Fluorescein labelling

Table I describes several of the fluorescent assays that have been used in our lab to study neutrophil activation. Fluorescein-labeled W-formylhexapeptide (FLPEP) has been used to characterize the ki- netics of ligand binding, dissociation, and internalization at 37°C (7,8). FLPEP is added to a suspension of cells, then receptor-bound and free FLPEP in solution are distinguished by adding antibody to fluorescein. This is a high-affinity antibody which binds free FLPEP within 1 s hut does not bind cell-bound FLPEP. When it binds the FLPEP, it quenches the fluorescein fluorescence. Hence the residual fluorescence after antibody addition represents FLPEP that is bound to the cell. Nonspecific binding is determined in cell suspensions that contain an excess of nonfluorescent peptide.

The stoichiometry of the recharged DNA/PLL/SPLL particles was studied using sucrose-gradient ultracentrifugation of fluorescently labeled polyion complexes in 25 mM HEPES buffer. Rhodamine-labeled DNA (Rh-DNA) and either fluorescein-labeled PLL (Fl-PLL) or SPLL (Fl-SPLL) were used to determine their relative amounts within DNA... 

Johnson et al. [186] measured diffusion of fluorescein-labeled macromolecules in agarose gels. Their data agreed well with Eq. (85), which combined the hydrodynamic effects with the steric hindrance factors. Gibbs and Johnson [131] measured diffusion of proteins and smaller molecules in polyacrylamide gels using pulsed-field gradient NMR methods and found their data to fit the stretched exponential form... 

Yang WC, Schmerr MJ, Jackma R et al (2005) Capillary electrophoresis-based noncompetitive immunoassay for the prion protein using fluorescein-labeled protein A as a fluorescent probe. Anal Chem 77 4489 1494... 

Farley RA, Tran CM, Carilli CT et al (1984) The amino acid sequence of a fluorescein-labeled peptide from the active site of (Na, K)-ATPase. J Biol Chem 259 9532-9535... 

Fig. 13.3. Fluorescence on the surface of H. contortus intestinal cells following incubation of transverse sections of the worm with fluorescein-labelled antibody probes.

A competitive fluorescence-polarization immunoassay method was described for the monitoring of 12 drugs including valproic acid [18]. Samples (serum or plasma) were deproteinated. Fluorescence from the fluorescein-labeled analyte used as tracer was excited at 488 nm and polarization of light emitted at 531 nm was measured. The calibration was stable for 4 weeks and the coefficient of variation was below 10%. A single measurement took 8-10 min. 

C. Grandjean, C. Rommens, H. Gras-Masse, and O. Melnyk, Convergent synthesis of fluorescein-labelled lysine-based cluster glycosides, Tetrahedron Lett., 40 (1999) 7235-7238. 

Fig. 13 Uptake of OVA-encapsulating y-PGA-Phe nanoparticles by DCs. DCs were incubated with Texas Red-labeled OVA (TR-OVA) alone (a) or TR-OVA encapsulated within fluorescein-labeled nanoparticles (TR-OVA/FITC-NPs) (b). The intracellular localization of OVA (red) and NPs (green) was observed by confocal laser scanning microscopy...

TRITC has been used in numerous applications involving fluorescence detection, including double-staining techniques with fluorescein-labeled probes (Mossberg and Ericsson, 1990), the synthesis of fluorescently labeled DNA probes (Smith et al., 1985), as a label in homogeneous... 

Preparation of a series of phycobiliprotein tandem dyes allows multiplexed analysis of different targets in a sample. In addition, since RPE can be excited by the argon-ion laser at 488 nm, a fluorescein-labeled probe can be used concurrently with RPE alone and RPE-tandem conjugates to create a multiplexed system of different fluorescent probes that can be used simultaneously. Table 9.3 shows the different combinations of dyes that can be used in this type of assay with RPE and APC. 

Friedman, M.L., and Ball Jr., W.J. (1989) Determination of monoclonal antibody-induced alterations in Na+/K+-ATPase conformations using fluorescein-labeled enzyme. Biochim. Biophys. Acta 995, 42. 

Plank, L., and Ware, B.R. (1987) Acanthamoeba profiln binding to fluorescein-labelled actin. Biophys. /. 51,985. 

