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Products for cytokine research are expanding beyond that stalwart old workhorse, the sandwich assay. Recent market entries include assays for mRNA and new competitive assays. But the sandwich assay isn't ready to be put out to pasture yet. It's more versatile than ever, thanks to new detection methods, matched antibody pairs, and kits for animal cytokines. The ancient maxim known as Occam's Razor, that the simplest explanation is the best, may have met its match in modern-day cytokine research. Even William of Occam would throw up his hands in despair at finding any simple way to organize or understand the cytokines, small messenger proteins whose number increase monthly, as do their known functions and interactions. Chaos plagues even their terminology. Decades ago, peptides that acted against viruses were dubbed interferons. Similar peptides that influenced cell growth were discovered in various other disciplines and were named lymphokines and monokines, colony stimulating factors, and growth factors. Today, scientists recognize all these as sister members of a family of regulatory peptides that act over short distances. Yet what name this family should bear--cytokines? growth factors? something completely new?--is still up in the air. The situation is somewhat better for the individual cytokines. Most have had multiple names because each discoverer of a function named the protein after that function. Yet, usually a single name gains favor once several proteins are discovered to have the same sequence and in fact be the same molecule. Helping to organize the cytokines further is that they can be sorted into families based on receptor use. Still, more than 150 cytokines have been cloned, with more being constantly discovered, so the Babel of multiple names is likely to continue. The stunning complexity of cytokine biology has stimulated research on several levels, with manufacturers producing products for all these levels. At the most basic level, scientists discover new cytokines, sequence and clone them, and study their functions. Another level of research is using cytokines to explore the immune system and tease apart the interplay of the various cytokines. Here again, confusion reigns. Whether a particular cytokine stimulates or inhibits the growth of a particular cell type, for example, depends on what other cytokines are nearby and in what concentrations. Clinical research is yet another level. Because cytokines play prominent roles in inflammation and many diseases, cytokines hold incredible promise as therapeutics and as therapeutic targets. But progress has been at turtle speeds. Most cytokines have so many functions that taming them to one task is a formidable challenge. Only a few cytokines with relatively narrow functions have passed through Food and Drug Administration approval processes and emerged as prescription drugs. Supporting these various levels of research are dozens of companies providing hundreds of products. Popular products include recombinant versions of the cytokines and their receptors and antibodies to the cytokines and receptors. Another popular product has been kits for performing sandwich assays, so called because the sample protein ends up sandwiched between layers of antibodies. In this assay, one antibody captures the cytokine and holds it in place, and the second antibody, which is labeled, binds to the cytokine and serves as the basis for detection. The most popular sandwich assays are enzyme-linked immunosorbent assays (ELISAs), although newer assays, such as those using flow cytometry or based on amplification of the sample or its signal, are challenging their dominance. This year witnessed some shakeups among major suppliers. In April, Becton Dickinson and Company (Franklin Lakes, NJ) signed an agreement to acquire PharMingen (San Diego) as a wholly owned subsidiary. In July, Genzyme General (Cambridge, MA) signed an agreement to sell its research products business to TECHNE Corp. (Minneapolis, MN), the parent company of R&D Systems. 
The Challenge Of Keeping Abreast
"I'm continually impressed by how fast the fields [related to cytokines] are moving and how exciting the research is," says Robert Goldman, president of Peprotech Inc. in Rocky Hill, NJ, which manufactures recombinant proteins, including cytokines. Goldman singles out two families of cytokines his company finds particularly exciting: chemokines and the tumor necrosis factor (TNF)--related ligands. "Chemokines are a whole group of relatively newly discovered small proteins that play a very important role in inflammation research, research into asthma, and more recently into HIV-related research," Goldman says. "In particular, chemokine receptors have been identified as important for the entry of the HIV virus into cells." These receptors must be present with another receptor called CD4 in order for HIV to bind to T-cells. "After that discovery, the interest in chemokines just exploded," Goldman says. Researchers are now trying to nail down how certain chemokine receptors allow HIV to breach the defenses of cells; pharmaceutical companies could then look for inhibitors that might fend off HIV infection. Peprotech sells more than 30 human chemokines, including not only RANTES, Exodus-2 (also called 6Ckine), lymphotactin, and monocyte chemotactic proteins (MCPs) 1 through 4, but also eotaxin and fractalkine. Eotaxin has attracted the notice of researchers because it appears to play a powerful role in allergies and asthma. Fractalkine is particularly intriguing because it is structurally unlike the other chemokines. Unlike other cytokines, fractalkine and its receptors can act as adhesion molecules. Usually, chemokines and other cytokines are similar enough from species to species that a researcher can use human cytokines in experiments with other animals, Goldman says. Still, some cytokines are species-specific, and some researchers prefer to use the actual cytokine for their animal model. So Peprotech sells many mouse and rat cytokines in addition to the corresponding human cytokines. Another area of great interest in cytokine research right now for which Peprotech has developed a product line is the TNF-related ligands and their receptors. The grandparents of this family, TNF-a and TNF-b , were first isolated in 1985 after a long search for factors that could act against tumors. Since then, more than a dozen TNF-related ligand-receptor pairs have been discovered as well as various "orphan" ligands and receptors. The members of the TNF family have functions well beyond their first-discovered role as antitumor agents. Several members share a "death domain," an aptly named sequence that triggers apoptosis (programmed cell death). The TNF family also seems to take part in inflammation and regulating the immune system.
