by Laura Lane
Wanted: Members of a notorious gang of molecules suspected of violating regulations of normal cell growth, inciting inflammatory reactions, spreading misery, and causing pain and suffering.
Cytokines can run but they can’t hide. With scientists in both industry and academia hot on the trail, this heterogeneous group of suspects will soon find their exploits revealed. To the uninitiated, the task seems deceptively simple. The experienced will tell of chasing one cytokine only to find, unexpectedly, that its actions also contribute to healthy processes. The truth is that the above advertisement could also read like this:
Help Wanted: Associates to relay messages between cells, facilitate immune system response to infection, oversee cell growth and programmed death, delegate tasks to appropriate receptors, and perform other tasks as needed to maintain the finely tuned balance of cell physiology.
Clearly, those willing to study the complex world of cytokines must share great purpose, great incentive, or a great sense of humor—or all of the above. These researchers must see the tremendous value in delineating the varied roles of each cytokine and discovering drugs to influence and mitigate such functions. So do the many companies that are offering a wide variety of technologies and tools to aid and abet cytokine-focused efforts. Developed with the wisdom collected over decades, today’s assays, antibodies, and accessories carry an ever-increasing promise of speed, sensitivity, and specificity.
Such improvements are becoming more necessary, as researchers face a growing list of cytokines, many of which could serve as drug targets. However, pinpointing those with the greatest potential is becoming more difficult, because cytokines circulate only in very low concentrations and can be bound to other proteins.
“It’s a challenge to measure low levels of cytokines in blood with a high degree of accuracy,” says Jehangir Mistry, director of research & development for Millipore Bioscience's drug discovery unit, pointing to the sensitivity limitations of currently available methods. This is coupled with complications arising due to interfering molecules present in the blood.
The lack of uniformity across various immunoassays also ranks high on researchers’ lists of frustrations, Mistry adds. “One of the major challenges right now is trying to measure cytokines across different platforms and different manufacturers.”
The problem is that each manufacturer uses its own kinds of antibody pairs and recombinant cytokine standards, resulting in assays with varying capabilities of assessing the diverse molecular forms of cytokines and eliminating or reducing the effect of interfering molecules in the blood.
“This leads to an important question: How does one harmonize the assays across different platforms and make use of data from one assay used in one lab with data from another lab with another assay,” Mistry says.
For some companies, the answer involves going back to basics. AbD Serotec uses technology from its parent company, Morphosys AG, which has developed the HuCAL (human combinatorial antibody library) technology that aims to produce antibodies identical to ones found naturally in the human body. Unlike traditional methods of using mouse hybridoma cells to produce monoclonal antibodies, the HuCAL technique is “analogous to the phage display-type technology, in which you select out binders,” says Andrew Lane, scientific affairs and licensing manager at AbD Serotec. “Key advantages are that you can really direct a system to block out unwanted cross-reactivities to closely related cytokines, and you can also easily develop pairs of reagents for sandwich assay applications.”
The company plans on releasing its first anti-cytokine antibodies later this year. Developed via the HuCAL technique, these antibodies should fit the bill for researchers who are “very reliant on antibodies detecting the native molecule,” Lane says. “That’s honestly what they’re interested in.”
Also seeking to fulfill that need is Apollo Cytokine Research. The company specializes in developing and manufacturing cytokines and soluble cytokine receptors using human embryonic kidney cells, which are also referred to as 293 cells. As human cells, they produce proteins with not only native structures, but also with the appropriate post-translational modifications, says Leanne Daly, the company’s president.
“Producing proteins that are identical to those in the human body is a huge step,”
Daly says. “You can’t do the same thing by making proteins in rabbits or mice and then humanizing them.”
While companies like Apollo are tackling the issue of specificity, other companies are seeking to overcome the challenge of sensitivity. Finding solutions is more important than ever as pharmaceutical and biotechnology companies use high throughput and high content screening methods to discover new drugs, says Krista Steger, director of marketing at Cisbio. The need for efficiency has lead to homogeneous assays that circumvent the step of washing.
“Then you lose sensitivity,” Steger says. “It’s inherent with any homogenous assay and no matter what technology you’re talking about. And then it gets more difficult with miniaturized assays and detecting even lower levels of cytokines.”
Efforts to ameliorate the predicament include developing probes that can minimize noise. Cisbio’s strategy has been their HTRF technology that combines the advantages of time-resolved fluorometry (TRF) and fluorescence resonance energy transfer (FRET). Taking from TRF, the company developed a donor fluorophore for europium cryptate, which has long-lasting emissions. While the donor perpetually produces emissions of 620 nm, the acceptor fluorophore, a modified allophycocyanin, produces emissions of 665 nm only with specific binding events, as is the case with FRET.
The company offers a wide assortment of antibodies that come labeled with the HTRF fluorophores, alone or packaged as cytokine assay kits. You can also purchase kits that enable you to label your own antibodies and create assays for whichever cytokines you’re currently studying.
Improved detection chemistries can also help with multiplexing strategies that both speed up drug target screening and enable the biomarker profiling of diseases. “The multiplexing platform has really opened the door for cytokine research where people can study various cytokines simultaneously and understand which are expressed in different disease states,” Millipore’s Mistry says.
Currently, the company offers assays that allow the simultaneous study of up to 30 cytokines. Based on the multiplexing Luminex platform, the assay kits provide beads that each have a different spectral signature and are tethered to an antibody against a specific cytokine. The bead reader detects both the signature of the bead and any signals resulting from antibody binding.
In the near future, Luminex will be introducing their multiplexing format with magnetic beads, Mistry says. The new beads could prevent clogging that can occur with extraneous components found in bodily fluids.
