Fluorescently-labelled antibody

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.

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. 

S. Ribrioux, G. Kleymann, W. Haase, K. Heitmann, C. Ostermeier, and H. Michel, Use of nanogold-and fluorescent-labeled antibody Fv fragments in immunocytochemistry. J. Histochem, Cytochem. 44, 207-213 (1996). 

R. Ekins, F. Chu and E. Biggart, Development of microspots multi-analyte ratiometric immunoassay using dual fluorescent-labelled antibodies, Anal Chim Acta 227, 73-96 (1989). 

As a technique for selective surface illumination at liquid/solid interfaces, TIRF was first introduced by Hirschfeld(1) in 1965. Other important early applications were pioneered by Harrick and Loeb(2) in 1973 for detecting fluorescence from a surface coated with dansyl-labeled bovine serum allbumin, by Kronick and Little(3) in 1975 for measuring the equilibrium constant between soluble fluorescent-labeled antibodies and surface-immobilized antigens, and by Watkins and Robertson(4) in 1977 for measuring kinetics of protein adsorption following a concentration jump. Previous rcvicws(5 7) contain additional references to some important early work. Section 7.5 presents a literature review of recent work. 

In the following sections, we describe the use of fluorescently labeled antibodies however, these protocols are applicable to fluorescent fusion proteins as well. 

Fluorescently labeled antibodies, which are conjugated with donor and acceptor dyes, respectively. 

K Shimura, BL Karger. Affinity probe capillary electrophoresis Analysis of recombinant human growth hormone with a fluorescent-labeled antibody fragment. Anal Chem 66 9-15, 1994. 

Rates of lateral diffusion of membrane components have also been determined using optical methods. The early experiments of Frye and Ediden16 demonstrated lateral motion of fluorescent-labeled surface antigens in heterokaryons of mouse and human cells. They observed intermixing of fluorescent-labeled antibodies against mouse cell and human cell antigens. Optical methods may also be characterized as either transient or steady state. The use of fluorescence correlation spectroscopy as... 

The resulting determination of the numbers of antireceptor antibody molecules bound/epithelial cell are shown in Table 2. The value for the control antibody represents the autofluorescence of the target cells in terms of the equivalent number of fluorescent-labeled antibody molecules, rather than binding of the control antibody. Thus, a similar value was obtained for target cells with and without control antibody (not shown). 

Confocal laser scanning microscopy can be used in conjunction with microwave heating for examining the three-dimensional structure and cellular interrelationships in sections of paraffin-embedded tissues (Boon and Kok, 1994). Tissues are fixed with Kryofix, a coagulant fixative containing 50% ethyl alcohol and polyethylene glycol (PEG molecular weight 300) for 90 sec in a microwave oven. The use of thick paraffin sections (15 (xm) and fluorescently labeled antibodies is preferred. 

Proteomic Fluorescence microscopy In situ visualisation of cellular/ECM protein with fluorescence-labelled antibody Multiplexing capability (typically 3 fluorophores). Extra- and intracellular antigen location on opaque biomaterials. Issues with bleaching and autofluorescence. Yes/no... 

The newly available Odyssey and Aerius infrared imaging systems make it possible to probe whole cells with two-color infrared fluorescently labeled antibodies (anti-phosphopeptide, for example) that are used to detect changes in intracellular kinase signaling (Chen et al., 2005). The advantage of using infrared-labeled probes lies in the increased sensitivity and dynamic range and consequent reduction in the use of reagents. 

A series of photomultiplier tubes (PMTs) will detect different wavelengths of light emitted by excited, fluorescently labeled antibodies on the surface of each cell as it passes through the laser. 

Fig. 5 NRL Array Biosensor mixed format immunoassays (modified from [119]). Schematic of (a) the sandwich and (b) the competitive immunoassay fonnats used in the detection of the Campylobacter jejuni and aflatoxin Bi (AFBi), respectively, (a) Sandwich format antigen captured by the immobilized antibody then quantified by passing a second, fluorescently labeled, antibody over the surface, (b) Competitive format competition for binding sites on the fluorescently labeled antibody occurs between the unlabeled antigen in solution and the surface-bound antigen analog, (c) Final charge-coupled devices image taken with the NRL Array Biosensor of a waveguide exposed simultaneously to the C. jejuni (5 x 10" cfu/mL) sandwich assay (SAND) and the aflatoxin Bj (AFBi-1 ng/mL) competitive assay (COMP) in various combinations...

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