Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
2000-07-18
2002-08-27
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C436S172000, C436S169000, C436S501000, C436S536000, C436S805000, C435S007100, C435S007720, C435S007900, C435S007920, C435S007940, C435S287100, C435S287300, C435S288500, C435S288700, C435S808000, C356S073100, C356S319000, C356S320000, C356S340000, C422S050000, C422S052000, C422S051000, C422S051000, C422S068100, C422S082080, C422S082110, C422S082050
Reexamination Certificate
active
06440748
ABSTRACT:
The subject application claims priority of Intentional Application PCT/DE98/03154 filed on Oct. 27, 1998 and German foreign application 197 47 572.8 filed on Oct. 28, 1997.
BACKGROUND OF THE INVENTION
The invention concerns a device and a method for carrying out fluorescence immunoassays, wherein from at least one light source light is directed onto a surface at one end of a light waveguide and with the light coupled into the light waveguide by evanescent field excitation at the surface of the light waveguide fluorescence of at least one labelling substance bound to a chemical or biochemical partner of a general receptor-ligand system is excited and the fluorescent light is partially coupled into the light waveguide and decoupled from the light waveguide from. the surface into which the exciting light was coupled and directed via an optical system onto an optical detector with which the intensity of the fluorescent light is measured.
With the invention the most varied biochemical assays can be carried out on general receptor-ligand systems such as erg. antibody-antigen. With the assays, chemical or biochemical substances are analysed quantitatively in liquid samples.
Thus antibodies can be labelled with a given labelling substance (fluorogens), wherein the respective labelling substance can be optically excited at a given exciting wavelength of the light, and the fluorescent light which is obtained by excitation and which in turn occurs with another wavelength is detected with a suitable optical detector and the intensity of the fluorescent light is used to determine the respective proportion of the chemical or biochemical substance from the sample liquid. For fluorescence excitation, the evanescent field which forms at an interface is used. Here, the known physical relationships of the evanescent field and at the same time in particular the necessary total reflection of the exciting light must be taken into consideration.
Thus from WO 97/10 506 is known an optical device for carrying out fluorescence immunoassays in which is used a light waveguide into which light from a light source is coupled and fluorescence of a sample is produced by evanescent field excitation. Here, the surface of the light waveguide before carrying out the respective assay, that is, introducing the sample liquid into a receptacle in which the light waveguide is also contained, is coated accordingly with a chemical or biochemical component.
For coupling the light into the light waveguide, a very elaborate and complicated optical system which consists of a plurality of individual optical components is necessary. Thus the light from the light source used is directed via a lens system onto a semi-transparent mirror and a portion of the light is coupled as a reference signal onto an optical detector and the other portion of the light is coupled via an additional lens into the fibre. In this case the end of the light waveguide opposite the coupling and decoupling surface is metallised, so that most of the exciting and fluorescent light is decoupled from the light waveguide again. As a result, the ratio of exciting to fluorescent light for evaluation with the photodetector is made worse and consequently the accuracy of measurement is undesirably impaired.
Another drawback of this known device lies in that the light is to pass through the lens arranged in front of the coupling surface of the light waveguide, so that light incidence at a precise main angle within a narrow angular range into the light waveguide which is advantageously required for evanescent field excitation can be achieved only with great difficulty, if at all.
The fluorescence immunoassays are then carried out according to this known solution in such a way that the prepared, coated light waveguide contained in the receptacle is brought into contact with the sample liquid by the fact that the sample liquid enters through perforations in the lid of the receptacle and binding to the complementary partner of the receptor-ligand system which is immobilised on the surface of the light waveguide can be effected. After binding, fluorescence is excited by irradiation of the light from the light source used and its intensity is measured with the optical detector.
Since for the binding process the respective transport of substances to the light waveguide surface is important and in this case convection and diffusion must be taken into consideration, in the solution known from WO 97/10 506 measurement errors occur because the sample volume respectively contained in the receptacle is constant and entry of the sample liquid through the perforations takes place very rapidly and hence binding is effected in a stationary liquid. In this case binding is influenced mainly by diffusion, which apart from other drawbacks also leads to prolonging the measuring time.
With this type of measurement without time resolution, background correction of the measurement signal is necessary, which was not taken into consideration in WO 97/10506.
Another essential drawback which is associated with this known solution lies in that only the exciting light is used as the reference signal in order to increase the accuracy of the measurement results. To improve the measurement accuracy and informativeness of the assay results as well as increased reproducibility, however, it is necessary to perform reference measurements more suitable for the immunoassays.
Furthermore in U.S. Pat. Nos. 4,909,990 and 5,492,674 are described solutions with which fluorescence immunoassays can be carried out. Here there is used a light waveguide transparent to exciting and fluorescent light, for example an optical fibre, which is at least partially guided in a capillary sleeve or held therein. Before carrying out the fluorescence immunoassay, a sample liquid passes into the gap between capillary sleeve and light waveguide, which has been suitably prepared biochemically beforehand and is held there. Transport of substances to the surface of the light waveguide is here effected almost entirely by diffusion, and after the capillary sleeve is suitably filled and the sample liquid has been brought into contact with the prepared surface of the light waveguide in the region of the capillary sleeve measurement of the fluorescent light is effected, which has been excited in a conventional manner by using the evanescent field of exciting light radiated into the light waveguide. A relatively small sample volume can be used owing to the small usable volumes of the capillary.
SUMMARY OF THE INVENTION
It is therefore the object of the invention to provide a possible way of carrying out fluorescence immunoassays with high accuracy of measurement at low cost within a short time.
The device according to the invention is based on the known state of the art already described and also uses at least one light source with which light, for fluorescence excitation by means of the evanescent field, is coupled into a light waveguide, and the intensity of the fluorescent light of a labelling substance which is bound to a partner of a general receptor-ligand system is determined with an optical detector. Here the fluorescent light and part of the exciting light are decoupled from the same surface into which the exciting light was coupled. The light waveguide is here held in a measuring chamber which is formed in a piston of a piston and cylinder unit. The measuring chamber of the piston here has an inlet, into the interior of the cylinder, which passes through the piston. Such a piston and cylinder unit can here be at least approximately constructed as a conventional syringe, wherein only the piston must be modified correspondingly with the measuring chamber.
A further improvement can be achieved by the fact that in the piston is formed an additional sample collecting chamber which communicates with the measuring chamber. This results in a unit in which incubation and measurement can be performed and after carrying out the respective assay the sample is reliably held and the corresponding piston and cylinder unit can be transport
Grawe Frank
Katerkamp Andreas
Key Göran
Kunz Ulrich
Institut für Chemo- und Biosensorik Münster E.V.
Nguyen Bao-Thuy L.
Padmanabhan Kartic
Young & Basile P.C.
LandOfFree
Process and device for carrying out fluorescence immunoassays does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Process and device for carrying out fluorescence immunoassays, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Process and device for carrying out fluorescence immunoassays will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2877316