MONOCLONAL ANTIBODIES

Antigens, by their nature as macromolecules having primary, secondary, tertiary, and quaternary structures, constitute a “mosaic” of antigenic determinants. An outgrowth of basic serologic principles and techniques has been the attempt to “purify” antigens to reduce the heterogeneity of antibodies developed against them. Antigen molecules with only a single epitope are rarely encountered; rather, hundreds or even thousands of potential antigenic determinants may exist on a cell surface or within the mix of other substances. When these mixed antigens are injected into an animal, an equal number of lymphocyte clones are stimulated. Even though each clone produces a specific antibody, the final result is a highly heterogeneous mixture of antibody molecules, the specificity and affinity of which are often unknown and difficult to control from batch to batch. When these polyclonal antisera are used in immunologic test systems involving infectious agents, cross-reactivity may be noted either because antigenic determinants are shared by different species or because mutations may have led to the evolution of epitopes sufficiently close in specificity to produce detectable cross-reactions. Attempts to produce pure antibodies through absorption with cross-reacting antigens or to prepare “clonal” from “polyclonal” antisera via techniques such as affinity column chromatography have only been partially successful.

As the science of serologic testing evolved, the view was held that the availability of an antibody having a high degree of molecular homogeneity and with specificity for a single, antigenic epitope without cross-reactivity would solve many of the problems encountered in the use of polyclonal antibodies. Highly specific monoclonal antibodies, the product of a single clone of lymphoid cells, gradually emerged as a by-product of the investigations in cell fusion and hybridoma technology conducted by Kohler and Milstein. Because of their discovery, it is now possible to isolate cloned lines of individual lymphocytes that produce unique, monospecific antibody molecules. Monoclonal antibodies refer to a uniform, homogeneous, molecular species of Ig, rather than to a heterogeneous array of Igs as is produced during the usual immune response. The principal feature of this technology was not that a single line of monoclonal antibody (MAB)-producing cells could be isolated, but rather that these mouse lymphocytes could be “fused” with mouse myeloma cells to produce hybrid cells having two inherent properties: (1) the capability of producing monospecific antibodies (acquired from the parent lymphocytes) and (2) the ability to grow permanently in culture (the characteristic “immortality” acquired from the transformed myeloma cells). Thus, individual monoclonal antibodies can be produced in a continuous and almost endless supply.

Monoclonal antibodies have been developed against clinically relevant antigens of many bacterial, viral, fungal, and parasitic agents, and reagents prepared with these antibodies are used in many commercially available EIA and immunofluorescence test kits. In addition to the direct detection of microbial structural antigens (e.g., capsular polysaccharides, outer membrane protein antigens), monoclonal antibodies have also been developed for the detection of microbial virulence factors, such as toxins produced by enterohemorrhagic E. coli (e.g., Shiga and Shiga-like toxins). This approach introduces a new way of evaluating the relationship of microorganisms and infectious diseases. Instead of the conventional focus on detection and identification of the organisms themselves, these reagents allow specific detection of microbial virulence factors that may be shared by several bacterial species associated with a given symptom complex. For example, it may be more important to know that an enteric toxin is the cause of hyperosmotic diarrhoea, rather than to receive the information that the patient is infected with Shigella species or an enterotoxigenic E. coli strain.

Procedure for Production of Monoclonal Antibodies.

1. Selection of Antigen

Monoclonal antibodies can be produced against any substance recognized as an antigen by the immune system of the animal being injected. Using a pure antigen is ideal. In fact, certain antigens, such as chemically purified drugs used for assays (e.g., digoxin), may be homogeneous. Even so, one can never guarantee that an antigenic determinant will consist of only one epitope. The fact that impure antigens can be used in MAB production is a chief advantage over conventional methods used to produce polyclonal antibodies.

2. Animal Immunization

The chief objectives in the immunization procedure are to prime the immune system of the animal to avidly recognize all antigens injected, to maximally stimulate B-lymphocyte clones, and to have the spleen cells divide at a high rate. In the production of monoclonal antibodies, the BALB/c mouse strain is most commonly used. The antigen is injected subcutaneously or intraperitoneally, with the simultaneous injection of Freund’s adjuvant. Injections are repeated at weekly intervals, and a final “booster” injection is given intravenously approximately 3 days prior to harvesting the spleen cells. At the end of the injection schedule, the animal is killed and the spleen is aseptically removed.

