Antibody fluorescent-labelled

Figure 11.2 Antigen-antibody fluorescent labeling scheme (Reaction 11.2).

Recently, polyclonal mouse antihuman IgG antibodies fluorescently labeled with Cy5 were used to detect the binding of human myeloma proteins to biotinylated monoclonal antibodies. Again, Cy5-labeled antibodies were used to determine bacterial, viral, and protein antigens bound to biotinylated IgGs. 

Another type of microchip uses bound proteins instead of DNA. These protein arrays are based on interactions between proteins and antibodies (Chapter 14). For example, antibodies to known diseases can be bound to the microarray. A sample of a patient s blood can then be put on the microarray. If the patient has a particular disease, proteins specific to that disease bind to the appropriate antibodies. Fluorescently labeled antibodies are then added and the microarray scanned. The results look similar to the DNA microarrays discussed previously. Figure 13.32 shows how this would work to identify that a patient had anthrax. This technique is growing in popularity and power, but is limited by whether purified antibodies have been created for a particular disease. 

The LANCE cAMP assay is a competitive assay in which cAMP produced by the cells competes with fluorescent-labeled acceptor cAMP for a cryptate tagged donor antibody. The principal of the assay is shown in Fig. 6. On the left strepta-vidin conjugated Europium binds to biotinylated cAMP. An antibody labeled with the fluorescent dye Alexa binds to the cAMP, bringing the donor and acceptor into close proximity, and energy transfer occurs. When the cell releases cAMP, it competes with the biotin-labeled cAMP for the antibody, and a signal decrease is observed. In the TR-FRET assay the antibody is directly labeled with either Eu or Tb. In this format an increase in cAMP also causes a decrease in signal. 

Indirect immunofluorescence assay (IFA) A laboratory test used to detect antibodies in serum or other body fluid. The specific antibodies are labeled with a compound that will make them glow a fluorescent green color when observed microscopically under ultraviolet light. 

The tuneable nature of the evanescent field penetration depth is critical to the effective operation of this sensor as it facilitates surface-specific excitation of fluorescence. This means that only those fluorophores attached to the surface via the antibody-antigen-labelled antibody recognition event... 

Use of sulfo-NHS-LC-SPDP or other heterobifunctional crosslinkers to modify PAMAM dendrimers may be done along with the use of a secondary conjugation reaction to couple a detectable label or another protein to the dendrimer surface. Patri et al. (2004) used the SPDP activation method along with amine-reactive fluorescent labels (FITC or 6-carboxytetramethylrhodamine succinimidyl ester) to create an antibody conjugate, which also was detectable by fluorescent imaging. Thomas et al. (2004) used a similar procedure and the same crosslinker to thiolate dendrimers for conjugation with sulfo-SMCC-activated antibodies. In this case, the dendrimers were labeled with FITC at a level of 5 fluorescent molecules per G-5 PAMAM molecule. 

Figure 7.21 Dendrimers that are fluorescently labeled as well as biotinylated create enhanced detection reagents for use in (strept)avidin-biotin-based assays. Large complexes containing multiple fluorescent dendrimers can bind to antigens and form a highly sensitive detection system that exceeds the detection capability of fluorescently labeled antibodies.

Fluorescent labels, by contrast, can provide tremendous sensitivity due to their property of discrete emission of light upon excitation. Proteins, nucleic acids, and other molecules can be labeled with fluorescent probes to provide highly receptive reagents for numerous in vitro assay procedures. For instance, fluorescently tagged antibodies can be used to probe cells and tissues for the presence of particular antigens, and then detected through the use of fluorescence microscopy techniques. Since each probe has its own fluorescence emission character, more... 

The spectral properties of four major phycobiliproteins used as fluorescent labels can be found in Tables 9.1 and 9.2. The bilin content of these proteins ranges from a low of four prosthetic groups in C-phycocyanin to the 34 groups of B- and R-phycoerythrin. Phycoerythrin derivatives, therefore, can be used to create the most intensely fluorescent probes possible using these proteins. The fluorescent yield of the most luminescent phycobiliprotein molecule is equivalent to about 30 fluoresceins or 100 rhodamine molecules. Streptavidin-phycoerythrin conjugates, for example, have been used to detect as little as 100 biotinylated antibodies bound to receptor proteins per cell (Zola et al., 1990). 

In some cases, the preparation of a fluorescently labeled antibody is not even necessary. Particularly, if indirect methods are used to detect antibody binding to antigen, then preparing a fluorescently labeled primary antibody is not needed. Instead, the selection from a commercial source of a labeled secondary antibody having specificity for the species and class of primary antibody to be used is all that is required. However, if the primary antibody needs to be labeled and it is not manufactured commercially, then a custom labeling procedure will have to be done. 

In order to facilitate analysis of FeBABE produced fragments, the prey protein or biomolecule is labeled at one end with a tag that can be detected after electrophoresis, usually in a transfer blot. The tag can be a fusion tag, such as 6X His, or any other group that can be targeted with an antibody and detected. Alternatively, radiolabels and fluorescent labels have been used with prey molecules, including the use of end-labeled DNA to study where DNA binding proteins dock onto the oligonucleotide sequence. 

---