ABC method

FIGURE 11.12 TSA method using the conventional ABC method, together with pretreatment procedures and optimal antigen retrieval. Source www.hmds.org.uk/ histology.html... 

Enzyme labels are usually coupled to secondary antibodies or to (strept)avidin. The latter is used for detection of biotinylated primary or secondary antibodies in ABC methods (see Sect. 6.2.1). Enzyme labels routinely used in immunohisto-chemistry are horseradish peroxidase (HRP) and calf intestinal alkaline phosphatase (AP). Glucose oxidase from Aspergillus niger and E. coli (3-galactosidase are only rarely applied. 

The addition of the secondary antibody step allows for signal amplification of the process. However, for low density antigens (>2 K, < 10 K molecules/cell) it is recommended to employ further amplification of the immunocytochemical signal with tyramide, with ABC method or with polymer-conjugated technology such as EnVision System developed by DakoCytomation (see Sect. 6.2). 

Avidin-biotin complex (ABC) is based on the high affinity that streptavidin (from Streptomyces avidinii) and avidin (from chicken egg) have for biotin. Biotin is a naturally occurring vitamin. One mole avidin will bind four moles biotin. ABC method affords a several-fold higher antigen detectability than those achieved in the standard indirect method. 

The Avidin-Biotin Complex (ABC) Method and Other Avidin-Biotin Binding Methods ... 

In the avidin-biotin (ABC) method a complex of avidin and biotinylated tracer containing the free avidin binding sites is applied to the biotinylated antibody. As a high... 

The avidin-biotin complex (ABC) method and the streptavidin-biotin (SAB) method are more sensitive than the peroxidase-antiperoxidase (PAP) method for histochemical techniques. The strong noncovalent attraction between biotin and avidin or streptavidin is exploited in many histochemical, immunohistochemical, and in situ hybridization... 

Streptavidin (from Streptomyces avidinii) and avidin (from chicken egg) both possess four binding sites for biotin. The biotin molecule is easily conjugated to antibodies and enzymes. In the avidin-biotin complex (ABC) method secondary antibodies are conjugated to biotin and function as links between tissue-bound primary antibodies and an avidin-biotin-peroxidase complex (5). 

EPOS, which contained primary antibodies, the EnVision system contained secondary antibodies with anti-mouse Ig and anti-rabbit Ig specificity. This universal reagent could be used to detect any tissue-bound primary antibody of mouse or rabbit origin. The utility of this method opened the door to a new family of polymer-based immunohistochemical methods. The sensitivity of these methods compared to LSAB and ABC methods was comparable or even slightly greater in most cases (7). With the latest development of EnVision FLEX+ the sensitivity has been improved even further. However, because of the large molecular size of the polymer conjugates, accessibility to certain epitopes was restricted, presumably due to steric hindrance, in a minority of cases. 

However, because a pharmacy contains hxmdreds or even thousands of items, it is difficult to use either method reliably, particularly if there are multiple vendors. In such cases, there is an added method to the periodic inspection called the ABC method of control (Figure 11.2). The ABC method of control prioritizes the items into three levels based on the theory that a small percentage of all the merchandise accounts for a large percentage of the dollar investment. A items typically comprise only 20% of the inventory items, but account for nearly 80% of the cost. Because one of the main objectives of inventory control is to minimize the inventory investment, it is... 

Use, for example, Vectastain ABC method (Vector Laboratories, Burlingame, CA), or CSA Dakor (catalyzed signal amplification system for mouse primary antibodies) Dako and APAAP mouse or rat primary antibody kits (Dako). 

FIGURE 1.12 (A) Radicalization of tyramine amplification. (B) Tyramine-reporter deposition. (C) Comparison with the ABC method. From Merz H, Ottesen K, Meyer W, et al. Combination of antigen retrieval techniques and signal amplification of immunohistochemistry in situ hybridization and FISH techniques. In Shi S-R, Ciu I, Taylor CR, eds. Antigen Retrieval Techniques Immunohistochemistry and Molecular Morphology. Natick, MA Eaton 2000 228-229. 

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