Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1993-07-28
1996-06-18
Spiegel, Carol A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 772, 435 791, 435 792, 435 8, 435 28, 435962, 435968, 435973, 436518, 436527, 436528, 436530, 436531, 436537, 436546, 436 56, 436172, 436825, G01N 3353
Patent
active
055276849
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a method of measuring the luminescence emitted in a luminescent assay, which makes it possible to correct certain perturbations due to the measuring medium.
BACKGROUND OF THE INVENTION
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 become increasingly important.
In fact, they have a number of advantages, including the sensitivity and rapidity of the measurement, the stability and safety of the reagents labeled with fluorescent compounds, and the relatively low cost.
It is known that detection methods which use fluorescence are intrinsically very sensitive and could permit lower detection limits than those achieved by immunoassays which use radiolabeled reagents, in particular if modulatable laser light sources are used (I. Wieder, Immunofluorescence and related staining techniques, 1978, Elsevier).
Numerous fluorescent molecules usable as tracers in assays of this type have previously been described; among these, rare earth complexes, which possess valuable properties, may be mentioned in particular.
"Tracer" is understood as meaning either a luminescent molecule emitting a direct luminescence, or a luminescent molecule capable of inducing a luminescent emission, it being possible for said molecule to be coupled with one of the reagents of the assay, and the emission of a direct or induced luminescence enabling the target analyte to be detected and/or determined.
The use of particular complexes, rare earth cryptares, is described for example in European patent applications 0 321 353, 0 180 492 and 0 232 348 or international patent application WO 90/04791.
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 is nevertheless limited by different interference parameters, among which the following may be mentioned: fluorescence, which is due especially to the interference emissions of the molecules present in the measuring medium and capable of being excited and of emitting at wavelengths close to those of the fluorescent tracer and/or with strong intensities; its absorption, which results in a loss of exciting light; and its light diffusion properties when the measuring medium is not clear; medium; and reflections caused by the equipment.
Together these interferences considerably affect the sensitivity and reproducibility of the measurement.
Some of these problems have already been solved by a variety of techniques.
In particular, the time-resolved methods of measuring fluorescence enable the problem of interference emissions (background) 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 and cryptates.
Nevertheless, the problem of the limitations due to the spectroscopic properties of the medium, and in particular to its absorption, has not been solved satisfactorily.
In fact, none of the proposed techniques for avoiding the filtering effect of the medium makes it possible to carry out the measurement easily and inexpensively and at the same time to obtain a high sensitivity and a very good reproducibility in the measurement.
In particular, the solution which consists in greatly diluting the sample detracts from the sensitivity of the detection.
Furthermore, the use of a double exciting beam system involves the use of expensive equipment and special measuring cuvettes which are difficult to standardize. Moreover, systematic measurement of the absorption of the medium prior to measurement of the fluorescence of the sample complicates the assay me
REFERENCES:
patent: 4100416 (1978-07-01), Hirschfeld
patent: 4341957 (1982-07-01), Wieder
patent: 4542104 (1985-09-01), Stryer et al.
patent: 4868103 (1989-09-01), Stavrianopoulos et al.
patent: 4876190 (1989-10-01), Recktenwald
W. P. Collins (ed), Alternative Immunoassays (John Wiley & Sons Ltd 1985) pp. 103-217.
Dumont Christophe
Jolu Etienne J.-P.
Mabile Michel
Mathis Gerard
Pouyat Dominique
Cis Bio International
Spiegel Carol A.
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