Antibodies, fluorophore-labeled

The commercially available dicyanomethylene squaraine dye Seta-670-mono-NHS showed extremely low blinking effects and good photostability when used in single-molecule studies of multiple-fluorophore labeled antibodies [113]. Seta-670-mono-NHS and Seta-635-NH-mono-NHS were covalently labeled to antibodies and used in a surface-enhanced immunoassay [114]. From the fluorescence intensity and lifetime changes determined for a surface that had been coated with silver nanoparticles, both labeled compounds exhibited a 15- to 20-fold... 

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... 

A common application for (strept)avidin-biotin chemistry is in immunoassays. The specificity of antibody molecules provides the targeting capability to recognize and bind particular antigen molecules. If there are biotin labels on the antibody, it creates multiple sites for the binding of (strept)avidin. If (strept)avidin is in turn labeled with an enzyme, fluorophore, etc., then a very sensitive antigen detection system is created. The potential for more than one labeled (strept)avidin to become attached to each antibody through its multiple biotinylation sites is the key to dramatic increases in assay sensitivity over that obtained through the use of antibodies directly labeled with a detectable tag. 

Fluorophore-labeled antibodies, 14 148 Fluorophores, commonly used, 14 148, 149t Fluoropolymers... 

Primary antibodies blot excess blocking solution from sections and incubate for 60 min at room temperature or overnight at +4°C with a mixture of correspondingly diluted unlabeled primary antibodies raised in two different host species (e.g., mouse and rabbit). When using fluorophore-labeled primary antibodies as in direct immunostaining method (one antibody layer), you may skip step (6) with secondary antibodies for indirect immunostaining method (two antibodies layers). Wash sections in PBS for 3 x 3 min. 

When using biotin-labeled secondary antibody, you have first to visualize biotin with a fluorophore-labeled (strept)avidin employing ABC technique (see Sect. 6.2.1), before proceeding to counterstaining (step 7). 

Another approach to this persistent problem relies on haptenylation of primary antibodies. Hapten (e.g., biotin, digoxigenin or any fluorophore) can be covalently bound to the antibody via A-hydroxysuccinimide esters (NHS-ES) (see Sect. 2.1), or conjugated employing monovalent IgG Fc-specific Fab fragments (see Sect. 2.2). Haptenylated primary antibodies can be subsequently visualized with the use of secondary antibodies recognizing the corresponding hapten (Fig. 8.5). Fluorophore-labeled primary antibodies can be directly visualized in a fluorescent microscope. 

Fig. 30 Representative scheme for the signal amplification concept by increasing the number of fluorophores per binding site in an antigen-antibody sandwich assay, (a) Binding of a labeled antibody to the target analyte yields a moderate fluorescence signal because the antibody is labeled with only few fluorophores (b) For the same binding event, the emission signal is dramatically amplified when using an antibody labeled with a nanoparticle that is doped with a large number of fluorophores...

FIA was originally developed as a histological technique to localize specific cellular sites using the specificity of an immunological reaction (23). The resulting fluorescent antibodies can be detected in animal tissues at levels as low as 1 /tg/mL of body fluid. Fluorophore-labeled antibodies have also been used widely for flow cytometry applications using fluorescein antibodies to cell surface markers to detect and quantify specific cells (24). 

Fluorescence polarization (anisotropy) version 2 (IMAP) Fluorophore labeled peptides bind to special detection beads coated with trivalent metal binding results in change in Brownian motion measured as with FP1 above Versatile without need for antibody Susceptible to compound interference peptide must be relatively small precludes use of protein substrates Turek-Etienne (2003a)... 

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