Uses Of Enzyme Immunoassays Growing In Laboratories And Clinics

Life Technologies P.O. Box 6009 8451 Helgerman Court Gaithersburg, Md. 20884 (301) 840-4150 Fax: (800) 331-2286 MGM Instruments Inc. 925 Sherman Ave. Hamden, Conn. 06514 (203) 248-4008 Fax: (203) 288-2621 Nordic Immunological Labs Drawer 2517 Capo Beach, Calif. 92624 (714) 498-4467 Fax: (714) 361-0138 Organon Teknika/ Biotechnology Research Institute 1330 Piccard Dr. Rockville, Md. 20850-4396 (800) 354-0809 Fax: (301) 840-2161 Oxford Biomedical Research Inc. P.O. Box 522 1858 Starr Batt Dr. Rochester Hills, Mich. 48309 (810) 852-8815 Fax: (810) 852-4466 Paracelsian 222 Langmuir Laboratories Cornell Technical Park Ithaca, N.Y. 14805 (607) 257-4224 Fax: (607) 257-2734 Perceptive Diagnostics 735 Concord Ave. Cambridge, Mass. 02138 (617) 499-1433 Fax: (617) 497-6927 Pharmacia Hepar Inc. 160 Industrial Dr. Franklin, Ohio 45005 (800) 447-3846 Fax: (513) 746-8055 Pierce Chemical Co. P.O. Box 117 Rockford, Ill. 61105 (800) 874-3723 Fax: (815) 968-7316 R & D Systems 614 McKinley Place, N.E. Minneapolis, Minn. 55413 (612) 379-2956 Fax: (612) 379-6580 Repligen Corp. 1 Kendall Square Building 700 Cambridge, Mass. 02139 (617) 225-6000 Fax: (617) 494-1786 Scimedex Corp. 400 Ford Rd. Denville, N.J. 07834 (201) 625-8822 Fax: (201) 625-8796 Serex Inc. 203 West Passaic St. diaMaywood, N.J. 07607 (201) 368-5700 Fax: (201) 368-7850 Syva P.O. Box 4901 San Jose, Calif 95161-9013 (800) 227-8376 Fax: (408) 239-2206 TAGO Inc. 887 Mitten Rd. Burlingame, Calif. 94010 (415) 692-4015 Fax: (415) 692-9004 Takara Biochemical Inc. 719 Allston Way Berkeley, Calif. 94710 (510) 649-9895 The Binding Site Inc. 5889 Oberline Dr. Suite 101 San Diego, Calif. 92121 (619) 453-9177 Fax: (619) 453-9189 Tosoh Medics Inc. 373 Vintage Park Dr. Foster City, Calif. 94404 (415) 578-2600 Fax: (415) 578-2626 USA Scientific Plastics P.O. Box 3565 Ocala, Fla. 34478 (904) 237-6288 Fax: (904) 351-2057 Vector Laboratories 30 Ingold Rd. Burlingame, Calif. 94010 (415) 697-3600 Fax: (415) 697-0339 Cistron Biotechnology 10 Bloomfield Ave. Pine Brook, N.J. 07058 (201) 575-1700 Fax: (201) 575-4854 Endogen Inc. 68 Fargo St. Boston, Mass. 02210 (617) 439-3250 Fax: (617) 439-0355 Exocell Inc. 3508 Market St. Suite 420 Philadelphia, Pa. 19104 (800) 234-3962 Fax: (215) 222-5325 Genzyme Diagnostics Division of Genzyme Corp. One Kendall Square Cambridge, Mass. 02139 (617) 252-7744 Fax: (617) 252-7600 Granbio Inc. P.O. Box 392140 Temecula, Calif. 92589 (909) 676-0049 Human Biologicals Inc. P.O. Box 50862 Phoenix, Ariz. 85076 (602) 893-8817 Fax: (602) 893-8817 Hycor 18800 Von Karman Irvine, Calif. 92715 (714) 440-2000 Fax: (714) 440-2222 ICN Pharmaceuticals ICN Plaza 3300 Hyland Ave. Costa Mesa, Calif. 92626 (714) 545-0113 Fax: (714) 641-7275 Idexx Laboratories One Idexx Dr. Westbrook, Maine 04092 (207) 856-0300 Fax: (207) 856-0346 Immuno-Dynamics Inc. P.O. Box 766 La Jolla, Calif. 92038 (619) 452-1270 Fax: (619) 458-3515 ImmunoSystems Inc. 4 Washington Ave. Scarborough, Maine 04074 (207) 883-9900 Fax: (207) 883-8088 ImmunoVision 1506 Ford Ave. Springdale, Ariz. 72764 (800) 541-0960 Fax: (501) 751-7002 Integrated Biosolutions Inc. 4270 U.S. Route 1 Monmouth Junction, N.J. 08852 (908) 274-1778 Fax: (908) 274-1733 Kronus 1000 Calle Amanecer San Clemente, Calif. 92673 (714) 366-9100 Fax: (714) 366-9300 Lampire Biological P.O. Box 170 Pipersville, Pa. 18947 (215) 795-2838 Fax: (215) 795-0237 The following vendors develop and/or market enzyme immunoassay kits and products for a variety of research and clinical laboratory uses. For more information about products, services, and prices, please contact these companies directly. 5 Prime - 3 Prime Inc. 5603 Arapahoe Rd. Boulder, Colo. 80303 (303) 440-3705 Fax: (303) 440-0835 AAI-Abtech P.O. Box 376 Yardley, Pa. 19067 (800) 233-2197 Fax: (215) 321-7099 AMAC Inc. 160 B Larrabee Rd. Westbrook, Maine 04092 (207) 854-0426 Fax: (207) 854-0116 American Qualex 14620 E. Firestone Blvd. La Mirada, Calif. 90638 (714) 521-3753 Fax: (714) 994-1203 Amersham Corp. 2636 South Clearbrook Dr. Arlington Heights, Ill. 60005 (708) 593-6300 Fax: (708) 437-1640 Bio-Rad Laboratories Life Sciences Group 2000 Alfred Nobel Dr. Hercules, Calif. 