Immunochemical Protocols

CHAPTER1 Production Jonathan of Polyclonal A. Green and Margaret Antisera M. Munson 1. Introduction All immunochemical procedures require a suitable antiserum or mono clonal antibody raised against the antigen of interest. Polyclonal antibodies are raised by injecting an immunogen into an animal and, after an appropriate time, collecting the blood fraction containing the antibodies of interest. In producing antibodies, several parameters must be considered with respect to the final use to which the antibody will be put. These include (1) the specificity of the antibody, i.e., the ability to distinguish between different antigens, (2) the avidity of the antibody, i.e., the strength of binding, and (3) the titer of the antibody, which determines the optimal dilution of the antibody in the assay system. A highly specific antibody with high avidity may be suitable for immunohistochemistry, where it is essential that the antibody remains attached during the extensive washing procedures, but may be less useful for immunoafbnity chromatography, as it may prove impossible to elute the antigen from the column without extensive denaturation. To produce an antiserum, the antigen for the first immunization is often prepared in an adjuvant (usually a water in oil emulsion containing heatkilled bacteria), which allows it to be released slowly and to stimulate the animal’s immune system. Subsequent injections of antigen are done with incomplete adjuvant that does not contain the bacteria. The species used to raise the antibodies depends on animal facilities, amount of antigen available, and the amount of antiserum required. Anot