Dissociation, enhanced

The DELFIA assay, the first effective lanthanide-based immunoassay, was developed and commercialized by the early 1980s.108-112 DELFIA (Dissociation Enhanced Lanthanide Fluoro-ImmunoAssay) is a heterogeneous assay which uses a lanthanide complex based on aminocarboxylate ligands such as EDTA, EGTA, or DTPA, linked to the antibody by reaction of appended isothiocyanate groups (e.g., complex (45)) with nucleophilic residues, particularly amines, on the protein surface (Figure 11). 

A number of dissociative enhancement immunoassays have been developed using Eu3+ or Sm3+ complexed to a proprietary chelator, A -f/j-isothiocyanatobenzyl]-diethylenetriamine-A1, A2, A3, A3-tetraacetate (Figure 14.7)/1 70 This chelator is cou-... 

Fig. 25. (A) DELFIA (Dissociation Enhanced Lanthanide Fluoro-ImmunoAssay) system. This heterogeneous immunoassay system uses a primary antibody bound to a solid support, to which a variable amount of unlabeled antigen is bound. The secondary antibody is labeled with a non-phospho-rescent lanthanide chelate, which becomes phosphorescent after dissociation from the antibody, due to the addition of an enhancement solution [which typically contains a mixture of sensitizer (typically a (1-diketonate) and micelle inducing surfactant (5). (B) Heterogeneous fluoroimmunoassay using a secondary antibody directly labeled with a phosphorescent lanthanide chelate.

The demonstration that MTs from a wide variety of fish species are recognized by an antiserum raised against one piscine MT has enabled the development of immunotechniques based on ELISA143 and radioimmunoassay (RIA) procedures144 for the quantification of these compounds. A competitive solid-phase assay based on dissociation-enhanced lanthanide fluoroimmuno-detection (DELFI A) of anti-MT monoclonal antibody bound to a solid phase has been reported.145 An electrochemical determination of MTs by square wave cathodic stripping voltammetry has also been developed and optimized.146... 

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. 

Schoket B, Doty WA, Vincze I, et al. 1993. Increased sensitivity for determination of polycyclic aromatic hydrocarbon-DNA adducts in human DNA samples by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). Cancer Epidemiology, Biomarkers Prevention 2 349- 353. 

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]. 

Crooks, S.R., Ross, P., Thompson, C.S., Haggan, S.A., and Elliott,C.T. (2000) Detection of unwanted residues of ivermectin in bovine milk by dissociation enhanced lanthanide fluoroimmunoassay. Luminescence, 15, 371 376. 

Fluoroimmunoassay makes use of the above behaviour. One of the common commercial methods is dissociation-enhanced fluoroimmunoassay (DELFIA). In this, a nonfluorescent Eu(III) EDTA-like complex is attached by a simple chemical reaction to an antibody or antigen, in a process called labelling. An immunoreaction is next initiated to bind the target, and then a (3-diketone and trioctylphosphine oxide (TOPO) mixture are added to the immunocomplex formed, at pH 3, to promote release of the Eu(III) from the antibody and its complexation as the strongly fluorescent complex [Eu((3-diketonate)3(TOPO)2], which is then measured by time-resolved fluorescence methods. The signal size relates to the amount of europium complexed, which in turns relates directly to the amount of the specifically formed target immunocomplex. This process is represented schematically in Figure 9.5. 

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