Before we offered the Biotin-Avidin System researchers used directly-labeled, enzyme-tagged primary and secondary antibodies for detection of tissues, blots, and microtiter plates (Figs. 1 and 2). These direct/indirect methods were followed by the PAP method (Fig. 3), which provided some amplified signal.
The Biotin-Avidin System
When we introduced the Biotin-Avidin (now also Streptavidin) System a substantial amplification over the earlier methods was achieved. Avidin is an egg-white derived glycoprotein with an extraordinarily high affinity (affinity constant > 1015 M-1) for biotin. Streptavidin is similar in properties to avidin but has a lower affinity for biotin. Many biotin molecules can be coupled to a protein, enabling the biotinylated protein to bind more than one molecule of avidin. If biotinylation is performed under gentle conditions, the biological activity of the protein can be preserved. By covalently linking avidin or streptavidin with different ligands such as fluorochromes, enzymes or EM markers, what we have termed the Biotin-Avidin System (or Biotin-Streptavidin System) can be utilized to study a wide variety of biological structures and processes. The Biotin-Avidin/Streptavidin System has proven to be particularly useful in the detection and localization of antigens, glycoconjugates, and nucleic acids by employing biotinylated antibodies, lectins, or nucleic acid probes.
Various Biotin-Avidin Methods
Over the years, several basic biotin-avidin/streptavidin techniques have evolved. One of the earliest and currently least used has been termed the “sandwich” or “bridge” technique and relies on the multiple biotin binding sites on avidin. Following the application of a biotinylated antibody, avidin is added, and then a biotinylated enzyme or EM marker. (This technique is not shown.)
The next technique that evolved has been called the “covalent conjugate” or “labeled avidin” (or “labeled streptavidin”) procedure (Fig. 4). Following the addition of a biotinylated primary or secondary reagent, a covalent conjugate between (strept)avidin and an enzyme, fluorochrome, or EM marker is added.
One example might be staining a histological section with monoclonal mouse primary antibody directed toward a specific determinant on the cells. After the primary antibody is applied, biotinylated anti-mouse IgG is added, followed by peroxidase conjugated avidin.
Development is accomplished by adding an appropriate substrate for peroxidase.
The most recent and the most widely used technique is our “ABC” or “preformed complex” method. After application of a biotinylated secondary or primary antibody, a preformed complex between avidin or streptavidin and a biotinylated enzyme is added (Figs. 5 and 6). This latest technique appears to be the most sensitive in many applications and is discussed more fully in the VECTASTAIN® ABC section of this catalog.
Although each technique has its merit, it is now widely appreciated that the Biotin-Avidin/Streptavidin System provides the highest sensitivity in fluorescence and enzyme-based detection, versatility that allows easy interchange or introduction of multiple markers, and the ability to localize or detect antigens which are difficult or impossible to see or measure with other systems.
Advantages of the Biotin-Avidin/Streptavidin System
The Biotin-Avidin/Streptavidin System has several advantages over direct coupling of the marker to an antibody, a lectin or a nucleic acid probe.
The Biotin-Avidin/Streptavidin System can improve sensitivity because of the potential for amplification due to multiple site binding.
Avidin or streptavidin can be prepared with high fluorochrome to protein ratios, and avidin and streptavidin conjugates are very stable.
Only a single labeled conjugate, namely avidin or streptavidin, need be kept on hand since it can be used with a variety of biotinylated lectins, antibodies or probes.
Biotin-Avidin/Streptavidin System reagents can overcome the problem of background fluorescence sometimes encountered in the use of heavily fluorescein-labeled or rhodamine-labeled antibody. These conjugates are sometimes “sticky” and adsorb nonspecifically to tissues, while fluorochrome-conjugated Avidin D or streptavidin do not.
The extraordinarily high affinity between avidin or streptavidin and biotin assures the user of a rapidly formed and stable complex between the (strept)avidin conjugate and the biotin-labeled protein.
Simultaneously localizing more than one antigen in the same tissue section can be performed even with two or three primary antibodies from the same species. By using either separate enzyme systems, two different substrates for the same enzyme, or assorted fluorochrome conjugates, more than one antigen can be localized in the same tissue section.
Applications of the Biotin-Avidin/Streptavidin System
Biotin-Avidin/Streptavidin System reagents have been found to be superior substitutes for many conventional, less sensitive methods. Just a few of these uses include:
Introducing multiple labels into tissues
Localizing hormone binding sites
Nitrocellulose and nylon transfer blot detection
In situ hybridization
Radio-, enzyme-, and fluorescent immunoassays
Purification of cell surface antigens
Coupling of antibodies and antigens to agarose
Examination of membrane vesicle orientation