Abstract
Although the presence of antibodies against double-stranded (ds) DNA is considered the serological hallmark of systemic lupus erythematosus (SLE), it is not detected in all SLE patients by routine laboratory tests. Looking at DNA–anti(ds)DNA interaction as one kind of DNA–protein interaction gave us the grounds for a novel type of assay, easy to perform, and providing a direct insight on DNA–anti(ds)DNA IgG interaction. The assay is an application of the electrophoretic mobility shift assay (EMSA) and is based on the observation that the electrophoretic mobility of a DNA–protein complex is typically less than that of free DNA. The EMSA, performed here with purified bacterial DNA and the purified IgG fraction of sera from systemic lupus erythematosus (SLE) or other patients as well as from healthy individuals, revealed itself to be more sensitive than the routinely used assays for the detection of anti-dsDNA in SLE and discoid lupus erythematosus (DLE) patients. In addition, besides providing a direct visualization of DNA–anti(ds)DNA IgG complexes, the assay offers the possibility to study in detail the nature of DNA–IgG interactions. In a further development, we showed that the assay could be performed successfully with sera.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF, Schaller JG, Talal N, Winchester RJ (1982) The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 25:1271–1277
Egner W (2000) The use of laboratory tests in the diagnosis of SLE. J Clin Pathol 53:424–432
Pisetsky DS (2016) Anti-DNA antibodies—quintessential biomarkers of SLE. Nat Rev Rheumatol 12:102–110
Riboldi P, Gerosa M, Moroni G, Radice A, Allegri F, Sinico A, Tincani A, Meroni PL (2005) Anti-DNA antibodies: a diagnostic and prognostic tool for systemic lupus erythematosus? Autoimmunity 38:39–45
McCloskey LJ, Christner P, Jacobs-Kosmin D, Jaskowski TD, Hill HR, Lakos G, Teodorescu M (2010) Myth and reality: practical test system for the measurement of anti-DNA antibodies in the diagnosis of systemic lupus erythematosus (SLE). J Clin Lab Anal 24:77–84
Lane D, Prentki P, Chandler M (1992) Use of gel retardation to analyze protein-nucleic acid interactions. Microbiol Rev 56:509–528
Hellman LM, Fried MG (2007) Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nat Protoc 2:1849–1861
Keyhani J, Servais G, Einollahi N, Keyhani E, Duchâteau J (1995) Plasma membrane DNA in autoimmune diseases. Transplant Proc 27:2756–2758
Keyhani J, Jafari-Far F, Ahadi M, Akbari K, Einollahi N, Keyhani E (1998) DNA-protein interaction as a tool in detecting and monitoring systemic lupus erythematosus (SLE). In: Ahmad F, Baumbach L, Bernstein P, Black S, Deutscher MP, Hassler S, Huijing F, Rudd KE, Whelan WJ (eds) Advances in gene technology: molecular biology in the conquest of disease, Miami, February 1998, Miami nature biology short reports, vol 9. Oxford University Press, New York, pp 117–118
Keyhani J, Jafari-Far F, Einollahi N, Ghadirian E, Keyhani E (1998) DNA-mobility shift assay and the detection of anti-DNA IgG in systemic lupus erythematosus patients. Immunol Lett 62:81–86
Keyhani J, Keyhani E, Servais G, Duchateau J (2011) In vivo and in vitro use of plasma membrane DNA in the detection and follow-up of systemic lupus erythematosus. J Mol Cell Biol 3:200–201
Keyhani J, Ahadi M, Keyhani E, Naraghi Z, Shahmohammadi S (2012) DNA mobility shift assay as a tool for the detection of anti-dsDNA antibodies in sera from discoid lupus erythematosus patients. J Dermatol 39:602–607
Bak G, Han K, Kim KS, Lee Y (2015) Electrophoretic mobility shift assay of RNA-RNA complexes. Methods Mol Biol 1240:153–163
Stefanovsky VY, Moss T (2015) The cruciform DNA mobility shift assay: a tool to study proteins that recognize bent DNA. Methods Mol Biol 1334:195–203
Parés-Matos EI (2013) Electrophoretic mobility-shift and super-shift assays for studies and characterization of protein-DNA complexes. Methods Mol Biol 977:159–167
Rowe SE, O’Gara JP (2016) Electrophoretic mobility shift assays. Methods Mol Biol 1373:155–167
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p 446
Levy JA, Virolainen M, Defendi V (1968) Human lymphoblastoid lines from lymph node and spleen. Cancer 22:517–524
Silhavy TJ, Berman ML, Enquist LW (1984) Experiments with gene fusions. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p 179
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p 438
Åkerström B, Björck L (1986) A physicochemical study of protein G, a molecule with unique immunoglobulin G-binding properties. J Biol Chem 261:10240–10247
Silhavy TJ, Berman ML, Enquist LW (1984) Experiments with gene fusions. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 137–139
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p 450
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Keyhani, J., Keyhani, E. (2019). Detection of DNA Autoantibodies by Electrophoretic Mobility Shift Assay. In: Houen, G. (eds) Autoantibodies. Methods in Molecular Biology, vol 1901. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8949-2_11
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8949-2_11
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8948-5
Online ISBN: 978-1-4939-8949-2
eBook Packages: Springer Protocols