Luminescence Developments Help Scientists See The Light

Biologists are constantly seeking more sensitive assays to detect the presence of organisms or telltale DNA, RNA, and proteins. Although radioactive tags incorporated into the target itself (or into a complementary strand)-and later detected by Geiger counters or film exposures-have traditionally given good sensitivity, the problems of waste disposal and laboratory monitoring have driven a search for alternative tags that have radioactivity's sensitivity but avoid its hazards. Fluorescent tags-bound directly to the analyte-are easily disposable, but these reagents are not always sensitive enough to meet a researcher's needs (J. Kling, The Scientist, April 14, 1997, page 14). As a result, radiolabels continue to be a mainstay in many smaller research laboratories. But where fluorescent tags fail, chemiluminescence and bioluminescence detection can often step in. These systems rely on a reporter enzyme-which signals the presence of the target and produces the luminescence-and a substrate, usually added separately. The substrate is processed by the reporter enzyme, resulting in a characteristic flash or glow. By "turning over" a large number of substrates, a reporter enzyme can produce a much more sensitive signal than could a single fluorescent tag. An instrument called a luminometer can detect and quantify that response and calculate the concentration of the target. The process also lends itself to automation, and that could portend big things for luminescence, says William W. Ward, an assistant professor of biochemistry at Rutgers University in New Brunswick, N.J. For instance, many novel chemicals are produced every year, but only a small fraction are tested to see if they cause cancer because the testing techniques involved take too long. "I can see the day when all compounds that are thought to interact at the DNA level will be screened automatically by [this kind of technique]. There will be no backlog of compounds waiting to be tested." Luminescence systems have been adapted to many of the protocols that use fluorescent and radiolabeled tags, such as tagging DNA and RNA probes, DNA/RNA sequencing, and chromosome imaging. In many applications, "bioluminescence is the only simple way to detect the event [turning on a gene] that you are interested in without killing the cell," says Eric Fairfield, president of Fairfield Enterprises, a Los Alamos, N.Mex.-based company that is developing alternatives to luminescence detection systems. The sensitivity and flexibility of luminescence systems have spawned other uses. For instance, the enzyme known as luciferase-responsible for the firefly's evening light show-modifies adenosine triphosphate (ATP) and its substrate luciferin, which produces light. Since ATP production is crucial to every known life form, companies like GEM Biomedical of Hamden, Conn., have developed systems to monitor sanitation in manufacturing plants. Gem's BG-P portable luminescence workstation is just 7 inches by 12 inches and 7.5 pounds, making it useful for "swab" tests of surfaces. The instrument indicates within minutes whether the surface just checked is sterile, according to the company. SCINTILLATING: EG&G Wallac's MicroBeta TriLux Luminescence and Liquid Scintillation Counter is a compact, automatic unit. FOR LUMINESCENCE DETECTION: EG&G Wallac’s Victor Multi-Label Reader Since the luciferase/ATP system can work with any cell type, it is often used to check cells in culture to see if they are still growing or to measure the toxic effects of particular chemicals on cells. EG&G Wallac Inc. of Gaithersburg, Md., sells its CytoLux cell viability assay with such uses in mind. The kit includes all the reagents necessary to first break open the cells to release the ATP, and then apply the luciferase and luciferin. Once the reagents are added, the solution can be transferred to a luminometer-such as Wallac's MicroBeta TriLux or Victor-for measurement. Since the isolation and cloning of the firefly luciferase gene in the 1980s, the enzyme has found widespread use as a reporter to monitor gene expression. Promoter regions of DNA, along with "enhancer" and "silencer" regions, help to regulate when the products of genes are manufactured, and when production is cut back or halted. To test whether an unknown sequence of DNA acts as a promoter, the sequence is often fused to a reporter gene-such as luciferase-that can be easily detected. Once the reporter gene is transfected into a cell line, the appearance of luciferase establishes the unknown strand as a promoter. The reagents in Wallac's GeneLux luciferase reporter gene assay have been optimized for best performance so that minor changes of the reagent concentrations in the reaction mixture have less of an influence, according to the company. The Luciferase Assay Kit-sold by Tropix Inc. of Bedford, Mass.