Refinements In Bioluminescence Assays Expand Technique's Applications

The following vendors develop and/or market bioluminescence assay kits, products, and instrumentation for a variety of research and clinical laboratory uses. For more information about products, services, and prices, please contact these companies directly. Accurate Chemical and Scientific Corp. 300 Shames Dr. Westbury, N.Y. 11590< (800) 645-6264 - Fax: (516) 997-4948 Analytical Luminescence Laboratory 11760 Sorrento Valley Rd., Suite E San Diego, Calif. 92121 (619) 455-9283 BATLE Luminetrics Enterprises Inc. 5601 N. Dixie Highway, Suite 215 Ft. Lauderdale, Fla. 33334 (800) 423-2402 - Fax: (305) 491-0869 Boehringer Mannheim Biochemicals Inc. 9115 Hague Rd. Indianapolis, Ind. 46250 (800) 428-5433 - Fax: (317) 845-2000 Calbiochem-Novabiochem Corp. 10394 Pacific Center Court San Diego, Calif. 92121 (619) 450-9600 - Fax: (619) 453-3552 Carolina Biological Supply Co. 2700 York Rd. Burlington, N.C. 27215 (800) 334-5551 - Fax: (919) 584-3399 Charm Bioengineering Inc. 36 Franklin St. Malden, Mass. 02148 (617) 322-1523 - Fax: (617) 322-3141 Coulter Corp. MC 32-A038 P.O. Box 169015 Miami, Fla. 33116-9015 (800) 526-6932 - Fax: (305) 380-5990 Dynatech Laboratories Inc. 14340 Sullyfield Circle Chantilly, Va. 22021 (703) 631-7800 - Fax: (703) 631-7816 Fine Science Tools Inc. 373 G Vintage Park Dr. Foster City,, Calif. 94404 (800) 521-2109 - Fax: (415) 349-3729 ICN Biomedicals ICN Plaza 3300 Hyland Ave. Costa Mesa, Calif. 92626 (714) 545-0113 - Fax: (714) 641-7275 Integrated Biosolutions Inc. 4270 U.S. Route 1 Monmouth Junction, N.J. 08852 (908) 274-1778 - Fax: (908) 274-1733 IN/US Systems Inc. 5809 N. 50th St. Tampa, Fla. 33610-4809 (800) 875-4687 - Fax: (813) 620-3708 Jasco Inc. 8649 Commerce Dr. Easton, Md. 21601 (410) 822-1220 - Fax: (410) 822-7526 JM Science Inc. 5820 Main St., Suite 300 Buffalo, N.Y. 14221-5734 (800) 387-7187 - Fax: (716) 634-1970 Labsystems Inc. 300 Second Ave. Needham Heights, Mass. 02194-2818 (800) 522-7763 - Fax: (508) 480-9999 LJL Biosystems 404 Tasman Dr. Sunnyvale,, Calif. 94089 (408) 541-8755 - Fax: (408) 541-8786 MGM Instruments Inc. 925 Sherman Ave. Hamden, Conn. 06514 (203) 248-4008 - Fax: (203) 288-2621 National Diagnostics Inc. 305 Patton Dr. Atlanta, Ga. 30336 (404) 699-2121 - Fax: (404) 699-2077 New England Biolabs Inc. 32 Tozer Rd. Beverly, Mass. 01915 (508) 927-5054 - Fax: (508) 921-1350 Perkin Elmer Corp. 761 Main St. Norwalk, Conn. 06859-0105 (203) 762-4681 - Fax: (203) 762-4997 Promega Corp. 2800 Woods Hollow Rd. Madison, Wis. 53711 (608) 274-4330 - Fax: (608) 273-6967 R&D Systems Corp. 614 McKinley Place, N.E. Minneapolis, Minn. 55413 (612) 379-2956 - Fax: (612) 379-6580 SeaLite Sciences Inc. 187 Ben Burton Circle Bogart, Ga. 30622 (800) 874-4471 - Fax: (404) 546-4962 Sigma Chemical Co. Catalog Department 3050 Spruce St. St. Louis, Mo. 63103 (800) 325-3010 - Fax: (314) 771-5750 Tropix Inc. 47 Wiggins Ave. Bedford, Mass. 01730 (617) 271-0045 - Fax: (617) 275-8581 Wallac Inc. (An EG&G Co.) 9238 Gaither Rd. Gaithersburg, Md. 20877 (800) 638-6692 - Fax: (301) 963-7780 Worthington Biochemical Corp. Halls Mills Rd.v Freehold, N.J. 07728 (908) 462-3838 - Fax: (908) 308-4453 Chemists, biologists, and medical researchers are continually looking for sensitive, nonradioactive assays. In the mid-1980s, many scientists turned to newly developed bioluminescence assays for their needs. Now, in just the past few years, the uses for these tests--based on the phenomenon of light emission from a biochemical reaction-- have increased dramatically in number, as the underlying technology has been refined and extended. Bioluminescence occurs in certain jellyfish, bacteria, mushrooms, fungi, crustaceans, fishes, worms, and beetles, including the familiar Photinus pyralis, a type of firefly. Harnessed for use in the laboratory, bioluminescence can be used to detect bacterial contamination in soft drink bottles; to predict the effect of chemotherapies on cancer cells; and as a reporter molecule to highlight gene expression in molecular biology experiments. More broadly, using recombinant technologies, researchers are incorporating bioluminescence into immunoassays and DNA probes. "There is a tremendous range of applications of bioluminescence," says Larry Kricka, a professor in the department of pathology and laboratory medicine at the University of Pennsylvania in Philadelphia. A bioluminescence experiment basically requires a paired set of biochemicals: an enzyme, variously called a luciferase or a photoprotein, and a substrate, which is a molecule that emits light upon interacting with the enzyme or photoprotein. Each bioluminescent system requires unique co- factors and buffers. "There are a lot of natural, biological, light-producing systems, and the enzymes in all of them are called luciferases," says Julie Molloy, business manager of Analytical Luminescence Laboratory, San Diego, which specializes in kits using firefly luciferase. The substrates for luciferases are called luciferins. Both terms were coined by German physiologist Emil Heinrich DuBois-Reymond near the end of the 