A simple Dot Blot Assay for population scale screening of DNA methylation

2018 ◽  
Author(s):  
Nelia Luviano ◽  
Sayuri Diaz-Palma ◽  
Céline Cosseau ◽  
Christoph Grunau
Keyword(s):  
Dna Methylation ◽  
Large Scale ◽  
General Rule ◽  
Enzyme Linked Immunosorbent Assay ◽  
Global Dna Methylation ◽  
Epigenetic Changes ◽  
Dot Blot ◽  
Wide Range ◽  
Dot Blot Assay ◽  
Experimental Populations

AbstractThe study of epigenetic changes in natural and experimental populations has increased the need to find a cost-effective and high throughput method to analyze multiple samples to effectuate a population-wide screening to study epigenetic changes triggered by biotic or abiotic stress. One of the most studied epigenetic marks is global DNA methylation, its measurement is used as a first step to differentiate methylation between individuals. There is a wide range of methods designed to detect genome-wide 5 methyl-cytosine (5mC) that differ in sensitivity, price, level of expertise required, but as a general rule, require large amounts of DNA and are relatively expensive. This is a limit for the analysis of 5mC in a large number of individuals as a prerequisite to population-wide testing of methylation markers. In this work, we evaluated a method based on antibody recognition of 5mC to measure the DNA methylation level of individuals of the species Biomphalaria glabrata, the intermediate host of schistosomiases, a neglected tropical disease. We validated the method to complete a large screening in the genome of B. glabrata snails treated with a chemical inhibitor of DNA methylation; however, the method can be applied to any species containing 5mC. The dot blot assay is a suitable method to perform a large-scale screening of global DNA methylation to compare 5mC levels between individuals from different natural or experimental populations. The dot blot method compares favorably with methods with an equivalent sensitivity such as the Enzyme Linked Immunosorbent Assay (ELISA) kit since it requires a smaller amount of DNA (30 ng) is less expensive and allows many more samples to be analyzed.

A Simple, Rapid and Non-Radiolabeled Immune Assay to Detect Anti-AChR Antibodies in Myasthenia Gravis

2018 ◽  
Vol 50 (3) ◽  
pp. 229-235 ◽  
Author(s):  
Suresh Bokoliya ◽  
Shripad Patil ◽  
Madhu Nagappa ◽  
Arun Taly
Keyword(s):  
Myasthenia Gravis ◽  
Case Control Study ◽  
Enzyme Linked Immunosorbent Assay ◽  
Test Results ◽  
Dot Blot ◽  
Dot Blot Assay ◽  
Healthy Control ◽  
Neurological Illnesses ◽  
Control Study ◽  
Immune Assay

AbstractObjectiveTo assess the practicality of dot-blot testing for rapid and sensitive detection of the antiacetylcholine receptor (anti-AChR) antibodies in myasthenia gravis (MG).MethodsIn this case-control study, we tested serum specimens of 85 patients with MG, 85 healthy control individuals, and 85 patients without MG who have other autoimmune and neurological illnesses. All the serum specimens were tested for anti-AChR antibodies using 3 assays: in-house enzyme-linked immunosorbent assay (ELISA), the dot-blot assay, and commercial ELISA.ResultsIn-house ELISA, commercial ELISA, and dot-blot test results were positive for anti-AChR antibodies in 65 (76.5%) patients with MG. The results of all 3 tests were negative for anti-AChR antibodies in healthy controls and patients without MG. We observed perfect concordance (K = 1, P <.001) between all 3 tests. In-house ELISA correlated significantly (r = 0.873, P <.001) with commercial ELISA. In-house ELISA and the dot-blot test demonstrated similar diagnostic performance in detecting anti-AChR antibodies.ConclusionsThe dot-blot assay is a simple, nonradioactive immune assay for rapid detection of anti-AChR antibodies in MG.

scholarly journals Generation and diagnostic application of monoclonal antibodies against Seneca Valley virus

2011 ◽  
Vol 24 (1) ◽  
pp. 42-50 ◽  
Author(s):  
Ming Yang ◽  
Rebekah van Bruggen ◽  
Wanhong Xu
Keyword(s):  
Monoclonal Antibodies ◽  
Indirect Elisa ◽  
Foot And Mouth Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot ◽  
Specific Antibody Response ◽  
Dot Blot Assay ◽  
Infected Cells ◽  
Seneca Valley Virus ◽  
Vesicular Disease

