New Immunoassay Products Let Users Put New Twists On Old Themes

Advanced Chemtech AFfinity Bioreagents Biodesign International Bio-Rad Laboratories BioSource International Boehringer Mannheim Biochemicals Cardinal Associates Inc. Immunochemistry Technologies Peninsula Laboratories Inc. Tropix Inc. Wako Chemicals USA Inc. Wallac Inc. A quick inventory of nearly any molecular biology laboratory these days will turn up a kudzu-like infestation of monoclonal antibodies, fluorescent-tagged secondary antibodies, and immunoassay kits. Immunoassays have become de rigueur for scientists studying cellular proteins. As a result, research supply companies are producing a variety of monoclonal and polyclonal antibodies to an ever-proliferating number of proteins. While each month seems to bring yet another protein into the immunoassay fold, new products are allowing today's scientists to put a new twist on some old themes. Using antibodies to identify, quantify, and isolate proteins may be a ubiquitous research activity, but new products have scientists turning these workhorses of the immune system into tools to probe the immune system itself. Improvements in nonradioactive detection methods now provide scientists with sensitive, nonhazardous immunoassays. Even some classic molecular biology techniques like reporter gene expression have been updated using immunoassay techniques. Immunoassay methods take advantage of the finely tuned system mammals possess for dealing with foreign substances. By and large, when the immune system of an animal -- be it rabbit, goat, or human -- discovers a non-self particle such as a viral coat protein or a bacterial membrane protein, it stimulates mature B lymphocytes to produce antibodies against the non-self particle or antigen. The antibodies bind only to the specific antigen that triggered the antibody production in the first place, making the interaction highly specific. It is the specificity of the antibody/antigen interaction that researchers exploit in immunoassay techniques. By creating antibodies that specifically bind to one protein, scientists can ultimately detect the quantities of that protein in a sample or locate it in a cell or on a western blot. However, this process requires a detection system. Classically, researchers performing immunoassays have relied upon radioactive isotope-labeled antibodies to visualize and quantify the amount of antibody-bound protein in any given sample. While the signal is readily detected by X-ray film or liquid scintillation counting, those procedures require laboratory personnel to be trained to handle radioactivity in addition to creating a fair amount of wet and dry radioactive wastes. However, heightened concerns over the safety of handling and disposing of radioactive materials as well as intensified regulations worldwide regarding uses of radioactive materials have led to an increasing demand for reliable nonradioactive means of detecting antibody-bound protein. FOR GREATER SENSITIVITY: The more than 50 Cytoscreen ELISA kits from Biosource International use an indirect conjugate of horseradish peroxidase with streptavidin and a biotin-linked detection antibody. For years, enzyme immunoassays (EIAs) and fluorescent tags have allowed researchers to forgo radioactive labels. By far the most common of these assays is the enzyme-linked immunosorbent assay, or ELISA. For these assays, the antibody is usually attached to the walls of a microtiter plate and the sample is added. Either horseradish peroxidase or alkaline phosphatase is usually linked to a secondary antibody to create a "sandwich," and the enzyme reacts with substrate such as biotin to produce a color change that can be read and quantified in a microtiter analyzer. In general, such strategies work well, but recently several companies have tweaked their systems to provide enhanced sensitivity. Earlier this month, Bio-Rad Laboratories in Hercules, Calif., introduced a chemiluminescent immunoblot kit called Immun-Star. The system uses alkaline phosphatase linked to a secondary antibody and the dioxetane substrate known as CDP-star, which is manufactured by Bedford, Mass.-based Tropix Inc., to produce flashes of light on a standard western blot that can be recorded on X-ray film. The kit provides sensitivity to the 10 picogram level, which rivals radioactive immunoblots. But Bio-Rad product manager Eric Larsen claims that his company's product has some advantages over conventional radioactive immunoblotting. "Immun-Star produces a long-lived signal that generates a clean, clear blot in 30 seconds to five minutes," he explains. "That's much faster than a radioactive immunoblot, which can take from an hour up to two days." Alain de Bruin, a veterinary medicine specialist from the Institute of Animal Pathology at the University of Bern in Switzerland, who performed beta-testing for Bio-Rad, comments that Immun-Star, "like many chemiluminescent systems, produces a very strong signal, but Immun-Star's signal is