Biotin-avidin method

The low-molecular-weight vitamin biotin is easily conjugated to antibodies and enzyme markers. Up to 150 biotin molecules can be attached to one antibody molecule, and the strong affinity of the biotin for the glycoprotein avidin allows its use as complex-ing secondary reagents. Biotin labeling of the primary (direct) or secondary (indirect) antibody can be used in the avidin-biotin methods. In the labeled avidin method the tracer is attached directly to the avidin molecule. In the avidin-biotin bridge method a biotinylated enzyme such as peroxidase is allowed to bind after attachment of avidin to the biotin-labeled antibody. 

The avidin-biotin system was developed for detecting antigens at the electron microscope level (Heitzmann and Richards, 1974). Later Heggeness and Ash (1977) proposed the use of this system for fluorescence immunohistochemistry. Guesdon et al. (1979) proposed a variety of labeled avidin-biotin methods which were further supported by Warnke and Levy (1980). The avidin-biotin methods used today are similar to the system described by Hsu et al. (1981). This system is a significant improvement over the previous immuno-histochemical techniques. The problem of endogenous biotin is discussed on page 98. 

The first aptasensor reported was particularly based on optical detection [66]. The 58-mer RNA aptamer selective to L-adenosine was immobilized onto the core of multimode fiber using avidin-biotin method. The detection was based on competitive binding of FITC-labeled L-adenosine with unlabeled analyte. This sensor also allowed to study the kinetics of binding and determine equilibrium constants. 

Avidin-biotin complex (ABC)—the most sensitive of all avidin-biotin methods (Figure 3.1.2)... 

In 1981 a new generation of immunohistochemical methods emerged with the advent of the avidin-biotin methods, which remains widely used today (4). All avidin-biotin methods rely on the strong affinity of avidin or streptavidin for the vitamin biotin. 

In current procedures using the avidin-biotin method, an excess of the quite expensive avidin is needed, i.e., either in the incubation step with avidin or in the production of preformed avidin-biotin-POase complexes (Hsu et al., 1981 Section 17.3.2,2.3). 

It is essential (Kendall et al., 1983 Yolken et al., 1983) to biotinylate correctly the antibody or enzyme, since there is a pronounced optimum (Section 3.2.2). However, current avidin/biotin methods may have to be changed with respect to biotinylation procedures to attain their full potential. The optimal dilutions of the biotinylated antibody, biotinylated enzyme and avidin solutions have to be determined and the test performed as detailed in Table 14.4. It is essential to use incubation periods of about 20 min (pronounced optimum) for the reaction between the biotinylated proteins and avidin (Kendall et al., 1983). 

Antibody-masking enzyme tag immunoassay Adenosine 5 -monophosphate S-Acetylmercaptosuccinic anhydride Alkaline phosphatase anti-alkaline phosphatase (enzyme-antibody) complex Alkaline phosphatase 5-Aminosalicylic acid Adenosine 5 -triphosphate Aa-Benzoyl-L-arginine ethyl ester (-f-)-Biotin bromoacetyl hydrazide (-b)-Biotin Y-aminocaproic acid A-hydroxy-succinimide ester Bis-diazotized benzidine -Galactosidase (-I- )-Biotin hydrazide (-I- )-Biotin-A-hydroxysuccinimide ester (-I-)-Biotin p-nitrophenyl ester Bridged avidin-biotin (method)... 

Indirect avidin-biotin method uses two additional incubation steps after the 2° antibody (Fig. 7.5). Avidin with no label is incubated after the 2° antibody. In fourth step, a labeled biotin is incubated with the complex and it binds to all unbound sites on the avidin. The avidin then serves as a bridge to attach the 2° antibody to the fluorophore with sequential incubations of these reagents. 

One advantage of indirect avidin-biotin method is an amplification of the detection system. For example, after biotin conjugated 2° antibody, avidin is incubated, binding to the available biotins. After the remaining free avidin is rinsed off, a biotin conjugated to 488 fluorophore is added and it binds to the remaining sites on the avidin. With this method, amplification occurs because any avidin can be attached to multiple biotins conjugated with either fluorescent or enzyme. This method requires two additional incubation steps of the indirect immunocytochemistry with fluorescent-labeled 2°. 

Direct avidin-biotin method - a three-step immunocytochemical procedure that uses biotin-labeled 2° antibody to bind the 1° antibody and in a third step, labeled avidin is bound to the biotin of a 2° antibody. 

Indirect avidin-biotin method - a four-step method with two additional incubation steps after the 1° antibody the 2° antibody with biotin is followed by incubation with an unlabeled avidin and finally a labeled biotin. 

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