Antibody microarrays encompass different platforms, sizes, and species, and may provide either a qualitative or quantitative readout. But one thing they all have in common is the ability to generate highly multiplexed data. Here, we explain how antibody microarrays are used and highlight the benefits they bring to scientific research. We also comment on the importance of high scan resolution to accurately discriminate between microarray features and background.
A technology that answers many different research questions
Antibody microarrays cover a wide variety of targets and are often designed based on protein class, signaling pathway, or research area. According to Adriana Lagraulet, applications manager at Innopsys, a main application of antibody microarrays is to evaluate the expression of protein biomarkers within a sample. “Using either proteome-wide or targeted antibody microarrays, it is possible to screen for protein biomarker signatures associated with different disease stages or to evaluate protein activation and cell signaling,” she says. “Antibody microarrays are also used for rapid identification of pathogens that share similar clinical presentation. Recently, researchers from the Smorodintsev Research Institute of Influenza at St. Petersburg, Russia, developed an antibody microarray capable of differentiating between six common viruses causing upper respiratory disease. This showed limits of detection slightly lower than those observed with classical ELISA.”
Detection of 10–2,000 targets in parallel
The number of targets detectable by antibody microarray varies considerably. Kelly Whittaker, senior technical support specialist, explains that RayBiotech’s antibody microarray offerings range from 10–2,000 targets and may be either semi- or fully-quantitative. “Semi-quantitative antibody microarrays compare relative changes in protein expression across different experimental conditions,” she says. “In contrast, fully quantitative arrays include a standard curve that is used to determine the concentration of the proteins detected. Oftentimes, researchers might use a large, semi-quantitative screening array to identify a smaller panel of interesting targets before moving to a more focused antibody microarray that may, or may not, be fully quantitative. Single-target ELISAs or other complementary platforms can be used to confirm results or investigate specific proteins in more detail.”
Bio-Techne’s antibody microarrays also enable researchers to perform large, semi-quantitative discovery screens and select targets for follow-up studies. “Our antibody microarray products allow for multiplexing from 25–119 analytes and follow a similar workflow to western blot detection,” reports Catherine McAvoy, product manager. “Samples are incubated on the antibody-spotted membranes, then a cocktail of biotinylated detection antibodies is added. Next, the captured proteins are visualized using streptavidin-HRP and chemiluminescent detection reagents, and the membranes are processed using film or a digital imager.”
Image: An antibody microarray workflow. Image provided by Bio-Techne.
RayBiotech’s nitrocellulose-based antibody microarrays also employ chemiluminescent detection, while the company’s glass-based products instead use a streptavidin-conjugated fluorophore to produce a fluorescent readout. An advantage of glass-based antibody microarrays is the reduced sample volume requirement; just 50 µL of sample is needed for quantification of up to 40 analytes. “A further benefit of glass-based antibody microarrays is that the fluorescent signal is very stable (unlike chemiluminescence, which decays in minutes) and offers a wider dynamic range. Additionally, the slides can be nested into a tray that matches a standard 96-well microplate, allowing for automated processing of 64 arrays simultaneously,” adds Whittaker. “The latest automated liquid-handling workstations can accommodate several trays at once, allowing hundreds of samples to be processed per day.”
Nitrocellulose is widely used as an antibody microarray substrate due to its well-documented protein binding properties. “Nitrocellulose binds antibodies non-covalently to retain the tertiary structure,” notes Ilenia Bertipaglia, Ph.D., technical support representative at Grace Bio-Labs. “This preserves the antigen-binding capability to ensure maximum sensitivity. Our nitrocellulose substrate-based products include microscope slides coated with an ultra-thin nitrocellulose film that is designed to reduce background fluorescence and maximize signal-to-noise ratios, and porous nitrocellulose film slides that comprise a microporous nitrocellulose film cast on a variety of solid surfaces. Porous nitrocellulose provides increased binding capacity to enhance detection of low abundance proteins.”
Benefits of antibody microarrays
Besides allowing multiple targets to be screened simultaneously, antibody microarrays offer many additional benefits. “A major advantage of antibody microarrays is that they minimize sample volume requirements,” says Whittaker. “Researchers using antibody microarrays can gain a broad overview of protein expression in a sample volume similar to that used for a single target assay such as ELISA. This can be especially important for species or samples where volume or protein content is restricted.” Bertipaglia adds that antibody microarrays are also incredibly sensitive as well as enabling high-throughput workflows. “Moreover, because of the low quantity of antibodies required, and the reduced amount of plastic, antibody microarrays are more economical and greener than many other methods,” she explains. “And, compared to techniques such as mass spectrometry (MS) proteomics analysis, antibody microarrays are much quicker and easier to implement and do not require a highly trained workforce.” Overall, these features of antibody microarrays make them a cost-effective tool that can save both time and money.
Importance of high scan resolution
With antibody microarrays often being used to study both rare and abundant proteins within the same sample, a highly sensitive scanner is essential. Innopsys’ InnoScan microarray scanners incorporate dedicated lasers and photomultipliers that increase sensitivity and allow for resolutions up to 0.5 µm pixel size. “Another key feature of the InnoScan systems is the extended dynamic range (XDR) option,” comments Lagraulet. “This allows for acquisition of 20-bit images with a dynamic range of 6 logs, which is of particular interest for antibody microarrays because one can find very heterogeneous expression of proteins in the same microarray. In classical 16-bit images, saturation often makes protein quantification and comparison between samples difficult. However, with XDR, the saturation point is enlarged to more than 1 million fluorescent units such that weak-expressed and high-expressed proteins can be accurately detected in a single step.” The value of XDR is demonstrated in a recent publication, where researchers used antibody microarrays to discover the link between leptin expression and COVID-19 severity in obese patients.