Chemistry: analytical and immunological testing – Optical result – With fluorescence or luminescence
Reexamination Certificate
1995-11-03
2001-03-06
Caputa, Anthony C. (Department: 1645)
Chemistry: analytical and immunological testing
Optical result
With fluorescence or luminescence
C422S082050, C422S052000, C435S007100, C435S007900, C436S537000, C436S800000, C436S805000, C436S807000
Reexamination Certificate
active
06197594
ABSTRACT:
DESCRIPTION
The present invention concerns a method for the detection of an analyte in a sample liquid by a luminescence measurement according to the principle of a ligand receptor assay, e.g. an immunoassay or a hybridization assay or a combination thereof, wherein a sample liquid is incubated with at least one receptor which carries a luminescent label and the presence or/and the amount of the analyte to be detected is determined in the sample liquid by luminescence measurement.
The use of luminescent direct labels such as e.g. isoluminol or acridinium compounds or enzyme-amplified luminescent labels such as e.g. horseradish peroxidase/luminol or alkaline phosphatase/stabilized dioxetans as a detection system in immunological test procedures has the advantage over other test systems such as radioactive marker groups of a higher sensitivity (see among others McCapra F. and Beheshti I. in Knox van Dyke (publ.): Bioluminescence and Chemiluminescence; Instruments and Applications, Vol. 1, 9-42 1985; Barnard G. J. R. et al. in Knox van Dyke (publ.): Bioluminescence and Chemiluminescence; Instruments and Applications, Vol. 1, 151-183, 1985; Weeks I. et al. in de Luca M. A. and McElroy W. D. (publ.): Methods in Enzymology, Vol. 133, 366-387, 1986; McCapra F., et al., Journal of Bioluminescence and Chemiluminescence 4, 51-58, 1989; Bronstein I. and McGrath P., Nature 338, 599-600, 1989; Thorpe G. H. and Kricka L. J., Journal of Bioluminescence and Chemiluminescence 3, 97-100, 1989).
However, a disadvantage of luminescent labels is their low signal strength. This is due to the fact that a signal multiplication is not possible in the luminescence measurement i.e. each direct label or each luminescent enzyme substrate molecule is consumed in a single light-generating reaction. Therefore for a long time attempts have been made to eliminate this disadvantage.
Thus for example it is known that the chemiluminescence from the peroxidase (POD)-catalyzed luminol oxidation can be amplified by benzothiazoles (Whitehead T. P. et al., Nature 305, 158-159, 1983), p-substituted phenols such as e.g. p-iodophenols (Thorpe G. H. et al., Clinical Chemistry 31, 1335-1341, 1985; Coyle P. M. et al., Annals of Clinical Biochemistry 23, 42-46, 1986), fluorescein (EP 0 228 046 B1), o- or p-thiazolyl phenols or o- or p-thienyl phenols (EP 0 455 471 A2), hydroxyfluorenones (WO 90/13665). These amplification molecules improve the co-ordination of the light-generating luminol oxidation.
In addition it is known that the alkaline phosphatase-catalyzed chemiluminescence of stabilized triggerable dioxetans can be amplified by polyvinylbenzyl(benzyldimethylammonium)chloride (U.S. Pat. No. 5,145,772 by Bronstein I. et al.; Tropix News Letter on Chemiluminescent Substrates, 1993), polyvinylbenzyl-trialkyl-phosphonium salts (EP 0 561 033 A1 by Schaap A. P.). These hydrophobic polymeric additives displace water from the immediate environment of the products which are formed in an excited state, stabilize these in a hydrophobic medium and thus increase the photon yield.
