Search Results for: arrays

Phosphorylation studies made with Antibody Arrays

Oropharyngeal squamous cell carcinomas (OSCCs) can be either Human papillomavirus (HPV)-positive or HPV-negative. Profiles of druggable Receptor Tyrosine Kinases (RTKs) are different in both groups, as shown in a paper by Cortelazzi, B. et al. The authors chose a cohort of 17 HPV-positive and 59 HVP-negative Formalin-Fixed OSCCs, in order to study E5 expression and RTK alterations. RTK activation was explored in further 12 Frozen OSCCs.

HPV-positive and HPV-negative OSCCs showed different RTK profiles, including differences in E5 and HER2 levels, as well as in HER3 activation and heterodimerisation (HER3/EGFR, also seen for HER2/EGFR). PI3KCA mutations/expression/increased gene copy number and PTEN mutations were found in both groups, whereas PTEN gene loss was only observed in the HPV-positive cases.

The authors stated that, for HPV-positive cases, it would be interesting to study the expression of E5, which may modulate EGFR turnover and activate VEGF and PDGFRβ. They also indicate that in HPV-negative cases, HER3 may be a promising druggable biomarker that would deserve further investigation. Finally, PI3KCA and PTEN alterations encourage the promising clinical evaluation of PI3K/mTOR inhibitor activity in OSCCs, particularly in HPV-positive/PI3KCA-mutated OSCCs.

This study was possible in part thanks to an approach based on arrays to detect multiple biomarkers in biological samples at the same time, followed by validation using simplex technologies. These experimental approaches are known for cytokine profiling but also exist for phosphorylation studies (RTKs, EGFR, mTOR phospho-pathways…).

To ensure top quality in the data obtained, you might outsource your biomarker profiling and validation to certified service providers fully trained. tebu-bio: European RaybioTech's Certified Laboratory service provider

In Europe, tebu-bio laboratories (France) were among the first laboratories in the world to be certified by Raybiotech in 2012 but also by Quansys BioSciences and FullMoon BioSystems technologies. The Biomarkers team will be proud to support you to publish novel discoveries by providing innovative discovery & validation tools.

Cytokine signature in airway inflammation with arrays

Chronic Mucosal Inflammation is the hallmark of common airway diseases (ex.  Allergic Rhinitis and asthma). Lipoxin B4(LXB4) is an endogenous mucosal protective mediator decreasing such diseases. LXB4 mechanisms of action remain poorly understood.

Cytokine Quantibody Arrays from Raybiotech and tebu-bio laboratories

Cytokine Quantibody Arrays from Raybiotech and tebu-bio laboratories.

In a recent paper in Mucosal Immunology (Karra, L. et al. (2015) 8; DOI:10.1038/mi.2014.116), Allergic Rhinitis  and asthma murine models have been described to better investigate the role of LXB4 in Mucosal Inflammation. The authors conclude that, in the upper airway, LXB4 significantly decreases nasal mucosal leukocytes and degranulation of Mast Cells (MCs) and Eosinophils. They also show that, in the lower airway, LXB4 significantly decreased airway inflammation, mucus metaplasia, and hyper-responsiveness.

Inhibition of MC degranulation in vivo by LXB4 is more potent than Dexamethasone (a well-known apoptosis inducing glucocorticoid) with unique profiles for cytokine regulation. This latter  is proved by  quantitative analysis of 20 murine cytokines in a single array (Quantibody Cytokine Array).

These findings indicate that LXB4 carries cell type selective and mucosal protective actions and need to be translated to human models. This publication opens the way to consider lipoxins as new therapeutical candidates.

tebu-bio: European RaybioTech's Certified Laboratory service provider

tebu-bio laboratories are the European RaybioTech’s Certified Laboratory service provider.

The Cytokine Arrays are available as ready-to-use assays. They can also be outsourced to Certified Service Laboratory Providers as fee-for-services. In that case, researchers just send their samples and in return receive the experimental data set. To ensure top quality in the data obtained, Raybiotech’s service providers successfully complete training and certification programs to receive the “RayBiotech Certified Array Service Providers” yellow award.

In Europe, tebu-bio laboratories (France) were among the first laboratories in the world to be certified by Raybiotech in 2012. tebu-bio’s services also cover Quansys BioSciences and FullMoon BioSystems technologies for outsourcing protein profiling and quantification.

Tips for data interpretation in profiling arrays

One of the questions we usually get in the Biomarkers team, once researchers or clinicians have done profiling arrays (e.g. for secretome and kinome), is how to interpret, biologically speaking, the obtained data.

It’s not the scope of this post to give a general overview of what has been published so far, but you can always have a look at publications using profiling arrays to see how other people have presented their results. The aim of this post is to give some general “rules” or ideas to help you analyse your data… even before you do your experiment. [Read more…]

Antibody arrays: a matter of species

Research is never easy. That’s why we either like doing it or supporting it.

That said, it’s easier for some researchers than others. If you are working on human, mouse or even rat models, you have quite a wide variety of tools available. Even if you work on zebrafish, at least, you have some validated zebra antibodies, guaranteed to work for your animal model.

What if you are working on other “exotic” animal models? Not by choice, but because they are really the relevant models for your research. Let’s take some examples. [Read more…]

Why multiplex histone binding arrays?

In the summer of 2013, my colleague Frederic Dubor, who is in charge of our services lab, mentioned to me that he had just been asked by 3 clients within a very short time-frame, whether our laboratory would  be able to run histone binding studies for them. In fact, it wasn’t about binding to single full length histones, but all 3 clients had asked for multiplex arrays, meaning they were interested in measuring binding of either antibodies (epitope mapping) or epigenetic readers (bromodomains and methyl readers) to a set of histone derived peptides, without and with a number of modifications such as acetylation and methylation,

At that time, neither Frederic nor I could help – but these requests did make us search for a supplier offering arrays which would meet these needs. Half a year later,  we were pleased to announce a new cooperation with Epicypher, one of the market leaders in epigenetic proteomics.

But I’m running ahead a little quickly… I’d first like to summarize the potential applications of the arrays we were not able to offer one year ago.

So, what precisely were researchers looking for when asking to “run histone binding studies”?

1. The determination of the exact modification on a specific histone recognized by an antibody raised against histones. In fact, this could be meant as a strigent quality control experiment for an antibody to be used to recognize specific histone modifications. Furthermore, such a specific antibody could be used to block out the binding of any other protein to the histone target sequence. As the 4 histones which make the nucleosome’s histone octamer (H2A, H2B, H3, and H4) can be epigenetically modified by a number of chemical reactions, such as acetylation, methylation, phosphorylation, ubiquitination, sumoylation, and ADP Ribosylation, theoretically thousands of modification patterns can be found on histones.Writers - erasers - readers in Epigenetics - Epicypher agreed 24.6.2014

2. The determination of specific binding sites of epigenetic “readers”, such as bromodomains (recognizing acetylated sites) and methyl readers. These “readers” can be domains of “writers” (enzymes modifying histones, e.g. methyl transferases and acetylases) or “erasers” (e.g. demethylases and deacetylases). Thus, these modifying enzymes can be targeted to their sites of action on histones.

3. Furthermore, a third application field for peptide arrays containing diverse modified and non modified histone sequences could be the analysis of  the substrate specificity of entire histone modifying enzymes.

All these applications could help in answering questions in research fields and diseases which are linked to epigenetic histone modifications such as cancer, aging, asthma, diabetes, cardiovascular diseases, neurodegeneration, autoimmune disorders, and developmental disorders.

We found arrays to answer all these questions: Precise histone binding studies are now possible with EpiCypher’s EpiTitan Histone Peptide Arrays

The EpiTitan™ Histone Peptide Arrays represent the next generation in histone peptide arrays, and are the gold standard against which all other histone peptide arrays are judged.

The arrays feature hundreds of highly purified and well-characterized histone peptides spotted onto the substrate. EpiTitan™ Histone Peptide Arrays are a dramatic improvement over arrays in which the peptides are synthesized on the substrate (SPOT synthesis), a technique that leads to peptides on the array that are of unknown quantity and quality. All the peptides on the EpiTitan™ Histone Peptide Arrays are purified by HPLC and validated by mass spectrometry prior to spotting.

