Dissociative enhancement principle

With the DELFIA chelates, the sensitivity is, furthermore, increased because of the dissociation-enhancement principle the lanthanide ion in the chelate is dissociated and a new highly fluorescent chelate is formed inside a protective micelle (Figure 2). 

Pro-inflammatory cytokines are important mediators of inflammation and tissue destruction. This section describes two cell-based assays that were used to screen for inhibitors of cytokine production and some of the compounds discovered using these screens. The two screens were important elements of a collaboration between Xenova Ltd and the Suntory Institute of Biomedical Research to find microbial metabolites with potential utility for the treatment of rheumatoid arthritis. Both screens were cell stimulatory assays with similar formats, the principle of which is illustrated in Figure 3. Treatment of cells with a particular stimulus activates a signal transduction pathway, one of the end results of which is production of a cytokine, which is secreted into the assay medium. After a separation step, the cytokine of interest is measured quantitatively in the supernatant by dissociation enhanced lanthanide fluorescence immunoassay (DELFIA) using a europium-labeled tertiary antibody. At the same time, cytotoxic properties of test substances are determined by assessing their effect on proliferation of the separated cells. 

The dissociation-enhanced lanthanide fhtoroimmunoassay (DELFIA) technique is based on the principle of TRF. This theoretical concept was reduced to practice in the early 1970s [1 3] and was subsequently commercialized by the scientific equipment manufacturer, LKB/Wallac, as time-resolved fluorometric immunoassay methodology in the early 1980s [4 6]. DELFIA represents the first ultrasensitive nonisotopic immunoassay. This technology was reviewed in detail by Soini and Lovgren [7]. 

The lanthanide is dissociated from the labelling chelate into a highly fluorescent beta-diketone chelate micelle. The principle of dissociation-enhancement, the basis for assays with both reagent stability and intensive, stable fluorescence. 

Fig. 1 Two different detection principles in a microtitration well (a) dissociation enhanced measurement of nonfluorescent europium(III) chelates and (c) surface read-out of bound nanoparticle-antibody bioconjugates from the bottom of the microtitration well. Explanation, (a) Europium(III) ions are released from the nonluminescent chelates attached to the labeled antibody (1) to enhancement solution establishing highly luminescent complexes (2). (b) The nanoparticle-antibody bioconjugates bound on an illuminated area (3) on the bottom of the well generate a signal (4), while the bioconjugates on the sides (5) are omitted in the surface read-out...

Different variations of time-resolved luminescence assays were patented in 1982 by Wieder [1], in 1983 by Soini and HemmUa [2], and in 1999 by Diamandis [3]. The Finnish company Wallac first commercialized the principle and introduced an assay reader for dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) in the beginning of the 1980s. The first (1982) DELFIA-based bioassay for diagnostic market was for Rubella antibodies, and it was the first sensitive nonradioisotope innnunoassay marking the beginning of a new era [4]. 

The rate constants in table 4 for Ru/AlaOs should be considered as initial rate constants since it was not possible to achieve a higher coverage of N— than 0.25. Furthennorc, it was not possible to detect TPA peaks for Ru/AlaOs within the experimental detection limit of about 20 ppm. Ru/MgO is a heterogeneous system with respect to the adsorption and desorption of Na due to the presence of promoted active sites which dominate under NH3 synthesis conditions. The rate constant of desorption given in table 4 for Ru/MgO refers to the unpromoted sites [19]. The Na TPD, Na TPA and lER results thus demonstrate the enhancing influence of the alkali promoter on the rate of N3 dissociation and recombination as expected based on the principle of microscopic reversibility. Adding alkali renders the Ru metal surfaces more uniform towards the interaction with Na. 

As for solvents, liquid ammonia or dimethylsulfoxide are most often used. There are some cases when tert-butanol is used as a solvent. In principle, ion-radical reactions need aprotic solvents of expressed polarity. This facilitates the formation of such polar forms as ion-radicals are. Meanwhile, the polarity of the solvent assists ion-pair dissociation. This enhances reactivity of organic ions and sometimes enhances it to an unnecessary degree. Certainly, a decrease in the permissible limit of the solvent s polarity widens the possibilities for ion-radical synthesis. Interphase catalysis is a useful method to circumvent the solvent restriction. Thus, 18-crown-6-ether assists anion-radical formation in the reaction between benzoquinone and potassium triethylgermyl in benzene (Bravo-Zhivotovskii et al. 1980). In the presence of tri(dodecyl)methylammonium chloride, fluorenylpi-nacoline forms the anion-radical on the action of calcium hydroxide octahydrate in benzene. The cation of the onium salts stabilizes the anion-radical (Cazianis and Screttas 1983). Surprisingly, the fluorenylpinacoline anion-radicals are stable even in the presence of water. 

In principle, one can induce and control unimolecular reactions directly in the electronic ground state via intense IR fields. Note that this resembles traditional thermal unimolecular reactions, in the sense that the dynamics is confined to the electronic ground state. High intensities are typically required in order to climb up the vibrational ladder and induce bond breaking (or isomerization). The dissociation probability is substantially enhanced when the frequency of the field is time dependent, i.e., the frequency must decrease as a function of time in order to accommodate the anharmonicity of the potential. Selective bond breaking in polyatomic molecules is, in addition, complicated by the fact that the dynamics in various bond-stretching coordinates is coupled due to anharmonic terms in the potential. 

Figure 7. Schematic energy level diagram showing the principle of the ionization method for detecting electron transfer in gas-phase adducts. Naphthalene cation (the hole donor) is formed by resonance-enhanced two-photon ionization of the neutral. A hole acceptor, whose ionization potential is lower than that of naphthalene, is not ionized, since its S level is not resonant with the UV photons used (vi). The vibrational levels of the ionic form of the acceptor are resonant with the naphthalene cation, and accept the hole easily. Detection is by photodissociation, using photons of different frequency (V2) that dissociate the naphthalene cation in a resonance-enhanced multiphoton absorption process. Charge transfer is detected by the diminution of the product ion signal in the presence of a suitable acceptor. Adapted from Ref. [32].

In one experiment (224), Zare and co-workers measured the j distributions of H2 and D2 desorbed from the Cu(110) and Cu(lll) surfaces. They found that although these distributions had a mean rotational energy somewhat less than kHTs (where kH is the Boltzmann constant and Ts is the surface temperature), they did not deviate significantly from a Boltzmann distribution. Since the measured rotational state distributions seemed to have enhance populations at low j, application of the principle of detailed balancing led Zare and co-workers to propose that rotation might slightly hinder dissociative chemisorption. 

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