-contains proprietary reagents to enhance light emission, while Tropix's Galacto-Star reporter gene assay system includes reagents for the ß-Galactosidase chemiluminescent reporter enzyme. Tropix's Dual-Light combined reporter gene assay can detect luciferase and b-Galactosidase in a single-extract aliquot, allowing the researcher to test two promoters at the same time. The kit is said to give results in one hour. ICN Pharmaceuticals Inc. of Costa Mesa, Calif., also offers reporter gene kits for b-Galactosidase and b-Glucoronidase (Aurora Gal-XE and Aurora GUS, respectively). Its secreted alkaline phosphatase kit (Aurora AP) overcomes one important limitation of many reporter gene assays: The cells must be broken open (or "lysed") to release the reporter gene product so that it can be detected. The secreted alkaline phosphatase is released from the cells soon after it is manufactured, giving confirmation of a promoter's activity without killing the cells. All of ICN's kits use the 1,2-dioxetane substrate for alkaline phosphatase, chosen because it emits a glow rather than a split-second flash. "There is plenty of time to measure the light produced using a luminometer, or to make multiple film exposures," says Jim Haug, vice president of marketing at ICN. Using northern and Southern blotting techniques, researchers can scan for specific fragments of RNA or DNA and isolate or quantify them. These assays once were the sole domain of radioisotopes, but luminescence systems are taking over in this area, too. Northern blots are used to detect messenger RNA (mRNA), which contains all of the genetic information but none of the non-coding introns that make up much of DNA. But northerns are time-consuming and difficult to quantify. With that in mind, Boehringer Mannheim Corp. of Indianapolis offers a chemiluminescent kit, called Northern ELISA, designed to give better results. The system uses a digoxigenin-labeled probe to tag the target mRNA, followed by an anti-digoxigenin/horseradish peroxidase complex that locks onto the probe. USES CHEMICAL LABEL: EG&G Wallac's Luminescence Labeling Kit for labeling antibodies, proteins, peptides, and DNA Other available kits focus on detecting the presence of target DNA sequences. Boehringer's Dig/Genius 3 nucleic acid labeling kit detects digoxigenin-labeled DNA probes with an anti-DIG/alkaline phosphatase conjugate that processes the blue-colored substrates NBT and BCIP. Amersham Life Science Inc. of Arlington Heights, Ill., sells the widely popular ECL 3« oligolabeling and detection system. The kit includes reagents to synthesize fluorescein-tagged probes, an anti-fluorescein/horseradish peroxidase conjugate, and detecting agents to pinpoint the probe. Additional kits available for luminescence detection of RNA and DNA include: ICN's Aurora Southern Blotting kit; Tropix's Southern-Light; and the BrightStar Psoralen-Biotin and BrightStar BioDetect kits from Ambion Inc. of Austin, Texas. Determining what genes are present in an organism is all well and good, but to find out what's really going on in a cell, the researcher must know what proteins are being manufactured and in what quantity. That's where western blotting techniques come in to play. But like their northern and Southern counterparts, these assays can be tedious and troublesome to quantify. Not surprisingly, luminescence has come to the rescue here, too. In these systems, the protein sample is separated through gel electrophoresis. The proteins of interest are pinpointed using a mouse, rabbit, or human antibody that recognizes the protein. Next, the researcher applies a second antibody that locks onto the mouse, human, or rabbit signature of the first antibody. The second antibody is linked to a reporter enzyme, and the final step is to apply the luminescent substrate of that enzyme. ICN Pharmaceuticals' Aurora Western Blotting system and the Western-Star kit sold by Tropix both rely on the alkaline phosphatase reporter enzyme, though each kit uses a different alkaline phosphatase substrate. Both give sensitivity equal to or better than traditional assays that incorporate radioactive iodine (125I). The dioxetane substrate used by Tropix gives a particularly long-lasting signal, according to Mark Roskey, senior product manager at Tropix. "In a western blotting application we can still get a good signal 12 to 24 hours after the addition of substrate, whereas the signal with competitive western blotting kits decays after as little as 30 minutes." The Phototope-HRP Western Blot Detection Kit-offered by New England BioLabs Inc. of Beverly, Mass.