DNA mobility shift assay as a tool for the detection of anti-dsDNA antibodies in sera from discoid lupus erythematosus patients

2012 ◽  
Vol 39 (7) ◽  
pp. 602-607 ◽  
Author(s):  
Jacqueline KEYHANI ◽  
Mashallah AHADI ◽  
Ezzatollah KEYHANI ◽  
Zahra NARAGHI ◽  
Safar SHAMOHAMMADI
Keyword(s):  
Lupus Erythematosus ◽  
Discoid Lupus Erythematosus ◽  
Mobility Shift ◽  
Dna Mobility ◽  
Mobility Shift Assay ◽  
Dsdna Antibodies

The clinical relevance of anti-dsDNA antibodies determined by the Elia™ dsDNA assay in patients with negative indirect immunofluorescence on the HEp-2 cell

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Christoph Robier ◽  
Maximiliane Haas ◽  
Franz Quehenberger
Keyword(s):  
Indirect Immunofluorescence ◽  
Lupus Erythematosus ◽  
Medical Records ◽  
Clinical Relevance ◽  
Solid Phase ◽  
Clinical Importance ◽  
Discoid Lupus Erythematosus ◽  
Combined Use ◽  
Significant Difference ◽  
Dsdna Antibodies

AbstractObjectivesData on the clinical importance of the detection of anti-dsDNA antibodies in patients with negative indirect immunofluorescence on the HEp-2 cell (IIF) are sparse and are especially not available for all common commercially available assays. This study aimed to assess the clinical significance of anti-dsDNA antibodies determined by the Elia™ dsDNA assay in patients with negative IIF.MethodsWe retrospectively examined the medical records of 234 consecutive subjects with detectable anti-dsDNA antibodies determined by the Elia™ dsDNA assay.ResultsA total of 124 subjects with detectable anti-dsDNA autoantibodies were IIF-negative, but yielded positive or borderline results in the Elia™ CTD screen assay for antinuclear antibodies (ANA). Within this group, 6/49 IIF-negative patients (12%) with ANA-associated systemic autoimmune rheumatic disorders (AASARD) and 118/185 subjects (64%) with various other diseases (Non-AASARD) were identified. There was no statistically significant difference with regard to the concentrations of anti-dsDNA antibodies (p=0.53) between the AASARD and the Non-AASARD group. Within the AASARD group, four patients diagnosed with systemic lupus erythematosus (SLE, treated), discoid lupus erythematosus (untreated), indetermined connective tissue disease (untreated) and polymyositis (treated) had positive anti-dsDNA autoantibodies, whereas two patients with treated SLE, thereby one in remission, had borderline concentrations of anti-dsDNA antibodies.ConclusionsOur findings suggest that the detection of anti-dsDNA antibodies in IIF-negative patients can be of clinical relevance in some cases. Our results further support the combined use of IIF and solid-phase assays in screening algorithms for ANA, in order to avoid overlooking potentially important autoantibody entities.

scholarly journals Apolipoprotein H Promoter Polymorphisms in Relation to Lupus and Lupus-related Phenotypes

2009 ◽  
Vol 36 (2) ◽  
pp. 315-322 ◽  
Author(s):  
SANGITA SURESH ◽  
F. YESIM K. DEMIRCI ◽  
ERIN JACOBS ◽  
AMY H. KAO ◽  
ELISA Y. RHEW ◽  
...  
Keyword(s):  
Lupus Erythematosus ◽  
Disease Risk ◽  
Gene Promoters ◽  
Nucleotide Polymorphisms ◽  
Promoter Polymorphisms ◽  
Mobility Shift ◽  
Association Analyses ◽  
Gene Assay ◽  
Mobility Shift Assay

Objective.Sequence variation in gene promoters is often associated with disease risk. We tested the hypothesis that common promoter variation in the APOH gene (encoding for ß2-glycoprotein I) is associated with systemic lupus erythematosus (SLE) risk and SLE-related clinical phenotypes in a Caucasian cohort.Methods.We used a case-control design and genotyped 345 women with SLE and 454 healthy control women for 8APOHpromoter single-nucleotide polymorphisms (SNP; –1284C>G, –1219G>A, –1190G>C, –759A>G, –700C>A, –643T>C, –38G>A, and –32C>A).Association analyses were performed on single SNP and haplotypes. Haplotype analyses were performed using EH (Estimate Haplotype–frequencies) and Haploview programs.In vitroreporter gene assay was performed in COS-1 cells. Electrophoretic mobility shift assay (EMSA) was performed using HepG2 nuclear cells.Results.Overall haplotype distribution of theAPOHpromoter SNP was significantly different between cases and controls (p = 0.009). The –643C allele was found to be protective against carotid plaque formation (adjusted OR 0.37, p = 0.013) among patients with SLE. The –643C allele was associated with a ~2-fold decrease in promoter activity as compared to wild-type –643T allele (mean ± standard deviation: 3.94 ± 0.05 vs 6.99 ± 0.68, p = 0.016). EMSA showed that the –643T>C SNP harbors a binding site for a nuclear factor. The –1219G>A SNP showed a significant association with the risk of lupus nephritis (age-adjusted OR 0.36, p = 0.016).Conclusion.Our data indicate thatAPOHpromoter variants may be involved in the etiology of SLE, especially the risk for autoimmune-mediated cardiovascular disease.

