Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Patent
1994-09-16
1997-05-06
Scheiner, Toni R.
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
435 71, 435 72, 435962, 436 81, 436800, G01N 33543, G01N 3320, G01N 3353
Patent
active
056270747
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method of reducing interference in a fluorescent assay.
At the present time, immunoassays are widely used for the qualitative and quantitative analysis of compounds in biological fluids.
Among the techniques in existence, fluorimetric assays have gained importance.
In fact, they have a number of advantages, including the sensitivity and speed of the measurement, the stability and safety of the reagents labeled with fluorescent compounds, and the relatively low cost.
It is known that methods of detection using fluorescence are intrinsically very sensitive and could permit lower detection limits than those attained by immunoassays using radiolabeled reagents, in particular by the use of modulatable laser light sources (I. Wieder, Immunofluorescence and related staining techniques, 1978, Elsevier).
Numerous fluorescent molecules usable as tracers in this type of assay have previously been described, among which rare earth complexes possess valuable properties.
The use of particular complexes, rare earth cryptates, is described in the following patent applications: EP 0 180 492, PCT/FR 86 00269, EP 0 321 353 or PCT/FR 89 00562.
These rare earth cryptates have the advantage of being very stable in a saline protein medium, this property being particularly important in the case of homogeneous immunoassays.
The sensitivity of the measurement can nevertheless be greatly affected by different types of interference resulting from the presence of various molecules in the medium in which the measurement is made.
This problem is particularly acute in the case of assays in a serum medium in which numerous molecules are capable of interfering.
For example, the measured signal can be subject to interference by emission from molecules capable of being excited and of emitting at the same wavelengths as the molecule used as the tracer.
The time-resolved methods of measuring fluorescence enable this disadvantage to be partially overcome. The principle of these methods consists in measuring the fluorescence emitted by a tracer molecule having a relatively long emission lifetime, the measurement being delayed in time beyond the emission lifetime of the other molecules present.
It is necessary in this case to use fluorescent tracer molecules with a relatively long lifetime, such as rare earth chelates.
The sensitivity of the measurement can also be affected by interference from molecules in the medium which are capable of perturbing the variation in fluorescence resulting from binding between the analyte to be detected and the labeled biospecific reagent. Patent application EP 0 324 323 describes the use of a modulator which stabilizes the rare earth chelate bound to the biospecific reagent, in such a way that the measured fluorescence is actually a function of the concentration of the analyte. The effect of this modulator is to prevent perturbation of the fluorescence of the rare earth chelate by the other molecules present in the medium. The measured variation in fluorescence is then a function only of the antigen-antibody reaction. The proposed modulators are macro-molecules, such as proteins and detergents, and have to be used in excess in the range from 0.1 to 10 g/l.
Nevertheless, the problem of the interference due to the molecules present in the measuring medium is not completely solved by any of these methods. In fact, an important source limiting the sensitivity of the fluorescent measurement is the existence of quenching processes due to molecules present in the medium which are capable of inhibiting the fluorescence of the fluorescent molecule used as the marker in the assay. In the case of rare earth complexes, these processes can result from proximity electron transfer mechanisms, where the inhibitor molecule occupies the coordination sites remaining free within the complex. In particular, there may be mentioned the redox reactions taking place between the fluorescent molecule and molecules present in the medium. These mechanisms are capable of varying the emitted fluorescence sign
REFERENCES:
patent: 4542104 (1985-09-01), Stryer et al.
Journal of the American Chemical Society, vol. 102, No. 7, 26 Mar. 1980, American Chemical Society, E.L. Yee et al.; "Electrochemical Society of Europium and Ytterbium Cryptate Formation in Aqueous Solution. Effects of Varying the Metal Oxidation State upon Crypatate Thermodynamics and Kinetics", pp. 2278-2285, see abstract; p. 2284, column 2.
Dumont Christophe
Jolu Etienne J.
Mathis Gerard
Cis Bio International
Eyler Yvonne
Scheiner Toni R.
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