A method to detect particle-specific antibodies against Ku and the DNA-dependent protein kinase catalytic subunit in autoimmune sera.
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.
Jafri
F
F
Program in Gene Regulation, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912, USA.
Hardin
J A
JA
Dynan
W S
WS
D000918:Antibody Specificity; D034961:Antigens, Nuclear; D001323:Autoantibodies / Q000097:blood*; D001324:Autoantigens / Q000737:chemistry; D001327:Autoimmune Diseases / Q000276:immunology*; D004265:DNA Helicases; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes / Q000737:chemistry; D006801:Humans; D000072200:Ku Autoantigen; D009687:Nuclear Proteins / Q000276:immunology*; D009698:Nucleoproteins / Q000737:chemistry / Q000276:immunology; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000276:immunology*; D021122:Protein Subunits
D034961:Antigens, Nuclear; D001323:Autoantibodies; D001324:Autoantigens; D004268:DNA-Binding Proteins; D000939:Epitopes; D009687:Nuclear Proteins; D009698:Nucleoproteins; D021122:Protein Subunits; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen