Search Results for: q-plex

8 criteria for selecting your ELISA kits

Biomarkers specialists are often asked to select an ELISA kit for researchers: with thousands of ELISA references available on the market, the choice can be tricky regarding proteins for which several kits available.

When researchers have to choose a new ELISA kit, the price is regularly the first parameter of selection. But my experience with long term projects shows that it should in fact be the very last one…

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Discovery of new biomarkers… 3 tips regarding controls

One of the recurrent questions that we get at the Biomarkers team at tebu-bio is on what controls should be included in a given experiment. Either if the experiment is done by researchers in their lab, or if we collect their samples and perform the analysis in our lab, a good design starts by using the most convenient controls.

One of the controls is related to the study itself. In this sense, definition of what a control population is, and how we want to study it vs. a cohort of patients has been discussed elsewhere in a proteomics post. Today, we will put our spotlight on the “technical” controls, i.e. those related to the technique itself.

Control # 1 – positive control

Obvious. We need to check that the technology we are using is able to detect what we want to detect. And before starting with unknown samples, we need to check that it works in samples we know well.Multicoloured wells - Blog Thumbnail

Ideally, a positive control should be as similar as the samples we want to analyse. In this sense, samples for a given health state (be it with a disease or not), are commercially available, or they can be found if not yet available. tebu-bio has a network of collaborations with private companies that can provide validated samples, fulfilling all ethical and clinical criteria.

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Background can be an issue…only if you are not able to detect it. If you can see it, then you can either modify your protocol, or discard that sample. Picture shows an example of what can be seen with slightly-hemolysed plasma.

Alternatively, we can use recombinant or chemically synthesised controls. If we take the example of recombinant proteins (either in pure form or spiked in biological samples), there are many which are commercially available, or they can be made upon demand. Here it is important that the recombinant protein is very similar to the one found in an organism, including glycosylations and other post-translational changes. In this sense, for many control proteins, HEK293 is preferred over E.coli as an expression system.

Control #2 – negative control

Water. Or PBS. Or not?

Ideally, a negative sample should be as similar as to our case samples as possible. Meaning that it has the same clinical and biological parameters than our samples of interest…

Commercially available samples mentioned previously can be a good approach. For immunological studies involving cell culture supernatants, it is important to include a control with the culture medium only, as FBS can affect the specificity of the assay and can render false-negatives due to background.

Background due to FBS is not detected by technologies such as ELISAs or bead-based, whereas it is detected in optical-based technologies such as arrays and Q-plex.

Control #3 – technical replicates

Every technology has an inherent coefficient of variation (CV).

Genomic technologies usually are under 5 % CV. Immunoassays are around 10 to 25 % (or even more). This means that, for some biomarkers where the difference between healthy vs. disease is small, CV may hide the relevance of these biomarkers. This is especially dramatic in studies related to signal transduction, where differences are usually very small.

6-plicates in an antibody array.

6-plicates in an antibody array.

A way to make sure about whether a result comes from real biology or artificial CV is the performance of replicates.

Triplicates (or even 4-plicates) have been popular with ELISA users. Nowadays, however, most researchers perform duplicates, and repeat the analysis of the sample if the results are very discordant. This approach is quite practical, and still allows to have accurate results in a sensible way (i.e. not doing 4-plicates for every sample!).

Antibody arrays in the market usually have replicates spotted onto the same slide (from duplicates to 8-plicates), which can be considered as semi-independent technical replicates. Therefore, there is no need, in most cases, to perform additional technical replicates.

In any case, every project is different, so we are continously advising our customers on what is the best approach for one given study. From its design to the technology best suited to get the best results, we are glad to contribute to the advance of the understanding of biomarkers in several diseases.

Wondering what controls to include in your experiment? Don’t hesitate to contact us!

More is not always better – Tech tips for ELISAs

Following our previous post on how to improve results obtained in ELISA, let’s focus today on one specific point, which is reducing background.

ELISA has many advantages, but one of the drawbacks is that, since we cannot “see” how the reaction works (in contrast to other optical-based technologies such as antibody arrays or Q-plex), high final Abs values may come from a specific signal… or be due to background.

Usually, incorporating sufficient controls in the ELISA plate will allow users to discriminate real positives from false positives (e.g. if you are using cell culture supernatant with FBS % over 1 %, it might be wise to include a medium-only control). FBS contains cytokines that can cross-react with antibodies, even if targeted to different species, in about 10 % of the cases (based on our experience at the Biomarkers team at tebu-bio).

