Preparation of Strept avidin Conjugates

Colloidal gold-labeled (strept)avidin can be used as highly sensitive detection reagents for microscopy techniques (Cubie and Norval, 1989) (Chapter 24). Finally, cytotoxic substances coupled to (strept)avidin can be used to direct cell-killing activity toward a tumor-cell-bound, biotinylated monoclonal antibody (or other targeting molecule) for cancer therapy (Hashimoto et al, 1984) (Chapter 21). 

The following sections discuss the main techniques used to make (strept)avidin conjugates and various biotinylated components. Chapter 11 and Chapter 18, Section 3 should be consulted for a complete overview of biotinylation reagents. 

Perhaps the most common conjugates of (strept)avidin involve attaching enzyme molecules for use in ELISA systems. As in the case of antibody-enzyme conjugation schemes (Chapter 20), by far the most commonly used enzymes for this purpose are HRP and alkaline phosphatase. Other enzymes such as (3-galactosidase and glucose oxidase are used less often, especially with regard to assay tests for clinically important analytes (Chapter 26). 

Other proteins commonly crosslinked to (strept)avidin are chromogenic or fluorescent molecules, such as ferritin or phycobiliproteins (Chapter 9, Section 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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Conjugates of (strept)avidin with these fluorescent probes may be prepared by activation of the phycobiliprotein with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) to create a sulf-hydryl-reactive derivative, followed by modification of (strept)avidin 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 (strept)avidin can be found in Section 3.1, this chapter. The procedure for SPDP activation of phycobiliproteins can be found in Chapter 9, Section 7. Reacting the SPDP-activated phycobiliprotein with thiol-labeled (strept)avidin at a molar ratio of 2 1 will result in highly fluorescent biotin binding probes. 

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

If a conjugate is required for immunization experiments, it is convenient to prepare not only the SAMA- or Cys-peptide, but also the corresponding l)iotinyl-peptide or an N-acetylated MAP-8 (Section 4). The biotinyl-peptide can be bound to (strept)avidin-coated microtitre plates to determine the antipeptide titre of the sera obtained. The MAP-8 can be coated directly onto regular plates. 

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