Antibodies Making Their Way From The Clinic To The Research Lab

if (n == null) The Scientist 9[18]:18, Sep. 18, 1995 Tools The Immune Response By Holly Ahern Imagine that you've just cloned a gene for a bacterial enzyme with unique structural properties and you want to find out more about it. What natural role does the enzyme play, you wonder, and do organisms other than bacteria produce it? To answer these questions, you could screen countless genomic libraries for clones bearing similar DNA sequences. Or you could perform Southern blots, a molecular biology technique in which DNA fragments are separated by electrophoresis and then transferred to a support membrane and "probed" for homologous sequences. Alternatively, you could inject your protein into a mouse, a rabbit, or a chicken and let the animal's immune system make you an antibody as specific for your protein as a key is for a lock. The antibody will detect mutant forms of the enzyme on a blot made from electrophoretically separated proteins, bind to a target protein and label it, precipitate it, or -- when hooked to a solid phase such as agarose beads or paramagnetic particles -- purify the protein from a protein mixture. "Researchers can utilize antibodies in several different types of tests," notes 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. Research techniques in which antibodies are used include immunoblotting to detect membrane-bound proteins, immunohistochemistry for labeling specific areas in fixed smears of cells or tissues, enzyme immunoassays such as ELISA (enzyme-linked immunosorbent assay) and ELIFA (enzyme-linked im-munoflow assay) and immunoprecipitation, in which an antibody-bound protein precipitates out of a solution. Antibodies, produced as natural protection against infection and disease, are making their way out of clinical laboratories, where they have been used for decades to quantify blood levels of diagnostic markers such as hormones, cancer antigens, and human antibodies against bacteria and viruses. They are moving into the laboratories of molecular biologists and microbiologists for biochemical studies on gene products, or to detect pathogenic organisms in food and in water supplies. Cell biologists and neurobiologists study signal transduction events in cells and the phenomenon of apoptosis (programmed cell death) with antibodies labeled with fluorescent or radioactive tags. Researchers can obtain antibodies from a number of companies. Some, such as Upstate Biotechnology Inc. of Lake Placid, N.Y., and the Berkeley Antibody Company (BAbCo) in Richmond, Calif., specialize in immunochemicals. Others, like Pierce Chemical Co.; St. Louis-based Sigma Chemical Co.; and Boehringer Mannheim Biochemicals in Indianapolis, carry a broader range of biochemicals and biotechnology products. One area of research for which antibodies are abundant is signal transduction. "Antibodies for signal transduction research are becoming increasingly available," observes Mary E. Morton, Charles A. Dana Faculty Fellow and an assistant professor of biology at the College of the Holy Cross in Worcester, Mass. Morton uses antibodies to study the distribution and developmental expression of calcium channels in cells and has licensed several antibodies to a commercial supplier. "Until recently, antibodies for neurotransmitter receptors have been hard to come by, in part because of the structure of the receptor molecule," she notes. "Antibodies such as these and the calcium channel antibodies are slowly becoming more available." Researchers consider using antibodies in their investigations because they make powerful laboratory tools. Antibodies recognize and bind to their target with an affinity unmatched in nature. Used in assays, antibodies are both specific and sensitive reagents. Antibodies are naturally produced by a specific subset of immune cells called plasma cells (mature B-lymphocytes) during an animal's response to foreign molecules and particles (antigens). The presence of an antigen triggers the immune system into producing antibody molecules that recognize only the antigen(s) that elicited the response. Once secreted by the plasma cells, antibodies circulate in the blood and lymph, where they bind to the targeted antigen. The antibody-antigen complexes set into motion additional immune reactions aimed at destroying the invading substance. The complexes are cleared from the body by macrophages, which en-gulf and ingest the offending particles. In the laboratory, antibodies are produced by teasing the immune system of an animal such as a mouse, rabbit, chicken, or goat into producing antibodies against a specific antigen. "Many different animals can be used