Avidin—enzyme conjugate

Avidin-enzyme conjugate formation through thioether bond... 

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. 

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). 

All incubations in antibody/avidin/enzyme conjugates described below are carried out at room temperature for 30 min unless otherwise stated. The substrate reactions are carried out at room temperature, and signal development monitored by light microscopy. The substrate incubation times are, therefore, determined empirically for each experiment. All slides are finally washed in distilled water, air-dried at 42°C, and mounted in glycerol jelly. 

Similar techniques can be used to devise avidin—biotin assay systems for detection of nucleic acid hybridization. DNA probes labeled with biotin can be detected after they bind their complementary DNA target through the use of avidin-labeled complexes (Bugawanefrz/., 1990 Lloyd etal., 1990). Direct detection of hybridized probes can be accomplished, in a manner similar to that for LAB, by incubating with an avidin-enzyme conjugate followed by substrate development. BRAB-like and ABC-like assays also can be utilized to further enhance a DNA probe signal (Chapter 17, Section 2.3). 

Avidin-Enzyme Conjugate Formation through Thioether Bond... 

However, the same basic principles can be applied (with appropriate modifications) to the labeling of antigens with other enzymes (peroxidase, alkaline phosphatase, -galactosidase). This is particularly true for those labeling methods involving the use of biotinylated antigens, since biotinylated enzymes as well as avidin-enzyme conjugates are available from numerous commercial sources. 

The second step required in the application of avidin-biotin technology is to prepare an appropriate avidin-associated probe or probes for the desired application (for general reviews, see refs. 1 and 2). For example, a fluorescent form of avidin can be used for fluorescence microscopy, fluorescence-activated cell sorting, and in some cases, for immunoassay. Likewise, an avidin-enzyme conjugate can be used for immunoblotdng, immunoassay, light microscopy, and in some cases, electron microscopy. An immobilized form of avidin can be used for isoladon purposes (see Note 1). 

The avidin-biotin technique is especially useful in double-labeling experiments. This can be done using two different enzymes in the final step, combined with two different andbody species (monoclonal and/ or polyclonal). One of the andbodies can be biodnylated and the other immunochemical reaction can be based on a different procedure (e.g., employing an antibody-enzyme conjugate). Alternatively, both antibodies can be biotinylated and the secondary reaction with the respective avidin-enzyme conjugate can be performed on different sides of an impermeant (e.g., plastic-embedded) tissue section. 

Stringent controls [e.g., employing native (nonbiotinylated) antibody in the primary step or biotin-blocked avidin-enzyme conjugate in the secondary step] should also be carefully implemented. 

Other avidin-enzyme conjugates that convert solid substrates to colored precipitates can be used. Likewise, fluorescent derivatives of avidin can be used, and the sample can be viewed with a fluorescent microscope two different fluorophores can be employed in double-labeling experiments (ieethis vol., Chapters 11 and 42). Preformed complexes containing avidin and biotinylated enzyme can also be used in place of the covalently coupled conjugate. 

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. 

To detect the positions of electrophoretically separated DNA fragments, would ethidium bromide or a biotinylated DNA probe (used in conjunction with an avidin-enzyme conjugate and activity stain) be expected to provide positional data for all DNA fragments present in the sample Which would provide selective data for DNA fragments containing a particular sequence ... 

Antibodies may be conjugated to enzymes such as alkaline phosphatase or horseradish peroxidase, or to other tags such as fluorescein or biotin (55). The fluorescein is measured by antifluorescein antibodies coupled with an enzyme, and biotin is measured by an avidin-enzyme conjugate, which binds with high affinity to biotin. Michalik et al.(47) used human albumin to tag their second antitoxin, and then used an anti-albumin antibody conjugated to peroxidase for detection of botulism toxins Aand B. They reported a sensitivity for this method of 100 and 300 MLD for toxin types A and B, respectively. 

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