Radioactivity using avidin-biotin

Important insights into the structure of the avidine-biotin complex have been obtained by SPR. The technique was useful also in determining the absolute amount of protein molecules on metallic films. For that purpose, a gold film on glass plate was coated with a dextran layer exposed to a solution containing certain monoclonal antibodies in a flow-through cell. Optical quantities determined in this way were calibrated by means of radioactively marked antibodies. 

Isotope Dilution Assay. An isotope dilution assay for biotin, based on the high affinity of avidin for the ureido group of biotin, compares the binding of radioactive biotin and nonradio active biotin with avidin. This method is sensitive to a level of 1—10 ng biotin (82—84), and the radiotracers typically used are p C]biotin (83), [3H]biotin (84,85) or an I-labeled biotin derivative (86). A variation of this approach uses I-labeled avidin (87) for the assay. 

The non-radioactive labeling utilizes fluorescence, chemiluminescence, or biotin/avidin interactions. Capillary electrophoresis with laser-induced fluorescence was first employed in PAL by Miller et al. [51]. Gilbert and Rando recently reported several biotin-containing heterobifunctional reagents and used them successfully [18] (Fig. 5). 

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). 

Probes prepared with either digoxigenin- or biotin-modified nucleotides can be hybridized to Southern blots to detect target nucleic acid sequences. These methods offer an attractive alternative to radioactively tagged probes in terms of safety, cost, and efficiency. Most previous nonradioactive strategies utilized the detection of the modified base by the use of a coupled antibody- or avidin-alkaline phosphatase. The blot with the bound alkaline phosphatase was then treated with a compound, such as Nitro Blue Tetrazolium (NBT), that was converted to an insoluble, colored compound at the site of hybridization, thus facilitating visualization of the hybridized probe. 

Yet, for routine laboratory practice the choice of the detection method is highly dependent on the biotin concentration range to be determined (Table 2). At high levels, the HABA methods remain the more convenient ones, since they require no radioactive materials or sophisticated detection techniques. The microbiological methods, in spite of their lack of accuracy, are still very useful for routine analysis. Perhaps the best methods rely on the use of HPLC separation coupled to a sensitive detection method (avidin-based or fluorimetric). The author s guess is that the enzyme-linked methods based on avidin-horseradish per-oxydase conjugate will be the future standard because they are compatible with the 96-well plate format and depend on visible spectrophotometry, being therefore easily automated. 

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