Monoclonal and polyclonal antibodies are powerful tools for addressing cell biological questions. These immunological reagents can be used to detect and analyze proteins and carbohydrates, characterize the subcellular distribution of proteins, and purify proteins. In addition, when taken up by cells, antibodies can be used as tags to monitor the intracellular pathways of proteins and to inhibit protein activity. These diverse applications of antibody technology have been facilitated by advances in the understanding of the molecular genetics of antibody molecules and their three-dimensional structures. The development of methods for measuring antibody binding activity and for isolating antibodies has contributed to their widespread use and extensive range of applications.
The development of monoclonal antibodies, unique and powerful reagents for detecting and measuring interactions with specific protein epitopes, has revolutionized the use of antibodies in cell biological research. These antibodies are obtained by fusing immune B cells from the spleen with tumor cells to produce hybridomas. Hybridomas each secrete a single type of antibody—the monoclonal antibody—that has precise specificity and often high affinity. Because the cloned hybridoma cell line is immortal, with appropriate care it can be maintained indefinitely and the antibodies it produces can be supplied in essentially limitless quantities. Preparations containing monoclonal antibodies include hybridoma supernatants, ascites fluid from a mouse inoculated with the hybridoma, and purified monoclonal antibody.
Polyclonal antibodies take less effort to prepare than monoclonal antibodies. The process consists simply of immunizing an animal of any one of a variety of species (including goat, horse, rat, mouse, and rabbit) with purified antigen and then, after the animal develops an immune response, isolating antibodies from its serum. Because polyclonal antibodies are essentially a collection of monoclonal antibodies with different epitope specificities and affinities, they are useful for analyses of denatured forms of a protein, for immunoprecipitation and immunoblotting. These types of analyses are often not successful with monoclonal antibodies because the single epitope recognized by the monoclonal antibody preparation may be destroyed during protein denaturation.
The choice of animal species for immunization depends in part on the amount of antiserum required for subsequent experiments. Mice, rats, and guinea pigs yield relatively low volumes of antiserum compared to rabbits and other larger animals. For this reason, rabbits are often the animals of choice. It is often desirable to produce polyclonal antibodies in other species, however, especially when an experiment requires two distinct types of antibodies that recognize different proteins (as is often the case in indirect immunofluorescence assays).
The term “antibody production” has both general and specific meanings. In the broad sense, it refers to the entire process of creating a usable specific antibody, including steps of immunogen preparation, immunization, hybridoma creation, collection, screening, isotyping, purification, and labeling for direct use in a particular method. In the more restricted sense, antibody production refers to the steps leading up to antibody generation but does not include various forms of purifying and labeling the antibody for particular uses.Successful antibody production depends upon careful planning and implementation with respect to several important steps and considerations:
1. Synthesizing or purifying the target antigen (e.g., peptide or hapten)；
2. Choosing an appropriate immunogenic carrier protein；
3. Conjugating the antigen and carrier protein to create the immunogen；
4. Immunizing animals using appropriate schedules and adjuvant formulae；
5. Screening serum (or hybridomas) for antibody titer and isotype；
Procedures for generating, purifying and modifying antibodies for use as antigen-specific probes were developed during the 1970s and 1980s and have remained relatively unchanged since Harlow and Lane published their classic Antibodies: A Laboratory Manual in 1988.