long-lived enough to allow us to detect it on chemiluminescent screen and digitize it on the computer as well as place it against X-ray film." Bio-Rad maintains that the signal is strong enough to measure 24 hours after activation and that the blot can be reactivated with substrate weeks later. The Immun-Star kit also provides an enhancer that allows researchers to use standard nitrocellulose rather than PVDF membrane. To ramp up the sensitivity of its streptavidin horseradish peroxidase (HRP)-based ELISA kits, BioSource International of Camarillo, Calif., used an entirely different method. Typical ELISA kits involve a microtiter plate coated with the antibody to the desired molecule, and a second antibody used for detection that is labeled with either enzyme or biotin. For its UltraSensitive Enzyme Immunoassays, BioSource labels the secondary antibody with several molecules of biotin and then attaches multiple HRP molecules to streptavidin, which binds to biotin. As a result, each bound antibody produces a greatly enhanced signal. BioSource vice president Madelyn Baran notes that the assay is capable of detecting various molecules at the range of 150 femtograms to 10 picograms, which "is within the range of certain cytokines in the blood." Gaithersburg, Md.-based Wallac Inc. has taken an entirely different tack. Instead of enhancing existing enzyme-based detection systems, the company has developed a direct labeling method that does not involve radioactive isotopes. With the radioactive labeling method, each antibody that binds to an antigen produces a signal, which allows for a direct measurement of antibody-antigen binding. With enzyme-based detection systems, the signal results from a chemical reaction that happens as a result of antibody-antigen binding rather than a direct measure of the binding. Instead of radioactivity, the Delfia Research System by Wallac measures the fluorescence of metals from the lanthanide series, including europium, samarium, and terbium, chelated to molecules that fluoresce. Using a time-resolved fluorescence, the Wallac system first excites the metal chelate and then measures the resulting fluorescence in the absence of background excitation. Fray Lindqvist, a Wallac product manager, notes that because the excitation wavelength and the emission wavelengths are so different, the Delfia Research System measures only the emissions from a metal chelate, much in the same way that a liquid scintillation counter measures the emission of alpha, beta, or gamma particles from a radioactive isotope. Scientists can label antibodies for immunoassays or DNA for hybridization using the metal chelates. "The Delfia Research System's great advantage over radioactive labeling comes from the fact that antibodies labeled with lanthanide chelates can be stored for up to a year," says Lindqvist. "With radioactive isotopes, you have to relabel every month." As compared with enzyme immunoassays, the Delfia Research System directly and accurately measures a much wider range of concentrations. Lindqvist notes that initial costs of Delfia are quite similar to those of radioactive isotope labels, but "when you factor in costs of waste disposal, monitoring, and training, labeling with lanthanide chelates is more cost-effective." Since the first monoclonal antibodies were constructed 20 years ago, scientists have been using immunochemicals to purify proteins and visualize cell components. While it seems as if every possible variety of monoclonal antibody has been produced, BioSource International discovered an unfilled niche in the market for monoclonals. "Last January, there was not a single immunoassay kit to a rat cytokine on the market," says BioSource's Baran. "And when we asked our customers what they needed, the answer was 80 percent or greater rat, rat, rat." The demand, according to Baran, comes from the fact that rats are fast becoming an important animal model of the immune system. "It's just easier to work with a larger animal," she points out. "And rats, surprisingly, are much more calm animals than mice." BioSource currently has ELISA kits available for rat Il-4, TNF-alpha, Il-1-beta, and interferon-gamma. Anna N. Taylor, a professor of neurobiology at the University of California, Los Angeles, and her research associate Delia Tio have been using the BioSource rat TNF-alpha and interferon-gamma kits to study the effects of fetal alcohol syndrome on the immune system. "We started using the rat kits simply because there weren't any others available," notes Taylor. Tio points out that the kits' strength lies in their simplicity and performance. These ELISAs give results in "an easy half-day's work," she says. Other companies have joined BioSource in producing rat immunoassays. Wako Chemicals USA Inc. of Richmond, Va., for example, produces rat Il-8, hemoglobin, and transferrin ELISA kits, among others. And several companies produce human and mouse cytokine ELISA kits, including Peninsula Laboratories Inc. of Belmont, Calif., Boehringer