19th century, in connection with his work on the glowing clam Pholas dactylus. Luciferases and luciferins are not interchangeable between bioluminescent species, where they may be involved in totally different reactions. For example, luciferase from Photobacterium fischeri, often just called bacterial luciferase, catalyzes reactions involving the molecules FMNH2 and NADH, and is used to trace certain metabolic reactions. The more commonly used firefly luciferase, by contrast, detects the biological energy molecule ATP (adenosine triphosphate). Bioluminescent proteins from sea organisms take part in yet a third type of reaction. Harnessing bioluminescence began in the 1940s, when Baltimore schoolchildren brought jars of fireflies to the Johns Hopkins University laboratory of William McElroy and Marlene DeLuca. Over the next three decades, this team isolated and described the reactants and enzymes of the bioluminescence reaction. They found that light emission occurs when firefly luciferin, which is an organic acid, reacts with ATP to form an intermediate compound, luciferyl adenylate. In the presence of oxygen and magnesium or manganese ions, luciferase catalyzes oxidation of the intermediate, producing a compound called oxyluciferin--and a flash of light. The rationale for using firefly bioluminescence in many of today's assays is simply that where there is life, there is ATP. Measuring the light produced in the bioluminescent reaction with ATP is, therefore, also a measure of metabolic activity. "But it wasn't until the late 1970s and early 1980s that there were active applications," says Molloy. "One of the first areas looked at was a rapid urine screen for urinary tract infections. The technique was also carried aboard the Viking spacecraft as one of the methods to detect potential life on Mars." DeLuca and McElroy eventually relocated to the University of California, San Diego, and founded the nearby Analytical Luminescence Laboratory in 1981. "Today people use firefly luciferase to test for ATP in all sorts of different applications," says Kricka. "It is a rapid microbiological test for bacterial contamination. For example, Coca Cola uses it to test beverages before they are bottled." In 1986, bioluminescence entered the age of molecular biology in an arresting figure of a glowing tobacco plant published by researchers in the departments of biology and chemistry at UC-San Diego (David W. Ow, et. al., Science, 234[4778]:856-9, 1986). They linked a complementary DNA (cDNA) product corresponding to the gene for firefly luciferase to a plant virus promoter (control sequence), then inserted this into a bacterial plasmid (a ring of DNA) and infected tobacco leaf cells in culture. Transgenic tobacco plants were regenerated from cultures that glowed when exposed to oxygen and luciferin. A wide variety of researchers now use the firefly luciferase gene as a reporter molecule by linking it to different genes of interest. An investigator is then able to follow gene expression by detecting bioluminescence in different cells and tissues at different stages of development in a transgenic organism. "The gene for firefly luciferase has become a powerful tool in genetic engineering and molecular biology, used instead of the [bacterial] CAT [chloramphenicol acetyltrans-ferase] gene as a reporter," says Kricka. "It is nonradioisotopic and very, very sensitive." Analytical Luminescence Laboratory offers firefly luciferin, luciferase, buffers, and standards in a variety of combinations, plus protocols for applications in genetic engineering and for detecting bacteria and yeast. Firefly luciferin and luciferase are also offered by Accurate Chemical and Scientific Corp., Westbury, N.Y.; Boehringer Mannheim Biochemicals Inc., Indianapolis; Calbiochem- Novabiochem Corp., San Diego; ICN Biomedicals, Costa Mesa, Calif.; Promega Corp., Madison, Wis.; R&D Systems Corp., Minneapolis; Sigma Chemical Corp., St. Louis; Worthington Biochemical Corp., Freehold, N.J.; and others. While Analytical Luminescence Laboratory gets its luciferin and luciferase from freeze-dried firefly abdomens shipped from an East Coast company, many of the other vendors obtain luciferase from a firefly luci-ferase cDNA cloned in E. coli bacteria. This source, as with most proteins derived from recombinant DNA technology, is purer than the direct biological source. And for teachers interested in a glowing lesson, the Carolina Biological Supply Co. of Burlington, N.C., sells freeze-dried firefly tails in kits with all materials needed for students to extract luciferin and luciferase and then conduct a bioluminescence reaction. BATLE Luminetrics Enterprises Inc. of Fort Lauderdale, Fla., is developing a very specific use of firefly bioluminescence. An in vitro TCA-100 Tumor Chemosensitivity Assay uses firefly luciferase to assess whether tumor cells respond to specific drugs in vitro, before testing the drugs on the patient. "You culture tumor cells, expose the cells to combinations of drugs, and estimate the numbers of cells by bioluminescence with firefly luciferase," says Kricka. Declining luminescence indicates