Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Means for analyzing liquid or solid sample
Reexamination Certificate
1999-07-15
2002-03-05
Snay, Jeffrey (Department: 1743)
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Means for analyzing liquid or solid sample
C250S458100
Reexamination Certificate
active
06352672
ABSTRACT:
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.
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 cryptates, 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:
the spectroscopic properties of the medium, and in particular its intrinsic 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;
the quenching of the emitted fluorescence by inhibitors present in the medium; and
the composition of the equipment, and especially the interference 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 method.
European patent application 355 849 describes a method and an automatic apparatus for checking the reliability of the fluorescent measurement of a sample and for correcting this measurement on the basis of an internal reference. To do this, measurement of the test sample has to be preceded by a measurement with 2 reference samples, one being a “blank” containing only the measuring medium, and the other containing the measuring medium and the fluorescent marker.
European patent application 91 126 relates to a fluorimeter which makes it possible to measure, in parallel with the fluorescence of the sample, the transmission of the sample and the fluctuations in excitation energy, in order to correct the measured fluorescence. This system requires a particular measuring cell which allows the exciting beam to pass through, since the transmission measurement has to be made in line with the exciting beam.
Other systems employ a system for splitting the exciting beam, as described for example in European patent applications 174 722 and 241 268.
The invention therefore relates to a method of measuring the luminescence emitted in a luminescent assay, which comprises employing at least one luminescent tracer compound and a luminescent compound used as an internal reference, which, when exposed to the same excitation wavelength, are capable of emitting at different wavelengths, &lgr;
2
and &lgr;
1
respectively, either by direct luminescence or by the induction of a luminescent emission, the luminescence of the reference compound reflecting the optical quality of the medium, and correcting the measurement of the luminescence emitted by the tracer compound at wavelength &lgr;
2
on the basis of the measurement of the luminescence emitted by the reference compound at wavelength &lgr;
1
.
“Reference compound” is understood as meaning either a luminescent molecule emitting a direct luminescence, or a luminescent molecule capable of inducing a luminescent emission, said emission of a direct or indirect luminescence not being perturbed by the reagent system of the assay.
The luminescent compounds which can be used in the method of measurement according to the invention can emit directly either at their emission wavelength or at another wavelength, as for example in the case of a spectral shift associated with the reagent system of the assay.
If appropriate, the luminescent compounds which can be used in the method of measurement according to the invention can emit indirectly by inducing a luminescent emission, especially as in the case of homogeneous energy transfer methods.
Advantageously, the luminescent emissions at wavelengths &lgr;
2
and &lgr;
1
are detected simultaneously.
The method according to the invention, which uses a single exciting beam, permits easy and reliable measurement of the luminescence emitted in a luminescent assay without the need for complex equipment, by eliminating the perturbations due to the spectroscopic properties of the assay medium.
Advantageously, the emission wavelengths of the luminescent tracer compound and luminescent reference compound, &lgr;
2
and &lgr;
1
, will be different but preferably similar (the difference being less than or equal to 100 nm, for example) so that the perturbation of the luminescent emission which is due to the absorption of the medium is produced in the same manner as regards the emission of the tracer compound and that of the reference compound.
It is pointed out that, advantageously, the method according to the present invention does not require the assay sample to be placed in a special measuring cell.
The luminescent emission of the reference compound at wavelength &lgr;
1
enables the measurement made at wavelength &lgr;
2
to be corrected. The correction can be effected for example by dividing the latter measurement by the measurement m
Dumont Christophe
Jolu Etienne Jean-Pierre
Mabile Michel
Mathis Gerard
Pouyat Dominique
CIS bio international
Snay Jeffrey
LandOfFree
Apparatus for measuring the luminescence emitted in a... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Apparatus for measuring the luminescence emitted in a..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Apparatus for measuring the luminescence emitted in a... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2855343