methods in many other fields of science, in recent years this methodology has been largely supplanted by the enzyme-linked immunosorbent assay (ELISA). For this reason, the first section of this chapter (units 2.1-2.3) primarily describes methods for assaying antibodies based on the ELISA. Using any of several variations on the ELISA, the investigator can quantitatively or qualitatively measure a particular antibody activity or use a defined antibody preparation to measure an antigen. A method for measuring antibody activity that is equally simple and versatile, but dependent on sophisticated equipment such as a flow cytometer, is presented in Chapter 5. Those who find a need for classical radioimmunoassay methods are referred to Cooper and Paterson (1993).
With simple and reliable assays for measuring antibody activity, the investigator can then proceed to producing antibodies as described in the second section of this chapter (units 2.4-2.6). The first question to be considered is whether polyclonal or monoclonal antibodies are required. The advantage of polyclonal antibodies (unit 2.4) is that they can be prepared with a minimum of effort from a wide variety of species. In addition, they are particularly useful for analyses of denatured forms of a protein, for immunoprecipitation, and for immunoblots. Polyclonal antibodies (in their unpurified form, polyclonal antisera) can also be raised against synthetic peptides if conjugated to appropriate carrier molecules (Chapter 9). The use of Freunds adjuvant remains the standard by which methods for immunization are measured. In recent years other antigen delivery systems have been developed, including the use of ISCOMs (immunostimulatory complexes; unit 2.11). Monoclonal antibodies (unit 2.5) mark a major technological revolution of modern immunology. With a sensible strategy and sufficient effort, a reagent of precise specificity and/or of high affinity can be generated. Because a monoclonal antibody represents the product of a cloned B cell immortalized by cell fusion to a myeloma (plasma cell) tumor, the cell line and thus the antibody it produces can be maintained indefinitely with appropriate care, and the antibody can be produced in essentially limitless quantities
With antisera or monoclonal hybridoma tissue culture supernatants, the next step for many applications is to purify and prepare fragments of the antibodies. Since problems of purification in part result from the great diversity of antibody sequences, protocols are described for purifying IgG (unit 2.7), IgM and IgD (unit 2.9), and IgA (unit 2.10B). Molecular fragments of antibodies are needed when monovalent binding activities are required (e.g., F(ab')^; unit 2.10A) or when antibodies lacking various portions of the molecule prone to generating particular experimental artifacts (such as Fc fragments; unit 2.8) must be eliminated. Fragmentation of antibodies requires first a knowledge of the structural subclass (or isotype) of the antibody in question (unit 2.2), then a pilot experiment to evaluate the precise conditions for the preparation of the required fragments, and finally, preparative-scale fragmentation and purification.
Frequently, an investigator will have a hybridoma line producing a murine monoclonal antibody and will need to manipulate the antibody genes, either to alter the constant (C) region with which the variable (V) regions are associated, or to introduce designed changes in the antibody combining site. Contemporary PCR methods make it feasible to clone the antibody V region genes encoding both the heavy and light chains of the hybridoma, to ligate these into plasmid expression vectors encoding constant regions of human or other species, and to express them after transfection in myeloma cell lines capable of high-level production. The steps in this process, leading to the production of transfectomas, are described in unit 2.12.
It is often desirable to produce antibodies containing two different specificities within a single molecule. Such bispecific antibodies have many clinical and experimental appli cations since they have been shown to redirect normal cellular or cytotoxic defense mechanisms to tumor cells. unit 2.13 describes current methods used to produce bispecific antibodies, including the use of chemical cross-linkers and the fusion of two antibody-producing hybridomas. The unit also includes several methods for screening and purifying bispecific antibodies.
unit 2.14 describes the relatively new approach to raising immune responses using antigen-expressing DNAs. Two methods of nucleic acid immunization are described: (1) injections with saline containing DNA expression vectors, and (2) injections of DNA using a gene gun. Protocols for preparing and storing DNA-coated gold beads used with the gene gun approach are included.
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