falling ATP production, which means that the cells are being killed. Company literature suggests that a physician can use TCA-100 along with other information to choose among recommended standard chemotherapeutic protocols for a particular tumor type, to try drugs on drug-resistant tumors, or to evaluate chemotherapies for cancers so rare that standard protocols don't exist. Several vendors also sell the key instrumentation for bioluminescence systems. Luminometers to detect bioluminescence can be purchased from Analytical Luminescence Laboratory, San Diego; Dynatech Laboratories Inc., Chantilly, Va.; Labsystems Inc., Shrewsbury, Mass.; MGM Instruments Inc., Hamden, Conn.; and others. Tapping the sea for bioluminescent organisms has ancient roots--Roman scholar Pliny the Elder (23-79 A.D.) wrote of a glowing slime that he would rub on a variety of objects, which was actually a jellyfish that lived in the Bay of Naples. Today, SeaLite Sciences Inc. of Bogart, Ga., is the sole company developing and commercializing recombinant bioluminescent proteins from sea life, based on discoveries made at the University of Georgia, Athens. SeaLite's AquaLite system uses a gene from the bioluminescent jellyfish Aequorea victori, produced in recombinant E. coli bacteria. It is quite different from the firefly luciferase system. The equivalent of luciferase is not an enzyme, but a photoprotein called aequorin. When a jellyfish version of luciferin binds oxygen and a precursor form of aequorin, called apoaequorin, the mature aequorin forms. Adding calcium ions alters the shape of the aequorin, triggering release of a flash of blue light. "Aequorin is different from conventional luciferases," says Lee Herron, president of SeaLite. "Most luciferases glow; the photoprotein is more of a very rapid flash." Aqua- Lite, the glowing product itself, consists of aequorin bound to its luciferin. The basis of the assay is that the conjugate flashes in the presence of calcium. The AquaLite system can be adapted to two major biotechnolo- gies--immunoassay and DNA probes--by covalently linking recombinant aequorin to antibodies or streptavidin, respectively. AquaLite offers advantages over enzyme-based immunoassays and fluorescence-based DNA probes in that it can detect analytes in small amounts and over a wide range of concentrations, making serial dilutions unnecessary. A bioluminescence immunoassay has two components-- polystyrene tubes coated with antibodies to an analyte, and recombinant aequorin covalently linked to antibodies to the analyte. When a body fluid sample is added to the tube, the aequorin-tagged antibody and the antibody coated on the tube form a sandwich around the analyte. The user washes unbound antibody from the tube, places the tube in a luminometer, and injects a calcium solution. As aequorin contorts, the characteristic flash appears--if the aequorin has been retained by the analyte. The light intensity is directly proportional to the amount of the analyte (and, therefore, bound aequorin) present, so a calibration curve can be set up to determine the concentration of the analyte in the sample. SeaLite offers a bioluminescence immunoassay kit to detect interleukin-6 (IL-6) in human serum, and plans to introduce kits for other cytokines soon. The company also uses the sandwich approach to detect and quantitate the pregnancy hormone human chorionic gonadotropin (hCG) and thyroid stimulating hormone (TSH). Bioluminescent DNA probes are also in development, with at least one currently available. Most DNA probes are now used in in vitro diagnostics (R. Lewis, The Scientist, Jan. 10, 1994, page 17). In many of these probes, the nucleic acid is linked to the vitamin biotin. It is the attraction of biotin to streptavidin, which is bound to an antibody or fluorescent molecule, providing a signal that permits detection of the probe. Used in such a probe, AquaLite binds to streptavidin. A flash of blue announces the attached probe's presence, such as in a body fluid harboring infectious bacteria or viruses. SeaLite sells aequorin bound to streptavidin or biotin. Generally speaking, bioluminescence is a type of chemiluminescence. In most labs, researchers have tended to more often use the strictly chemical systems, such as those based on alkaline phosphatase. "This was because bioluminescent reagents until recently were extracted [only] from biological sources," says Kricka. "There were questions of purity, quality, and reliability, whereas chemiluminescence, based on an organic compound with a well-defined stability, didn't have the same problems." Bioluminescence is now filling its own niches as well as competing with chemiluminescence. ATP detection via firefly luciferase, for example, has no chemiluminescent counterpart, Kricka says. And aequorin, used in bioimmunoassay and DNA probes, directly competes with well- established chemiluminescent approaches. Ricki Lewis, a freelance writer based in Scotia, N.Y., is the author of several biology textbooks.

Refinements In Bioluminescence Assays Expand Technique's Applications

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