A method to detect particle-specific antibodies against Ku and the DNA-dependent protein kinase catalytic subunit in autoimmune sera

Farahdiba Jafri, John A. Hardin, William S. Dynan

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Sera from patients with systemic lupus erythematosus, polymyositis, scleroderma, and mixed connective tissue disease are frequently characterized by the presence of high levels of autoantibodies directed against linked sets of nuclear proteins. One of these autoantigen systems is made up of Ku and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), proteins that are essential for double-strand DNA break repair and for the related process of V(D)J recombination. Ku and DNA-PKcs bind avidly to DNA ends in vivo and in vitro and form an active protein kinase complex. One hypothesis is that this assembled nucleoprotein particle, rather than its component proteins, is a primary trigger for the autoimmune response and thus a major target for the resulting autoantibodies. To screen for particle-specific antibodies, we developed an assay in which the fully native nucleoprotein particle is reconstituted in vitro and is tethered to the surface of an ELISA plate via a streptavidin-biotin linkage. These particles are recognized efficiently by monoclonal antibodies and by autoantibodies present in patient sera. The assay may detect a broader spectrum of epitopes than a conventional ELISA in which Ku and DNA-PKcs are adsorbed directly to a plastic surface. The method will be advantageous for high-throughput screening for antibodies and other ligands that bind the assembled DNA-dependent protein kinase complex.

Original languageEnglish (US)
Pages (from-to)53-61
Number of pages9
JournalJournal of Immunological Methods
Volume251
Issue number1-2
DOIs
StatePublished - May 1 2001
Externally publishedYes

Fingerprint

DNA-Activated Protein Kinase
Catalytic Domain
Autoantibodies
Nucleoproteins
Antibodies
Serum
Enzyme-Linked Immunosorbent Assay
V(D)J Recombination
Mixed Connective Tissue Disease
Polymyositis
Streptavidin
Double-Stranded DNA Breaks
Autoantigens
Biotin
Nuclear Proteins
Autoimmunity
DNA Repair
Systemic Lupus Erythematosus
Protein Kinases
Plastics

Keywords

  • Autoimmunity
  • DNA-dependent protein kinase
  • ELISA
  • Ku protein
  • Particle-specific antibodies

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

Cite this

A method to detect particle-specific antibodies against Ku and the DNA-dependent protein kinase catalytic subunit in autoimmune sera. / Jafri, Farahdiba; Hardin, John A.; Dynan, William S.

In: Journal of Immunological Methods, Vol. 251, No. 1-2, 01.05.2001, p. 53-61.

Research output: Contribution to journalArticle

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