Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
2000-01-21
2004-11-09
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C436S514000, C436S523000, C436S524000, C436S528000, C436S531000, C436S513000, C435S962000, C435S973000, C435S974000, C435S805000, C435S810000, C435S007500, C435S007900, C435S007920, C435S007930, C435S007940, C435S007950, C435S005000, C422S051000
Reexamination Certificate
active
06815217
ABSTRACT:
The invention concerns a solid phase with at least one test area to detect an analyte which additionally comprises at least one control area to detect interferences. Furthermore the invention concerns a method for the detection of one or several analytes using a solid phase according to the invention in which interfering reactions can be detected and corrected if necessary.
When analytes are detected by binding assays interferences in the detection reaction occur with some samples which lead to false test results. This phenomenon is usually referred to as a matrix effect of the sample. The presence of an interference cannot in general be indicated in conventional test formats. It is attempted to prevent matrix effects of the various samples or to reduce them as far as possible by elaborate optimization of the solid phase, test buffers and detection reagent. However, such a reduction of interference in detection methods is complicated and expensive. Moreover one cannot completely rule out the possibility that an adequate reduction of interference does not occur for certain samples despite test optimization since it is unfortunately not possible to completely suppress matrix effects. In addition new interferences which were unknown during the development of the detection method can occur which also lead to false results. Consequently there is a problem that in the known detection methods false test results can be obtained without the user being aware of this. This circumstance is particularly tragic in the case of qualitative tests for the detection of an infectious disease. Thus for example a false positive sample resulting from matrix problems has considerable consequences for a HIV test.
U.S. Pat. No. 4,558,013 describes a test strip which contains a non-defined uncoated negative control region in addition to test regions coated with specific test reagents. The value measured in the test region is corrected by subtracting the unspecific binding in the control region. However, such a procedure can only partially correct for interferences since the unspecific binding of interfering components to the test region usually differs considerably from the binding of interfering components to the uncoated control region.
U.S. Pat. No. 5,356,785 describes a solid phase with several test areas which each contain different amounts of a solid phase receptor for the detection of an analyte. The solid phase additionally contains a reference area which generates a detectable signal of known intensity with the test reagent. Control areas to determine unspecific interactions between the sample and the solid phase are not disclosed.
U.S. Pat. No. 4,916,056 (Brown III et al.) describes a solid phase for the qualitative or quantitative determination of an analyte, in particular of an antigen, antibody or DNA segment in a sample. The solid phase contains, in addition to a test area, a reference area which yields a positive signal in the test and a non-defined negative control area which contains the positive reference area and the test area. A disadvantage of this device is that the surface of the uncoated control region differs too greatly from the test area to achieve an effective correction for interferences.
Hence an object of the invention was to provide devices and methods for the detection of analytes which enable a direct indication of the presence and optionally the type of interferences such that these interferences can be taken into account when evaluating the test results.
This object is achieved according to the invention by a solid phase having at least one defined test area for the detection of an analyte in a sample which is characterized in that the solid phase additionally comprises at least one defined control area for the detection of interferences. In this connection the term “defined test areas” on a solid phase is understood to mean that the test areas comprise defined regions of the solid phase which are preferably spatially separated from other test areas by inert regions. The defined test areas preferably have a diameter of 10 &mgr;m to 1 cm and particularly preferably 10 &mgr;m to 5 mm. Miniaturized test areas with a diameter of 10 &mgr;m to 2 mm are most preferred. Solid phases with several test areas are preferred which are also referred to as array systems. Such array systems are for example described in Ekins and Chu (Clin. Chem. 37 (1995), 1955-1967) and in U.S. Pat. Nos. 5,432,099, 5,516,635 and 5,126,276. An advantage of array systems is that several analyte and control determinations can be carried out simultaneously on one sample. The use of control areas to detect unspecific binding and/or interfering samples can considerably improve the reliability of the results especially with miniaturized array test systems.
In this connection the detection of interferences and unspecific binding in qualitative tests and in particular in those which have stringent requirements for specificity such as tests for infections (e.g. HIV) are of particular interest. The indication of an interference and correction of the test result enables false-positive results to be considerably reduced thus leading to an enormous improvement of specificity.
The solid phase according to the invention is any conventional support for detection methods, preferably a non-porous support e.g. a support with a plastic, glass, metal or metal oxide surface. Porous supports such as test strips are also suitable. Spatially discrete regions (test areas) are located on this support. Immobilized solid phase receptors are applied to these test areas. The solid phase receptors are immobilized by known methods e.g. by direct adsorptive binding, by covalent coupling or by coupling via high affinity binding pairs e.g. streptavidin/biotin, antigen/antibody or sugar/lectin. The presence or/and the amount of the analyte in a sample can be determined by specific binding of components from the detection medium e.g. of the analyte to be determined or of an analyte analogue to the solid phase receptor.
The detection of the analyte and the presence of interfering reactions is achieved in the method according to the invention in a known manner by using suitable marker groups e.g. fluorescent marker groups. Alternatively with suitable solid phases it is possible to also detect the interaction of components of the detection medium with the test and control areas by determining the layer thickness of the respective area e.g. by plasmon resonance spectroscopy.
With array systems in which several analytes from a sample are detected simultaneously, it is preferable to use a “universal” marker group which enables a simultaneous detection of several different analytes to different test areas. An example of such universal marker groups are marker groups which carry a receptor that can specifically interact (e.g. by means of a high-affinity binding pair such as antibody/antigen or streptavidin/biotin etc.) with a complementary receptor on a test reagent e.g. a soluble receptor for an analyte to be determined or for an analyte analogue.
The application of such a universal marker group is exemplified in FIG.
1
. In this case a fluorescent latex bead which is coupled with an anti-digoxigenin antibody (<Dig> label) is used for three different test formats on a single solid phase i.e. to determine HIV antibodies, HBs antigen and anti-HBc antibodies. In the case of the anti-HIV antibody determination an immobilized HIV antigen and a digoxigenylated soluble HIV antigen are used which form an immobilized immune complex with the anti-HIV antibodies to be detected. The marker group can bind to the digoxigenin groups present on this immune complex. For the determination of HBs antigen, an immobilized antibody and a digoxigenylated soluble antibody that can interact with the marker group are used in a corresponding manner. A competitive test format is used for the anti-HBc determination in which anti-HBc antibodies present in the sample compete with a digoxigenylated anti-HBc antibody for immobilized HBc antigen. The quantity of marker
Berger Johann
Finckh Peter
Hornauer Hans
Karl Johann
Krause Friedemann
Brinks Hofer Gilson & Lione
Roche Diagnostics GmbH
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