10 - Antibody Modification and Conjugation

Abstract

The employment of antibody molecules in immunoassay, targeting, or detection techniques encapsulates a broad spectrum of applications affecting nearly every field of the life sciences. The availability of relatively inexorbitant polyclonal and monoclonal antibodies of exacting specificity has made plausible the design of reagent systems that can interact in high affinity with virtually any conceivable analyte. The directed specificity of purified immunoglobulins provides powerful tools for constructing immunological reagents. These specific antibodies can be modified to permit easy tracking in complex mixtures by using a number of conjugation and modification techniques. An antibody molecule labeled with an enzyme, a fluorescent compound, or biotin provides a detectable complex that can be quantified or visualized through its tag. To maintain specificity in antibody conjugates derived from polyclonal antisera, only affinity-purified immunoglobulins should be used. Such purified preparations are isolated from antisera by affinity chromatography using the corresponding immobilized antigen. These preparations thus constitute only the population of antibody molecules that have the desired antigenic specificity. Modification or conjugation of whole immunoglobulin fractions should be avoided since other antibody populations are present and can cause considerable nonspecificity in the resultant activity of the reagent.