Seneca Valley virus (SVV), a member of the Picornaviridae family, was implicated in a suspicious vesicular disease discovered in pigs from Canada in 2007. Because any outbreak of vesicular disease in pigs is assumed to be foot-and-mouth disease (FMD) until confirmed otherwise, a test for diagnosing the presence of SVV would be a very useful tool. To develop the diagnostic tests for SVV infection, 5 monoclonal antibodies (mAbs) were produced from mice immunized with binary ethylenimine (BEI)-inactivated SVV. Using a dot blot assay, the reactivity of the mAbs was confirmed to be specific for SVV, not reacting with any of the other vesicular disease viruses tested. The mAbs demonstrated reactivity with SVV antigen in infected cells by an immunohistochemistry assay. An SVV-specific competitive enzyme-linked immunosorbent assay (cELISA) was developed using BEI-inactivated SVV antigen and a mAb for serodiagnosis. The cELISA results were compared to the indirect isotype (immunoglobulin [Ig]M and IgG) ELISA and the virus neutralization test. All SVV experimentally inoculated pigs exhibited a positive SVV-specific antibody response at 6 days postinoculation, and the sera remained positive until the end of the experiment on day 57 (>40% inhibition) using the cELISA. The cELISA reflected the profile of the indirect ELISA for both IgM and IgG. This panel of SVV-specific mAbs is valuable for the identification of SVV antigen and the serological detection of SVV-specific antibodies.

scholarly journals Effect of Immunization with Recombinant OspA on Serologic Tests for Lyme Borreliosis

2001 ◽  
Vol 8 (1) ◽  
pp. 79-84 ◽  
Author(s):  
Paul T. Fawcett ◽  
Carlos D. Rose ◽  
Sandra M. Budd ◽  
Kathleen M. Gibney
Keyword(s):  
Western Blot ◽  
Lyme Borreliosis ◽  
Vaccine Recipient ◽  
Enzyme Linked Immunosorbent Assay ◽  
Test Results ◽  
Dot Blot ◽  
Western Blot Assay ◽  
Serologic Tests ◽  
Human Volunteers ◽  
Dot Blot Assay

ABSTRACT This study evaluated the effects of vaccination with OspA on the use of serologic tests as aids in the diagnosis of Lyme borreliosis. Sera from control and OspA-immunized mice and from OspA-immunized human volunteers were tested for serologic reactivity to Borrelia burgdorferi. Testing was performed with samples obtained prior to administration of vaccine and at 30 days following administration of an initial and a second dose of OspA vaccine. The assays used to assess serologic reactivity included an in-house-developed enzyme-linked immunosorbent assay (ELISA), an in-house-developed Western blot assay, two commercial Western blot tests, and a commercially available dot blot assay. Data obtained from this study demonstrate that immunization with the OspA vaccine will cause ELISA to yield positive results (as reported previously) for the majority of vaccine recipients. Results obtained from Western blot analysis indicate that vaccination with recombinant OspA induces production of antibodies which bind to several different borrelial proteins. The degree of reactivity detected by Western blotting varied greatly between the three assays used. The in-house assay showed the least reactivity, while one commercial Western blot test actually yielded positive test results for infection with B. burgdorferi. The usefulness of all three Western blot assays for the diagnosis of potential infection in a vaccine recipient is severely limited by the extensive reactivity caused by vaccination alone. Antibodies produced in response to OspA vaccination did not significantly affect the performance of the dot blot test; thus, it could provide a reliable means to test for infection withB. burgdorferi in OspA vaccine recipients.

scholarly journals Relationships between Global DNA Methylation in Circulating White Blood Cells and Breast Cancer Risk Factors

2017 ◽  
Vol 2017 ◽  
pp. 1-25 ◽  
Author(s):  
Nayha Chopra-Tandon ◽  
Haotian Wu ◽  
Kathleen F. Arcaro ◽  
Susan R. Sturgeon
Keyword(s):  
Breast Cancer ◽  
Risk Factors ◽  
Dna Methylation ◽  
Breast Cancer Risk ◽  
Cancer Risk ◽  
The Other ◽  
Global Dna Methylation ◽  
Cancer Risk Factors ◽  
Wide Range ◽  
Breast Cancer Risk Factors

