Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-06-25
2001-12-18
Huff, Sheela (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S007200, C435S007210, C435S007230, C435S007920, C436S512000, C530S300000, C530S326000, C530S329000
Reexamination Certificate
active
06331402
ABSTRACT:
The present invention concerns an immunological process for the detection of an analyte in a sample, in particular of tumor markers, wherein substances containing a peptide sequence derived from the framework regions of the variable domain of the antibodies to be detected or the antibodies used for immune therapy or scintigraphy is added to the test preparation. The invention also concerns the use of such substances for the reduction of interference of immunoassays, a suppressive agent and a process for the reduction of interference of immunoassays by the substances mentioned.
In the field of diagnostics especially immunological detection processes have become very important during the last years. By these processes analytes can be detected in biological samples. These analytes are for example medicinal drugs, hormones, proteins, infectious agents, microorganisms and antibodies directed against these analytes. In particular in the diagnostics of cancer diseases the tumor antigens or tumor markers like e.g. CEA (carcinoembryonic antigen), PSA (prostate specific antigen) or CA 125 are detected immunologically depending on the disease.
All immunological detection reactions include a specific binding reaction between the substance to be detected (analyte) and at least one specific binding partner which specifically reacts with the analyte or which specifically binds to it. The analyte and the specific binding partner form a specific binding pair which generally is a complex between an antigen and an antibody or an antibody fragment. More than one analyte or more than one binding partner can react with each other in each reaction.
There are different possibilities how to detect these specific binding reactions. In general one of the binding partners of the specific binding reaction is labeled. Usual labelings are chromogens, fluorophores, substances capable of chemi- or electrochemiluminescence, radioisotopes, haptens, enzyme labels or substances which can form another specific binding pair such as biotin/streptavidin.
An essential problem of immunoassays is related to the possible unspecific binding reactions and undesired interactions between the specific binding partners of the immunoassay and the sample components. Such interactions generally lead to an increase in the background signal and to a stronger signal variance and consequently to a reduced sensitivity and specificity of the corresponding test. Depending on the kind of interference provoked by unspecific interactions false positive or false negative test results can occur.
This is the case when interference factors occur in human sera and particularly interact with immunoglobulin reagents of an immunoassay frequently used as binding partners and may thus have an effect on the immunoassay. Especially in immunoassays with two monoclonal antibodies considerable measuring errors with serious consequences for the further treatment of a patient (Kinders and Hass (1990) Eur. J. Cancer 26 (5), 647-648; Boscato and Stuart (1988) Clin. Chem. 34 (1), 27-33) may occur.
A large number of these interference factors can be classified as HAMA (human anti-mouse antibodies) which are antibodies present in the sample to be tested and directed against the specific antibodies used as reagents. Often the name HAMA-interference is also used for antibody interferences which were not provoked by contact with mouse immunoglobulin or which were not strictly specific for mouse immunoglobulin. By this HAMA interference unspecific cross-linking of the antibody bound to the solid phase with the labeled antibody to be detected can for example occur in a conventional sandwich assay despite the absence of the analyte. As a consequence, a false positive signal results.
This problem has become bigger since recently immunoscintigraphy has been used more and more frequently in the diagnostics and therapy of tumors. For this purpose radioactively labeled monoclonal antibodies are injected in the bloodstream of the patient. Then the labeled antibodies specifically bind to the relevant tumor tissue. By subsequent scanning of the radioactivity, e.g. with a scintillation camera the tumor can be localized exactly. Immunologically stimulating therapies—even with monoclonal antibodies—are also used frequently to stimulate the formation of tumor-specific antibodies for the control of the tumor in the patient's organism.
When using these processes very high and partly very specific HAMA titers occur, as mentioned above, with increasing frequency in the patient sera (see for example Kath. et al. (1996) Oncology 2, 287-296; Holz et al. (1996) Clin. Immunther. 5 (3), 214-222; White et al. (1997) Europ. J. of Cancer 33(5), 40; Baum et al. (1993) Hybridoma 12(5), 583-589; Donnerstag et al. (1995) Int. J. Oncology 6, 853-858; Livingston et al. (1995) Vaccine Research 4 (2), 87-94; Jeweid et al. (1996) Cancer 78 (1), 157-168). They can also provoke interferences in tests involving chimeric antibodies. An important example of antibodies approved in the tumor therapy of colon carcinoma in Germany is the antibody 17-1A commercially named Panorex (Kath et al. (1996), supra; Holz et al. (1996), supra). In addition the list of the antibodies used in clinical studies includes a large number of further therapeutic reagents containing antibodies which very probably will be approved. Antibodies against CEA, CA125 and CA72.4 are already clinically applied in the immunological scintigraphy and it is expected that they will be used with increasing frequency too.
For state of the art elimination of these interference factors, often unspecific immunoglobulins or fragments thereof are used which are derived from the same strain of animal species as the antibodies used as reagents in the test. Thus alternative binding sites are offered to the interference factors where they can be taken up so that the specific immune reaction between the analyte and the antibodies is no more disturbed. For elimination of unspecific reactions in immunoassays using monoclonal mouse antibodies as reagents the addition of mouse or rat serum and, respectively, ascites is described in EP-A-0 174 026. A further possibility to avoid such interference is the addition of purified monoclonal Fab fragments or IgG molecules derived from the mouse which especially in a polymerized form develop a good suppressive effect according to U.S. Pat. No. 4,914,040.
To avoid false positive reactions for the detection of CEA the addition of a mixture of IgG molecules of different classes (IgGI, IgG2a, IgG2b) is recommended in the WO 91/16627. The provision of different immunoglobulin preparations in the quantities known and of a constant quality requires, however a lot of experimental time and mainly concerns the constant regions of the antibodies.
The reduction of the interference potential in the constant part of the antibody can also be achieved by the use of Fab fragments instead of intact immunoglobulins since the mainly occurring Fc interferences do not get a chance here due to the lacking Fc part. An optimized version are humanized antibodies with an Fv region composed of one mouse and one human part so that the susceptibility to interference can be further reduced even in the variable part (Kuroki et al. (1995) J. of Immunolog. Methods 180, 81-91). The use of chimeric antibodies with a variable mouse part and human constant regions can also reduce the susceptibility to interference of diagnostic tests.
According to the state of the art the addition of a protein and in particular of antibodies immunologically related to the antibodies used as detection reagents to avoid unspecifically increased or decreased values in the detection of an analyte is described too (EP-A-0 296 544). Due to the similarity of the antibodies added to the reagent antibodies used the substance interfering with these antibodies is captured selectively. For the provision of the suppressive antibodies it is recommended in the EP-A-0 296 544 to cleave off the antigen-binding structures of the reagent antibodies used, to block the antigen-binding s
Lenz Helmut
Moessner Ellen
Nussbaum Sabine
Praast Gerald
Amick Marilyn
Harris Alana M.
Huff Sheela
Roche Diagnostics Corporation
Roche Diagnostics GmbH
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