Fluorescein isocyanate

Albert H. Coons (Fig. 1.2) was the first who attached a fluorescent dye (fluorescein isocyanate) to an antibody and used this antibody to localize its respective antigen in a tissue section. The concept of putting a visible label on an antibody molecule appeared both bold and original. His initial results were described in two brief papers in the early 1940s (Coons et al. 1941,1942), but the research was halted while he joined the army and spent the next 4 years in the South Pacific. His later studies (Coons and Kaplan 1950) contributed immensely to the use of the fluorescent antibody method in a wide variety of experimental settings. In our time, the use of antibodies to detect and localize individual or multiple antigens in situ has developed into a powerful research tool in almost every field of biomedical research (http //books.nap.edu/html/biomems/acoons.pdf). 

Albert H. Coons was the first to attach a fluorescent dye (fluorescein isocyanate) to an antibody and to use this antibody to localize its respective antigen in a tissue section. Fluorescein, one of the most popular fluorochromes ever designed, has enjoyed extensive application in immunofluorescence labeling. For many years, classical fluorescent probes such as FITC or Texas red (TR) have been successfully utilized in fluorescence microscopy. In recent decades, brighter and more stable fluorochromes have continually been developed (see Table 14.1). Marketed by a number of distributors, cyanine dyes, Cy2, Cy3, Cy5, Cy7, feature enhanced water solubility and photostability as well as a higher fluorescence emission intensity as compared to many of the traditional dyes, such as FITC or TR. 

If a compound is nonfluorescent, it may be converted to a fluorescent derivative. For example, nonfluorescent steroids may be converted to fluorescent compounds by dehydration with concentrated sulfuric acid. These cyclic alcohols are converted to phenols. Similarly, dibasic acids, such as malic acid, may be reacted with j8-naphthol in concentrated sulfuric acid to form a fluorescing derivative. White and Argauer have developed fluorometric methods for many metals by forming chelates with organic compounds (see Ref. 23). Antibodies may be made to fluoresce by condensing them with fluorescein isocyanate, which reacts with the free amino groups of the proteins. NADH, the reduced form of nicotinamide adenine dmucleotide, fluoresces. It is a product or reactant (cofactor) in many enzyme reactions (see Chapter 24), and its fluorescence serves as the basis of the sensitive assay of enzymes and their substrates. Most amino acids do not fluoresce, but fluorescent derivatives are formed by reaction with dansyl chloride. 

Polygalacturonases.—The (endo-)polygalacturonase from Pseudomonas cepacia has been labelled with fluorescein isocyanate so that its penetration into onion (Allium cepa) tissues could be followed. ... 

The reaction of aromatic amines with phosgene to produce isocyanates has wide applicability. The example of the preparation of p-nitrophenyl isocyanate illustrates the general procedure. Even complex aromatic amines such as fluorescein amine may be subjected to the reaction to produce fluorescein isocyanate, which has found some application in biochemical research. 

The antibodies produced against double-helical complexes of polynucleotides have also been used to demonstrate the presence of double-stranded RNA in cells. The presence and the localization of double-stranded reovirus RNA has been revealed in cells infected by this virus using anti-poly I poly C — MBSA antibodies marked with fluorescein isocyanate (Silverstein and ScHUR, 1970). 

Extrinsic fluorescence is used whenever the natural fluorescence of a macromolecule is inadequate for accurate fluorescence measurement. In this case, one can attach a fluorescent reporter group by using the reactive isocyanate or isothiocyanate derivatives of fluorescein or rhodamine, two intensely fluorescent molecules. One can covalently also label a protein s a- and e-amino groups with dansyl chloride (/.e., A,A-dimethylaminonaphtha-lenesulfonyl chloride). Another useful reagent is 8-ani-lino-l-naphthalenesulfonic acid (abbreviated ANS). This compound is bound noncovalently by hydrophobic interactions in aqueous solutions, ANS is only very fluorescent, but upon binding within an apolar environment, the quantum yield of ANS becomes about 100 times greater. 

More often, carbamates of dextran are prepared by one-step conversion of the polysaccharide with isocyanates or isothiocyanates. This path is especially useful for the conjugation of fluorescent dyes. Fluorescent-marked dextrans are commercial products today. Fluorescent dextran derivatives with different molecular weights and substituents are available as invaluable tracers for studies on microcirculation and vascular permeability in health and disease [8]. Fluorescein isothiocyanate (FITC) dextran (actually the thiocarba-mate) has been well established in this area of research since the 1970s [358]. The isothiocyanate of fluorescein is covalently bound to dextran leading to... 

To date, the most popular fluorescent labels for FIA have been those derived from the long-wavelength, strongly emitting xanthene dyes fluorescein isothiocyanate (FTTC) and lissamine rhodamine B (RB200).F The isothiocyanates or isocyanates of these fluorophores can be used to label primary and secondary aliphatic amines in aqueous solutions by simple procedures. 

With particularly bulky substituents, the PSM process can be limited to the crystal surfaces. For example, Metzler-Nolte and coworkers investigated the reaction of IRMOF-3 with fluorescein isothiocyanate (ArNCS, equation 1) and used confocal laser scanning microscopy to show that fluorescence originated only from a very thin layer at the outer surfaces of the crystals. The steric bulk of the fluorescein group prevented the isocyanate from penetrating into the bulk of the crystals. ... 

---