Immunoassay Techniques Proven To Be Outstanding In Several Fields

SIDEBAR: Selected Suppliers of Immunoassays and Assay Systems Immunoassays combine principles of immunology and chemistry into tests that are used by scientists in practically every discipline, including fields as diverse as molecular biology and environmental science. In research applications, immunoassays such as immunoblotting (detecting membrane-bound proteins), immunohistochemistry (cell or tissue staining), and enzyme immunoassays provide a sensitive and specific means of detecting target substances. Some investigators are even taking the technology on the road. Scientists with several federal agencies-including the Environmental Protection Agency (EPA), the Food and Drug Administration (FDA), and the United States Department of Agriculture-are developing immunoassays to detect toxic chemicals at hazardous waste sites, identify minute amounts of pesticide in water, and rapidly recognize bacterial pathogens in food. Immunoassays are simple to perform and portable enough to take to a testing site, which might be a former industrial landfill or a polluted stream. Although the various types of immunoassays are performed differently, they have one thing in common-they all involve antibodies. "The basis for the immunoassay is the specificity of the antibody-antigen reaction," states Keld Sorensen, senior research scientist at Pierce Chemical Co. in Rockford, Ill., and an adjunct assistant professor of biochemistry and immunology at the University of Illinois College of Medicine at Rockford. Used in an appropriate immunoassay system, specificity leads to sensitivity. "Proteins such as adenylate cyclase that participate in the signal-transduction [cell-signaling] process occur only rarely in cells," says Sorensen. "To detect these substances, you must have an extremely sensitive detection system. That's why the immunochemical methods are so important." While immunoassay techniques vary, the essential component of every immunoassay is an antibody. In nature, antibodies are produced by a type of immune cell known as a plasma cell (a mature B lymphocyte) in response to a foreign particle or substance (an antigen). The antibodies produced during an immune response recognize and bind only to the antigen(s) that elicited the response. Once coated with antibody molecules, antigens are targeted for destruction by other immune system cells. In the laboratory, researchers have harnessed the power of the immune response to produce antibodies of their own design. Injected into a mouse, rat, chicken, or goat, cells or substances such as protein or polysaccharide induce the animal's immune system to produce antibodies. "An immunized animal will produce antibodies against the antigen which can be collected, purified, and used as a reagent in an immunoassay," explains Mary E. Morton, Charles A. Dana Faculty Fellow and an assistant professor of biology at College of the Holy Cross in Worcester, Mass. Once an animal is immunized, antibodies are removed from a body fluid, usually the liquid fraction of the animal's blood (the serum or plasma). In some cases, antibodies are purified from an animal's milk, the yolk of its eggs, or-in the case of monoclonal antibody-producing mice-a type of peritoneal fluid called ascites. Because animal serum contains many other types of proteins in addition to antibodies, the immunoglobulin molecules (antibodies) are generally purified from the immune serum before they are used as an immunoreagent. According to Morton, "there are many different ways to purify antibodies," including affinity chromatography with immobilized protein A, where the antibody solution is passed through a column with a solid-phase material cross-linked to the immunoglobulin receptor protein A. Researchers can create and purify their own antibodies, or purchase them from several companies that produce antibodies commonly used in the lab. For example, Pierce's ImmunoPure line of antisera consists of affinity-purified antibodies against a wide range of antigens, including cell receptors, enzymes, hormones, and human immunoglobulins (H. Ahern, The Scientist, Sept. 18, 1995, page 18). The list of techniques in which antibodies are used as either a primary or a secondary reagent includes immunoblotting, in which a single protein is identified on a Western blot of electrophoretically separated proteins; immunoprecipitation, which involves the precipitation of antibody-bound antigens; and immunohistochemistry, whereby fixed cells are stained with antibodies bearing enzymatic or fluorescent