for the production of antibodies," explains Jordan Fishman, president of Quality Controlled Biochemicals Inc., a Hopkinton, Mass., company that provides custom peptide services and anti-peptide antibodies as well as cytokines and other research biochemicals. "Making antibodies in chickens is an up-and-coming technique. Chickens seem to be quite adept at bringing out tough antigens, and the antibodies are recovered from the yolks of their eggs." In most cases, however, blood is collected from the immunized animal, and the liquid portion, called plasma or serum, is separated from the red blood cells. Once the presence of the antibody in the serum is confirmed, the serum is called antiserum. Antisera are widely used by clinicians and researchers as reagents in immunological assays such as immunoprecipitation, in ELISA, and for the detection of proteins separated by electrophoresis and transferred to a support membrane by Western blot. Antisera can be either polyclonal or monoclonal, a distinction based on the method of production. When an antigen such as a purified protein is injected into an animal, the different parts of the protein molecule, called epitopes or determinates, serve as targets for the production of antibodies. Polyclonal antibodies are produced when several different plasma cell lines, called clones, secrete antibody molecules, each one specific for a different epitope. The result is a heterogeneous antiserum that may contain several different types of antibody molecules directed against the various determinants on the antigen. Conversely, monoclonal antibodies are secreted by a plasma cell clone derived from a single B-lymphocyte precursor; thus, the antibody molecules are all the same. Unlike polyclonal antisera, which can be collected and purified from the blood of laboratory animals, monoclonal antibodies are produced by plasma cells grown in cell culture. Lymphocytes, however, cannot be kept indefinitely in culture, and the loss of a cell line also means loss of the antibody. To keep antibody-producing cells alive, the hybridoma method developed in 1975 by immunologists George Köhler and Cesar Milstein (Nature, 256:495-7, 1975) is often used. In this technique, spleen cells from an immunized mouse are fused with human myeloma cells, which are immortal. The resulting hybridoma cells grow in culture, forever secreting their epitope-specific antibodies. The antibodies can be collected from the supernatant of hybridoma cultures, or from ascites, a fluid extracted from mice with tumors induced when hybridoma cells are injected into the mouse's peritoneum. "As long as the hybridoma cell line is maintained, there will be a virtually unlimited supply of monoclonal antibodies," Morton points out. As a primary reagent, one that binds directly to a target antigen in an immunoassay, monoclonal antibodies offer a high degree of antigen-binding specificity with little variability. "With monoclonal antibodies, you essentially know what epitope the antibody reacts with and that over time the antibody will remain the same," says Morton. The specificity of monoclonals is important for detecting target molecules, such as neuropeptides or enzymes involved in signal transduction, which may be present in small quantities. Because monoclonal antibodies are epitope-specific, they generally do not cross-react with other proteins in immunological assays. For immunostaining cells with fluorescently tagged antisera, for example, monoclonal antibodies often work very well because their specificity for the target antigen decreases the amount of nonspecific background staining that can block out the desired image. Often, monoclonal antisera are used in combination with polyclonal antisera that function as a secondary reagent, detecting the primary antibody bound to the antigenic target. Secondary antibodies, usually produced against a particular class of immunoglobulin such as IgG or IgM, are conjugated to substances that change color or light up, indicating the presence or concentration of an antigen, or its location on a Western blot. In an ELISA test to detect apolipoprotein A-1, for example, an investigator might use mouse monoclonal anti-apolipoprotein A-1 as a primary antibody to detect the protein, followed by a polyclonal antiserum, produced in a goat against mouse IgG molecules, conjugated to an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). The enzyme that tags the target protein catalyzes a colorimetric or chemiluminscent reaction, yielding a visible result of color or light. "Using a secondary antibody in immunoassays amplifies the reaction of the primary antibody with the target molecule," explains Pierce's Sorensen. Pierce