Streptavidin enzyme conjugation

For identification the lectins must be labeled. The best labels are biotin (streptavidin-enzyme conjugate) or digoxigenin (anti-digoxigenin antibody-enzyme conjugate), but in the case of Concanavalin A, direct labeling with HRP is possible (see below). 

Figure 18-5 Use of streptavidin-enzyme conjugates and biotinylated antibodies in the detection of antigens on Western blots.

Add streptavidin-enzyme conjugate solution (50 pL/well) and incubate the plates for 30 min. 

Secondary label Biotin Binds avidin-enzyme and streptavidin-enzyme conjugates (activity)... 

PCR can be used to introduce labels that can then be used for detection. The ability to add to the 5 end of the primers sequences not complementary to the target template, which then becomes incorporated into the double-stranded PCR product, allows the introduction of labels. Thus, the addition of biotin to the 5 end of the primer allows detection of hybridized PCR product with streptavidin or avidin-enzyme conjugates (B4). Other labels such as digoxigenin can be added to the 5 end of the primer, amplified, and detected either colorimetrically or by chemiluminescence (F3). 

Incubate with the corresponding enzyme conjugate (e.g., anti-digoxigenin AP conjugate or streptavidin-HRP) for 30 min, wash carefully and perform the staining as described in Protocols 2.5.5 and 2.5.4. 

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 (3-galactosidase and glucose oxidase are used less often, especially with regard to assay tests for clinically important analytes (Chapter 16). 

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. 

The use of biotinylated secondary antibodies in conjunction with enzyme-conjugated streptavidin (or avidin, extravidin) both increases sensitivity and saves time in that a further step is eliminated from the assay and therefore another step of optimization is not required. 

In a similar method the labeled streptavidin-biotin (LSAB) method also utilizes a biotinylated secondary antibody that links primary antibodies to a streptavidin-peroxidase conjugate (6). In both methods a single primary antibody is subsequently associated with multiple peroxidase molecules, and because of the large enzyme-to-antibody ratio, a considerable increase in sensitivity is achieved compared to direct peroxidase-conjugate methods. 

In order to avoid nonspecific binding of native egg-white avidin with cell surface lectins, streptavidin- or nonglycosylated avidin-enzyme conjugates or complexes can be employed in its stead. 

Second probe In instances where biotin-lectin conjugates are used, an enzyme conjugate of streptavidin, avidin, or antibiotin antibody is required and should be suitably diluted in a quench buffer. 

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