Radioactive labeling and detection

Holtke, H.-J., Seibl, R., Burg, J., Miihlegger, K., and Kessler, C. (1990) Non-radioactive labeling and detection of nucleic acids II. Optimization of the digoxigenin system. Mol. Gen. Hoppe-Seyler 371, 929-938. 

Figure 9. View of the essential parts of the crossed beam apparatus using short-lived radioactive labeling and detection (23) Ay radioactive beam source By scrubber-furnace C, LN -cooled collimator D, shut-off plug Ey nozzle beam furnace and cryopump F, gate valve G, hodoscope H, LN -coohd beam trap 7, calibrated beam monitor /, silicon surface barrier detectors K, halogen crossed beam L, radioactive beam M, rotary feed-through used to close the source stopcock.

Next, detection and quantitation of tumor cells adherent to specific substrates can be done by either radioactive or nonradioactive methods. For radioactive methods, labeling and detection has been already described in Section 2.3.3.5. Alternatively, tumor cells can also be labeled with fluorescent dyes, and quantitated accordingly (see same section). We will describe below only nonradioactive methods. 

FIGURE 2.10 Detection of TP53 exon 6 mutations using singlestrand conformation polymorphism (SSCP) analysis. PCR products of seventeen tumor samples were amplified with a pair of primers flanking exon 6, with one primer being radioactively labeled, and electro-phoresed in polyacrylamide gel. Sample 2 shows an additional abnormally migrating band (arrow), which is indicative of a mutation. 

M8. Martin, R., Hoover, C., Grimme, S., Crogan, C., Hdltke, J., and Kessler, C., A highly sensitive non-radioactive DNA labeling and detection system. BioTechniques 9, 762-768... 

In the binding assay technique, various tRNA molecules, one of which is radioactively labeled with are mixed with ribosomes and synthetic trinucleotides bound to a filter. If the radioactive label is detected on the filter, then it is known that the particular tRNA bound to that triplet. The binding experiments can be repeated until all the triplets are assigned. 

Krypton, Kr, is an elemental, colorless, odorless, inert gas. It is noncombustible, nontoxic, and nonreactive however, it is an asphyxiant gas and will displace oxygen in the air. Krypton 85 is radioactive and has a half-life of 10.3 years. The four-digit UN identification number for krypton is 1056 as a compressed gas and 1970 as a cryogenic liquid. These forms of krypton are not radioactive. Radioactive isotopes of krypton are shipped under radioactive labels and placards as required. Its primary uses are in the activation of phosphors for self-luminous markers, detecting leaks, and in medicine to trace blood flow. 

Traditionally probe DNA was labeled radioactively, most commonly by means of 2p labeled triphosphates. The quite short half life of the isotope and the requirement of security devices favours the use of nonradioactive labeling and detection techniques, especially for those, who do not apply such methods very often. 

The measurement of low concentrations of compounds by fluorimetry is normally the main purpose of fluorescence labelling of compounds which have no native properties that allow sensitive detection. Where there is fluorescence quenching by known or unknown components of the sample solutions or a lack of specificity of the fluorescence measurements, the application of alternative methods for the determination of fluorescent derivatives may be necessary. In such cases fluorescence may be used to monitor the chromatographic separation even though alternative quantitation methods are applied. Radioactivity measurement and mass spectrometry are frequently used. If a reagent is selected for a certain analytical purpose, besides its reactivity and the fluorescence and chromatographic characteristics of its derivatives, its accessibility to radioactive labelling and its suitability for qualitative and quantitative mass spectrometry should therefore also be considered. 

In repair replication experiments, cells are irradiated and then incubated in medium containing 3H-5-bromodeoxyuridine ( H-BrdUrd). After a suitable repair period the cells are harvested, and the DNA is extracted and centrifuged to equilibrium in cesium chloride. When the gradient fractions are collected, it is possible to follow the new DNA (semiconservatively synthesized during repair and at hybrid density) by counts. The new DNA is half substituted with bromouracil (BrUra) and sediments to a lower point on the gradient than the old or parental DNA, which is detected by absorbancy at 260 nm or by incorporation of a second radioactive label and is, of course, not density labeled. The appearance of counts in the old DNA region is indicative that repair has occurred and that the new incorporation is truly in the parental... 

The ability of SPR to probe both kinetic and thermodynamic processes, as well as to provide micro-structural information, make it a very important component of the experimental methodology available to probe molecular interactions occurring at surfaces. Furthermore, it allows some of the limitations of other techniques to be overcome. For example, other methods often require one of the partners to be labelled in some way in order to allow it to be detected. Fluorescent probes, radioactive labels, and attachment of independently detectable molecules (e.g. enzymes) have all been used for this purpose. These suffer from the drawback that they may interfere with the binding of the labelled partner to the unlabelled one, or cause unwanted structural perturbations. SPR observations can be based solely on the dielectric properties of molecules, or their intrinsic light absorption characteristics, and thus require no specific labelling. 

ImmunO lSS iy. Chemiluminescence compounds (eg, acridinium esters and sulfonamides, isoluminol), luciferases (eg, firefly, marine bacterial, Benilla and Varela luciferase), photoproteins (eg, aequorin, Benilld), and components of bioluminescence reactions have been tested as replacements for radioactive labels in both competitive and sandwich-type immunoassays. Acridinium ester labels are used extensively in routine clinical immunoassay analysis designed to detect a wide range of hormones, cancer markers, specific antibodies, specific proteins, and therapeutic dmgs. An acridinium ester label produces a flash of light when it reacts with an alkaline solution of hydrogen peroxide. The detection limit for the label is 0.5 amol. 

Physical methods Physical methods include photometric absorption and fluorescence and phosphorescence inhibition, which is wrongly referred to as fluorescence quenching [1], and the detection of radioactively labelled substances by means of autoradiographic techniques, scintillation procedures or other radiometric methods. These methods are nondestructive (Chapt. 2). 

Physical detection methods are based on inclusion of substance-specific properties. The most commonly employed are the absorption or emission of electromagnetic radiation, which is detected by suitable detectors (the eye, photomultiplier). The / -radiation of radioactively labelled substances can also be detected directly. These nondestructive detection methods allow subsequent micropreparative manipulation of the substances concerned. They can also be followed by microchemical and/or biological-physiological detection methods. 

The scintillators are a special type of fluorescence indicators they are employed for the fluorimetric detection of radioactively labelled substances. They are stimulated by ) -radiation to the emission of electromagnetic radiation and will be discussed in Volume 2. 

In-vitro models can provide preliminary insights into some pharmacodynamic aspects. For example, cultured Caco 2 cell lines (derived from a human colorectal carcinoma) may be used to simulate intestinal absorption behaviour, while cultured hepatic cell lines are available for metabolic studies. However, a comprehensive understanding of the pharmacokinetic effects vfill require the use of in-vivo animal studies, where the drug levels in various tissues can be measured after different dosages and time intervals. Radioactively labelled drugs (carbon-14) may be used to facilitate detection. Animal model studies of human biopharmaceutical products may be compromised by immune responses that would not be expected when actually treating human subjects. 

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