Radiolabelling quantification

A variety of assays have been developed to quantify phagocytic activity. These include direct microscopic visualization (2,3), spectrophotometric evaluation of phagocytized paraffin droplets containing dye (4), scintillation counting of radiolabeled bacteria (5), fluorometric (6), and flow cytometric analysis of fluorescent particles (7-13). The flow cytometric assay offers the advantage of rapid analysis of thousands of cells and quantification of the internalized particle density for each analyzed cell. The assay may be performed with purified leukocyte preparations (7-13) or anficoagulated whole blood (14,15). 

CAM invasion 3-D from 2-D Monitors tissue invasion and metastasis to organs in a physiological microenvironment Accurate quantification difificult, poor correlation of results with metastasis in patients, radiolabeling of cells, laborious, requires animal tissue, variability, cannot be used for HT (128, 129)... 

Saguy, I. S., Gremaud, E., Gloria, H., and Turesky, R. J. (1997). Distribution and quantification of oil uptake in french fries utilizing a radiolabeled 14C palmitic acid. J. Agric. Food Chem. 45, 4286-4289. 

Although the radiolabeled nucleic acid precursor assays described above produce accurate and reproducible quantification of the number of viable cells in a sample, Mosmann [187] sought to develop a more rapid assay capable of handling large numbers of samples. A colorimetric technique was developed based upon the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-zolium bromide, or MTT. Early studies of MTT by Slater et al. [188] determined that when the MTT tetrazolium salt interacts with the dehydrogenase enzymes... 

Zhao, W., Zhang, H., Zhu, M., Warrack, B., Ma, L., Humphreys, W. G., and Sanders, M. (2006). An integrated method for quantification and identification of radiolabeled metabolites Application of chip-based nanoelectrospray and mass defect filter techniques. In Proceedings of the 54th ASMS Conference on Mass Spectrometry and Allied Topics, Seattle, WA. 

Zhu, M., Zhang, D., and Skiles, G. L. (2005). Quantification and structural elucidation of low quantities of radiolabeled metabolites using microplate scintillation counting techniques in conjunction with LC-MS. In Identification and Quantification of Drugs, Metabolites and Metabolizing Enzymes by LC-MS (Chowdhury, S. K., Ed.). Elsevier, Amsterdam, pp. 195-223. 

The described method is suitable to compare up to 50 drugs in one experiment. The price for this is the limited quantification, as the staining is not strictly stoichiometric, and does not allow the distinction between matrix synthesis and degradation. For more detailed assessment, radiolabeling is the better choice. The limitation of these primary culture assays lies in the elaborate preparation and isolation of the chondrocytes. Several attempts to immortalize this differentiated mesenchymal cell type have so far resulted in the loss of certain cartilage-specific properties. 

Quantification. Radioimmunoassay. In plasma sensitivity 200 pg/ml (comparison with methods using radiolabelled drug)—... 

A9.5.2.3.9.4 If the BCF in terms of radiolabelled residues is documented to be > 1000, identification and quantification of degradation products, representing > 10% of total residues in fish tissues at steady-state, are for e.g. pesticides strongly reeommended in the OECD guideline No. 305 (1996). If no identifieation and quantifieation of metabolites are available, the assessment of bioeoneentration should be based on the measured radiolabelled BCF value. If, for highly bioaccumulative substanees (BCF > 500), only BCFs based on the parent compound and on radiolabelled measurements are available, the latter should thus be used in relation to classification. 

Together with details of sample preparation and storage, an appropriate analytical method of known accuracy, precision, and sensitivity must be available for the quantification of the substance in the test solution and in the biological material. If these are lacking it is impossible to determine a true BCF. The use of radiolabelled test substance can facilitate the analysis of water and fish samples. However, unless combined with a specific analytical method, the total radioactivity measurements potentially reflect the presence of parent substance, possible metabolite(s), and possible metabolized carbon, which have been incorporated in the fish tissue in organic molecules. For the determination of a true BCF it is essential to clearly discriminate the parent substance from possible metabolites. If radiolabelled materials are used in the test, it is possible to analyse for total radio label (i.e. parent and metabolites) or the samples may be purified so that the parent compound can be analysed separately. 

Quantification of DNA adducts in samples at very low exposure levels, and after administration of very low quantities of isotopes/ radiolabeled compounds (as low as lpCi/40kBq). 

A sensitive, reliable, and rapid technique for quantification of homoharringtonine in plasma or serum by HPLC with amperometric detection has been developed by Chan and co-workers (107). Previous determinations of homoharringtonine in plasma entailed the use of radiolabeled alkaloids (108,109). Tang et al. have used HPLC for the determination of harringtonine in human plasma (110). 

There are several ways to determine the quantity of plasmid in cells following transfection. We present here a fairly simple procedure in which radiolabeled plasmid complexed to cationic lipids is used to transfect cells, followed by quantification of the plasmid at various timepoints (119). 

In the case of cationic lipid/DNA complexes, the lipid markers that have been used are often the same as those used for conventional liposomes (i.e., CHE). This may not be appropriate. Therefore, we believe it is best to use a radiolabeled form of the lipids used to prepare the carrier system (133). Ideally, carrier cationic lipid and plasmid DNA quantification should be performed on the same tissues within in the same experiment. It may also be useful to analyze the fate of the neutral lipid components of the carrier. Radiolabeled DOPE is available, for example. It may also be useful, in certain types of analyses, such as pharmacokinetic and biodistribution studies, to label both the cationic and neutral components of the lipid carrier. Without some kind of tag, however, the process of detection may be more complex because of the need to efficiently extract the carrier lipid(s) from cells or tissues prior to analysis. The presence of endogenous lipids may make this difficult. For cationic lipids, such as DOTAP or DODAC, for example, it may be recommended that quantification of tissue levels, such as by HPLC analysis, be performed by those specializing in lipid analysis (Northern Lipids, Vancouver, BC, Canada). 

The most prominent of these assays are radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). In the case of RIA, the analyte sample is either extracted or used directly, and mixed with a constant amount of antibody and radiolabeled analyte [tracer mostly fi-emit-ters (e.g., H, " C) or y-emitters (e.g., I)]. After equilibration and separation of the free and bound antigen, radioactivity is measured for quantification of the analyte. In the case of sandwich-RIA, two antibody preparations are used the first serves as the binding partner of the analyte, whilst the second - which is radiolabeled - is directed either against the analyte or the first... 

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