Biotinylation of antigen

Dissolve 1 mg of NHS-SS-biotin in 1 ml of sterile distilled water, resulting in a stock solution of 1.65 nM/pl. The NHS reagent should dissolve readily in water, and if it does not dissolve within 10 min it may have absorbed water due to inappropriate storage and will not couple effectively. Alwas s tiy and use fresh reagent. 

Add an equimolar amount of peptide to the NHS-SS biotin to give a reaction volume of around 200 xl. 

Add 50 xl of 1 M NaHCOs pH 8.5 and make up the total reaction volume to 1 ml with sterile distilled water. 

Incubate on ice for 2 h. The sample can be used without removal of unincorporated biotin if coupling is performed at low density, since a vast excess of streptavidin is used when capturing the phage. If desired, however, imincraporated biotin can be removed from the sample using, for example, column chromatography. 

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Biotinylation may be done before or after liposome formation, but having a stock supply of biotin-modified PE is an advantage, since it can then be used to test a number of liposomal recipes. In addition, only a very small percent of the total lipid should be biotinylated to prevent avidin-induced aggregation in the absence of antigen. It is difficult to control precisely... 

Figure 22.18 Biotinylated liposomes may be used in immunoassay systems to enhance the signal for detection or measurement of specific analytes. The liposome components may be constructed to include fluorescent molecules to facilitate detection of antigens within tissue sections.

If cellular localization of the antigen-antibody complex is not required, enzyme immunolabeling can be performed on cells adherent to a microtiter plate, and the color change resulting from the enzymatic reaction can be detected as a change in absorbance with an automatic plate reader (see Chapter 28). Biotinylation of antibodies and the use of the avidin-biotin complex has further extended the versatility and sensitivity of the enzymatic techniques (see Chapters 7 and 25-27). Most recently, the principles behind these techniques have been applied in combination with in situ hybridization techniques. Using nucleic acid-antibody complexes as probes, specific DNA or RNA sequences can be localized (see Chapters 46 9). 

The above represent the past and present of the most common enzyme-mediated methods of antigen detection. There are alternate procedures available, involving such methods as antibiotin antibody steps that combine the avidin-biotin systems with a further antibiotin/antienzyme sandwich for still greater sensitivity. Also, there are methods that follow a PAP procedure with a biotinylated antibody to the PAP immunoglobulin followed by ABC detection (15). The obvious problem created with this approach is the tremendous... 

Figure 18-5 Use of streptavidin-enzyme conjugates and biotinylated antibodies in the detection of antigens on Western blots.

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. 

Reagents for biotinylation of proteins and peptides are commercially available and allow the fast and efficient derivatization of antibodies and enzymes without the loss of enzyme or antibody activity. Several reagents are also available for the biotinylation of sulfhydryl groups aldehydes, nucleic acids and carbohydrates. Hence, in addition to proteins and peptides, a variety of antigens can be labeled with biotin. 

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). 

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