94547 (510) 741-1000 Fax: (510) 741-1055 BioSource International Inc. 887 Mitlen Rd. Burlingame, Calif. 94011 (415) 698-4015 Biotecx Laboratories Inc. 6023 South Loop East Houston, Texas 77033 (713) 643-0606 Fax: (713) 643-3143 BioWhittaker Inc. 8830 Biggs Ford Rd. Walkersville, Md. 21793 (301) 898-7025 Fax: (301) 845-4491 Boehringer Mannheim Biochemicals 9115 Hague Rd. Indianapolis, Ind. 46250 (800) 428-5433 Fax: (317) 845-2000 Calypte Biomedical 1440 Fourth St. Berkeley, Calif. 94710 (510) 526-2541 Fax: (510) 526-5381 Cayman Chemical Co. 690 KMS Place Ann Arbor, Mich. 48108 (313) 662-6756 Fax: (313) 662-6896 Ciba-Corning Diagnostics 115 Norwood Park South Norwood, Mass. 02062 (508) 359-7711 Fax: (508) 359-3599 Many biological researchers who need to identify an interesting protein or determine its concentration in a sample say that enzyme immunoassays are their tool of choice. Using these versatile detection tests, they can, for example, pinpoint a specific gene from an organism's genome or analyze the kinetics of an enzyme. Clinical investigators can use enzyme immunoassays to detect circulating antibodies to disease-related antigens, distinguish among different Salmonella cell wall proteins, or identify substances in body fluids that signify the occurrence of a heart attack. Also, new automated enzyme immunoassay systems can now speed the diagnosis process dramatically. Researchers in both the clinic and the lab are calling enzyme immunoassays a major improvement over radioimmunoassays, widely used in past years for many of the same purposes. Users who are changing their systems cite the elimination of radioisotopes with their inherent health risks and disposal problems as a key advantage. "Enzyme immunoassays are at least as sensitive as radioimmunoassays," says Keld Sorensen, a senior research scientist for Pierce Chemical Co. in Rockford, Ill. "And any time you can change a procedure to get radioactivity out of the lab without affecting sensitivity, it's an improvement." The essential components of an enzyme immunoassay are an antibody specific to a target substance and an enzyme that makes detection of the bound antibody possible. Immunoassays performed in a solution, for example, respond to the initial reaction of the antibody and its target antigen, which then modulates the catalytic activity of the enzyme, allowing detection. Enzyme immunoassays combine the specific recognition of antibodies for their targets with the catalytic power of enzymes into a single sensitive and relatively simple test. The sensitivity of an enzyme immunoassay depends on the ability of antibodies to home in on a particular antigenic target, such as a protein, a bacterial or viral antigen, or other antibodies. Coupling this specificity to the catalytic ability of some enzymes to convert colorless chemicals to brightly colored products results in a detection system that can be adapted to a wide range of applications. With the growing emphasis on nonradioactive detection in molecular biology applications, enzyme immunodetection kits are becoming increasingly popular for use with Western blot (protein detection), Southern blot (DNA detection), and other procedures. In these tests, researchers first separate their samples by gel electrophoresis and then blot them to a support matrix made of nitrocellulose or nylon. After applying a primary antibody specific for the target, an enzyme-conjugated second antibody binds to the primary antibody, catalyzing a chromogenic reaction that leaves behind a dark precipitate on the support membrane. The sensitivity of these nonisotopic detection systems--such as the Genius kit for DNA detection from Indianapolis-based Boehringer Mannheim Biochemicals and the Immun-Blot assay kits from Bio-Rad Laboratories of Hercules, Calif., used in Western blotting procedures--is comparable to that of the more conventional radioisotopic detection schemes. By far the best known of the enzyme immunoassay techniques is ELISA (enzyme-linked immunosorbent assay). Because most ELISAs involve several washing steps to remove excess reactants before the next reagent is added, the tests are performed in the solid phase, which means that some component