When it comes to detecting specific proteins in biological fluids, the gold standard is ELISA.
A simple “sandwich” immunoassay, the “enzyme-linked immunosorbent assay” uses a plate-based “capture” antibody to pull down the protein of interest and a second “detection” antibody to quantify it.
The assay is fast, simple and high-throughput. What it is not, is multiplexed: A traditional ELISA quantifies one, and only one, analyte. Increasingly, however, researchers need more from their samples, especially when they’re handling precious samples, such as biopsies.
Fortunately, there exists a range of multiplexed immunoassay formats to help. Based on the same sandwich-format principles, the platforms differ in terms of hardware, plex level and throughput. But all enable researchers to maximize the value of their samples, squeezing ever more information from ever smaller volumes.
Multiplexing platforms
Multiplexing serves a number of useful purposes, says Thao Sebata, senior product manager for Thermo Fisher Scientific’s immunoassay portfolio. Most obviously, by producing more data points per reaction, it saves researchers time, money and reagents. Instead of running 30 different ELISAs, for instance—a process that could take weeks—a researcher could quantify 30 proteins at once using a multiplexed platform.
But the biggest advantage, she says, is in sample usage. One 30-plex assay consumes as much material, more or less, as one ELISA—a 30-fold savings in material. That may not matter if your sample is an abundant cell-culture supernatant. But “if you have limited, precious samples, such as tear or saliva, or [you’re] using a mouse model, where you can only squeeze so much blood out of a mouse, that’s really the biggest advantage,” she says.
Another benefit, says Angelica Olcott, a product manager at ProteinSimple, a division of Bio-Techne, involves confidence in the resulting data. Many biological phenomena operate through pathways involving multiple proteins, Olcott explains. “Seeing a response from multiple targets can give more confidence [that the observed response] is a real response.”
Researchers can exploit those advantages using any of several different platforms, ranging from low-throughput but high-plex to relatively low-plex but high-throughput.
Among the highest-plex immunoassays are antibody microarrays <https://www.biocompare.com/Molecular-Biology/7473-Antibody-Microarrays-Antibody-Arrays/>, in which dozens to hundreds of capture antibodies are arrayed on either a flexible membrane or a glass substrate. The RayBiotech Human Cytokine Array G4000, for instance, arrays 274 capture antibodies on five glass slides, and MilliporeSigma’s Panorama Antibody Array – Gene Regulation I contains 112 antibodies on a single slide.
Such assays provide a broad overview of how protein abundance changes from sample to sample, says Scott Van Arsdell, vice president of immunoassay technology at Aushon BioSystems. But they are qualitative rather than quantitative, and they’re impractical for large sample numbers. “You’re not running standard curves, you’re looking for effects and trends. It’s a screening tool to identify biomarkers that are affected by a treatment.”
They also are relatively inflexible in terms of customization, as any change in layout requires printing and testing a new array.
At the other extreme are ELISA variants in which multiple antibodies are spotted per well of a microtiter plate. Aushon BioSystems’ Ciraplex ULTRA multiplexed assays, for instance, include up to 12 antibodies, arrayed in a circle around the perimeter of the well. Detection is imaging-based, using a chemiluminescent reaction.
Why circular? As plates rotate on shaking platforms, fluid naturally is pushed towards the edge. “You get better fluid distribution with a circular array pattern than with a grid arrangement,” Van Arsdell explains.
Similarly, Meso-Scale Diagnostics’ MULTI-ARRAY assays include up to 10 capture antibodies per well of a 96- or 384-well plate. Detection is based on electrochemiluminescence, a process that uses electrical current to stimulate enzymatic light emission.
Occupying the middle ground are solution-based bead assays, such as those that run on the Luminex xMAP® platform. Luminex assays use a series of polystyrene or magnetic beads, each coded with a unique blend of different fluorescent dyes, such that each assay (e.g., IL-2 or IFN-gamma) is assigned to a unique color channel. A second fluorescent antibody quantifies the reaction, which is read out using either a specialized flow cytometer or a fluorescent imager. Because dozens to hundreds of beads of each type are quantified per sample (that is, each data point is replicated many times), the resulting data are highly robust, says Daniel Braunschweig, global product manager for Bio-Rad Laboratories’ Bio-Plex assays, which are based on xMAP.
(Other vendors have developed similar tools for standard flow cytometers, such as Miltenyi Biotec’s MACSPlex assays and the BD™ CBA Flex Set assays from BD Biosciences.)
The xMAP assays permit 100 distinct color channels per reaction, but the assays usually top out well below that because of incompatibilities in assay conditions or antibody cross-reactivity. For instance, says Anthony Saporita, an R&D scientist at MilliporeSigma, some assays require undiluted (“neat”) samples, whereas others expect less concentrated ones. Vendors can usually skirt such issues, Saporita notes, by creating smaller subpanels—splitting one 40-plex panel into, say, two 20-plex assays.
Bio-Rad, with 454 singleplex assays available, has as its largest panel a 40-plex human chemokine array, says Braunschweig, though a 48-plex panel is in development. Similarly, MilliporeSigma’s largest MILLIPLEX panel quantifies 41 human cytokines and chemokines, and Thermo Fisher Scientific’s largest panel profiles 35 human cytokines. (According to Sebata, cytokine/chemokine assays tend to be among the most popular Luminex assays, thanks to growing recognition of the importance of the immune response and inflammation in neurological disease, cancer and other illnesses.)
ProteinSimple offers another multiplex format. Called Simple Plex, the assay runs four ELISA assays in miniature “glass nano reactors” (GNR) housed in a microfluidic plate, says Olcott. Because the targets are kept separate, the assay isn’t truly multiplexed—each GNR performs only one reaction, though they occur in triplicate within the channel; but users can test as many as 16 samples simultaneously, for a total assay throughput of 64 results.
With all reagents preloaded on the plate, and all incubations and wash steps automated, user-to-user variability is minimized using Simple Plex, Olcott says. “Variability is in the range of 7% CV or less”—a degree of reproducibility that should make the assay particularly attractive for use in late-stage clinical trials, she says.
(Bio-Techne offers Luminex-based assays through its R&D Systems division.)