t is difficult to name a class of molecules that has had more impact on biomedical research than cytokines and growth factors. As the pharmaceutical and biotechnology industries train their sights on cancer, AIDS, and various autoimmune diseases, the machinations of the immune response are slowly being revealed, and it is clear that cytokines and growth factors play key roles in orchestrating defense.
But the work they perform is still poorly understood. The term "cytokine" was originally coined to distinguish growth factors that influence immune function from those that affect other cell types. And in fact, some varieties of cytokines are released by nearly all known cell types, making this class of molecules a popular target for research.
Cytokines are released by immune cells in response to all manner of insult or injury (real or perceived), stimulating activation, growth, and differentiation among the immune cells they target. But with hundreds of varieties of cytokines and growth factors so far known, the tangled cascades of activation and response that regulate the immune function have eluded most attempts to tease them apart.
But that's not to say that folks aren't trying. In fact, research in the area continues to intensify as immunotherapies-such as Immunex's impending antirheumatoid arthritis drug ENBREL-spur efforts to understand the immune response.
And there is no shortage of cytokine and growth factor assays to choose from, as the accompanying table suggests. The majority of cytokine and growth factor assays (let's label them CGF) are based on the ELISA (Enzyme-Linked Immunosorbent Assay) methodology, with samples first applied to antibody-coated plates. A second antibody-selected to bind to a different region of the molecule and itself linked to a reporter enzyme-is added, followed by the enzyme substrate. This last additive is processed by the enzyme, producing a signal that can be quantified using a fluorometer, a luminometer, or measured visually. This technique has largely replaced the older radioimmunoassay-which relies on radiolabels to reveal the presence of the molecule-because it has a longer shelf life and produces no radioactive waste.
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To paraphrase an oft-quoted maxim: Your ELISA is only as good as your antibody. "You probably shouldn't start an ELISA unless you know the antibodies are appropriate... the capture antibody [should have] a high affinity for the ligand you're measuring," says Jeffrey Hasday, director of the University of Maryland at Baltimore Cytokine Core Laboratory (Baltimore, Md.). He also suggests purchasing a pair of antibodies that have been developed together, by the supplier, so that you can be sure they have the appropriate binding affinity. Hunting down individual antibodies from different catalogs to find the best deals could result in failed experiments and wasted time.
High background noise can often be traced to insufficient washing between addition of sample and addition of antibodies, or to incomplete blocking of the nonspecific binding sites on the plate. Low signal, on the other hand, may indicate a problem with the enzyme conjugate, low antibody concentration, or poor buffer composition.
Hasday suggests assigning one person to do all of your cytokine and growth factor assays: "When you have someone picking up a kit every few months, you're much more likely to have errors." The UMAB Core Laboratory takes it one step further, with an automated workstation set up for the assigned assayist.
But if you choose to scatter the assays among the members of your research team, check up on their performance once in a while. Keep track of the coefficient of variance (standard deviation divided by the mean) to see how scattered the results are with different users. It should be less than 5 percent. "Don't believe what they tell you [about reliability] in the kit... [especially] in the pharmaceutical industry, you need to use good laboratory protocol, including keeping very close track of performance and calibration," says Hasday.
And as it has seemingly everywhere DNA chip technology is having an impact on cytokine and growth factor assays. Gene fragments attached to high density oligonucleotide arrays allow the researcher to simultaneously probe a cell line to see which genes are currently being expressed. The technique does pose a cost problem, however, and like their less sophisticated probe cousins, DNA chips also fail to demonstrate the presence of protein.
These assays complement-and occasionally supplant-the more traditional bioassays that were initially developed to discern the presence and function of immune system signalling molecules. Bioassays are based on cell lines that respond to one particular factor or another, so that a sample containing that molecule will stimulate a measurable response among the cells, such as proliferation, chemotaxis, cytotoxicity, colony formation, cellular degranulation, or the secretion of other molecules.
Although it takes longer to perform and requires long-term maintenance of sometimes finicky cell lines, the bioassay has at least one major advantage over ELISA: it gives some measure of the activity of the cytokine or growth factor, rather than merely pointing out its presence or absence. But a growth response in the cell line could be due to other cytokines or growth factors-known or unknown-teeming within the sample, begging for additional experiments to confirm the effect.
In today's era of white-hot research competition, speed is often of the essence, and that quality seems to have vaulted ELISA to the forefront of CGF assays.
But like bioassay results, the results from ELISAs can be ambiguous. "The assays tell you that the part of the molecule that the antibody was raised against is present, but they don't tell you that it is intact. You could have fragments and still get a signal," says Jeffrey Hasday, director of the University of Maryland at Baltimore Cytokine Core Laboratory (Baltimore). Immunoblotting techniques-such as Western blotting-can at least confirm that the molecule is intact.
Other problems arise in CGF assays (see sidebar), says Hasday, who has logged many hours debugging detection experiments in his role as lab director: naturally occuring inhibitors-such as soluble receptors-are especially good at mucking up cytokine assays. Since the inhibitors usually target the binding site of the molecule, antibodies grown up against some other region are generally immune from inhibitor interference.
Immunoassays are also difficult to standardize, says Horst Ibelgaufts, an assistant professor at the Gene Center at Ludwig-Maximilians-Universität (Munich, Germany). "[In some cytokines], the same ELISA used in different laboratories yields comparable results, whereas different ELISAs usually detect the highest levels in the same samples but yield different absolute values. Such observations highlight the necessity of establishing international standards for all immunoassays," he maintains.
The concentration value that comes from an assay is based on a standardization curve, but unless that curve is calibrated to a universal standard (such as that of the World Health Organization [WHO]), you will likely get varying meaurements with the same sample using different ELISA assays. "In fact, even when the systems claim to be calibrated against WHO standards, you still get varying results using different ELISAs," Hasday says. "It's a very good idea not to switch standards from a line of experiments."
The technology for cytokine and growth factor assays doesn't seem due for an overhaul anytime soon. Ease of use and low cost have given ELISAs the dominant position in the toolbox, although as the methods are worked out and the cost falls, DNA chip technology will almost certainly become a standard technique in high-powered research labs.
Most of the tinkering with the ELISA technique is in the final visualization step. A variety of linked enzyme and reporter substrate concentrations are currently in vogue, with others being developed. Chemiluminescence, for example, promises a stronger signal and greater sensitivity than fluorescence technology.
Other developments are aimed at boosting the signal generated by the existing enzyme/reporter substrate pair, such as the use of secondary antibodies that are linked to multiple enzymes.
So, fear not. The kit you buy today won't be obsolete tomorrow. But tread lightly before you buy. The broad range of choices available makes it imperative that you are sure of your experimental parameters before you start going through assays.
"[Some] people work with cells and have no idea whether the experimental model system is going to produce the cytokine of interest or not," says Hasday, from experience. "They don't know the stimulus, what dose to use, or the time points. You can have a very nice, rigorous approach to sort that out by first looking at time points and then finding the optimal time to dose and measure the response. That can save a whole lot of assays and money."
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