Preparation of Monoclonal and Polyclonal Antibodies against Specific Protein(s)
Contents Antibodies are small proteins produced in animals and have long been used as molecular probes in several aspects:1 (1) detection, measurement, and purification of biological molecules of interest; 0-8493-0815-1/04/$0.00+$ 1.50
(2) in situ immunocytochemical localization of specific protein/enzyme in cells and tissues; (3) immunoscreening of expressional cDNA (complementary deoxyribonucleic acid) libraries to identify cDNAs of interest; and (4) treatment of some human diseases. The preparation of antibodies is based on the fact that the immune system of an animal has specific immune responses to and produces antibodies against foreign substances (antigens) such as carbohydrates, nucleic acids, and proteins. The antibodies produced are then secreted into the serum by lymphoid cells in several organs including bone marrow, spleen, and lymph nodes.2 The serum can be obtained by bleeding an immunized animal. There are two typical types of antibodies:1-5 (1) monoclonal antibodies raised in mice, and (2) polyclonal antibodies that are usually raised in rabbits. Monoclonal antibodies contain a single antibody specificity, a single affinity, and a single immunoglobulin isotype. However, a preparation of polyclonal antibodies has a mixture of antibody molecules directed against the antigen as well as antibodies that do not react with the antigen of interest. These differences account for the fact that monoclonal antibodies are more specific as compared with polyclonal antibodies. This chapter describes the preparation of both monoclonal and polyclonal antibodies against a protein/enzyme of interest. However, these procedures are not designed for the production of antibodies for clinical use.