tags. Also included are RIA (radioimmunoassay) and a host of EIAs (enzyme immunoassays), such as ELISA (enzyme-linked immunosorbent assay) and ELIFA (enzyme-linked immunoflow assay). To detect and/or amplify the initial antibody-antigen reaction in an immunoassay, antibodies must be labeled. Antibody labels include radioisotopes; enzymes, such as horseradish peroxidase (HRP) and alkaline phosphatase (AP); biotin (a plant ligand); and fluorescent dyes, such as fluorescein or rhodamine. Antibodies come already conjugated to a label; alternatively, researchers have the option of labeling the antibody on their own. Antibody conjugates are available from Pierce, which carries affinity-purified antibodies conjugated to colloidal gold, biotin, fluorescein, rhodamine, HRP, and AP. PerSeptive Diagnostics Inc. in Cambridge, Mass., also offers several types of antibody conjugates, including antibodies labeled with the radioisotope 125I. Investigators can label their own antibodies using products such as the Fluorescein Labeling Kit and the QuickTag FITC Conjugation Kit from Indianapolis-based Boehringer Mannheim Biochemicals Inc., for labeling antibodies with the dye fluorescein. Pierce's ImmunoPure Plus Activated Peroxidase Kit allows researchers to prepare HRP-labeled antibodies for immunoblotting and ELISAs. In a direct immunoassay, the antibody used as the primary reagent is given the fluorescent or enzymatic tag. In indirect immunoassays, the secondary antibody-usually polyclonal antisera produced by a goat or a rabbit against human immunoglobulins-carries the label. When a secondary antibody is used, the initial immune reaction between the primary antibody and the target antigen is amplified, producing a more readily detectable signal. Labels and detection systems vary according to the application. "The choice of a label really depends on the type of assay you are performing," explains Morton. For immunochemical staining of cells to study the distribution of calcium channels in cells, for instance, Morton prefers to use antibodies labeled with a fluorochrome such as fluorescein. Western blots of electrophoretically separated proteins (immunoblots), on the other hand, are generally probed with antibodies labeled with an enzyme or a radioisotope such as 125I. Many research labs, however, are following a general trend that leads away from using isotopes, because radioactivity is considered more dangerous to work with and more expensive to use. "When you add up all of the costs associated with using radioactivity, in terms of disposal and laboratory safety issues, it is far less expensive to use an enzyme label with a chromogenic or chemiluminescent substrate than it is to buy radioisotopes," says Sorensen of Pierce. "And with chemiluminescence in particular, the sensitivity is at least as good," he adds (L.J. Kricka, Clinical Chemistry, 37[9]:1472-81, 1991). "The new chemiluminescent assays are at least as sensitive as methods using a radiolabeled antibody," agrees Morton. THE KIT CONCEPT: New England BioLabs' PhosphoPlus MAP Kinase Antibody Kit detects the phosphorylation status of MAP kinase, an enzyme involved in the signal transduction. The sensitivity of an immunoassay refers to the level at which an antibody-antigen reaction is detected. In an enzyme immunoassay, for example, the enzyme label on the antibody catalyzes the chemical conversion of a substrate into something that can be observed. Enzymes such as HRP and AP catalyze chromagenic reactions, in which a colorless substrate is converted into a colored compound, and also chemiluminescent reactions where light is emitted. Bio-Rad Laboratories of Hercules, Calif., has chromogenic substrate kits available for alkaline phosphatase, horseradish peroxidase, and TMB peroxidase (TMB is tetramethylbenzidine, the substrate in this case). Boehringer Mannheim also has several enzyme substrates for immunoassays available. They include ABTS (2,2+-azino-di-3-ethylbenzthiazoline sulfonate) and TMB (tetramethylbenzidine), which are used with HRP; and 4-nitrophenyl phosphate and 5-bromo-4-chloro- 3-indolyl phosphate (BCIP) for immunoassays in which an alkaline phosphatase-conjugated antibody is used. Immunoblotting, in particular, is one area in which the chemiluminescent substrates are edging out the detection systems based on enzymes or radioactivity. Tropix Inc. of Bedford, Mass., was the first company to develop a chemiluminescent substrate for EIA applications. Immunodetection systems from Tropix (Western-Light and ELISA-Light) are for use with alkaline phosphatase as the antibody