scholarly journals Centromere protein B assembles human centromeric alpha-satellite DNA at the 17-bp sequence, CENP-B box.

1992 ◽  
Vol 116 (3) ◽  
pp. 585-596 ◽  
Author(s):  
Y Muro ◽  
H Masumoto ◽  
K Yoda ◽  
N Nozaki ◽  
M Ohashi ◽  
...  
Keyword(s):  
Nuclear Extract ◽  
Affinity Column ◽  
Alpha Satellite ◽  
Mobility Shift ◽  
Alpha Satellite Dna ◽  
Centromere Protein ◽  
Alphoid Dna ◽  
Dna Mobility ◽  
Mobility Shift Assay ◽  
Protein B

We purified 15,000-fold from HeLa cell nuclear extract the centromere antigen that reacts specifically with the 17-bp sequence, designated previously as CENP-B box, in human centromeric alpha-satellite (alphoid) DNA by a two-step procedure including an oligonucleotide affinity column. The purified protein was identified as the centromere protein B (CENP-B) by its mobility on SDS-PAGE (80 kD), and reactivities to a monoclonal antibody raised to CENP-B (bacterial fusion protein) and to anticentromere sera from patients with autoimmune diseases. Direct binding by CENP-B of the CENP-B box sequence in the alphoid DNA has been proved using the purified CENP-B by DNA mobility-shift assay, Southwestern blotting, and DNase I protection analysis. The binding constant of the antigen to the CENP-B box sequence is 6 x 10(8) M-1. DNA mobility-shift assays indicated that the major complex formed between the CENP-B and the DNA contains two DNA molecules, suggesting the importance of the CENP-B/CENP-B box interaction in organization of higher ordered chromatin structures in the centromere and/or kinetochore. Location of DNA binding and dimerization domains in CENP-B was discussed based on the DNA mobility-shift assays performed with a protein fraction containing intact and partial cleavage products of CENP-B.

scholarly journals H-NS-Dependent Regulation of Flagellar Synthesis Is Mediated by a LysR Family Protein

2000 ◽  
Vol 182 (16) ◽  
pp. 4670-4672 ◽  
Author(s):  
Minsu Ko ◽  
Chankyu Park
Keyword(s):  
Escherichia Coli ◽  
Protein Binding ◽  
Genetic Screening ◽  
Transcriptional Regulator ◽  
Mobility Shift ◽  
Transposon Insertion ◽  
Family Protein ◽  
Dna Mobility ◽  
Mobility Shift Assay ◽  
Lysr Family

ABSTRACT H-NS regulates the flagellar master operon (flhDC) and thus is necessary for flagellation of Escherichia coli. However, the molecular mechanism of its regulation has remained unknown. Genetic screening of a transposon insertion abolishing the H-NS effect revealed a previously unidentified gene, namedhdfR, encoding a LysR family protein. Binding of purified HdfR to the flhDC promoter was demonstrated by a DNA mobility shift assay, indicating that HdfR is a transcriptional regulator for the flagellar master operon. Furthermore, the expression of the hdfR gene was shown to be negatively regulated by H-NS.

Snapshot blotting: The transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to EM grids

1992 ◽  
Vol 50 (1) ◽  
pp. 762-763
Author(s):  
Stephen D. Jett
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nucleic Acid Binding ◽  
Mobility Shift ◽  
Carbon Coated ◽  
Nucleoprotein Complexes ◽  
Mobility Shift Assay ◽  
Protein Nucleic Acid ◽  
Gel Mobility Shift ◽  
Nucleic Acid Interactions

The electrophoresis gel mobility shift assay is a popular method for the study of protein-nucleic acid interactions. The binding of proteins to DNA is characterized by a reduction in the electrophoretic mobility of the nucleic acid. Binding affinity, stoichiometry, and kinetics can be obtained from such assays; however, it is often desirable to image the various species in the gel bands using TEM. Present methods for isolation of nucleoproteins from gel bands are inefficient and often destroy the native structure of the complexes. We have developed a technique, called “snapshot blotting,” by which nucleic acids and nucleoprotein complexes in electrophoresis gels can be electrophoretically transferred directly onto carbon-coated grids for TEM imaging.

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