patients with Felty's syndrome had elevated levels of anti-eEF1A-1 antibodies. The cloning of this antibody-antigen pair should permit rational evaluation of any pathogenicity resulting from the interaction and its significance in neutropenia.
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