Nonradioactive detection method

FcR-mediated cytotoxicity assays are variable and difficult to perform, especially the ADCC assay, which uses primary cells from peripheral blood. However, recently these assays have been simplified and used as a bioactivity assays for characterization of therapeutic proteins and as a routine potency assays. Some laboratories in industry are able to use cloned NK cells as effector cells and the established cell lines or transfected cells as target cells in simplified ADCC assays. The CDC assay is used as a potency release assay for several therapeutic proteins. The use of cell lines instead of the primary cells in ADCC assays and nonradioactive detection methods, adenylate kinase (AK) or ATP with luminescent readouts, for example, have a potential to improve the performance of these assays and make them more suitable for the routine use. 

The reagent also has been used in a unique tRNA-mediated method of labeling proteins with biotin for nonradioactive detection of cell-free translation products (Kurzchalia et al., 1988), in creating one- and two-step noncompetitive avidin-biotin immunoassays (Vilja, 1991), for immobilizing streptavidin onto solid surfaces using biotinylated carriers with subsequent use in a protein avidin-biotin capture system (Suter and Butler, 1986), and for the detection of DNA on nitrocellulose blots (Leary et al., 1983). 

Enzymes useful for detection purposes in ELISA techniques (Chapter 26) also can be employed in the creation of highly sensitive DNA probes for hybridization assays. The attached enzyme molecule provides detectability for the oligonucleotide through turnover of substrates that can produce chromogenic or fluorescent products. Enzyme-based hybridization assays are perhaps the most common method of nonradioactive detection used in nucleic acid chemistry today. The sensitivity of enzyme-labeled probes can approach or equal that of radiolabeled nucleic acids, thus eliminating the need for radioactivity in most assay systems. 

Meyne, J. (1993) Chromosome mapping by fluorescent in situ hybridization. In Methods in Nonradioactive Detection (G.C. Howard, ed.), pp. 263-268. Appleton Lange, Norwalk, Connecticut. 

Colorimetric methods were developed as a nonradioactive alternate method of detection. Enzymes such as HRP or AP, which are usually conjugated to the secondary antibody, can convert substrates to colored precipitates that accumulate on the blot and generate a colored signal. Such substrates include 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) for AP and 3,3 -diaminobenzidine... 

Three types of detection methods can be used for in situ hybridization radioactive, fluorescent, and chromogenic. Radioactive detection techniques are the most commonly used primarily because they are more sensitive, but also because the other techniques are relatively more recent in terms of the development of the appropriate chemistry and the techniques that enable in vitro labeling of probes by these nonradioactive means. 

There are currently several methods for analysis of the amplified target DNA. For HIV-1, liquid hybridization with radioactively labeled probes is used (K12). Tests for HLA genes and sickle cell anemia utilize the reverse dot-blot format with a nylon membrane (S3). Each clinical research format has a well-characterized detection method defining the optimum probe concentration, the hybridization times and temperatures, as well as the concentrations of indicator reagents. Table 5 describes the optima and tolerances of a nonradioactive dot-blot assay that uses biotinylated probes and detection by a chemiluminescent substrate and a strepta-vidin-HRP conjugate. 

Biotinylated PCR products For purposes of nonradioactive detection, a single biotin molecule is incorporated into the 5 end of one or both of the PCR primer pairs. The biotinylated PCR products are used in a chemiluminescent detection system as described below. Attachment of biotin molecules to oligonucleotides has been described in detail (12). For an alternative method of biotinylating PCR products, see Note 3. 

Marvik O.J., Isaksen M.L., Roza L., Lindqvist B.H. Photoimmimodetection a nonradioactive labeling and detection method for DNA. Biotechniques 1997 23(5) 892-896 Mayes A.G., Mosbach K. Molecularly imprinted polymers useful materials for analytical chemistry Trac-TrendAnal. Chem. 1997 16(6) 321-332... 

Kurzchalia, T.V., Wiedmann, M., Breter, H., Zimmermann, W., Bauschke, E., and Rapoport, T.A. (1988) tRNA-mediated labeling of proteins with biotin. A nonradioactive method for the detection of cell-free translation products. Eur. J. Biochem. 172, 663-668. 

In addition to biotin, a digoxigenylated derivative of dUTP was also synthesized. This derivative of dUTP can be incorporated into DNA by Pol I (or the Klenow fragment of Pol I). Therefore, digoxigenin-labeled DNA probes can be prepared by nick translation or random primed-labeling methods developed for the biotin system. It is almost certain that more nonradioactive alternatives to biotin and digoxigenin will be developed in the future. Chemiluminescent methods for nonradioactive probe detection are now widely being used... 

Nucleic acids can also be biotinylated by nonenzymatic methods with photobiotin, a photoactivatable biotin analog (6), which can be commercially obtained from BRL, Sigma, and other commercial sources I have not compared the suitability of this method of biotin incorporation with that reported here, but expect that the method would be fully acceptable FMC (Rockland, ME) markets an alternate nonradioactive sequence detection kit known as Chemiprobe. The basis of this system is a chemical modification of cytosine residues m the probe DNA. After hybridization, the probe is detected by means of a monoclonal antibody that specifically recognizes the sulfonated DNA. Detection of the bound monoclonal antibody is achieved by means of an alkaline phosphatase-conjugated second antibody. 

The controversy over the degree to which radioactive probes are more sensitive has not been fully resolved. In any case, microwave pretreatment enhances ISH signal detection of RNA and DNA whether radiolabeled or nonradioactive probes are used both methods are presented later. Furthermore, a number of approaches is available to increase the sensitivity of the nonradioactive ISH procedures (for a review, see Komminoth and Werner, 1997) some of these approaches are discussed below. 

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