Mannheim Biochemicals of Indianapolis, and BioSource. One of the more exciting new immunoassay techniques allows researchers to directly test the function of the immune system. BioSource hosted its United States launch of a product known as Dynamix on September 3. Using whole blood, the kit stimulates immune cells ex vivo to produce cytokines and measures the levels using immunoassay techniques. Because there is no need to isolate the cells, researchers studying autoimmune diseases, new drugs, and HIV can directly measure the effects these diseases and drugs have on the immune system. For example, the TNF-alpha Dynamix system can be used to track the clinical course of multiple sclerosis (MS). Several studies evaluating TNF-alpha production indicate that the levels of TNF-alpha are greatest immediately before and during relapses of MS. By tracking TNF-alpha production, physicians and researchers may be able to predict relapses and evaluate new drug therapies. "The principal advantage of the system is its speed," states Nicholas Topley, a lecturer in cell and molecular biology at the Institute of Nephrology at the Cardiff Royal Infirmary in Wales. "You can finish the whole experiment in two or three hours, so you can study three or four patients in one day and see the results at the end of the day. If we had to isolate the cells, it would be several weeks before we had the results." Topley notes that by using peritoneal fluid rather than whole blood, he had to optimize the system somewhat before it produced reliable results; nonetheless, he reports, it remains a rapid way to measure the function of the immune system. Cardinal Associates Inc. of Santa Fe, N.Mex., is also focusing on measuring the function of the immune system using standard immunoassay techniques. The company's products focus on studying the oxidative burst-the killing potential of white blood cells known as neutrophils. Using Cardinal's specifically sized immunomagnetic particles, called MagNIM, researchers can isolate naturally functioning neutrophils from whole blood. Once the cells are isolated, they are stimulated to produce oxygen free radicals. The free radical production is measured using chemiluminescence. Cardinal Associates' president, Ross U. Robinson, points out that this ex vivo assay procedure could be very helpful in evaluating arthritis remedies because "activated neutrophils actually contribute to arthritic pain by generating free radicals." Probing Cellular Processes Immunoassays have long been used to study the workings of cells. New kits available today allow researchers to easily probe the cell's inner activities. Boehringer Mannheim uses immunoassay techniques to update the classic chloramphenicol transferase (CAT) assay, which measures transfection efficiency and promoter activity, with its CAT ELISA assay. The CAT assay originally used carbon-14 to measure CAT production. Instead, the CAT ELISA starts with an anti-CAT antibody coated microtiter plate. Cell extracts containing CAT are added to the microtiter wells. Another anti-CAT antibody that is also conjugated to digoxigenin binds to the CAT. An anti-digoxigenin antibody conjugated to peroxidase binds digoxigenin, and a substrate is added to quantify CAT. In addition to eliminating radioactivity from this conventional molecular biology assay, Brian Holoway, product manager for Boehringer Mannheim, notes that "depending on your needs, you can use colorimetric, chemiluminescent, or fluorescent detection systems with this assay." Chemiluminescent and fluorescent detection methods measure a wider range of values, from 10 picograms per milliliter to 50 nanograms per milliliter. For those whose immunoassay needs aren't being met, a number of companies offer custom assay service. Advanced Chemtech of Louisville, Ky., Biodesign International of Kennebunkport, Maine, and Immunochemistry Technologies of Minneapolis all offer peptide and antibody production and purification, assay development, and technical support to those working on more esoteric scientific questions (H.G. DeYoung, The Scientist, Sept. 16, 1996, page 18). Going custom certainly isn't always necessary. A number of companies produce antibodies by trying to determine where the market is going. Affinity Bioreagents of Golden, Colo., is one such company. The firm's antibodies include those against heat shock proteins, protein trafficking peptides, and leptin (the product of the ob gene) as well as steroid receptors. "Immunochemistry has been out there for a really long time, but then again, so has the car, and that is still an incredibly useful tool," says Affinity Bioreagents president James Stiehr. "The technology isn't really new. But as long as new proteins are being discovered and looked at, there will be a need for immunochemicals." Lisa Seachrist is a freelance science and medical writer based in Bethesda, Md.

New Immunoassay Products Let Users Put New Twists On Old Themes

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