offers a line of ImmunoPure reagents, consisting of monoclonal antibodies as well as polyclonal antisera that researchers can use as secondary reagents in immunoassays. Pierce also carries antibodies already labeled with fluorescent dyes, like fluorescein and rhodamine; enzymes such as horseradish peroxidase and calf intestinal alkaline phosphatase; biotin; and even the precious metal gold. Gold-labeled antibodies are particularly useful to prepare tissue slices for scanning electron microscopy, because the gold particles strongly emit the important image-forming electrons. Researchers can use either a monoclonal or a polyclonal antiserum for immunoprecipitations, in which an antibody-bound protein is separated from a mixture during a precipitation reaction. After the target protein is bound, the antibody-antigen complexes are usually collected with bacterial cells carrying an antibody molecule receptor (protein A) or with protein A-bearing agarose or acrylamide beads. Large antibody-antigen-bead complexes form and precipitate when the mixture is centrifuged. SEPARATION WITHOUT ANXIETY: PerSeptive Diagnostics' BioMag superparamagnetic particles, including BioMag CD34, may be used for cell sorting, lympocyte isolation, and stem-cell purification. An interesting twist on the immunoprecipitation scheme is to chemically link the antibody to magnetic particles. After the antibody binds the target protein, the protein-bead complex is isolated with a special magnet. "We have available both magnetic beads and a whole range of primary and secondary antibodies," says Howard Kartstein, sales manager at PerSeptive Diagnostics Inc. in Cambridge, Mass. Researchers "could also utilize the particles with their own antibody," he adds. PerSeptive's BioMag CD34 Selection Set includes beads with an antibody directed against the cell surface marker CD34, present only on stem cells. A magnet attracts the paramagnetic beads (beads with no magnetic memory) bound to cells bearing the CD34 marker, selectively removing those cells from a pool of different cell types. CD34 cells are used in research as well as in the clinic, where physicians return the separated cells to chemotherapy patients to repopulate their depleted bone marrow with stem cells. Antisera, both monoclonal and polyclonal and specific for immeasurable antigens, are widely available. Sigma Chemical Co. carries a range of monoclonal antibodies -- everything from monoclonal and polyclonal anti-albumin (a serum protein) to monoclonal anti-zearalenone (a fungal toxin). Life Technologies Inc. of Gaithersburg, Md., manufactures a line of immunology reagents that includes monoclonal antibodies and secondary antibodies such as monoclonal anti-mouse CD3, which researchers can obtain as purified antisera or conjugated to biotin, fluorescein, R-phycoerythrin, or RED613. Life Technologies also carries culture media and biochemicals such as amino acids and growth factors for growing hybridomas and producing monoclonal antibodies. For biomedical researchers studying cardiovascular diseases, antibodies from Boehringer Mannheim Biochemicals that are specific for various apolipoproteins allow investigators to correlate blood levels of lipoprotein, a suspected villain in human coronary disease, to a person's risk of developing arteriosclerosis. The anti-apolipoprotein antisera are applicable to immunoelectrophoresis pro- cedures such as radical immunodiffusion (RID) and rocket immunoelectrophoresis (IEP), in which the antibody-antigen reactions occur in agarose gels, as well as applicable to ELISA and immunoblotting procedures. Boehringer Mannheim reports that its monoclonal anti-apolipoprotein E, used in Western blot analysis of electrophoretically separated serum samples, detects only apolipoprotein E and none of the other lipoprotein fractions, such as AI, AII, or B. In addition to forming the basis for innumerable biomedical and clinical assays, antibodies have advanced basic research in several areas, particularly for neuroscientists and biochemists studying the phenomenon of signal transduction. "Signal transduction researchers study how cells convey a signal from their surface to their interior, a process that involves several interrelated biochemical pathways," explains Sorensen of Pierce. "Drug-discovery laboratories in particular are looking for ways to manipulate signal-transduction pathways and perhaps get a handle on conditions such as cancer that appear to involve a loss of a cell's regulatory capabilities." In ELISAs and Western blots, investigators use antibodies to detect the participating enzymes directly, or to look for the occurrence of signal-transduction events, such as phosphorylation, the