conjugate. These systems use the chemiluminescent substrates CSPD and AMPPD, which are 1,2-dioxetane molecules that emit light when decomposed by an enzyme. "These chemiluminescent substrates show glow kinetics, which means that light is given off steadily for a time and then diminishes, rather than as a rapid flash of light," comments Philip Weiland, technical support specialist with Tropix. To quantify the flash of light, the immunoblot is placed in contact with X-ray film, which is exposed only in the areas of light. This forms dark bands on the film, similar to the results obtained with a radioactive label. "Having a permanent record on film is an advantage of chemiluminescence over chromogenic substrates," in which the hard-copy result is a membrane bearing the precipitated chromagen, according to Morton. The membranes are flimsy and the signals fade over time. Additionally, with a "glowing" substrate, a researcher could reexpose immunoblot films where the signals were too faint, or use a new piece of film to correct overexposures. Chemiluminescent substrates are available from other companies. Pierce, for example, produces the SuperSignal CL-HRP Substrate, an enhanced chemiluminescent substrate for horseradish peroxidase. This system detects specific proteins on immunoblots with a sensitivity that rivals radioactivity (reportedly to picogram levels). When the chemiluminescent substrate is applied to membrane-bound proteins on an immunoblot, an instantaneous but long-lasting flash of light is produced. Together with the stability of chemiluminescent substrates (up to one year), the sensitivity, and the factor of no hazardous waste to deal with, chemiluminescent enzyme immunoassays are quite appealing. This is especially true for scientists studying signal transduction, in which the participating peptides and enzymes occur in cells in very low concentrations. For practically any research application, an investigator may choose to purchase the antibodies, conjugates, blocking reagents, substrates, and solid-phase supports separately and assemble an immunoassasy system on his or her own. However, for an initial venture into immunology, a molecular biologist or a neuroscientist might prefer to purchase all of the various components of an immunoassay together in a kit. Molecular biologists, for example, might consider using the picoBlue immunoscreening kit from Stratagene Cloning Systems in La Jolla, Calif., to screen genomic libraries constructed in plasmids or in bacteriophage (bacterial viruses) by blotting bacterial colonies or phage plaques to a membrane and probing with antibodies specific for the proteins produced by a target gene. This kit involves an AP-labeled secondary antibody to tag the clones containing the gene of interest. Cell biologists and other scientists interested in signal transduction use the PhosphoPlus Antibody Kits from New England BioLabs Inc. of Beverly, Mass., to detect phosphorylation status of proteins, especially MAP kinase and cdc2 kinase, two enzymes involved in signal transduction. The kits contain the antibodies and Phototope-Star chemiluminescent western blot detection system to assay the phosphorylation status of proteins. Peninsula Laboratories Inc. of Belmont, Calif., and R&D Systems Inc. in Minneapolis both offer several immunoassay kits for signal-transduction research. The Quantikine and Quantikine HS immunoassay kits from R&D Systems are for measuring cytokines and soluble cytokine receptors in serum. The Quantikine HS kit, in particular, measures cytokines present in extremely low concentrations. Peninsula laboratories has several EIA and RIA kits for detecting peptide portions of hormones and other proteins, and EIA kits for cytokines including the interleukins, interferon-a and TNF (tumor necrosis factor). FOR FINE-TUNING SAMPLES: Molecular Devices' SEPCTRAmax 340 tunable microplate reader allows users to tune in the optimal wavelength foir a particular microplate assay. Boehringer Mannheim Biochemicals has several immunoassay kits available. For researchers studying apoptosis (the phenomenon of cellular suicide), the company offers ELISA kits for cell death detection and to observe cellular DNA fragmentation. "The Cell Death Detection ELISA allows researchers to measure apoptosis quantitatively by detecting the release of nucleosomes [a complex of histones and DNA fragments] into the cytoplasm," an event that occurs during apoptosis, explains Paul Clark, technical-support specialist for Boehringer Mannheim. The Cellular DNA Fragmentation ELISA, also from Boehringer Mannheim, contains