It is not yet clear whether white blood cell DNA global methylation is associated with breast cancer risk. In this review we examine the relationships between multiple breast cancer risk factors and three markers of global DNA methylation:LINE-1, 5-mdC, andAlu. A literature search was conducted using Pubmed up to April 1, 2016, using combinations of relevant outcomes such as “WBC methylation,” “blood methylation,” “bloodLINE-1methylation,” and a comprehensive list of known and suspected breast cancer risk factors. Overall, the vast majority of reports in the literature have focused onLINE-1. There was reasonably consistent evidence across the studies examined that males have higher levels ofLINE-1methylation in WBC DNA than females. None of the other demographic, lifestyle, dietary, or health condition risk factors were consistently associated withLINE-1DNA methylation across studies. With the possible exception of sex, there was also little evidence that the wide range of breast cancer risk factors we examined were associated with either of the other two global DNA methylation markers: 5-mdC andAlu. One possible implication of the observed lack of association between global WBC DNA methylation and known breast cancer risk factors is that the association between global WBC DNA methylation and breast cancer, if it exists, is due to a disease effect.

scholarly journals Evaluation of Diagnostic Applications of Monoclonal Antibodies against Avian Influenza H7 Viruses

2010 ◽  
Vol 17 (9) ◽  
pp. 1398-1406 ◽  
Author(s):  
Ming Yang ◽  
Alfonso Clavijo ◽  
Jill Graham ◽  
John Pasick ◽  
James Neufeld ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Influenza A ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot ◽  
Western Blots ◽  
Positive Antibody ◽  
Dot Blot Assay ◽  
Diagnostic Applications ◽  
Ha Protein ◽  
H7 Subtype

ABSTRACT A panel of monoclonal antibodies (MAbs) was generated from mice immunized with binary ethylenimine (BEI)-inactivated H7N1 (A/TK/ON/18-2/00) virus. Using a dot blot assay, six of seven MAbs reacted with viruses of the H7 subtype, but not with any of the other 15 hemagglutinin (HA) subtypes tested. Four of the seven MAbs reacted with 14 different H7 isolates, indicating that the MAbs binding epitopes are conserved among viruses of the H7 subtype. The binding epitopes of all seven MAbs were conformational and reacted with the HA1 fraction of the HA protein in Western blots under nonreducing conditions. Applications of these MAbs in the development of rapid tests for H7 subtype viruses were evaluated. The MAbs demonstrated reactivity with AI virus H7 antigen in immunofluorescence and immunohistochemistry assays. Monoclonal antibody 3 showed a very strong immunostaining in the formalin-fixed and paraffin-embedded tissue from the H7N3 virus-infected chicken. A double-antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) was developed using two of the MAbs. The DAS ELISA specifically detected all H7 strains tested in this study. A competitive ELISA (cELISA) for the detection of H7-specific antibodies was evaluated using one MAb and BEI-inactivated H7N1 virus as the antigen. All infected birds showed positive antibody responses at 7 days postinfection. The sensitivity of this cELISA was comparable with that of an influenza A nucleoprotein-based cELISA. This panel of MAbs is valuable in the development of various immunoassays.

scholarly journals Alterations in CNS Functions and DNA Methylation in Rats after 24 h Exposure to Peat Smoke

Toxics ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 342
Author(s):  
Vera A. Vokina ◽  
Larisa M. Sosedova ◽  
Mikhail A. Novikov ◽  
Viktor S. Rukavishnikov ◽  
Ekaterina A. Kapustina ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Blood Cells ◽  
Combustion Products ◽  
Albino Rats ◽  
Global Dna Methylation ◽  
Bioelectrical Activity ◽  
Epigenetic Changes ◽  
Behavioral Disturbances ◽  
Natural Fire ◽  
Peat Smoke

The use of a developed experimental model of a natural fire made it possible to assess the consequences of 24 h exposure to peat combustion products in albino rats. Peat smoke exposure leads to behavioral disturbances in rats, characterized by an increase in locomotor activity and an increased level of anxiety. Indicators of brain bioelectrical activity of the exposed animals supported the state of anxiety and psychoemotional stress. Epigenetic changes in the blood cells of exposed animals were revealed under 24 h exposure to peat smoke, characterized by a decrease in the level of global DNA methylation.

scholarly journals Assessment of DNA Methylation and Oxidative Changes in the Heart and Brain of Rats Receiving a High-Fat Diet Supplemented with Various Forms of Chromium

Animals ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1470
Author(s):  
Wojciech Dworzański ◽  
Ewelina Cholewińska ◽  
Bartosz Fotschki ◽  
Jerzy Juśkiewicz ◽  
Piotr Listos ◽  
...  
Keyword(s):  
Dna Methylation ◽  
High Fat Diet ◽  
Protein Carbonyl ◽  
Global Dna Methylation ◽  
Standard Diet ◽  
Epigenetic Changes ◽  
Oxidation Reactions ◽  
High Fat ◽  
Repair Enzymes ◽  
The Brain