-uses the horseradish peroxidase reporter enzyme. EASY AS ABC: Wako BioProducts' High Sensitivity Immunoblotting Kit’s Avidin/Biotinylated enzyme Complex is claimed to result in a greater magnitude of binding ability. The use of secondary antibody-enzyme conjugates is fairly straightforward experimentally, but the secondary antibodies involved do not always bind to the first antibody with high specificity, increasing the background signal. The High Sensitivity Immunoblotting Kit sold by Wako BioProducts of Richmond, Va., uses the more involved ABC (Avidin/Biotinylated Enzyme Complex) system to improve the sensitivity. In the ABC approach, the primary antibody is localized to the target, and the secondary antibody is linked to numerous biotins. Finally, a streptavidin-biotin-horseradish peroxidase complex, formed separately, is introduced and binds to the biotin through the streptavidin portion. The entire complex is then exposed to the chemiluminescent substrate. That mixture includes thiazole, which enhances the chemiluminescent signal. HIGHLY SENSITIVE: Vector Laboratories’ Vectastain Elite ABC is suitable for low concentrations of antigen. Other products are more general, capable of tagging nucleic acids, proteins, or any other molecule of interest, in many cases. For instance, the Vectastain ABC kits, offered by Vector Laboratories Inc. of Burlingame, Calif., involve the same ABC method described previously, except they use avidin rather than streptavidin. Each kit includes avidin and a biotinylated secondary antibody. The four kits in the series offer alkaline phosphatase, horseradish peroxidase, or glucose oxidase as the developing enzymes. The Vectastain Elite ABC horseradish peroxidase system is five- to tenfold more sensitive than the regular Vectastain ABC peroxidase kit, making it the choice for applications in which the concentration of the antigen is low, according to Craig Pow, manager of technical services at Vector. The kits can be used to detect any substance that has an appropriate primary antibody bound to it. The Luminescence Labeling Kit sold by Wallac relies on a chemical label-made up of an acridinium ester-that can be attached to any molecule with a primary amine group (including nucleic acids, proteins, and some peptides). Exposed to hydrogen peroxide under basic conditions, the acridinium ester decomposes to form N-methyl acridone, which emits light at a wavelength of 430 nm. Although luminescent enzymes can turn over unlimited amounts of their substrate molecules, they fail to deliver on the promise of unlimited sensitivity. At some point, exposing the system for a longer period produces diminishing returns on sensitivity. One reason for this limitation-especially in tube- or microplate-based assays-is that water can quench the luminescence reaction, reducing the light output. How this happens is not well understood, but that hasn't stopped companies like Tropix from developing, through trial and error, reagents that counter the effect. The company's Sapphire, Emerald, Ruby, Sapphire-II, and Emerald-II enhancers work, with the 1,2-dioxetane alkaline phosphatase substrate, increasing signal emission two- to three-fold while having a minimal effect on light-emission kinetics. "This can be quite important, particularly if you are measuring near the limit of detection of your light-measurement device," says Roskey. The products tune the wavelength of light emission. Sapphire and Sapphire-II shift the light emission maximum to 461 nm from 475 nm, observed when dioxetane is used alone. Emerald and Emerald-II shift the maximum to 542 nm, and Ruby shifts it to 620 nm. The Emerald enhancers give the greatest signal intensity, while the Sapphire series produces a wider dynamic range. Luminescent enzyme/substrate systems give excellent sensitivity, but the multiple steps involved