The technique used in this array is quite easy.

  • Biotinylated peptides are spotted 12 times each per array on a surface covered with streptavidin to catch the peptides.
  • Each array slide comes with two subarrays, thus two samples per slide can be evaluated.
  • The protein to be investigated is added and binds to its specific target sequence and modification pattern.
  • The detection of binding either starts with an antibody against the protein of interest or an anti tag antibody, should the protein carry the respective tag, and can be visualized using fluorescence scanning or standard ECL techniques – by the way the EpiTitan™ Histone Peptide Array is the only array available on the market which supports both detection methods.
  • Protocols for qualitative as well as quantitative analysis of the data are provided.

Array layout

EpiCypher's EpiGold™ Histone Peptide Arrays

Design of the Arrays and experimental steps

Interested in more information or using EpiTitan™ Histone Peptide Arrays yourself?

Just get in touch if you’d like to check the list of histone peptides and the user manual. I’ll be happy to get back to you to tell you more about these Histone arrays and companion reagents (Epigenetic readers and erases or bioactive small molecules…).

While reading this blog you were maybe already thinking about how you could apply this array in your research project…

Then I have good news for you. Our partner Epicypher just started a grant program – please have a look at our previous blog: 5 fully funded Epigenetic Grants offered by Epicypher.

The principle of the grant program is very easy – you supply a brief outline of a study which involves the role of histone modifications in physiological or pathological processes – Epicypher supplies arrays free of charge to five applicants chosen by an independent scientific committee.

 

 

 

A new dimension in biomarker profiling: miRNA 3D-arrays by TORAY

Blood sample

Circulating miRNA from blood

miRNA are implicated in a number of diseases and cancers. Despite the fact that their different roles in physiological and pathological cell processes are still under investigation, there is already more and more interest in considering them as future key diagnostic and prognostic biomarkers. Indeed, they are conveniently present in blood, and furthermore, their small size makes them relatively robust compared to messenger RNAs. They are well conserved into poor quality samples such as FFPE samples allowing the best hopes when exploring biobanks of tissues.

Accurate and convenient technologies aimed at profiling miRNA expression from such biobanks and blood opens up a promising era in biomarker discovery for personalized healthcare.
But still, we need to find an efficient profiling approach to cover the full miRnome that contains about 2000 Human miRNA… [Read more…]

Innovation in Life Sciences: Bead Arrays for Antibody and Complement Profiling by Ayogly et al.

Ayogly et al. have designed an innovative suspension bead-array experimental approach for studying in one assay both antigen-specific antibody reactivity and complement activation induced C3 deposition.

The benefits of such a scalable approach is based on its capacity to measure multiplex antigen-specific complement activation in the context of an autoimmune disease (ex. Rheumatoid Arthritis) and viral antigen detection (ex. Epstein-Barr virus encoded Nuclear Antigen-1 (EBNA-1)).

The combination of antibody reactivity profiling and induced complement activation in one assay open new perspectives in autoantibody- and virus-induced immune diseases.

Want to know more?

Ayoglu B. et al. “Bead Arrays for Antibody and Complement Profiling Reveal Joint Contribution of Antibody Isotypes to C3 Deposition (5 May, 2014) PLoS ONE, CrossRef DOI Link to Publisher-Maintained copy: http://dx.doi.org/10.1371/journal.pone.0096403

Congratulations to the authors, who turned to tebu-bio’s range of products when selecting their antigens (ex. EBNA-1 protein) coupled to beads to determine the optimal assay conditions for measuring complement activation.

Histone Peptide Arrays’ contribution in Epigenetic studies

EpiGold™ Histone Peptide Array Data-FluorescenceDNA methylation and histone post-translational modifications are key epigenetic factors.

They regulate gene expression by interfering with the chromatin structure and the binding of effector proteins to post-translational modified histones (1).

