Preparation of Avidin or Streptavidin Conjugates

The following sections discuss the main techniques used to make avidin (or streptavidin) conjugates and various biotinylated components. Chapter 8, Section 3, should be consulted for a complete overview of the biotinylation reagents currently available. 

Perhaps the most common conjugates of avidin or streptavidin involve attaching enzyme molecules for use in ELISA systems. As in the case of antibody-enzyme conjugation schemes (Chapter 10), by far the most commonly used enzymes for this purpose are horseradish peroxidase and alkaline phosphatase. Other enzymes such as P-galactosidase and glucose oxidase are used less often, especially with regard to assay tests for clinically important analytes (Chapter 16). 

Other proteins commonly cross-linked to avidin or streptavidin are chromogenic or fluorescent molecules, such as ferritin or phycobiliproteins (Chapter 8, Section 1.7). These conjugates can be used in microscopy techniques to stain and localize certain antigens or receptors in cells or tissue sections. 

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In addition to preparing the avidin or streptavidin conjugates necessary to develop avidin—biotin-based systems, the process of modifying targeting molecules with a... 

Conjugates of avidin or streptavidin with these fluorescent probes may be prepared by activation of the phycobiliprotein with SPDP to create a sulfhydryl-reactive derivative, followed by modification of avidin or streptavidin with 2-iminothiolane or SATA (Chapter 1, Section 4.1) to create the free sulfhydryl groups necessary for conjugation. The protocol for SATA modification of avidin or streptavidin can be found in Section... 

A variation of the above method can be used, wherein the enzyme is first activated with SMCC and conjugated to a thiolated avidin or streptavidin molecule. This approach probably is the most common way of preparing avidin—enzyme conjugates, and since the preactivated enzymes are readily available (Pierce), it also may be the easiest. 

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