How to combine advantages of ELISAs and Arrays – Target validation with Quantibody®
On your journey to Biomarker profiling, you will reach the point where you need to quantify the proteins of interest identified along the way (eg. validation of semi-quantitative array results; biomarker discovery with an initial hunch on which pathway is involved).
At this stage, you will have narrowed down the number of targets you want to look at. However, the use of ELISAs is likely to be still too costly, too time consuming, and may require too much sample volume.
The Quantibody® technology offers a cost effective alternative method requiring small sample volume, without the need for specific equipment. It combines the high specificity and sensitivity of ELISA with the high throughput of the glass chip-based array. It’s available as Ready-to-Use assays or lab services by tebu-bio (certified service provider). .
While introducing this technology, which is an ideal companion for Target discovery and validation programs, the aim of this post is also to guide you in your choice of Quantibody reference. However, talking to one of our specialists may often be the best option (!).
Quantibody® main features…
- Low sample volume requirement: 50 µL or less
- Results on the same day (6-hour processing time)
- More cost-effective than ELISA
- No interference between capture antibodies (unlike bead-based multiplex assays)
- High specificity and system reproducibility
- No dedicated equipment required
- Customizable – create a custom array from our list of targets
- Q Analyzer software for one-step computation
A little bit about how it works…
Like a traditional sandwich-based ELISA, Quantibody uses a matched pair of antibodies for target protein detection. A panel of capture antibodies is printed in multiple identical arrays on a standard slide. After a blocking step, samples are incubated with the arrays. Nonspecific proteins are then washed off, and the arrays are incubated with a cocktail of biotinylated detection antibodies, followed by a streptavidin-conjugated fluor. Signals are then visualized using a fluorescence laser scanner.
To quantify target protein concentrations, the array-specific protein standards, whose concentrations have been predetermined, are provided to generate an 8-point standard curve of each target protein. By comparing signals from unknown samples to the standard curve, the unknown cytokine concentration in the samples will be determined.
What should you consider when choosing the right Quantibody for your project?
1) Your proteins of interest are involved in known pathways, diseases or physiological processes. In such case, you may find the array suited to your need in the Quantibody® catalogue offer, whether it be related to:
- Obesity, Angiogenesis or MMP
- Cytokine, Chemokine or Inflammation
- or Immune checkpoint, Immune response or Th1/Th2/Th17 pathways
- Discover more pathway focused arrays at this link
Within each thematic, combinations of arrays are likely to cover the range of interest for you. We can help you identify the best selection for the array of proteins you are interested to look at.
2) You have identified a selection of potential targets, however they are involved in various pathways or families for which no catalogue array is available. A custom array is the simple cost effective solution, available within just one or 2 weeks extra time compared to the catalogue items. Just provide me with the list of analytes, we’ll get back with compatiblity and pricing for you.
3) You are still left with quite a high number of proteins of interest after your initial profiling.
You may decide to still go for an absolute quantitation method using the Quantibody technology.
An example of arrays with larger numbers of analytes: the Cytokine Quantibody 9000 includes 400 cytokines.
However, with such high number of proteins to analyze you are likely to have quite a few not falling within the assay detection range. Absolute quantitation may not be the best option at this stage. It may be more time and cost effective to perform relative quantitation by running a broad profiling array. An example of straightforward tools to analyze over 1000 analytes would be the L-Series from Raybiotech to study the secretome, or FullMoon arrays for signalling pathways (SET100) or phosphorylation exploration (PEX100).
When (and why) should you decide to outsource your assay?
Apart from the obvious considerations (lack of human resources to perform your project, need to hurry up your experiments to match a deadline, lack of equipment..), there may be other reasons to rely on tebu-bio’s lab expertise to perform your biomarker quantification for you. If you are on a one shot experiment where you want to optimize your results and benefit from our expertise, outsourcing may be a better choice than doing it yourself. Although the technology is quite simple, tebu-bio’s lab is very experienced and is likely to generate better quality data than you would in a single trial.
Just one word about equipment…
You need a laser scanner to run the assay and most machines on the market are compatible with the Quantibody technology. I can provide you with the list of compatible scanners, and recommended settings. Just leave me a message below if you would like me to send it to you.
As commented in this post, the advice of a product specialist may be useful to help you select the right product for your specific project. Don’t hesitate to leave me a message below and I will get back to you shortly with my suggestions.
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