3. Fusion of Splenic Lymphocytes and Myeloma Cells

The animal spleen is placed in sterile culture medium containing antibiotics. The splenic tissue is teased to release cells and to form a slurry. This material is passed through a mesh to obtain single cells. Ficoll is added, and the slurry is centrifuged to remove RBCs. Polyethylene glycol (PEG) is added to the slurry to reduce cell-to-cell surface tension; this brings the cells into close proximity to one another, allowing their membranes to fuse. Dimethylsulfoxide (DMSO) is added to the fusion mixture to maximize cell contact even more. Finally, the cells are packed into a pellet by gently centrifuging the mixture for 5 minutes. Thus, at the end of these steps, the preparation consists of unfused myeloma cells, unfused lymphocytes, and a few fused hybrid lymphocyte-myeloma cells (It should be recognized that splenic lymphocytes and myeloma cells fuse with a frequency of only about 1 per 105 or 106 cells.).

4. Selection of Hybrid Lymphocyte-Myeloma Cells

Unfused myeloma cells rapidly outgrow the hybrids and must be removed in some manner. The myeloma cells used for fusion are grown in the presence of 8-azaguanine, a drug that causes the cells to permanently switch off the production of hypoxanthine phosphoribosyl transferase (HPRT), an enzyme that is needed to continue growth. If these HPRT-negative cells are suspended in a medium containing hypoxanthine, aminopterin, and thymidine (HAT medium), only the hybridoma cells will grow successfully. The hybridoma cells inherit HRPT from the splenic lymphocytes with which they have fused and will survive. The unfused myeloma cells, unable to synthesize DNA because of inability to produce HPRT, will be killed by the aminopterin in the selective HAT medium. It should also be remembered that unfused splenic lymphocytes do not survive beyond a few days in culture medium; therefore, the fused hybrid lymphocyte-myeloma cells alone survive in the HAT medium.

5. Cloning the Hybridoma Cells

The single hybrid cells producing the desired antibody must be isolated and grown as a clone. Two techniques can be used: (1) limiting dilution and (2) growth in an agar gel medium. In the limiting or doubling dilution technique, the suspension of hybrids (after maximum growth) is diluted and distributed into a series of sterile wells in a microtiter plate. The dilutions are so calculated that each well contains an average of only one cell that can then be replaced as a single antibody-producing clone. In the alternative method, using agarose gel supplemented with serum, amino acids, and antibiotics, the dividing hybrid cells form tiny, sphere-like clusters. These spheres can be selected with a Pasteur pipette and transferred to microtube wells for further culture and ultimately for assay to determine whether the desired antibody is being produced.

6. Screening for Desired Antibodies

In the fusion step of the procedure of producing monoclonal antibodies, many lymphocytes other than those producing the desired monoclonal antibodies may have fused. In fact, less than 5% of the hybrid cells out of those selected actually produce the desired specific antibodies. Thus, assays of the selected cell lines are required to determine if the desired antibody is being produced. Radioimmunoassays, enzyme-linked immunosorbent assay (ELISA), precipitin techniques, and blotting techniques can be used for this phase of the procedure.

7. Mass Production of Monoclonal Antibodies

Once the desired clone of hybrid cells has been selected, the next step is the production of large quantities of monoclonal antibodies. The peritoneal cavity of mice, preferably the same strain that was used for the initial immunization step, can be used to grow the selected hybrid cell clone. First, the peritoneal cavity is injected with an organic irritant, such as pristane, to produce a chemical peritonitis. Next, the selected hybrid cell line is injected into the peritoneal cavity. Within days, a tumor known as a hybridoma develops. This tumor produces large quantities of monoclonal antibodies that can be harvested by aspirating the ascitic fluid from the mouse’s peritoneal cavity. A tumor-bearing mouse will survive for 4 to 6 weeks, during which time large quantities of antibody can be harvested. Hybridomas can also be grown in tissue cultures in which highly purified antibodies are produced without the potential for contamination from serum, nonspecific interference from ascites proteins, or cross-reactivity of histoincompatibility antibodies derived from the mouse tissues.