Direct labeling method

In the direct labeling method, mRNA is converted into a labeled cDNA population. This is achieved by copying the transcripts into cDNA molecules with a reverse transcriptase while incorporating a modified Cy dye nucleotide. 

Among the direct labeling methods, reduction of the antibody by a thiol reagent, such as mercaptoethanol or dithiothreitol, results in high labeling yields (Reilly 1993 Schwarz et al. 1988 Thakur et al. 1991). Table 2.3.2 compiles Tc-labeled antibodies approved as radiopharmaceuticals in the United States and the European Union. 

Figure 10)). This method has a distinct advantage over the direct labelling method as it gives almost the same high sensitivity and wide dynamic range as the non-competitive assay format. 

Fig. 13. Schematic representation of direct labelling methods using (A) primary antibody coupled to gold or (B) Fab fragment coupled to gold and indirect labelling methods using (C) protein A-gold, (D) secondary antibody coupled to gold or (E) the streptavidin-biotin system, showing clearly the difference between resolution and sensitivity.

There are two common forms of heterogeneous assays used to detect and quantify disease markers the direct-labeling method, in which the proteins in a sample are labeled with a detectable tag and isolated from a complex sample by means of a bed of immobilized antibodies, and the indirect-labeling method, in which two antibodies are used for each marker. Sandwich-type immunoassays, such as the enzyme-linked immunosorbent assay (ELISA), require two antibodies an immobilized antibody used to capture a molecule of interest - a protein, a virus, or a small molecule (e.g., a hormone. 

Direct labelling with deuterium is another approach, and three promising methods have been reported. One, described by Koch and Stuart and coworkers,41 is based on the specific exchange of hydrogen attached to hydroxylated carbon atoms by deuterium atoms that occurs on treatment with Raney nickel-D20. Although no exchange was observed with soluble starch or glycogen, the H-2, H-3, and H-6 atoms of... 

Primary antibodies blot excess blocking solution from sections and incubate for 60 min at room temperature or overnight at +4°C with a mixture of correspondingly diluted unlabeled primary antibodies raised in two different host species (e.g., mouse and rabbit). When using fluorophore-labeled primary antibodies as in direct immunostaining method (one antibody layer), you may skip step (6) with secondary antibodies for indirect immunostaining method (two antibodies layers). Wash sections in PBS for 3 x 3 min. 

This assay method (RIPA) is used primarily in research. It is too technically demanding for routine use in clinical laboratories. HIV is cultured in radio-labeled cells, or viral proteins are directly labeled with a radioisotope. The virus is disrupted and then exposed to the test specimen. Specific antigen-antibody complexes are concentrated and isolated by immunoprecipitation. After exten-... 

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