Normalisation assays in biomarker studies – all’s well that starts well

The normalisation of quantitative assay data is critical when interpreting effective biological system status. With cells grown in culture and lysed, a simple total protein determination such as the Bradford assay (developed by Marion Bradford at the University of Georgia in 1976) can be enough by giving an estimate of the total cellular proteins. However, this type of measurement, along with Lowry and other dye binding assays, can be prone to errirs due to various factors such as detergent, chelators… Standardization also involves the use of a protein of interest. Here again, the protein used is crucial for the accuracy of the overall assay.

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Sensitive ELISA for Oxytocin quantification

The neuropeptides, Oxytocin and Vasopressin, were isolated and synthesized by Vincent du Vigneaud at Cornell Medical College in 1953, work for which he received the Nobel Prize in Chemistry in 1955. Oxytocin is a neurohypophysial peptide which is produced in the paraventricular nuclei of the hypothalamus and stored in the posterior pituitary. The molecule consists of nine amino acids linked with a disulfide bond and a semi-flexible carboxyamidated tail. Recent studies have defined Oxytocin’s role in various behaviors like social recognition, pair bonding, anxiety, and maternal behaviors (1-4) but also, male reproductive physiology. (5)

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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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Basics about insulin and dedicated research tools

Diabetes is a major health concern. And its research can be a nightmare sometimes. tebu-bio strive to offer a comprehensive range of research tools and services to study Obesity, Diabetes, and Metabolic syndrome (including pancreatic islet cells), and tools to unravel signaling mechanisms in insulin secretion. Anyhow, it might be good, though, to go back to the basics from time to time. Let’s remember our graduate courses (more or less years ago) about Insulin and its biological roles.

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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!

Ready-to-Use ELISAs to study Transcription Factors DNA binding

TFACT™ DNA-BINDING ELISA KITS » OCT2 TFACT™ DNA-BINDING ELISA

TFACT™ DNA-BINDING ELISA KITS » OCT2 TFACT™ DNA-BINDING ELISA by Assay Biotechnology. Source: tebu-bio.

Gene expression is regulated by different mechanisms. One of them is the binding of Transcription Factors (TF) to DNA sequences.

Traditionally, the study of TF-DNA interactions is made by several time-consuming and cumbersome: Electrophoretic Mobility Shift Assays (EMSA), Chromatin Immunoprecipitation, Western blotting, and expression of fused target and reporter genes.

ELISA-based formats now allow to have a more precise TF-DNA interaction study in addition to an ease of use.

These new tools significantly reduce the necessary runtime (within one day) and eliminate the need for harmful radioactive labeling. High sensitivity and signal-to-noise ratio are also guaranteed.

The TFact™ product-line belongs to these new ELISA-based Transcription Factor DNA binding assays. These indirect ELISAs allow an easy the detection and qualitative determination of the effects of phosphorylation on transcription factor activation profiles in a variety of nuclear and cell lysates from human, mouse and rat.

TFACT™ DNA-BINDING ELISA KITS » AML1 TFACT™ DNA-BINDING ELISA: AML1 (Phospho-Ser435)

The TFact™ AML1 DNA-Binding ELISA detects active AML1 in Hela Nuclear Extract. The Hela cells were grown 3 days in DMEM with 10% FBS and harvested for nuclear extract. The Hela cells were stimulated by PMA (200nM) before harvest.

TFACT™ DNA-BINDING ELISA Kits are available for various Transcription Factors and well-defined phosphorylated sequences: AML1, Jun, Androgen & Estogen Receptors, ATF2, CREB, FOXO, NFkB, STAT, p53, myb, SMAD, Sox …

Looking for simple but robust methods for studying TFs and the effect of phosphorylation in their activity?

Contact me for any further assistance!

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…]

From osteoarthritis to bladder cancer… hyaluronic acid is not just cosmetics!

Hyaluronic Acid (HA) is important in many biological processes such as wound repair, tissue hydration and inflammation. HA is also a potential biomarker for diseases such as osteoarthritis, liver cirrhosis and bladder cancer. So its importance as a prognostic / diagnostic / predictive biomarker is still to be elucidated… but it might be present even when you don’t expect it!

Hyaluronan (HA) is a linear polysaccharide comprised of a repeating disaccharide of N-acetylglucosamine and D-glucuronic acid. The major function of HA is to provide structural support of tissue as part of the extracellular matrix (ECM). Thus, HA is widely presented in connective tissue in higher animals. The size of HA varies from 100 kD to 10,000 kD and is responsible for different functions. [Read more…]

D-dimer and Cardiovascular Disease

Fibrinogen is the main protein of the blood coagulation system. It consists of two identical subunits that contain three polypeptide chains: alpha, beta and gamma. The process of blood coagulation results in the activation of fibrinogen into fibrin by thrombin and fibrin polymerization. Fibrin clot is then digested by plasmin, and fibrin degradation products of different molecular weights are released into the bloodstream.

D-dimer is one of these fibrinogen degradation products, and is a biomarker for Cardiovascular damage. Therefore, it is widely used in many detection systems in the market, including ELISAs and lateral flow tests.

Clone 8D3, which has been used in many of these detection systems, is nolonger available. So unless you have the hybridoma in your facility, if you were using 8D3, you will have to consider switching to another monoclonal. [Read more…]

Get your ELISA results before lunch!

ELISAs are widely used in biomarker-related studies, especially when a high number of samples is involved, and only a few biomarkers need to be quantified. That said, incubations can be somehow cumbersome, and some days one would like to get results fast (and accurate), and this is not always possible. [Read more…]