In addition to coming out with new cytokines and receptors as they are discovered, Peprotech has also focused on developing and releasing matched-pair antibody sets. Each set is a pair of antibodies to the same cytokine that have been tested to work well together in sandwich assays. One antibody is usually monoclonal and can be used as the capture antibody, and the other is polyclonal and can be used as the marker antibody. "Paired antibodies are essential for detecting the presence of cytokines in blood, serum, or culture media," Goldman says. For almost every cytokine, Peprotech has a corresponding polyclonal antibody. The company is now developing monoclonal antibodies to many of its cytokines, and "we are in the process of pairing monoclonal and polyclonal antibodies to check for compatibility for detection systems," Goldman says. He pinpoints three challenges that face companies that manufacture cytokines. One is to make the most exciting of the newly discovered cytokines and receptors available to researchers as soon as possible. He says Peprotech can usually have a product ready 4 to 6 weeks after a cytokine's sequence has been published, but some difficult cytokines take longer. Goldman singles out these difficult cytokines, which may require special expression systems or some special technology to purify the active protein, as manufacturers' second challenge. And finally, he points to the sheer volume of new cytokines. "Research is going so quickly," he says. "There are so many publications of new cytokines every week. Just keeping up with the science is another challenge."
Building A Better Mousetrap Improving on that mainstay of cytokine research, the ELISA sandwich assay, is a continual challenge for manufacturers in the cytokine field. One new approach to a better mousetrap is the CytoTrap Cytokine Stimulation Assay produced by BioSource International, Inc., of Camarillo, CA, which makes human, mouse, and rat cytokines and detection kits for cytokines and their genes. The CytoTrap is an ELISA that takes place entirely in a microwell and that has an unusual sample: cells. "With most ELISA kits, you're measuring a supernatant from a cell line or serum sample or whole blood," says Valerie Bressler-Hill, BioSource's Director of Marketing. "In the CytoTrap, you add your sample with the cells in it directly to the assay and then stimulate the cell to produce the cytokine of interest." "Where this is particularly interesting is in short-lived cytokines, such as IL-2 or IL-4," says Kevin Reagan, Vice President of Immunology. The CytoTrap assay measures the protein as soon as it's produced, so there's little time for it to be soaked up by soluble receptors or bound by other cells. In contrast, a regular ELISA run on serum for IL-4 generally detects nothing because IL-4 is so quickly absorbed. The CytoTrap assay takes about the same length of time as a regular ELISA. Currently, CytoTrap assays are available for mouse IL-2 and mouse IL-4 (human kits are also available).
"The swine is extremely important in the transplantation field because they are the most closely related model to humans," Reagan says. "In the transplantation area, there's a lot of cytokine work that's going on because the elicitation of certain cytokines predicts rejection." Another recent BioSource product is the CytoXpress line of assays for quantifying cytokine mRNA (rather than the cytokine protein itself). Reagan says that measuring protein can present a problem. Assays may not pick up some protein, either because the amount is below the sensitivity of the assay or because the protein that's produced is not released or is bound immediately. By measuring mRNA, the CytoXpress assay reveals gene expression closer to the start of the process. In the CytoXpress assay, the scientist converts the mRNA to cDNA and spikes the sample with a known quantity of a standard. Then the mixture is amplified by the polymerase chain reaction (PCR). The standard is designed so that the PCR primers bind both it and the sample. As a result, when the number of cDNA molecules at the start is between 20 and 20,000, the standard and sample amplify at the same rate. After the PCR reaction, the standard and sample are measured. Because the starting number of copies of the standard is known and because the standard and the sample amplify with equal efficiency, the relative optical density of the two allows easy calculation of the number of cytokine cDNA molecules at the start. Kits are available for measuring human and mouse IL-1b, -2, -4, -6, and 10; TNF-a; and IFN-g. Rat kits are available for most of these as well. For the future, Reagan and Bressler-Hill believe that current assay technologies just cannot be tweaked into doing everything that researchers need. Rather, companies will need to develop completely new technologies to overcome current limitations. For example, the four hours it takes to run even the fastest current assays is painfully slow when researchers want to monitor minute-by-minute changes in cytokine concentrations. And current assays that measure only one or two cytokines at a time can't reveal the intricate dance of interactions between cytokines. Placing further demands on future assays, Reagan says, is that increases in speed and number of cytokines detected shouldn't come at the expense of sensitivity. Larger samples would then be needed, something that's just not practical for researchers working with tiny animals such as mice.
