Chemistry: analytical and immunological testing – Biospecific ligand binding assay
Reexamination Certificate
1999-06-04
2002-01-08
Horlick, Kenneth R. (Department: 1656)
Chemistry: analytical and immunological testing
Biospecific ligand binding assay
C436S513000, C436S514000, C436S518000, C435S006120, C435S007100, C435S091100, C435S091200, C435S283100, C435S285100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330, C210S656000, C210S635000, C210S348000, C210S500260
Reexamination Certificate
active
06337214
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an apparatus and method for the detection of test materials in small concentrations, especially the detection of pathogen indicators. In particular it relates to the detection of DNA, RNA and proteins in serum.
BACKGROUND TO THE INVENTION
Historically, the diagnosis of diseases has depended upon clinical manifestations. However, new techniques of detecting diseases have been developed with the advent of nucleic acid and monoclonal antibody detection methods. The detection of nucleic acid has been used for diseases associated with abnormal gene products, such as anemia, Huntington's disease and certain thalassemia mutations. In addition, the detection of nucleic acid has been used for bacterial and viral diseases, such as Human Immunodeficiency Virus (HIV). Moreover, monoclonal antibody detection methods have gained acceptance for the identification and differentiating of certain diseases such as cancers.
As appreciated by those skilled in the art, the detection of a pathogen indicator has applicability to the detection of certain diseases associated with abnormal genes, certain diseases associated with the presence of an identifiable nucleic acid sequence and certain diseases associated with the immune system. The pathogen indicator described herein includes DNA, RNA, antibody, antigen, and other proteins.
Known manual pathogen indicator detection methods in research and clinical laboratories tend to have low accuracy, low sensitivity and are subject to human error, both in carrying out the methods and in interpreting the results. Other methods, e.g. culturing methods, are not suitable for many diseases. For example, tuberculosis has a very slow growth rate, which makes detection not easy or even not possible.
U.S. Pat. No. 5,753,439 to Smith et. al. describes a method to detect characteristic dinucleotide and trinucleotide acid sequences, to determine target sequences and to screen for genetic defects and disorders associated with the sequences. The assays are conducted on solid surfaces allowing for multiple reactions to be conducted. However, the method does not provide a control process to provide assurances that the results are accurate and sensitive to determining if there is an error in the method.
U.S. Pat. No. 5,824,478 to Muller describes a method wherein a sample is contacted with a detector probe and a capture probe to form a detector probe-analyte-capture probe complex which is used to detect a target analyte in a sample. The target analytes, capture probes, and detector probes can be nucleic acids and polypeptides. However, the method does not provide a control process to provide assurances that the results are accurate and sensitive to determining if there is an error in the method.
In a standard enzyme ELISA method for immunoassay, a tray with a plurality of wells, e.g. 96 wells, containing appropriate antibodies is used. One method to eliminate error in this ELISA is to use a control. One of the wells can be used as a positive control (with a positive antigen), while the remaining wells can be used for testing patient's sera. After addition of the serum samples, the wells are washed and a second antibody, which carries an enzyme, is added to the wells. After washing again, a substrate is added. The substrate and enzyme react, with a color reaction. The color yield from the reaction is associated with the presence of an antigen. The method is rife with possibilities for error. Human error can lead to some wells being washed twice or not at all, having reagents added twice or not at all, or wells being inadvertently contaminated with extraneous materials. For example, over washing tends to flush all the components and create a false negative result, while an incomplete wash will provide detection from non-binding materials and yield false positive results. The control well can give no assurance that the results from any other well is indicative of the presence or otherwise of the pathogen indicator under investigation. Additionally, color differences from well to well give additional uncertainties with respect to interpretation of the results.
Most of the previous tests are demanding of time, skill and concentration. So much so, that in many jurisdictions the number of tests that can be conducted by one technician is limited by regulation. This serves to raise the cost of testing, as it is so labour dependent.
For all the above reasons, and more, a new method, apparatus and a kit for detecting a pathogen indicator is desirable, which is accurate, reproducible, and is sensitive to determining if there is an error in the method.
SUMMARY OF THE INVENTION
The present invention provides a method for detecting the presence of a test material in a test sample. The method comprises the steps of: (a) introducing a test sample and a control material into a test column, wherein the column has at least two snares, one of said snares having a control capture material; at least one of said snares thereon having a target capture material specific to a corresponding test material in the test sample for which the detection is being sought, so that the control capture material will bind with the control material to form a bound control material; and the target capture material will bind with the corresponding test material to form a bound material; (b) washing the test column to remove any materials which have not been bound to the capture materials; and (c) detecting the presence of bound materials on each of the snares. The method can further comprise adding a label material for each of the bound materials to form labeled bound materials and then detecting the presence of the labeled bound materials.
In another embodiment, the present invention provides a method for detecting the presence of a DNA sequence in a test sample. The method comprises the steps of: (a) denaturing a test sample to form a single strand target DNA sequence for which detection is being sought; (b) introducing the test sample and a first control single strand DNA sequence into a test column which has at least two snares, one of said snares having a first control single strand capture DNA sequence; at least one of said snares thereon having a target single strand capture DNA sequence specific to the corresponding target DNA sequence in the test sample; and wherein the target single strand capture DNA sequence will bind with the corresponding target DNA sequence in the test sample to form a double strand DNA sequence, and the first control single strand capture DNA sequence will bind with the first control DNA sequence to form a double strand control DNA sequence; (c) adding a wash solution to the column to remove unbound DNA; (d) adding an enzyme to the column to destroy single strand DNA; (e) adding a denaturing solution to separate the formed double strand DNA sequences, then adding a wash solution to remove denatured non-capture single strand sequences, so that the single strand capture DNA sequences re-form on each snare; (f) adding DNA probes to provide detectable labels for single strand capture DNA sequences formed in step (e); (g) adding a wash solution to the column to remove unbound DNA probes; and (h) detecting any signals from each snare. In step (b), the first single strand control DNA sequence can be added into said test sample prior to introducing the sample into the test column, or can be added into the test column separately from the test sample. Moreover, the method can further include adding a substrate which reacts with the labels to give off detectable signals.
Additionally, the method further comprises introducing a second control single strand DNA sequence into the test column; wherein the test column has a control snare thereon having a second control single strand capture DNA sequence.
In a further embodiment, the snares have more than one single strand capture DNA sequences on one single snare, and the labels are different for different single strand capture DNA sequences on one single snare so that different DNA se
ACGT Medico Inc.
Horlick Kenneth R.
Li Yi
Siew Jeffrey
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