of the system is hooked to a solid support material. An investigator's choice of solid phase varies with the application, but the choices include microtiter plate wells, plastic or glass beads, emulsions of latex particles, and paper or nylon membranes. Scientists can design ELISAs in various ways to accommodate their specific application. The initial step is to adsorb one of the reactants--either the primary antibody, in order to capture the target antigen, or the antigen itself--to the solid phase. After the excess is washed away, the second component is added and reacts specifically with the first. "Linking the test system to a solid phase allows for the sequential amplification of the initial antibody's recognition of the target substance," says Paulette McCormick, a cell biologist at the State University of New York, Albany. The final step is the chromogenic reaction of the enzyme with its substrate that identifies the complex bound to the solid phase. The amount of colored end product is proportional to the amount of target substance in the test. In what is called an ELISA sandwich assay, a suitable antibody is adsorbed first to the solid phase, often the wells of a microtitration plate, and the excess washed away. Then a sample, which might be a patient's serum or a mixed solution of proteins, is applied to the wells. If the target substance is present in the sample, it will be specifically bound by the antibody on the well's surface. Unbound substances are washed away. Next, a second enzyme-labeled antibody is added, which binds to another epitope on the antigen, leaving an enzymatic tag. When the chromogenic substrate is added, the enzyme converts it to a brightly colored product that signifies the antigen's presence. Variations on the sandwich theme abound. Antigen can be bound to the solid phase in an antibody capture assay, where it competes with a sample antigen for recognition of the enzyme-labeled antibody. Capture assays can also be used in reverse, with the primary antibody attached to the solid phase competing for sites on the antigen with a labeled second antibody. To detect antibodies rather than antigens in an ELISA system, a sample antibody reacts with an antigen attached to the solid phase. In this method, termed an indirect antibody capture assay, a second antibody carrying the enzymatic tag is added, which binds to the captured target antibody. This form of ELISA is typically used by scientists screening clones for important antibody-secreting hybridomas. Laboratories that screen for specific antibodies in serum from immunized animals also use this technique. The choice of ELISA enzyme label is highly dependent on an investigator's application. The two most common enzyme choices are horseradish peroxidase (HRP) and alkaline phosphatase (AP). "Approximately 80 percent of all commercial ELISAs incorporate HRP in the system, and most of the rest use AP," says Pierce's Sorensen. A few other enzymes, such as acetylcholine esterase or a- galactosidase, are sometimes used. The popularity of HRP and AP, however, stems from their stability and the wide range of chromogenic substrates available for each enzyme. According to Sorensen, HRP is preferred over AP because, as a plant product, it is more easily produced and less expensive. Most ELISA kits developed are for use in human diagnostics. Using ELISA, investigators can detect hepatitis C virus in samples of body fluids, for example; discover contaminants such as Salmonella in foods before they are consumed; determine if a woman is pregnant; and identify people at risk for certain cancers. Physicians can perform diagnostic tests in their offices or clinics. Other types of enzyme immunoassays offer tests for therapeutic drugs or drugs of abuse or to assess a patient's thyroid function. Recently, enzyme immunoassays have been incorporated into automated testing systems, leading to additional applications. "Enzymatic immunoassays lend themselves to automation," says immunologist Gary Kitos, laboratory director at Allergy Testing Laboratory in Fort Lauderdale, Fla. "Most of the assay steps can be completed with robotics." A number of companies, including Ciba-Corning Diagnostics in