antibodies directed against BrdU (5-bromo-2+-deoxy-uridine)-labeled DNA that detect fragments produced as a result of programmed destruction of the nucleus. Both of these kits are photometric ELISAs and provide researchers with quantitative results similar to those achieved with radioactive assays. In addition to electrophoresis equipment and Western blotting apparatus for immunoblotting applications, Bio-Rad has ELISA kits to assay monoclonal antibodies, including the Clone Selector kit to screen mouse cell clones and the Mouse Typer isotyping kit for identifying the class, subclass, and the type of light chain (an immunoglobulin subunit) of mouse antibodies. An advantage of performing EIAs, particularly ELISA, is the potential for automating all or part of the immunoassay process. Generally, ELISAs are performed in 96-well plastic microtiter plates, and the various steps-adding antigen or antibody and washing the plates between additions, for example-can be accomplished with robotics. Although automated instruments for EIAs are used in clinical laboratories, which perform huge volumes of diagnostic EIAs, most research laboratories use manual or semimanual techniques. Operations that are time-consuming and labor-intensive, such as the washing steps between reagent additions in ELISAs, can be accomplished robotically. Microplate washers involve a one-time expenditure of a few thousand dollars; however, they significantly decrease the hours of labor involved in the process. The Model 1550 Automatic/Manual Microplate washer from Bio-Rad, for example, can be programmed to add buffer, soak the wells, and aspirate the wash buffer from all 96 wells of a microplate simulta-neously. While ELISAs and other EIAs can be analyzed manually, microplate readers that spectrophotometrically measure the optical density or absorbance of reactions as they occur in the microplate wells provide a much more quantitative analysis. Bio-Rad's Model 3550 and 3500-UV (for applications requiring an ultraviolet light source) automatic microplate readers collect the data, analyze the results, and even print out reports. Both microplate readers read single plates and can be converted to automatic multiple-plate operation, in which up to 25 plates can be loaded at one time and analyzed. The SPECTRAmax 340 microplate reader from Molecular Devices in Sunnyvale, Calif., is a "tunable" system, which means that the operator can tune in the optimal wavelength for a particular microplate assay. Instruments traditionally used for analytical chemistry techniques such as HPLC and capillary electrophoresis (CE) are sometimes utilized in immunoassays to detect complexes of antibodies and target substances such as proteins and peptides. CE instruments equipped with a UV-vis (detects light in the ultraviolet and visible ranges) or a laser-induced fluorescence-detection system allow analysts to perform immunoassays with detection limits in the nano- to picomolar range. Examples of such units are the P/ACE 5000 Series from Beckman Instruments Inc. of Fullerton, Calif., and the 270A-HT system from Perkin-Elmer Corp. Applied Biosystems Division in Foster City, Calif. DOUBLE DUTY: Beckman Instruments' P/ACE 5510 Capillary electrophoresis system with laser-induced fluorescence detector, right and P/ACE 5000 can be used in immunoassays. Typical solid-phase techniques such as ELISA are both specific and sensitive, but they take time, particularly in the washing and detection steps. With a CE format, the immune reactions are carried out in capillary tubes in the liquid phase. Complexes of antibody and target antigen form in the liquid phase, and the complexes are separated by the CE instrument. When a laser-induced fluorescence-detection system is used, fluorescently labeled antigens, both complexed with antibody and unbound, are detected when the fluorescent dye is activated with a laser. This information is translated into a quantitative estimation of the target protein or peptide. CE immunoassays could prove an important analytical resource for the diagnostic and biopharmaceutical industries, in which the goal of many researchers is to measure minute amounts of analytes in a sea of serum proteins. SIDEBAR: Selected Suppliers of Immunoassays and Assay Systems Holly Ahern is a science writer and an assistant professor of biology at Adirondack Community College in Queensbury, N.Y. (The Scientist, Vol:9, #19, pg.18-19 , October 2, 1995) (Copyright © The Scientist, Inc.)

Immunoassay Techniques Proven To Be Outstanding In Several Fields

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