The aim of the study was to determine how feeding rats a high-fat diet supplemented with various forms of chromium affects DNA methylation and oxidation reactions as well as the histology of heart and brain tissue. The rats received standard diet or high-fat diet and chromium at 0.3 mg/kg body weight (BW) in form of chromium (III) picolinate, chromium (III)-methionine, or nano-sized chromium. The content of malondialdehyde (MDA), protein carbonyl (PC), and 8-hydroxydeoxyguanosine (8-OHDG), the level of global DNA methylation and the activity of selected DNA repair enzymes were determined in the blood. In the brain and heart, the content of MDA, PC, 8-OHDG, and levels of global DNA methylation were determined. The brain was subjected to histological examination. The use of a high-fat diet was found to intensify epigenetic changes and oxidation reactions in the heart and brain. It was concluded that epigenetic changes and oxidation of lipids, proteins, and DNA in the heart and brain of rats resulting from the use of a high-fat diet cannot be limited by supplementing the diet with chromium. It was established that the use of chromium to supplement a high-fat diet intensifies the negative epigenetic and oxidative changes in the heart and brain, especially in the case of chromium nanoparticles.

scholarly journals Hypo-methylation mediates chromosomal instability in pancreatic NET

2017 ◽  
Vol 24 (3) ◽  
pp. 137-146 ◽  
Author(s):  
I Marinoni ◽  
A Wiederkeher ◽  
T Wiedmer ◽  
S Pantasis ◽  
A Di Domenico ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Chromosomal Instability ◽  
Global Dna Methylation ◽  
Epigenetic Changes ◽  
Knock Down ◽  
Alternative Lengthening ◽  
Increased Risk ◽  
Pancreatic Net ◽  
G0 Phase

DAXX and or ATRX loss occur in 40% of pancreatic neuroendocrine tumors (PanNETs). PanNETs negative for DAXX or ATRX show an increased risk of relapse. The tumor-associated pathways activated upon DAXX or ATRX loss and how this event may induce chromosomal instability (CIN) and alternative lengthening telomeres (ALT) are still unknown. Both DAXX and ATRX are involved in DNA methylation regulation. DNA methylation of heterochromatin and of non-coding sequences is extremely important for the maintenance of genomic stability. We analyzed the association of DAXX and/or ATRX loss and CIN with global DNA methylation in human PanNET samples and the effect of DAXX knock-down on methylation and cell proliferation. We assessedLINE1as well as global DNA methylation in 167 PanNETs, and we found that DAXX and or ATRX-negative tumors and tumors with CIN were hypomethylated. DAXX knock-down in PanNET cell lines blocked cells in G1/G0 phase and seemed to increase CIN in QGP-1 cells. However, no direct changes in DNA methylation were observed after DAXX knock-downin vitro. In conclusion, our data indicate that epigenetic changes are crucial steps in the progression of PanNETs loss and suggest that DNA methylation is the mechanism via which CIN is induced, allowing clonal expansion and selection.

scholarly journals Rapid Screening of the Epidermal Growth Factor Receptor Phosphosignaling Pathway via Microplate-Based Dot Blot Assays

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Amedeo Cappione ◽  
Janet Smith ◽  
Masaharu Mabuchi ◽  
Timothy Nadler
Keyword(s):  
Large Scale ◽  
Solid Phase ◽  
Growth Factor Receptor ◽  
Cost Effective ◽  
Protein Microarrays ◽  
Rapid Screening ◽  
Dot Blot ◽  
A431 Cells ◽  
Dot Blot Assay ◽  
Multiple Samples

Expression profiling on a large scale, as is the case in drug discovery, is often accomplished through use of sophisticated solid-phase protein microarrays or multiplex bead technologies. While offering both high-throughput and high-content analysis, these platforms are often too cost prohibitive or technically challenging for many research settings. Capitalizing on the favorable attributes of the standard ELISA and slot blotting techniques, we developed a modified dot blot assay that provides a simple cost-effective alternative for semiquantitative expression analysis of multiple proteins across multiple samples. Similar in protocol to an ELISA, but based in a membrane bound 96-well microplate, the assay takes advantage of vacuum filtration to expedite the tedious process of washing in between binding steps. We report on the optimization of the assay and demonstrate its use in profiling temporal changes in phosphorylation events in the well-characterized EGF-induced signaling cascade of A431 cells.

Sign in / Sign up

Export Citation Format

Share Document