DELFIA system

Product and services of interest include genomics, proteomics, custom research and services, and instruments, accessories, consumables and software. PerkinElmer proprietary technologies include time-resolved fluorescence which is employed in the sensitive Wallac DELFIA system and Wallac LANCE homogeneous assay system, and fluorescence polarization, [FP]2 , a robust fluorescence-based technique for receptor binding assays that is both fast and easy-to-use. 

Fig. 1. Assay principle of DELFIA system using a non-luminescent I ll label.

Research is going on to improve the DELFIA system , because of drawbacks such as the time-consuming conversion of the non-fluorescent RE label into a luminescent complex, or the system vulnerability to contamination by RE due to the excess of the reagents ntfa and topo. An alternative is the use of a -diketone that can be covalently bonded to proteins such as 5-(4,4,4-trifluoro-l,3-dioxobutyl)-2-thiophenesulfonyl chloride (ctta) . Since the stability of the RE + complexes formed by this ligand is quite low, a large excess of RE + has to be used to shift the equilibrium to the rare-earth complex. More stable europium complexes can be obtained by the use of tetradentate fi-diketonates, such as 7a-7d, anchored on a functionalized o-terphenyl skeleton, or 8a-8c, anchored on a biperfluorobutadiene skeleton . ... 

Pharmacia Diagnostics. (1990). Pharmacia DELFIA system B-2-microglobulin kit insert. Uppsala, Sweden Pharmacia Diagnostics. 

Figure 9.3 Comparison data of fluorescence detection to elemental detection of an hCG immunoassay. Through collaboration with North York General Hospital, the DELFIA system was compared directly with ICP-MS detection. In this assay, DELFIA sandwich ELISA for patient hCG was quantitated as directed by the manufacturer using Eu-tagged anti-hCG antibodies. After analysis using the DELFIA fluorometer, the assay was diluted for elemental analysis by ICP-MS with 50 pL concentrated HCI containing an internal standard of 1 ppb Ho. Cal n stds calibration standards low med hi stds low, medium, and high standards.

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 idea of the use of luminescent lanthanide chelates in bioanalysis can be traced back to the 1980s, when Hemmila (1985) reported a time-resolved fluorometric system using an Eu labeling reagent, which was commercially produced by Wallac Oy company. The system was intended for immunoassay and included the spectrometer for time-resolved measurements. The basic principle of the immunoassay system (DELFIA ) is shown in fig. 1. 

P2. Pamham, A. J., and Tarbit, I. F., Delfia and Amerlite. Two sensitive nonisotopic immunoassay systems for assay of thyrotropin compared. Clin. Chem. (Winston-Salem, N.C.) 33, 1421-1424 (1987). 

Application of assay format is attractive because it allows a mass-screening of the samples for rapid and inexpensive monitoring of the important classes of herbicides. Three assay systems based on specific properties of D1 protein have been tested, such as ELISA-type assay, DELFIA fluoro-mettic assay and assay based on the liposomes incorporated D1 protein. 

Presented experimental results suggest that application of herbicide-binding protein in sensor technology has a high potential. Several detection systems were tested in combination with D1 protein electrochemical (amperometry and cyclic voltammetry), optical (surface plasmon resonance and ellipsometty) and assays (ELISA and D1 protein- containing liposomes and DELFIA fluori-metric assay). The main mechanisms of D1 action are either on the ability of herbicides to replace the plastoquinone molecule in D1 protein and in this way change the electrochemical and optical... 

The use of fluorescent labels has been very successful for inorganic complexes such as the europium chelates, which are used today in the Delfia commercial system. In contrast, fluorescence has rarely been employed in the area of organometallic tracers, and only the immunoassay developed by Lakowicz uses such a tracer [88]. This is a homogeneous competitive immunoassay, the tracer being the complex (Re-L) -HSA, 48 obtained as shown in Scheme 8.20 and the detection method fluorescence polarization (FP). This compound 48 displays highly polarized emission (with a maximum polarization near 0.4 and maximum anisotropy near 0.3) in the absence of rotational diffusion and a long average lifetime (2.7 ps) when bound to proteins in air-equilibrated aqueous solution. 

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