Antibodies fluorescein isothiocyanate

Figure 7 (A) Electropherogram of FlTC-antibody (fluorescein isothiocyanate-antibody) to human IgA, (B) electropherogram of succinyl FlTC-antibody to human IgA, and (C) electropherogram of reaction mixture of human IgA with succinyl FlTC-antibody to human IgA. (Adapted from Ref. 74.)...

The most used EIA reagents conjugate a fluotophote such as fluorescein-isothiocyanate (EITC) or thodarnine—isothiocyanate to antibody (or antigen) free amino groups. Examples of other commonly used fluotophotes for EIA and their spectral characteristics ate presented in Table 3. EIA assays ate available in sandwich and competitive formats similar to EIAs. Unlike EIA kits which can be used directly with visual color deterrnination, EIAs require a fluorometer, and thus ate primarily laboratory-based. 

In addition to the wide range of commercial probes, many other fluorescent molecules have been synthesized and described in the literature. Only a handful, however, are generally used to label antibody molecules. Perhaps the most common fluorescent tags with application to immunoglobulin assays are reflected in the main derivatives produced by the prominent antibody manufacturing companies. These include derivatives of cyanine dyes, fluorescein, rhod-amine, Texas red, aminomethylcoumarin (AMCA), and phycoerythrin. Figure 20.16 shows the reaction of fluorescein isothiocyanate (FITC), one of the most common fluorescent probes, with an antibody molecule. 

Fischer D, Kissel T (2001) Histochemical characterization of primary capillary endothehal cells from porcine brains using monoclonal antibodies and fluorescein isothiocyanate-labelled lectins Implications for drug delivery. Europ J Pharm Biopharm 52 1-11... 

Figure 4 Increased percentages of CXCR3+CD4+ T cells in immunopotentiating reconstituted influenza virosomes (IRIV) stimulated peripheral blood mononuclear cells (PBMC). Healthy donors PBMC were cultured in the absence of stimuli (A), in the presence of liposomes [1 50 flnal dilution, (B)], or IRIV [1 50 final dilution, (C)]. After six days, culture cells were phenotyped for the expression of CXCR3 and CD4 by phosphatidylethanolamine and fluorescein isothiocyanate labelled monoclonal antibodies respectively. Source. From Ref 6.

Figure 7 Immunopotentiating reconstituted influenza virosomes (IRIV) mediated adjuvance in cytotoxic T-cell induction requires CD4+ T cells. CD8+ and CD14+ cells were cultured in the presence of autologous intact or irradiated CD4+ cells. These cultures were stimulated with influenza matrix (IM)58 66 (1 Pg/mL) alone (A) or supplemented with IRIV (1 50) (B). After seven days of incubation both cocultures were restimulated with irradiated IMss-ee pulsed CD14+ cells and cultured for six further days in the presence of interleukin-2 [see Materials and Methods ]. Six days after restimulation, cultures were stained with HLA-A0201 /IM58-66 PE-specilic tetramers and anti-CD8 fluorescein isothiocyanate monoclonal antibodies. Source. From Ref 6.

The second strategy uses combinations of different antibodies coupled to fluorochromes with distinct emission maxima (5,9). The most relevant fluoro-chromes for combined antigen detection are fluorescein isothiocyanate (FITC abs. max. 494 nm, emiss. max. 517 nm), rhodamine isothiocyanate (TRITC ... 

Abnova FISH probes and Dako FISH probes are labeled with fluorescein isothiocyanate (FITC) and Texas Red (sulforhod-amine lOI acid chloride) haptens, and the probe hybridization sites can be visualized with a BISH detection kit including anti-FITC and anti-Texas Red antibodies. 

The dissociation constant (Kd) of a monoclonal antibody with fluorescein isothiocyanate- (FITC)-labeled insulin and unlabeled insulins from several species were measured using CE with laser-induced fluorescence detection (CE-LIF) (9). Kd determinations were made by separating free FITC-labeled insulin and its complex with the antibody in equilibrated solutions in 6 s or less (Fig. 3). Dissociation and association rates for insulin, FITC-insulin, and the antibody are fast enough to reach equilibria in less... 

BrdU/DNA flow cytometry offers flexibility and diversity in the study of cell kinetics from cells in culture to human tumors in vivo. The essence of the procedure is to pulse label with BrdU by a short-term incubation in vitro or by a single injection in vivo samples are then taken at time intervals thereafter and stained after fixation in ethanol. The cells are then stained with a monoclonal antibody against BrdU that can be either directly conjugated to a fluoro-chrome (usually fluorescein isothiocyanate [FITC]) or, alternatively, bound to a second antibody conjugated with FITC. The cells are then counterstained with propidium iodide (PI) to measure the DNA content and analyzed on the flow cytometer. The results are displayed as linear-red fluorescence on the x-axis vs linear or log-green fluorescence on they-axis. 

