by Caitlin Smith
It is not surprising that cytokine analysis is a busy field—dysregulation of cytokines is known to lead to serious immune pathologies such as cancer, chronic inflammation, and autoimmunity. But perhaps few people know how complicated this field is. “Cytokine biology is exceedingly complex," says Brett Burkholder, manager for marketing and business development at RayBiotech. "Redundant and overlapping functions, synergistic and pleiotropic effects, temporal expression, all of these complicate the research. I see cells in the rest of the body being as complex as neurons, receiving multiple analog inputs but generating a digital output." Here are some current technology offerings that are aiming to make the complexity easier to handle.
Cytokine sample processing
ProImmune
offers a number of cytokine-related services that let you outsource some of your work—and help you process samples. ProImmune also provides high-throughput, validated ELISPOT analysis, a sensitive technique for measuring IFNg, IL-2, and granzyme B. Clinical trials occurring at multiple locations pose a sample processing challenge in ensuring that samples are collected and shipped consistently. “Sample logistics and preparation is probably the most important step in setting up successful assays for monitoring cell-mediated immune responses,” says Amanda Turner, marketing operations manager at ProImmune. “We have the experience and the capability to collect your samples wherever you are in North America and Europe and process them for maximum uniformity.”
Sampling handling is probably a bigger issue than most people realize, according to Burkholder. "A big challenge to improving cytokine analysis are sample matrix effects, and differences in apparent cytokine concentrations in samples due to different methods of collection, preparation, and storage of samples," he says. "No one has done more than a cursory study looking at consequences of these important considerations on the relative stability of cytokines." RayBiotech offers hundreds of sandwich-based ELISA assays for cytokine targets from several species, pre-printed antibody arrays, as well as custom array kits. RayBiotech has three assay formats: the C series (semi-quantitative, sandwich ELISA-based assay), the G series arrays (semi-quantitative, sandwich-based assay on a glass slide or chip with fluorescence detection), and the Quantibody assay (quantitative multiplex ELISA format with high-thoughput applications).
The antibodies used in RayBiotech’s G Series arrays are also used in their multiplex ELISA and single-target ELISA assays—a boon for those interested in biomarker studies. "Potential biomarker targets and target panels can be validated more quickly and easily using the exact same antibodies used to discover them," says Burkholder. "This can shave years off the time needed to proceed from identifying potential biomarkers to validation. Plus, many biomarker signatures work best when using several markers rather than a single one. The general consensus in the biomarker field is that multiple markers may fare better than single markers. As such, multiplex immunoassays may be essential for applying these new biomarker panels."
Another tool in sample processing concerns the types of media and reagents used—especially if you are using animal-free reagents for cells in culture. An increasing number of companies are developing animal-free reagents to meet this demand. For example, PeproTech offers a new line of animal-free recombinant proteins, and animal-free, serum-free media supplement kits for growing mammalian cell lines.
Cytokine screening
Cisbio Bioassays has developed a line of HTRF (homogeneous time-resolved fluorescence) cytokine detection assays for highly sensitive cytokine screening. “These assays have been used for both primary and secondary screening because they offer a number of cost-effective advantages over ELISA, multiplexed, and other classic methods in terms of throughput, robustness, sensitivity, and ease-of-use with extremely streamlined experimental setups,” says François Degorce, director of marketing and communication at Cisbio Bioassays. “Compared to ELISAs, our cytokine assays involve rapid, easily miniaturized protocols that can be run in a single plate from cell stimulation through assay measurement. They can be run as cell-based assays under the same experimental conditions, and are particularly adapted for being run as functional biomarkers or readout—for example, the modulation of a cell-surface receptor can induce a cellular response that can be seen through the secretion of a given cytokine (TNFalpha, IL1beta for instance).”
Degorce says that Cisbio is increasingly receiving requests for mouse cytokine ELISAs to be converted into their HTRF assays. “This is an area where our service offering is very appreciated, as we can provide anything from a simple antibody pair labeled with HTRF dyes, to a completely optimized HTRF assay,” says Degorce. “Our technical staff ensures the conversion of conventional heterogeneous immunoassays to an optimized high-throughput screening format, and ensures that this transition runs smoothly.”
Sometimes success in screening begins with smaller sample sizes. Life Technologies’ TaqMan® Protein Assays Open Kit enables you to make assays to many proteins, including cytokines. "The assay combines traditional immunoassay-style antibody binding with quantitative PCR for highly sensitive quantitative measurements, and enables analysis with much less sample than other protein assay technologies available today," says Tina Settineri, TaqMan protein assays product manager, lead genomic assays and molecular biology systems at Life Technologies. “This assay requires only 2 uL of starting sample (such as a cell lysate) for analysis, while other similar assays require 25-100 uL." Settineri says that the biggest challenges their customers face in cytokine analysis are sensitivity and sample size. "The TaqMan Protein assay technology, which uses antibody binding combined with proximity ligation and qPCR, addresses these challenges very well."
Wrestling with antibodies
A wide range of cytokine assays is offered by Gentel Biosciences, with over sixty array kits, high-density screening arrays, and quantitative sandwich assays. “The kits, instrument, and software are integrated to be ‘plug-and-play,’” says Dan Clutter, VP of commercial development at Gentel Biosciences. “The protocol is similar to ELISA in terms of ease-of-use, time-to-complete the assay and instrument price.”
Clutter says that while “we now have very high-quality quantitative 40-plex assays, and 100-plex assays for profiling, we are now looking forward to better tools for detecting post-translational modifications, and are developing new methods for investigating phosphorylation, glycosylation, and ubiquitylation of proteins.” For example, most antibodies recognize primary or secondary structure characteristics of proteins; however, a protein’s tertiary structure and post-translational modifications are of great importance biologically, yet are not recognized by most antibodies. “Cytokine assays will need to not only quantify the total number of cytokine polypeptides, but [also] the number that are phosphorylated, glycosylated, or ubiqutinated; that will require significantly improved assay performance and multiplex capability,” says Clutter. “In order to make the next technological leap, vastly improved affinity reagents will be required, and these will likely include non-antibody based reagents. Even though the number of antibodies is growing exponentially, they are not exponentially adding value due to the limiting characteristics of antibodies and the methods used to discover and manufacture them.”
Burkholder believes that a needed advance in cytokine research is finding methods of detection that don’t rely on antibodies, or at least immunologic methods for measuring absolute instead of relative concentrations. “The dirty little secret of antibody-based detection is that it does not measure absolute concentration of the antigen in solution, rather it measures the proportion of antibodies bound to antigen, which is based upon the concentration of the antigen with respect to the affinity of the antibody,” says Burkholder. For example, he notes that ELISA kits from three different manufacturers would probably use three different sets of antibodies with different affinities for the same antigen. You will likely measure three different concentrations if you analyze the same sample with the three kits. “Depending upon the epitopes recognized and the differences in affinities and standards, these differences can be 2-fold, 10-fold or 100-fold,” says Burkholder. "The sensitivities may be so different that only 1 of 3 kits will even detect the protein in your samples.”
Burkholder also questions the use of prokaryotically expressed recombinant antibodies for ELISA standards. "Post-translational modifications of proteins can have profound effects on the function and activity of proteins," he says. "I believe that comparison of results of immunodetection of native vs. recombinant proteins is suspect and introduces unnecessary and
intolerable uncertainty of results so obtained, but this is accepted as standard practice.” Burkholder remarks that there are more scientists today trying to use aptamers and other antigen-specific methods of detection rather than antibodies. “Proteins are messy,” he says. "DNA is a bit more predictable. Perhaps aptamers are the answer.” Time—and results—will tell.