Dissociation-enhanced lanthanide fluorescent

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. 

On this basis, the dissociation-enhanced lanthanide fluorescence immunoassay was developed. Mostly a two-site assay format is used whereby antibodies are immobilized at microtiter plates and after applying the sample antigen, europium-chelate-labeled antibodies bind to the sample. A commonly used chelating reagent is Nl-(p-isothiocyanato-benzyl)-diethylene triamine. After the addition of a S-diketone as a dissociation enhancer, at acidic pH, europium is released and fluorescence of Eu is measured. Other TRFIAs use stabilized lanthanide chelates or stable chelates. 

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]. 

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). 

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. 

Since the mid 1970 s many different structures and classes of lanthanide chelates have been synthesised (Rgure 5). For high sensitivity assays the dissociation enhanced system is currently the best approach, however for screening assays where the sensitivity or detection limitation is not an issue the fluorescent chelates can be used to simplify the assay. Some of these chelates can be even used to develop homogeneous and non-separation assays. This assay format is essential for the measurement of a reaction where the components have only a weak binding affinity thus cannot withstand a wash or separation step. 

Other techniques, such as C.D. spectral change, have been used to demonstrate the presence of octa coordination for lanthanide ion in a system containing Eu(FOD)3 and alcohols or ketones (28). However, the anionic tetrakis complexes e.g. Eu(acac)i, Eu(benzac)i, Eu(DBM)i, Eu(BTFA)4, tend to dissociate into the tris-complex and L in alcoholic solution. The degree of dissociation depends on the complex as well as the polarity of the medium. In alcohol-DMF medium the dissociation is enhanced compared to the alcoholic solutions (29). The end product of these dissociation reaction may well be an octacoordinated species. Fluorescence emission from the coordinated europium ion was also helpful in estabhshing the nature of the species in solution 29). 

In the DELFIA type of chelate the labelled protein as such is practically non-fluorescent. After the immunoreactions, however, europium or samarium is efficiently dissociated from the labelled compound within a few minutes by the low pH of the enhancement solution. Free lanthanide ions rapidly form a new and highly fluorescent chelate inside a protective micelle with the components of the... 

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