Polyclonal antiserum

Fig. 14.4. Expression of subunits of the pyruvate dehydrogenase complex during the development of A suum. Homogenates of different A suum larval stages and adult tissues were immunoblotted with polyclonal antisera prepared against individual subunits of the A suum PDC isolated from adult muscle, as described in detail in Klingbeil etal. (1996). UE, unembryonated egg M, adult body wall muscle p45, E3-binding protein (E3BP).

Pow, D.V., and Crook, D.K. (1993) Extremely high titre polyclonal antisera against small neurotransmitter molecules Rapid production, characterization and use in light- and electron-microscopic immuno-cytochcmistry. J. Neurosci. Meth. 48, 51-63. 

TABLE 18.10. Advantages and Disadvantages of Monoclonal Antibodies Compared to Polyclonal Antisera... 

Carefully selected monoclonal antibodies against apo(a) are well suited for most immunoassays (LI, L2, W17). Precipitation techniques such as nephelome-try or turbidimetry require monospecific polyclonal antisera. 

Polyclonal antisera are usually prepared either from isolated antigens, such as proteins or from synthetic peptides derived from the protein sequence. The antisera derived from small synthetic peptides are regio-specific with respect to the cognate protein and resemble in some way the monoclonal antibodies, but they are much easier to prepare. 

Cooper HM, Paterson Y (2000) Preparation of polyclonal antisera. Current protocols in molecular biology. Wiley, Unit 11.12 Green JA, Manson MM (1992) Production of polyclonal antisera. In Man-son MM (ed.) Immimochemical protocols. Methods in molecular biology, vol. 10. Humana Press, Totowa, N.J., p 1 Harlow E, Lane D (1988) Antibodies. A laboratory manual. Cold Spring Harbor Laboratory, p 92... 

A rapid avidin/biotin ELISA has been developed for the determination of bovine somatotropin in blood and milk (150). The method uses affinity-purified polyclonal antisera raised in rabbits to immobilize bovine somatotropin from... 

A simple technique for raising highly specific polyclonal antisera against minor or insoluble proteins would be of considerable value. 

PEG precipitation works well for IgM, but is less efficient for IgG, and salt precipitation methods are usually recommended for the latter. PEG precipitation may be preferred in multistep purifications that use ion-exchange columns, because the ionic strength is not altered and therefore does not require dialysis before ion-exchange chromatography. Furthermore, it is a very mild procedure that usually results in little denaturation of antibody. This procedure is applicable to both polyclonal antisera and most MAb-containmg fluids. 

The generation of polyclonal antibodies to an antigen of interest is an important technique applicable to many areas of biological research. In this chapter, we describe a basic immunization procedure designed to generate polyclonal antisera in rabbits and two methods that are commonly employed in the subsequent preliminary characterization of antipeptide antibodies raised in this way. 

There is no one procedure or combination of procedures that is applicable to all cases. The class of antibody to be purified has to be considered. Conventional procedures for the production of polyclonal antisera will generally result in immunoglobulin G, and procedures for the generation of monoclonal antisera may result in any of the immunoglobulin classes. Immunoglobulins from different species have broadly similar properties, but protein A, for example, will not bind all of the subclasses of human IgG, nor is it particularly effective in the purification of IgG from rat, sheep, or goat (2). Thus a certain amount of trial and error may be required to reach the best protocol for a particularly demanding application, but the methods described here should provide an adequate purity for most purposes. 

Epitope mapping can be used to identify areas of a protein that an antibody recognizes and binds to. Monoclonal antibodies are easier to characterize, but epitope maps can also be produced for polyclonal antisera. 

Gonzalez-Martinez, M.A., R. Puchades, A. Maquieira, I. Ferrer, M.P. Marco, and D. Barcelo. (1999). Reversible immunosensor for the automatic determination of atrazine Selection and performance of three polyclonal antisera. Anal. Chim. Acta, 386 201-210. 

In more practical terms, titers may vary from 1 100 to 1 2000 for polyclonal antisera, from 1 10 to 1 1,000 for monoclonal antibodies in cell culture supernatants, and up to 1 1,000,000 for monoclonal antibodies in ascites fluid. These dilutions may likely be... 

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