Norwood, Mass., and Tosoh Medics Inc. in Foster City, Calif., have developed random-access immunoassay analyzers that automate the various enzyme immunoassay steps. Automated immunoassays for drugs, antigens, and hormones are already on the market, with more expected to follow shortly. Investigators also are finding that ELISA can be made to order. The only qualifier is the requirement for a suitable antigen or an antibody, which can be conjugated to an enzyme label in the lab. Companies like Pierce and Bio-Rad market various ELISA components, including primary and enzyme- conjugated antibodies, blocking reagents, and conjugating reagents and enzymes, which enable individual researchers and commercial manufacturers to design their own specific immunoassay systems. For many applications, a large array of kits containing all of the necessary components is available. A number of kits that are small and self-contained have allowed investigators to take ELISA into the field. One area in which the simplicity and lower cost of enzyme immunoassays have resulted in the availability of many new tests is environmental analysis. Using enzyme immunoassays, environmental scientists can detect contaminants such as pesticide residues, industrial chemicals, and microbial toxins in samples of water, soil, or air. Many of these types of tests are portable enough to take directly to a suspected hazardous waste site. For environmental testing to detect the presence of pesticide residues, including triazines such as atrazine, propazine, and simazine, in water samples, Millipore Corp. in Bedford, Mass., markets the EnviroGard test kit, used either in the laboratory or in the field. The kit, developed by Millipore subsidiary ImmunoSystems Inc. of Scarborough, Maine, is "complementary to the conventional methods of gas chromatography and HPLC," according to Jim Fecteau, group manager at ImmunoSystems. The test, which is based on an antigen-capture method, is performed in antibody-coated test tubes. Using the kit, field investigators can detect the presence of contaminants at a site and determine their approximate concentration simply by adding a sample and kit reagents to the supplied test tubes. "In some cases we can detect chemical contaminants in the parts-per-trillion range," adds Fecteau. Animal scientists can benefit from compact ELISAs in their field work, as well. Idexx Corp. in Westbrook, Maine, produces membrane-based ELISA kits that allow such specialists to perform diagnostic ELISAs in the field--a barn, for example--as well as in their clinics. Enzyme immunoassays are also being used in quality and safety testing of foods. Immunoassays that detect numerous food components and contaminants--including amino acids and sugars, pesticide residues, and antibiotics--are becoming increasingly available. Current methods of testing for microbial contaminants in foods based on conventional microbiological techniques can take up to five days to complete. To speed the detection of bacterial agents in food, Organon Teknika/Biotechnology Research Institute in Rockville, Md., has developed an ELISA in which whole bacterial cells are captured by antibodies attached to a solid phase. Tests for Listeria species and E. coli are also in developmental stages at Organon Teknika. Use of ELISA tests by the food industry could decrease the time necessary to identify a bacterial contaminant to 24 to 48 hours. Although testing of this kind is not yet widely accepted by food-safety critics, enzyme-based immunoassays are clearly a positive step. In-solution assays, an alternative to the solid phase testing, are limited mostly to analyses of small molecules to maximize the effect of the reaction between the antibody and the enzyme. The best known of these is EMIT (enzyme- multiplied immunoassay technique) from Syva Co. in San Jose, Calif., often used by clinicians to detect drugs of abuse and for therapeutic drug monitoring. Holly Ahern is a freelance science writer based in Albany, N.Y. She teaches cell and molecular biology at the State University of New York, Albany.

Uses Of Enzyme Immunoassays Growing In Laboratories And Clinics

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