Blue fluorescent antibodies

Fig. 13 Biomolecules used as enantioselective fluorescence sensors, (a) Blue fluorescent antibody 43 interacting with stilbene derivatives (structure from protein data bank, entry 1FL3 [124] (b) sensing of adenosine enantiomers by the DNA aptamer 44...

Matsushita M, Yoshida K, Yamamoto N et al (2003) High-throughput screening by using a blue-fluorescent antibody sensor. Angew Chem Int Ed 42 5984—5987... 

Matsushita H, Yamamoto N, Meijler MM et al (2005) Chiral sensing using a blue fluorescent antibody. Mol Biosyst 1 303-306... 

A brilliant emitter. Certain naphthalene derivatives exhibit a weak yellow fluorescence when they are in a highly polar environment (such as water) and an intense blue fluorescence when they are in a markedly nonpolar environment (such as hexane). The binding of e-dansyl-lysine to specific antibody is accompanied by a marked increase in its fluorescence intensity and a shift in color from yellow to blue. What does this finding reveal about the hapten-antibody complex ... 

Diels-Alder reaction releasing, besides nitroxyl which can be converted to nitric oxide, a strongly blue fluorescent anthracene product 64 (Scheme 22) [99]. This reaction can therefore be followed sensitively in cell culture supernatants. Using early screening we have isolated seven different catalytic antibodies for the reaction out of approximately 12,000 individual cell culture wells resulting from fusions with ten different immunized mice. Due to early screening, the experiment was completed in a matter of weeks and comprised cloning of only a handful of antibodies [100]. 

The deoxynudeotide analogue-stilbene conjugate was incorporated into nascent DNAby DNApolymerase activity and its fluorescence properties were investigated [62]. It was shown that the blue fluorescence of stilbene was detected only upon binding of an antibody that spedfically recognizes the stilbene-modified nudeotide (Figure 3.16). 

One example is the fluoroimmunoassay for the routine detection of buprenorphine in urine samples of persons suspected of Temgesic abuse. Buprenorphine antibody is labelled with pseudobuprenor-phine, the dimer of buprenorphine. In this case, pseudobuprenorphine has a higher affinity for the antibody than that of FITC-norbuprenorphine. Pseudobuprenorphine shows an intense blue fluorescence with maximum at 435 nm when excited at 326 nm. The minimum detectable dose of buprenorphine by the fluoroimmunoassay is calculated to be 20 ng mL-i. 

The results reported showed that V450 6a is as fluorescent as, or more fluorescent than, the existing fluorophores with similar spectral characteristics (e.g., Pacific Blue 5), whereas two of the three antibody clones tested demonstrated that a 20-30% gain in signal could be obtained by using V450 6a. 

Fluorophore absorbs ultraviolet light (or violet, blue or green) and emits light of longer wavelength. Fluorophores are used in immunohistochemistry for labeling primary or secondary antibodies in direct and indirect immunostain-ing methods, respectively. They can be visualized in fluorescence microscopy using special filter sets. 

The fluorescence enhancement and the shift to blue indicate that water is largely excluded from the combining site when the hapten is bound. Hydrophobic interactions contribute significantly to the formation of most antigen-antibody complexes. 

Fig. 5.15 Antibodies against perinuclear antigens of granulocytes (pANCA). Antigenic substrate granulocytes. Counter-staining with Evans blue. Green fluorescence of the perinuclear cytoplasm of the granulocytes, red colouring of the lymphocytes (100 x) (134)...

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