Amplification of the chemiluminescence of acridinium compounds can be achieved by encapsulation of additionally hydrophobized acridinium ester molecules in liposomes composed of dipalmitoyl phospholipids and cholesterol in order to achieve an accumulation (Law S-J. et al., Journal of Bioluminescence and Chemiluminescence 4, 88-98) or by addition of quaternary phosphonium salts (EP 0 534 380 A1) or micelles of cetyltrimethylammonium bromide to achieve a signal amplification (McCapra F., Accounts of Chemical Research 9, 201-208, 1976). With regard to the mechanism of action, a preference for the light reaction over the dark reaction has been discussed in this case i.e. an increase in the yield of chemical product in the excited state and thus capable of luminescence.
A disadvantage of the above-mentioned amplification measures is that each of these is specific for one particular luminescence system. Therefore the object of the present invention was to provide a method that can be used independently of the respective luminescence system used and if desired in combination with other amplification methods, which leads to an improvement of signal detection in luminescent reactions.
This object is achieved according to the invention by a method for the detection of an analyte in a sample liquid by measurement of luminescence according to the principle of a ligand-receptor assay wherein a sample liquid is incubated with at least one receptor which carries a luminescent label and the presence or/and the amount of the analyte to be detected in the sample liquid is determined by measurement of luminescence which is characterized in that the luminescence is measured in a measuring medium containing dispersed components which cause a randomization of the light produced in the luminescent reaction and if desired results in the formation of a preferred direction in the light scattering.
This randomization or reflection of the light generated in the luminescent reaction caused by the presence of dispersed components surprisingly leads to a considerable increase in the sensitivity and the precision of the luminescence measurement. In addition it is possible by for example immobilizing the luminescent-labelled receptor on a solid phase to achieve a preferred direction for the light scattering due to the layer thickness relationships above and below the light source. These effects are independent of the respective luminescence system used and can therefore be used for a wide range of applications. A further advantage of the method according to the invention is that it can be carried out simply and cost-effectively.
The measurement medium in which the luminescence measurement is carried out in the process according to the invention usually comprises a liquid phase with gaseous, liquid or/and solid components dispersed therein. The dispersion preferably has an adequate stability so that no significant segregation of the dispersed components occurs during the measurement process e.g. by phase separation or sedimentation. When the method according to the invention is carried out in an automatic measuring instrument it is preferable that the dispersion is stable for at least one day, particularly preferably for at least one week and most preferably for at least 3 weeks. Examples of such dispersions are given in the following.
In a preferred embodiment the measuring medium comprises a suspension or a colloidal solution (sol) of solid particles which preferably have a mean diameter of 10 nm to 3 &mgr;m. The solid particles particularly preferably have a mean diameter of 100 nm to 800 nm. Most preferably the solid particles have a mean diameter of 150 nm to 600 nm. The amount of solid particles during the measurement is preferably 0.01-2.5% (mass/vol.) relative to the measuring medium and particularly preferably 0.05-1.5% (mass/vol.).
For reasons of dispersion stability it is preferred that the specific weight of the solid dispersed particles does not differ significantly from the specific weight of the measuring medium and preferably by no more than 25% and particularly preferably by no more than 10%. Examples of this are for instance dispersions of organic polymer particles, e.g. acrylic polymers, styrene polymers e.g. sulfate latices, amidine latices, zwitterion latices which may be functionalized. Further examples are ethylene, propylene, butadiene, vinyl and urethane polymers and copolymers of the above-mentioned polymers. Specific examples are shown in the following Table:
specific density
Polymer
(g/cm
3
)
polymethyl methacrylate
1.19
polystyrene
1.05
polyvinyltoluene
1.027
styrene/butadiene 95/5 (% w)
1.05
styrene/butadiene 60/40 (% w)
0.99
vinyltoluene/t.-butylstyrene
1.00
63/37 (% w)
When selecting the dispersed particles one should also take care that reactive groups that may be present on the particles are compatible with the respective luminescence system used.
In another preferred embodiment of the present invention the measuring medium comprises an emulsion or colloi
Hornauer Hans
Weindel Kurt
Caputa Anthony C.
Gucker Stephen
Nikaido Marmelstein Murray & Oram, LLP.
Roche Diagnostics GmbH
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