Several experimental strategies to analyze such binding have recently been designed; histone modified peptide arrays being one of them. [Read more…]

miRNAs: potent biomarkers in cancer research?

journal.pone.0118220.g002

Expression signals of validated miRNAs that differentiated pancreato-biliary cancer from non-malignant abnormalities (A), or from cancers of other types (B).

A recent paper by Kojima, M. et al. has found a signature of miRNAs to identify patients with pancreato-biliary cancers who could benefit from surgical intervention.

Namely, a combination of eight miRNAs (miR-6075, miR-4294, miR-6880-5p, miR-6799-5p, miR-125a-3p, miR-4530, miR-6836-3p, and miR-4476) achieved a sensitivity, specificity, accuracy and AUC of 80.3%, 97.6%, 91.6% and 0.953, respectively.

In contrast, CA19-9 and CEA gave sensitivities of 65.6% and 40.0%, specificities of 92.9% and 88.6%, and accuracies of 82.1% and 71.8%, respectively, in the same test cohort. This diagnostic index identified 18/21 operable pancreatic cancers and 38/48 operable biliary-tract cancers in the entire cohort.

Finding of this eight miRNAs was possible by using Toray’s 3D profiling technology. This finding is especially important, as it is difficult to detect pancreatic cancer or biliary-tract cancer at an early stage using current diagnostic technology.

microRNAs are stably present in peripheral blood, and are therefore a good candidate for finding prognostic or diagnostic biomarkers.

Studying only the miRNAs available in the literature may limit the novelty of the biomarkers found, so for a wide variety of diseases, a profiling is mandatory in order to find really specific circulating biomarkers.

Toray’s technology is available in Europe as fee-for-services via tebu-bio Laboratories (see Press Release). By exploring the full miRnome (composed by 2,000 miRNAs) with Toray’s 3D-Gene® technology, one might identify slight miRNA expression level changes (including low abundance miRNAs) in blood samples, biopsies FFPE specimens…

Interested in miRNA profiling in cancer research? Leave a message below or browse tebu-bio’s 3D-Gene® miRNA profiling platform web page!

Array profiling to study PPAR-alpha in progenitor cell

Peroxisome proliferator-activated receptor alpha (PPAR-alpha also known as NR1C1)  regulates a myriad of biological processes. It is a key modulator of lipid metabolism.

Raybiotech C-Series Arrays by tebu-bio (ready-to-use kits and lab services)

Raybiotech C-Series Arrays by tebu-bio (ready-to-use kits and lab services).

Vergori, L. et al. have shown in murine models how PPAR-alpha regulates endothelial progenitor cell maturation and myeloid lineage differentiation via a NADPH oxidase-dependent mechanism. (1) All the data described in this publication suggest that PPAR-alpha, in murine models, is a critical regulator of recruitment, homing and maturation of Bone Marrow-derived progenitor cells.

This conclusion was made (in part) possible with the analysis of secretome markers by using C-Series profiling arrays for the analysis of bone marrow-derived cells in PPAR-alpha wild-type vs. KO mice.

RayBio® Membrane-Based Antibody Arrays (C-Series) are tools for screening and comparing expression levels of many cytokines between samples. C-series Arrays are available as ready-to-use kits or lab services. To ensure top quality in the data obtained, Raybiotech’s service providers successfully complete training and certification programs to receive the “RayBiotech Certified Array Service Providers” yellow award.

In Europe, tebu-bio laboratories (France) were among the first laboratories in the world to be certified by Raybiotech in 2012.

tebu-bio’s services also cover Quansys BioSciences and FullMoon BioSystems technologies for outsourcing protein profiling and quantification.

Interested in studying the secretome biomarkers relevant in your model for Cardiovascular diseases?

Leave your comment or contact us!tebu-bio: European RaybioTech's Certified Laboratory service provider

Source:
(1) Vergori L. et al. “PPARa regulates endothelial progenitor cell maturation and myeloid lineage differentiation through a NADPH oxidase-dependent mechanism in mice” (2015) Stem Cells – 33 (4) :1292-303. DOI: 10.1002/stem.1924.