Anyway, if you suspect that you are obtaining a high background in your ELISA, and would like to improve it for future experiments, be sure to follow these guidelines (thanks to Daniel at Raybiotech, Inc. for helpful tips & tricks!). [Read more…]

More data with chemiluminescence imaging platform

Quansys Q-View Imager Pro. Source: tebu-bio.

Quansys Q-View Imager Pro. Source: tebu-bio.

Depending on the phase where your biomarker-related research program is, imaging of protein or antibody arrays has different requirements. For R&D purposes (incl. translational and preclinical ones), a standard imager is more than enough. However, for clinical assays at the late stages, a higher resolution may be needed, especially in view of a future file submission.

Known for the development of the Q-plex platform, which is being used by translational and clinical laboratories worldwide (e.g. to detect Pneumococcus), Quansys has released a new imager, designed for higher throughput (96- and 384-well plates) and clinical-grade research. [Read more…]

Biomarkers of micronutrient malnutrition

Unfortunately, and still in the XXIst century, micronutrient (MN) malnutrition is a problem in all countries, in all segments of society. While part of the world starves, another part is obese. And a balanced diet is still far from being real for many people. Something to think about…

This is a real public health concern, and policies in this area are aimed at preventing, among others, mild, moderate an severe MN malnutrition. Deficiencies in MNs such as iron, vitamin A (VA), iodine, zinc and folic acid are associated with adverse health outcomes, especially in pregnant women and children. For example, VA is critical for embryonic development, adult growth and development, cellular differentiation, immune function, reproduction and vision. On the other hand, low iron levels lead to reduced physical activity in adults and impaired brain development in children. [Read more…]

Tumour microenvironment – ameloblastomas

Following our post on tumour microenvironment and glioblastoma, we will focus today on ameloblastomas.

Ameloblastomas are benign tumours that occur in the jawbone, and invade bone. This type of tumour is treated by surgery and can cause various problems, including changes in facial countenance and mastication disorders.1-s2.0-S0006291X14X00355-cov150h

Ameloblastomas have abundant tumor stroma, including fibroblasts and immune cells. Cell-to-cell interactions in ameloblastoma have not been fully investigated yet. A recent publication by Fuchigami et al.  has investigated the soluble factors (i.e. secretome) involved in the formation and progression of ameloblastoma.

Using the Q-plex technology in a human ameloblastoma cell line (AM-3), as well as human fibroblasts (HFF-2) and primary-culture fibroblasts from human ameloblastoma tissues, they analysed the effect of ameloblastoma-associated cell-to-cell communications. Q-plex was used to study the cytokine secretion, namely IL-1alpha, IL-6, IL-8. Fuchigami et al. conclude that ameloblastoma cells and stromal fibroblasts behave interactively via these cytokines to create a tumour microenvironment (TME) that leads to the extension of this type of tumours.

32_Q_Plex__multiplex_ELISA_by_QuansysWould you like to know more about the Q-plex technology? We recently organised a webinar on this technology. Contact us if you want to receive a link with the recorded version or the PDF with the presentation!

 

Biomarker profiling or multiplex quantification for everyone

Many researchers would be keen to identify new targets for their research project: add a new cytokine to the classical inflammatory panels, find the missing link between 2 phosphorylation pathways, dig into the miRNA to find a new therapeutic target…
They expect they’ll need dedicated (and expensive) new equipment. Not necessarily! Let’s take a look at assays that use existing and quite common readers, or that can easily be outsourced to reliable labs…

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Novel biomarkers for esophageal cancer

As discussed in previous posts, use of new tools allows the finding of new disease-related biomarkers. Today, we want to put our spotlight on esophageal cancer.32_Q_Plex__multiplex_ELISA_by_Quansys

A recent alliance between Quansys Biosciences and Allegheny Health Network has allowed the development of a novel 4-plex assay for use in the early diagnosis of esophageal cancer. Though esophageal cancer is rare, it often has deadly outcomes. [Read more…]

Sex and the City-okines

The trend lately is to add “omics” to most of the research projects. Genomics, Proteomics, Metabolomics…there is no end. The latest trend seems to do “sexomics” studies. Meaning to study the differences in response to treatment, disease progression, etc, based on the sex of the animal model or the patients.

For years now, most of the studies took this variability into account, more or less formally or more or less obviously. In a recent publication, Allen et al. have studied the  different effects in male and female mice for early postnatal exposure to ultrafine particulate matter air pollution. [Read more…]

New tools to fight against Pneumococcus

Pneumococcus, or scientifically speaking, Streptococcus pneumoniae, is a pathogen responsible for a number of illnesses, including pneumonia, ear infections, sinus infections, meningitis and bacteremia. Some of these illnesses can be life-threatening, and therefore, an early and accurate identification of the pathogen causing them is crucial.

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