make the assays time-consuming and ill-suited for the kind of large-scale, automated assays that are being developed for drug screening and toxicological studies. If luminescence systems are manual transmission vehicles, the autofluorescent green fluorescent protein-which fluoresces on its own-may be a new line with automatic transmission. "You don't have to add [a substrate]-that cuts out a tremendous amount of work," says Ward, who is a pioneer in the development of the green fluorescent protein (GFP). The gene for GFP, isolated from the jellyfish Aequorea victoria, can be linked to so-called promoter regions, which "turn on" the expression of the genes. The promoters are themselves stimulated by the action of proteins or small molecules. By linking a known oncogene promoter to GFP and transfecting the complex into a cell line, a researcher can rapidly determine if exposure to a synthetic chemical nudges the promoter. The appearance of the characteristic fluorescence of GFP serves as a red flag; exposure to the same substance could be unleashed the dangerous oncogene in a normal cell. A luminescence enzyme linked to a promoter could do the job, too, but the substrate would produce light outside the cell. In a mixed population of cells, "if you want to isolate the cells [that respond], you've got a lot of work to do," according to Ward. Using GFP, the affected cells express the stable, fluorescent protein, allowing the investigator to separate them using fluorescence-activated cell sorting. "Now you've not only found what you're looking for, but you've purified it, and you've done it all with robotics in minutes," explains Ward, who envisions a battery of cell lines with various promoters linked to autofluorescent proteins. Such a system could rapidly distinguish which promoters a drug candidate or chemical interacts with, and even give hints at the mechanism of how a substance brings about its biological effect, he says. Quantum Biotechnologies Inc. of Laval, Quebec in Canada offers green, red-shifted, and blue autofluorescent proteins (AFPs). The company sells constructs of AFPs with various promoters that reportedly are useful for a wide variety of applications. SELECTED SUPPLIERS OF BIOLUMINESCENCE AND CHEMILUMINESCENCE PRODUCTS: Ambion Inc. Amersham Life Science Inc. Boehringer Manheim Chemicon International Inc. EG&G Wallac Inc. GEM Biomedical ICN Pharmaceuticals Inc. New England BioLabs Inc. Quantum Biotechnologies Inc. SeaLite Sciences Inc. Tropix Inc. Vector Laboratories Inc. Wako BioProducts SeaLite Sciences sells kits that use conjugates of the protein called AquaLite that is responsible for the bioluminescence exhibited by jellyfish. Similar to GFP, this protein avoids all the variables associated with enzyme reactions, including addition of substrate, incubation times, and temperature variations during product development, according to David F. Smith, vice president of research and development at SeaLite. The researcher needs only to add calcium ions, which cause conformation change that activates the enzyme. That done, "one simply places the microtiter plate or test tube in a luminometer," says Smith. The five kits available consist of conjugates of the AquaLite protein to streptavidin or biotin, or to anti-fluorescein, anti-digoxigenin, or anti-dinitrophenol antibodies. Chemicon International Inc. of Temecula, Calif.-which has licensed AquaLite from SeaLite Sciences-also offers AquaLite conjugated to a number of antibodies, including anti-bromodeoxyuridine, anti-phycoerythrin, anti-mouse IgG, anti-rabbit IgG, anti-human IgG, and anti-human IgM. The battle of the tags goes on: Radiolabels give way to fluorescent tags; fluorescence makes way for luminescent systems; and some believe that autofluorescent proteins will outdo all of them and revolutionize biotechnology research. But each of these methods is likely to find a niche in specific applications carried out in specialized laboratories, whether the two-person lab of a struggling biochemistry professor or the large-throughput automated labs of major pharmaceutical companies. That won't stop developers from constructing faster, brighter, and cheaper detection systems, however. For the researcher, that's all to the better. James Kling, a science writer based in Bellingham, Wash., is online at jkling@nasw.org.

Luminescence Developments Help Scientists See The Light

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