On The Cytokine Frontier Chemokines are generating great excitement right now at R&D Systems Inc. of Minneapolis, MN, a company that focuses on cell surface receptors and their ligands. "I can't overemphasize the amount of interest in chemokines and chemokine receptors," Thomas Detwiler, R&D's Vice President for Scientific Affairs, says. "We consider ourselves the dominant supplier of reagents in the chemokine field." In fact, R&D first came out with a line of chemokines, antibodies, and ELISA kits several years ago--and they bombed. "We were ahead of the curve," Detwiler says. Only after the discovery that chemokine receptors allow HIV to infect cells did the current explosion of interest in chemokine research--and the corresponding boom of sales of chemokine-related products--take place. Currently, R&D sells about 330 chemokine-related products, including human, mouse, and rat chemokines; monoclonal and polyclonal antibodies (some biotinylated and some labeled directly with a fluorophore) to human, mouse, and rat chemokines; matched antibody pairs; and immunoassays for human and mouse chemokines. Of these products, about a quarter debuted in the past year. Another research area that continues to flourish, Detwiler says, is the transforming growth factor-b (TGF-b) superfamily. TGF-b was first identified in 1981, and its name reflects its ability to prod certain normal cells to transform into cancerous ones. Its roles in cells have turned out to be startlingly broad, however, and TGF-b has turned out to be a member of an extensive family of proteins, including the activins, the inhibins, and the bone morphogenic proteins (BMPs). TGF-b and its superfamily are involved in nearly everything in the body. R&D has recently started to expand its product line related to the TGF-b superfamily. Already out are polyclonal antibodies to human BMP-2; other activin, inhibin, and BMP products are in the works. R&D's cytokine product line has been growing in other directions as well. In particular, the Quantikine sandwich ELISA kit line is branching into two new areas. One area is to supply a greater range of kits for mouse and rat cytokines. Among the new kits, called M Quantikine kits, are 21 kits for mice and two for rats. The other branch is the Quantikine Ab kits for detecting autoantibodies to cytokines. "There is an increasing use of cytokines, especially hematopoietic growth factors, in therapeutic treatments," Detwiler says. "Some treated patients make antibodies against some of these, and some apparently healthy people who have never received therapeutic cytokines make autoantibodies against them." R&D Systems expects an increasing number of researchers to turn to studying the circumstances of development of these antibodies and their physiological consequences. The first two Quantikine Ab kits are for detecting antibodies to human IL-1a and human IL-6. For situations in which Quantikine assays are not sensitive enough, require too much sample, or do not have enough dynamic range, R&D Systems recently introduced a new line of chemiluminescent sandwich immunoassays called QuantiGlo. "These assays are based on an enhanced luminol reaction with horseradish peroxidase," Detwiler says, which gives off light. "This gives you an inherently greater sensitivity and range than is possible with measurement of light absorbance." The emitted light is read with a luminometer. QuantiGlo standard curves typically span from less than 1 pg/mL to more than 2000 pg/mL, and the assay takes only four hours. Currently, QuantiGlo assays are available for six human cytokines-IL-1b, IL-4, IL-6, IL-12, TNF-a, and endothelin--with more assays under development. "Diagnostic assays are being explored, but this [area] has been a disappointment to us and, I think, to many others as well," Detwiler says. "The relationships among cytokines are so complicated that claims for simple diagnostic applications are difficult."
Besides the kits for endostatin, VEGF, and bFGF, CytImmune also sells Accucyte kits assays for human IL-1a, -1b, -2, -3, -4, -6, -7, -8, -10, -13, and -15; epidermal growth factor; IFN-a; leptin; MIP-1a; RANTES; TNF-a; and TNF-b as well as six kits to measure mouse cytokines. Because new cytokines and new functions of existing cytokines are constantly being discovered, companies' plans for the future are necessarily hazy. Who knows what lies around the next bend? Still, it seems clear that both improving the sandwich assay and developing alternatives to it will be high on many companies' lists of priorities. The many advantages that have boosted the sandwich assay into its current central position in cytokine research likely will keep it there some time yet. But the assay's limitations are chafing more and more. Scientists are eager to have assays that are faster, that can measure several cytokines at once, that aren't confounded by protein-bound cytokines, and that work with unusual sample types, and companies will be eager to oblige. --Shauna S. Roberts Shauna S. Roberts is a science and medical writer in New Orleans |
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