by Caitlin Smith
Do cells in an organism email each other? Yes, in a mass mailing sort of way. Cells use protein or peptide messages, rather than electronic ones, to regulate functions of cells elsewhere in the organism. These cytokines are chemical messengers that, in contrast to hormones, carry information over a wider distribution, show a greater change in concentration in response to stimuli such as infection, and are produced by many types of cells. “There is a greater understanding of the role that cytokines play,” says Debra MacIvor, product manager for biomarkers and immunoassays at Millipore. “Not only in inflammation and immune response, but also in the development and progression of cardiovascular disease, metabolic syndrome, and cancer.”
Sensitivity, specificity, and dynamic range
Several attributes of cytokines render designing assays for them fairly difficult – namely, their sometimes low abundance, and wide concentration ranges. “Most cytokine levels are very low in normal conditions, but they can increase to very high levels because of inflammation or challenge, such as by lipopolysaccharides,” says Xiao Qiang, R&D manager for biomarkers and immunoassays at Millipore. “Therefore, the assays need to be sensitive and dynamic ranges need to be broad. We will be evaluating a new Luminex system (FlexMAP 3D), which may generate assays with broader dynamic ranges.” Sample availability or scarcity can also compound these problems. “Sensitivity is a key issue and dynamic range can be a key issue for analyzing cytokines, especially when working with very small sample sizes,” says Sara Howland, product portfolio leader for drug discovery reagents at PerkinElmer. “One of the key steps forward with [PerkinElmer’s] AlphaLISA [platform] is that we have been able to develop ‘all-in-one well’ cellular assays. This means that we can grow cells up and detect all in the same well without any transfer or wash-steps...even when working with complex samples such as serum and plasma.”
Because good antibodies are a key component to cytokine analysis, specificity is also a concern with the newer multiplex assays. “As a researcher, I might want to know how multiplex cytokine analysis differs from traditional single ELISA assays,” says Amy Willson, technical product manager for Thermo Fisher Scientific. “For antibody-based assays (which most are), the quality of the antibodies is key. An antibody pair with excellent affinity and specificity is required to create a highly-sensitive, highly-specific assay. For multiplex assays, vendors must seek quality antibody pairs that work well together ... so there is some compromise.” Willson notes that this tradeoff also plays a role in optimizing buffers. “For single assays, buffers and procedures can be optimized for maximum sensitivity and sample recovery for each cytokine,” she says. “In a multiplex system, one buffer set is used with a variety of antibody pairs. Though the performance of some assays in the multiplex platform may not be quite as good as the single assays, the ability to obtain quantitative results for twelve cytokines from only 50 ul of serum is typically a worthwhile trade off.” Thermo’s newest platforms, the SearchLight Plate-based Protein Arrays and the ExcelArray Slide Arrays, are geared toward applications such as cytokine analysis. “The SearchLight platform is a multiplex, plate-based ELISA assay that utilizes a chemiluminescent substrate to analyze up to 16 different proteins using a small volume of sample,” says Willson. “SearchLight chemiluminescent arrays can be imaged in under 5 minutes, compared with about one hour for some other platforms.” Their new ExcelArray assays give quantitative results for 12 different cytokines per run.
Bender MedSystems offers Instant ELISA plates for cytokine detection. “The designation ‘Instant’ signifies that the addition of sample is all that is required to start the assay,” says Michael Krischke, director of marketing and sales at Bender MedSystems. “In contrast to the conventional ELISA plates, the Instant ELISA® plate contains, in a lyophilized form, the coating and detection antibody, streptavidin-HRP and sample diluent. Additonally, wells [with] the ready-to-use standard curve contain standard protein.” The resulting reduction of pipetting steps can save you time and decrease the chance of error or unnecessary variation. Krischke says that requests to refine their ELISA technology further led to the development of Bender MedSystems High Sensitivity (HS) ELISAs. “Assays with increased sensitivity are important for specialized purposes, or the evaluation of drug effects,” says Krischke. “The assay’s key feature, its high sensitivity, is backed up by an amplification technology.” Another means for detecting cytokines is PBL InterferonSource’s new iLite™ Human IFN-Alpha Kit, which measures the amount of Type I interferon in the medium of cultured cells. PBL InterferonSource also offers ELISA kits, various cytokines, and antibodies to them.
Cytokine origins
Recombinant cytokines are available from many several sources, such as PBL InterferonSource, Cell Sciences, and Millipore among others. Millipore’s vast collection includes cytokine panels for canines and non-human primates, along with humans, mice, and rats. More species are planned for 2009, according to Debra MacIvor, product manager for biomarkers and immunoassays at Millipore. “In December we will be launching two new mouse cytokine/chemokine panels, and in January we will launch three new human cytokine/chemokine panels. This will expand our total number of cytokines offered to over 90.”
But have you ever wondered from where your cytokines came? Most recombinant cytokines are produced by non-human expression systems, e.g. bacteria or CHO cells. However, non-human cells cannot perform the correct post-translation modifications, and sometimes cannot complete the proteolytic processing needed for some cytokines. This can have dramatic biological effects, so new expression systems in human cells are now emerging to remedy this.
HumanZyme makes cytokines in their HumaXpress™ human cell culture system. “Ours are suspension cultures, no serum, and no protein; so purifications start with a supernatant with virtually no other factors,” says Frederic Rhoads, VP of sales and marketing at HumanZyme. “This allows us to make an authentic, pure protein preparation without His tags or other types of modifications.” He notes that most scientists don’t think about the fact that cytokines produced in other expression systems – for example, insect cell or CHO cell expression systems – lack important post-translational modifications that are biologically relevant. According to Rhoads, cytokines expressed in human cells are more potent (up to 50,000 times) and easier to express, partly because human cells ensure proper folding, glycosylation, and other modifications. “We encourage scientists to discover how primary, secondary, tertiary, and quaternary structures that result from production in human cells increase the biological relevance of a protein.” For example, Rhoads says that in their hands, the noggin protein is ten times more active than other noggin proteins on the market.” In addition, Rhoads says that protein activity assays don’t always reflect biological relevance. “We saw biomarkers showing up with our noggin that didn’t show up with other noggins. So this biological relevance is not accounted for by activity assays.”
Symansis also makes recombinant cytokines in a human cell expression system, and avoids purification labels. Leanne Daly, sales, marketing and business development executive at Symansis, believes that expressing cytokines in human cells represents an advance because it “will allow researchers to understand more what is happening to proteins in the human body. There have been significant attempts to chemically modify non-human cell derived cytokines to be more human-like, but there is a growing awareness that in many instances, particularly in therapeutics, cytokines should mimic those found in the human body as closely as possible.” The closer the recombinant messages resemble the biological ones, the better the communication.