Fluorescence forms the basis of a wide variety of assays, particularly those that are im-munologically based. IgG has been quantitated in bovine milk using a solid-phase agglutination assay using fluorescein isothiocyanate (FITC)-labeled rabbit anti-bovine IgG as the detection antibody (Losso et al., 1993). Such assays are both repeatable and rapid. 

The IFA is an immunoassay in which the antibody or antigen are labeled with a fluorescent probe. These can be direct or indirect (8, 41, 57). The IFA technique is usually used to locate cellular constituents. The commonly used fluorescent probes are rhodamine B isothiocyanate and fluorescein isothiocyanate. The sensitivity of the assay ranges in the ng/ml range. 

The sections are washed in PBS for 5 min and incubated for 30 min in fluorescein isothiocyanate (FICT)-labeled swine antirabbit immunoglobulin conjugate at 1 20 dilution in PBS. After being washed in PBS for 5 min, the sections are mounted with an aqueous mounting medium. The sections are observed under an epiilluminating fluorescent microscope. The negative control is not incubated with the primary antibody, and a frozen section from a patient with the known condition is used as the positive control. 

The cells are incubated in the primary antibody at an appropriate dilution for 30-60min at 4°C or room temperature, depending on the type of cells or antigens. After being washed in PBS, the cells are incubated in a fluorescein isothiocyanate (FTTQ-con-jugated goat antimouse IgG (or sheep antimouse IgG) for 30 min at 4°C in the dark. The cells are washed twice in PBS and resuspended in —200 (xl of 1% fetal calf serum or ISOTON II for flow cytometric analysis. 

The cells and nuclei (0.5-1.0 X 106) are aliquoted into polystyrene tubes. Approximately 10 jjlI of normal horse serum (Vector Laboratories, Burlingame, CA) is added to block any nonspecific binding. The suspension is incubated with biotinylated antiestrogen monoclonal antibody (1D5, Dako Corp., Carpinteria, CA) at 1 25 dilution for 1 hr at 37°C. Aliquots are stained with 10 p,l of fluorescein isothiocyanate (FITC)-conjugated strepta-vidin (Dako buffer containing 0.1% Triton X-100). 

Fluorescein Fluorescein is a fluorescent dye that can be readily linked to proteins and that is therefore useful, when conjugated to specific antibodies, for lighting up cells with particular phenotypes. It is sometimes abbreviated as FITC because fluorescein isothiocyanate is the chemically active form of the molecule that is used in the conjugation process. 

Species-specific anti-immunoglobulin antibodies and their fluorescent conjugates, Cy3, Cy5, or fluorescein isothiocyanate (FITC) (Sigma, St. Louis, MO BD-Phar-Mingen, San Diego, CA). 

Indirect immunofluorescence studies are performed on 3 11 m-tliick cryostat sections which are air dried and incubated with anti-rat intercellular adhesion molecule-1 (ICAM-1) antibody (Tamatani and Miyasaka 1990) for 60 min at room temperature. After washing the antibody binding is visualized by incubating the sections for 30 min with fluorescein isothiocyanate-labeled goat anti-mouse IgG. 

Fluoroimmunoassay These assays rely on the use of fluorescence-emitting labels. Typically, antibodies labelled with afluorophore are used, for example, fluorescein isothiocyanate. After washing, measured fluorescence relates to the amount of antigen. Another useful variant is called the homogeneous fluorescent polarization immunoassay that is commonly used to measure dmgs and analytes. 

The most frequently used fluorogenic substance for protein labeling is fluorescein-isothiocyanate, most likely owing to its availability and easy way of labeling. Detailed procedure for protein labeling (and especially antibody labeling) are described elsewhere 324)... 

Protein A is a specific protein isolated from the cell wall of Staphylococcus aureus whose characteristic property is the ability to react and to form precipitates with a variety of IgG molecules from several species. This interaction is reminia nt of the formation of antigen-antibody complexes, and has been used to study different aspects of immune response as well as cell surface structure and function For easier detection, Protein A is covalently coupled to fluorescein isothiocyanate (FITC). The commercial preparations of FITC-Protein A contain an avarage of 6 FITC substituent groups per molecule of protein. Such a degree of labeling does not affect the biological properties of the native protein. 

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