A CL ISH assay for the detection of human papillomavirus (HPV) DNA was developed, in which the hybridization reaction was performed using either digoxigenin-, biotin-, or fluorescein-labeled probes [64], The hybrids were visualized using AP as the enzyme label and a highly sensitive 1,2-dioxetane phosphate as chemiluminescent substrate. This assay was applied to biopsy specimens from different pathologies associated with HPV, which had previously proved positive for HPV DNA by polymerase chain reaction (PCR). The analytical sensitivity was assessed using samples of HeLa and CaSki cell lines, whose content in HPV DNA is known (10-50 copies of HPV 18 DNA in HeLa cells and 400-600 copies... 

Fig. 19.14 (a) Emission from dye DNA complexes Fluorescein labeled DNA at 60 pM (Top) Cy3 labeled DNA at 60 pM (Middle) After DNA hybridization at 30 pM for each cDNA sample (Bottom), (b) Emission from a probe MB (Top) Emission after adding a target cDNA (Bottom)... 

Rao et al.20 demonstrated a fluorescence polarization immunoassay for evaluating serum concentrations of tricyclic antidepressants (amitriptyline, imipramine, clomipramine, and doxepin) with respect to nonresponse, compliance, therapeutic window, and influences of age, sex, substance abuse, and toxicity. Abbott Laboratories TDx/TDxFLx Toxicology Tricyclic Assay FPIA (fluorescence polarization immunoassay) was used. This assay of 50 /uL samples contained tricyclic antidepressant antibodies raised in rabbits and fluorescein-labeled tricyclic antidepressant as a tracer. The assay was calibrated with imipramine in the range of 75 to 1000 fig/L (268 to 3571 nmol/L). Intra-assay and inter-assay coefficients of variation for internal quality control samples from the manufacturer were 4.2 and 4.7%, respectively. The limits of detection were 72,71,64, and 72 nmol/L for amitriptyline, imipramine, clomipramine, and doxepin, respectively. This high-throughput immunoassay was easy to use although amitriptyline, dosulepine, desipramine, and nortriptyline showed cross-reactivities ranging from 74 to 100%. 

Experiments using fluorescein-labelled fMet-Leu-Phe indicate that the majority of re-expressed receptors that appear on the plasma membrane within 4-10 min after stimulation arise from the mobilisation of internal pools. Subcellular fractionation studies indicate that the pools of these receptors are the membranes of specific granules, although it is possible that these are on other membranes (e.g. on gelatinase-containing granules or se-... 

Oligonucleotides bearing nido-carborane have been synthesized as phospho-diesters using an automated DNA synthesizer (see Scheme 2.2-14) [39]. These oligophosphates are homogeneous, very hydrophilic and are readily taken up into cells. Fluorescein-labeled nido-carboranyl oligomeric phosphate diesters accumulate in the cell nucleus [40]. 

Like FRET, today BRET is predominantly used in biological sciences, especially in the monitoring of protein-protein interactions such as hormone-receptor interaction [223, 224] and protein-DNA interaction in living systems. However, BL resonance energy transfer can also be applied in immunoassays by using for instance a peptide-tagged luciferase and a fluorescein-labeled antipeptide antibody [225]. The development of more BRET assays for small-molecule analytes is thus awaited. 

Liu B, Bazan GC (2007) Tetrahydrofuran activates fluorescence resonant energy transfer from a cationic conjugated polyelectrolyte to fluorescein-labeled DNA in aqueous media. Chem Asian J 2 499-504... 

Fluorescein-labelled deoxyribonucleotide triphosphates may be used in place of those labelled with 32P. Once the DNA sequences are separated by electrophoresis, the resulting DNA bands fluoresce and are analysed by a flu-orogram imager, which produces a picture of the fluorescent bands similar to the autoradiography produced when using 32P-labelled nucleotides. 

Fluorescence resonance energy transfer has also been used for ionic strength measurements.(95) Fluorescein labeled dextran (donor) and polyethyleneimine-Texas Red (acceptor) were placed behind a dialysis membrane. The polymer association is ionic strength dependent and the ratio of intensities (F o/Fw) was used as the measured parameter. Since both the donor and acceptor are fluorescent, this kind of sensor may allow expand the sensitive ionic strength range by shifts in observation wavelength, as was discussed for pH probe Carboxy SNAFL-2 (see Section 10.3). 

In a particle concentration fluoroimmunoassay system that is based on microtiter plates with 0.22-/ filters on the bottoms, microparticles are used with a flowthrough wash system. Either fluorescein-labeled antibodies or cells serve as labels.(41) Car-boxyfluorescein diacetate, which crosses the cell membrane, is hydrolyzed by cytoplasmic esterases, thereby trapping carboxyfluorescein within the cell. The method is also useful for screening and isotyping antibodies. 

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