enzymatic process of adding phosphate groups to a substrate. Because phosphorylation appears to play a key role in cell signaling, several companies -- including Pierce, Boehringer Mann-heim, and Life Technologies -- sell antibodies specific for phosphorylating enzymes such as protein kinase C and tyrosine kinase. Pierce also carries antibodies against phosphorylated substrates, like phosphotyrosine (a phosphorylated amino acid), to detect the actual phosphorylation event. Apoptosis, the phenomenon of programmed cell death, is another area of research to which antibodies can be applied. "Antibodies specific for Fas antigen [involved in apoptosis] are in demand," says Ken Plaia, technical specialist for Oncor Inc. of Gaithersburg, Md., which carries antibodies for signal-transduction and apoptosis research. "Apoptosis is becoming a very big field." Some types of cells, such as those involved in immune responses, are apparently programmed for destruction as early as the stem-cell stage of development. At the conclusion of apoptosis, the apoptotic cell's DNA is degraded into fragments that can be detected in agarose gels. With the ApopTag apoptosis detection kit from Oncor, digoxigenin-labeled nucleotides are detected by peroxidase-conjugated anti-digoxigenin antisera, which stain the apoptotic cells. Oncor also carries antibodies specific for proteins involved in signal transduction, as well as proteins such as the p53 gene product, implicated in cancer. Another supplier of antibodies for signal-transduction research, Upstate Biotechnology Inc., intends to broaden its product line to accommodate the growing demand for apoptosis reagents. "Over the next few months we are expanding our antibody line to include a greater number of products for apoptosis research," notes Andrea Besse, technical representative for Upstate Biotechnology. Antibodies conjugated to fluorescent dyes and used to stain tissue or samples of cells pinpoint the cellular location of receptors and transducers. The fluorescence pattern of the cells, usually detected by fluorescence microscopy or by spectrometry, indicates how cells send and receive biochemical signals. Molecular Probes Inc. of Eugene, Ore., provides secondary antibodies linked to fluorescent dyes for fluorescence-microscopy applications. Molecular Probes also provides protocols and reagents for researchers who want to fluorescently label antibodies to suit their particular applications. Although the varieties of antisera number in the thousands, there are still some applications for which a commercial antibody does not exist. Investigators who can't find suitable antisera from among the myriad of available products have the option of producing an antibody themselves. This could involve purchasing rabbits or mice and learning the ins and outs of animal care. Alternatively, a researcher can send an antigen to a contract company, such as BAbCo, where the antibody will be custom-made. This way, a molecular biologist or biochemist with little expertise in immunology can still gain the antibody advantage. Quality Controlled Biochemicals will also custom-produce antibodies against peptides. An investigator who knows the amino acid or nucleotide sequence of a protein can synthesize a peptide deduced from sequence. Used as an antigen, the peptide will stimulate the production of specific antibodies that will recognize the native protein from whence the peptide came. "To study protein -- perhaps a receptor involved in signal transduction or a protein involved in a key regulatory pathway -- all you need to know is the sequence of the gene [or protein]," explains Fishman, the company's president. "From the sequence, regions are identified which have immunogenic amino acids and a peptide antibody can be made." Antipeptide antibodies are at least as specific as monoclonal antibodies for particular epitopes, because an animal is immunized with only the part of the antigen for which an antibody is sought. "One of the big advantages of using antipeptide antibodies is that you don't to go through the hassle of producing a monoclonal antibody," says Fishman. He believes that the key to making peptide antibodies that can be used successfully in research is in the quality of the peptide and the method of immunization: "If the peptide is properly made and purified in the best manner possible before an animal is immunized, the number of successes will continue to go up." Holly Ahern is a science writer and an assistant professor of biology at Adirondack Community College in Queensbury, N.Y.

Antibodies Making Their Way From The Clinic To The Research Lab

The Immune Response

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