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Exploring Multiplex Technologies: Finding The Right System For Your Research
Buying Tips
Oct 8 '04
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Introduction |
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 Once upon a time, the enzyme linked immunosorbant assay (ELISA) was the way to measure analytes in solution. In its most common form, a plate-bound antibody captures an antigen. A second antibody recognizes another epitope (or other epitopes) on the antigen, forming a sandwich. This secondary antibody can be directly linked to an enzyme, or it can be recognized by a third antibody which is linked to an enzyme. (The latter technique can both amplify the signal as well as enable researchers to avoid having to label every secondary antibody they use.) The rate of catalysis, indicated by a colorimetric product, tells how much labeled antibody was in the well. That, in turn, tells how much of the antigen was there for it to stick to. Regardless of whether the primary and secondary antibody are purchased commercially or generated by the laboratory, it is crucial that they not interfere with one another – that is, they need to work as a “matched pair”.
Considerable variations exist on the basic singleplex ELISA theme, from bypassing the primary antibody and directly plating a cell lysate, to using a chemiluminescent substrate or a fluorescently-labeled secondary (or tertiary) antibody.
Several technologies now on the market -- with others hot on their heels – have taken the ELISA one step further, allowing researchers to simultaneously track several analytes from a single sample measurement. These differ from each other in several respects – from how much capital investment or expertise is required, to the ability to quantitate and the adaptability to high-throughput, to the number of parameters that can be measured. Some utilize beads as the solid substrate, while others emulate DNA arrays by printing antibodies onto modified glass slides. Yet they have at least one thing in common: they are based almost exclusively on an antibody interacting with the antigen, points out Dan Remick, professor of pathology at the University of Michigan. So the quality, sensitivity, and specificity of the assay are driven principally by the quality of the antibody – despite claims to the contrary, he adds.
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Consideration #1: Capital Outlay |
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| Before investing in a system, assess what kind of equipment you already have or have access to. Is there a flow cytometer? A DNA slide reader? A cooled CCD camera? How about an arrayer? Robotic handling equipment? If you’re not planning to make multiplexing your lab’s exclusive work, you may want to consider making use of some of these. Similarly, you may want to purchase equipment that can do double-duty. Many manufacturers even claim that no special equipment is needed to use their technology. You can then shop around for the consumables and kits you need for the application you’re interested in. But keep in mind that oftentimes considerable expertise is required to set up, optimize, troubleshoot, and use these piecemeal systems.
The alternative is to choose a more turnkey system. These – together with specialized reagents - tend to be optimized to work as a unit, and add-ons are designed to integrate seamlessly with the basic unit. They are often fairly straightforward to operate. But you are often limited in your source of reagents, and these systems may be good for little else than what they were designed to do.
And though it’s often impossible to know, you may want to try and predict where your research will be, and what advancements might be made in technology in the foreseeable future. Will a heavy investment now pay off when that new post-doc joins that lab? Will that piece of equipment you’ve been eyeing – due out next year – work with what you’re planning to buy?
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Consideration #2: Consumables |
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| For ease of use, nothing beats a pre-optimized kit and most systems offer a variety of these to measure the most common proteins (usually in sets like “pro-inflammatory cytokines”). Even if what you’re looking for isn’t available neatly packaged, you may be able to have kits custom made. But beware, notes Scott Van Arsdell, associate director of research for Pierce Biotechnology: not all platforms are supported equally. While some manufacturers let you choose from among close to 200 antibody pairs, the selection from others is far more limited. If the proteins you need to measure aren’t supported by your preferred technology, check to see if kits can be custom-made from your own reagents or those from a third party before giving up on that technology. |
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Consideration #3: How many analytes? |
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| Which system to choose may hinge, as much as anything else, on how many analytes you’re surveying. Some platforms are designed to examine only a few proteins at a time, while others can simultaneously perform 100 assays or more on a single sample, points out Grant Gibson, director of technical marketing at Luminex. When using certain systems – or kits designed to for them – you’ll be looking at a set number of analytes whether you need to or not, and that is reflected in the price of the assay. Each “answer”, notes Remick, typically costs about $1: you may end paying a lot for unnecessary tests. |
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Consideration #4: How many samples? |
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| A typical singleplex ELISA is set up in a 96 well plate, with the 50 microliter samples run in duplicate, or in serial dilution, along with a standard curve. Most multiplex assays are set up in a similar way, with each well assaying the presence of more than one analyte. The principle exception is the slide-based “chip” arrays that typically have 8 or 16 wells per slide. Depending on the specific slide system, these may be grouped together to allow up to 64 wells (56 samples) to be processed simultaneously, says John L. Tonkinson, marketing manager for array products and services at Schleicher & Schuell BioScience.
Whether each platform is capable of determining how much protein is present in the sample is still a matter for debate, but there is no quarrel that quantitation requires that some sample space is devoted to a standard curve. Thus, if this is an important consideration, make sure to choose a system with enough real estate to handle it.
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Consideration #5: Throughput |
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| Planar systems can generally be read in about 5 minutes (or less) because the entire slide or plate is scanned at once (or in just a few sections) says Brendan Yee, business manager for multiple analyte solutions at Beckman Coulter. On the other hand, microfluidic-based systems – that is, bead arrays – take 45-60 seconds to read a single sample, and thus require an hour or so to read a full plate. Given the 4 or 5 hours necessary to set up and run the assay, this is probably not much of a consideration if you’re just looking at a few (or even a few dozen) samples at a time. Yet for labs screening hundreds or thousands of samples, the speed at which your system can get through a plate or slide matters. Also at issue is the number plates or samples your system can handle at once: if you’re looking for higher throughput, consider a system with robotic handling capabilities. |
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Consideration #6: Comfort level |
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| Most multiplexing technologies are variations on the ELISA. They require roughly the same type of blocking, washing, and incubation steps (with adjustments for attached vs. suspended capture of the antigen, for example). Differences appear in the substrate the capture antibody is attached to: a polystyrene plate, a (coated) glass slide, a polymer bead, or even single-stranded RNA. They may differ, too, in the readout: -- a change in color, fluorescence, or light output, resulting from an enzymatic reaction or a change of fluorescence following laser excitation. And the signal may be detected by film, CCD camera, or photomultiplier tube. Analysis software – the final step – is often pre-packaged with the system, but in many cases can be purchased separately according to the user’s needs.
While some of these technologies have been around for a long time, others are revolutionary. Assess whether you’re an on-the-cutting-edge type of researcher, or whether you prefer the tried-and-true, before committing to a system. Along the same lines, assess the level of expertise needed to operate (and innovate with) the system, and the amount of resources you’re willing to commit to training – the learning curve for something you’re familiar with isn’t quite as steep.
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Conclusions |
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| There are a lot of good products on the market now that can answer a lot of questions at once. It’s tough to sort out claims and counterclaims made by competing companies, and it’s tough to know exactly how each fit with your particular needs. But when all is said and done, probably the two best ways to assess a technology are word of mouth and hands-on experience. Ask several real users what they like and dislike about a platform, especially with regard to any particular concerns you might have. And ask if you can try it out yourself, with your own samples (this may be pricey, so perhaps this is better asked of the company reps). And if worse comes to worse, make use of contract services until you’re ready to decide. |
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Josh P. Roberts
Contributing Writer
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