Measuring Cytokine Levels

USING ELISAs

The enzyme-linked immunosorbant assay (ELISA) is the most popular way to detect cytokines and forms the basis for most other methods in use today. In its most basic form, a "capture" antibody on a solid support, generally one well of a 96-well plate, pulls cytokines out of a biological fluid such as serum. A secondary antibody to a different epitope on the cytokine is then added to produce an antibody-antigen-antibody sandwich.

This second antibody is the key to cytokine detection and quantitation. In some cases, it is conjugated to an enzyme that converts a substrate to a measurable product in proportion to the amount of cytokine bound. Alternatively, the antibody is conjugated to a ligand such as biotin, which can capture a streptavidin-coupled detection reagent. Or, fluorophores can be attached in place of enzymes, bypassing several steps of the traditional ELISA protocol.

Ready-made kits for a variety of human, mouse, and (to a lesser extent) other species' cytokines exist and are generally optimized for compatibility of the antibodies with each other and the assay format. Antibodies must recognize the native cytokine, for example, and should not compete for the same epitope.

But ELISA needn't be limited to one analyte tested per well; assays can be made to squeeze considerably more information from the same precious sample volume through multiplexing. SearchLight Proteome Arrays from Pierce Biotechnology of Rockford, Ill., for example, are essentially ELISA kits with up to nine antibodies bound in each well in array format. Meso Scale Discovery of Gaithesburg, Md., offers a multiplex platform in which proteins are captured by antibodies deposited onto built-in electrodes. Secondary antibodies labeled with ruthenium organometallic complexes enable detection of the bound cytokine by a proprietary reader, which applies current to the electrodes and reads the resulting electrochemiluminescence.

BEAD IT

Other cytokine-detection systems are variants of the ELISA theme. One popular approach has the reaction occur on the surface of an addressable bead, rather than in a microtiter plate. Among these options, Luminex of Austin, Texas, is "probably the best single worked-out platform" for multianalyte analysis, says Calman Prussin, chair of the NIH's Cytokine Interest Group. The system, says Valerie Bressler-Hill, vice president for marketing of Biosource International of Camarillo, Calif., a Luminex partner, "is just fantastic" for scientists who want to profile a disease state, for instance, or an animal model.

The Luminex system can distinguish among 100 different polystyrene spheres. The company supplies raw, internally dyed 5.6-micrometer beads to "value-added resellers" such as Biosource, which modifies them and creates kits for end users, says Grant Gibson, Luminex's director of technical marketing. Assays are set up like a fluorescent ELISA, with beads acting as the solid support. The bead's color indicates the identity of the cytokine, while the amount of secondary fluorophore reveals the quantity captured. Instruments and reagents are available only through resellers, sometimes under a private label: the LiquiChip from Qiagen, Netherlands, and the Bio-Plex platform from Bio-Rad Laboratories, Hercules, Calif., for example, are rebranded Luminex 100 systems.

The Cytokine Bead Array (CBA), from the Pharmingen division of San Jose, Calif.-based BD Biosciences, employs a similar concept for flow cytometers. But while Luminex uses a flexible, open system, CBA kits "are built around a particular physiological motif," enumerating Th1 and Th2 cells, for example, or inflammation, says BD's marketing director Dara Grantham Wright.

Manufacturers such as Bangs Laboratories of Fishers, Ind., market internally dyed beads with streptavadin or goat-antimouse IgG for investigators wishing to create their own flow cytometry bead assays.

CHIPS AHOY

Array-based systems have also been developed. Biosource's PVDF membrane-based Cartesian Array provides a simultaneous, qualitative measure of 18 cytokines in a sample. But because it yields only a quick snapshot of the presence or absence of cytokines, it is not the main method of screening for cytokines, says Bressler-Hill. Researchers are looking for quantitative measures, she says, and a number of companies have begun offering tools to provide exactly that.

Keene, NH-based Schleicher & Schuell BioScience's FAST Quant, for example, measures nine cytokines in 56 samples and "combines the concept of a microarray with an ELISA standard curve," says John Tonkinson, marketing manager for array products and services. The arrays match or exceed the sensitivity of an ELISA, he says.

Other solid-state arrays, such as EMD Biosciences' ProteoPlex, promise both high sensitivity and quantitative measurements as well. That turnkey kit, which David Smaller, vice president for corporate development of the Merck affiliate in Darmstadt, Germany, calls "an ELISA on a slide," measures 12 cytokines in quadruplicate, in each of the slide's 16 wells.

Finally, Zyomyx of Hayward, Calif., offers a complete Protein Profiling Biochip System, consisting of a chip-processing station, a chip reader, and antibody arrays capable of measuring 30 cytokines in six samples simultaneously. Both human and murine cytokine chips are available.

INTERNAL AFFAIRS

ELISA, array, and bead-based techniques measure secreted cytokines. But several companies make flow-based kits that allow researchers to permeabalize and stain cells for the presence of cytokines before they leave the cell. Cells can be counterstained for cell-surface markers as well.

Going one step further, Miltenyi Biotec of Bergisch-Gladbach, Germany, offers flow-based Cytokine Capture Assays that essentially glue cytokines to the cells. They are "designed to analyze cytokine-secreting cells while they're still viable," says Miltenyi technical scientist Roger Burger.

In the plate-based ELISPOT assay, secreted cytokines remain attached to an antibody-coated plate after the cells are washed away. "Now what you're actually measuring is the number of cells in a population that secreted the cytokine," says Ann Nielson, senior technical correspondent at R&D Systems of Minneapolis.

OTHER TOOLS

Scientists use a variety of other tools to detect cytokines in more specialized circumstances. In many cases, old techniques work best. Immunohistochemistry, for example, remains the preferred method to localize proteins in sectioned tissue. And since most immunoassays detect both active and inactive cytokines, including those bound to a soluble receptor, some researchers still rely on the exquisitely sensitive, "good, old-fashioned bioassay," in which cytokine is required for the growth (or death) of an indicator cell line, explains Prussin.

But new techniques continue to emerge. Finkelman, for instance, noting that cytokines do not normally tend to accumulate in serum, has devised a way to measure in vivo cytokine production by injecting an animal with anticytokine immunoglobulin, yielding a stable complex that can be retrieved by bleeding. BD-Pharmingen markets a version of this test for detecting interferon-gamma in mice.

Josh P. Roberts tcwriter@msn.com is a freelance writer in Minneapolis.