Avidin-Biotin Method Advantages

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°. 

Another simple detection method is the polymer-based detection system. This detection system does not depend on the avidin-biotin interaction for localization of the marker enzyme to the antigen but bases on an enzyme conjugated polymer backbone vith attached secondary (link) antibody. This method has the advantage to join both secondary antibody step and the detection complex incubation step in a single step. Consequently, it is a more rapid method and the blocking of the endogenous biotin activity is not necessary. 

A few methods based on fluorescence have been described for biotin and avidin determinations. A first one is based on the quenching of the avidin tryptophan fluorescence by biotin upon binding (71). A second one involves the increase of the fluorescence of avidin labeled with fluorescein isothiocyanate upon binding of biotin (72). The latter technique has been applied to HPLC postcolumn detection of biotin (see below). The sensitivities are, respectively, 20 and 0.5 ng. Another method is based on the variation of the fluorescence polarization of a biotin-fluorescein derivative upon interaction with avidin (73). Minimal detectable concentrations reported were 5 ng for avidin and 0.1 ng for biotin (73). Mock et al. reported another technique relying on the displacement by biotin of the fluorescent probe 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) (Fig. 10) when bound to avidin (74). The advantage of this method is obviously the large increase of fluorescence of 2,6-ANS when bound to avidin as compared to the unbound form in water solution. The detection limit was around 1 ng. This technique has also been applied to postcolumn detection of biotin (see below). 

NPH HCl) with l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC HCl) as a coupling agent [593,594], The advantage is that with hydrazines it is possible to carry out the derivatization in milder conditions. An efficient postcolumn derivatization is obtained using avidin or streptavidin. In fact, these two proteins react in a very specific way with biotin, therefore they are bound with a fluorescent marker, such as fluorescein 5-isothiocyanate (FITC) to obtain fluorescent derivatives. Some authors [595] report the use of MS or MS/MS for biotin detection, but this method seems to be less sensitive than FLD. 

In addition to the detection of antigens and antibodies, EIA will, undoubtedly, play an increasingly important role in molecular biology. For example, the bio-blot method (Leary et al., 1983) for the detection of DNA-DNA or DNA-RNA duplexes on nitrocellulose membranes offers important advantages over conventional procedures in which radioactive probes are used and autoradiographic detection. In this method, biotinylated DNA probes are prepared by nick translation (Rigby et al., 1977) in the presence of biotinylated analogs and hybridized with the DNA or RNA on filters. Biotin is then detected by avidin-labeled enzyme (Section 3.1). 

There are three attributes of the TSA procedure that makes the method very powerful. First, the activated tyramide reacts quickly with tyrosine and cannot diffuse far from the HRP-labeled 2° antibody. Second, is that the tyramide is bound to the tissue and is not free to move after the reaction is completed. These advantages are compared to development of many chromogens by HRP where the reaction product is not bound to the tissue and is able to move. Third, there are a wide variety of label compounds that can be attached to tyramine, including both fluorophores and biotin. It is possible to use tyramide bound to biotin and then an avidin HRP, so that a chromogen can be used to localize the 1° antibody. 

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