Chemistry: analytical and immunological testing – Biospecific ligand binding assay
Reexamination Certificate
1998-06-08
2001-01-16
Jones, W. Gary (Department: 1656)
Chemistry: analytical and immunological testing
Biospecific ligand binding assay
C435S006120, C435S007100, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330, C210S635000, C210S636000, C210S658000, C210S663000, C422S105000, C436S501000
Reexamination Certificate
active
06174733
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the detection materials in small concentrations, especially the detection of pathogens. In particular it relates to the detection of proteins, DNA and RNA in serum.
BACKGROUND TO THE INVENTION
Known manual pathogen detection methods in research and clinical laboratories tend to have low accuracy, low sensitivity to pathogens 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 pathogens. For example, tuberculosis has a very slow growth rate, which makes detection not easy or even not possible.
One immunologic method, which identifies an organism with a known antiserum, is widely used for pathogen detection. The accuracy of the method is relatively high but the sensitivity is relatively low, e.g. it needs about one million antigen-antibody complexes to clearly indicate the results. There are a number of other disadvantages of this method, which are known.
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 of the wells is used as a positive control (with a positive antigen), while the remaining wells are 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 colour reaction. The colour yield from the reaction is indicative of the presence of the pathogen. 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, overwashing 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 under investigation. Additionally, colour 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 of detecting pathogens 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 at least two predetermined known materials in a test sample, wherein at least one of the predetermined known materials is a control material, wherein the method comprises:
a) introducing the test sample, which contains at least one control material, into a test column which has a snare for each predetermined known material, each snare having a capture material specific to the associated predetermined known material, which will bind with the associated predetermined known 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.
In one embodiment, the predetermined known materials, which are not control materials, are pathogens.
In another embodiment, step c) comprises adding a label material for each of the bound materials to form labelled bound materials and then detecting the labelled bound materials.
In a further embodiment, the method is for detecting the presence of at least two predetermined known DNA materials, wherein the capture materials are single strand capture DNA materials and the test sample has been denatured so that any predetermined known DNA materials are in single strand form prior to addition to the test column, and detection of the bound materials is accomplished by adding a label material for each of the bound materials to form labelled bound materials and then detecting the labelled bound materials.
In another embodiment, the label material for the control DNA material and the non-control DNA material is selected such that the label material is a single label material.
Another aspect of the invention provides a method for detecting the presence of a pathogen, comprising the steps of:
i) adding a sample, which contains at least one control material, to a column which has at least one control snare and at least one test snare, each of the control snares having thereon a first capture antibody for binding to the control material, and each of the test snares having thereon a pathogen capture antibody for binding to the pathogen for which detection is being sought, so that the control material binds with the first capture antibody to form a bound control material, and any pathogen present binds with the pathogen capture antibody to form a bound pathogen;
ii) adding a wash solution to the column to remove any unbound control material and any unbound pathogens;
iii) adding sufficient primary antibodies to the column, to bind with the control material and any bound pathogens, said primary antibodies having labels thereon;
iv) adding a wash solution to the column to remove any unbound primary antibodies;
v) adding a substrate which reacts with the labels to give off a detectable signal; and
vi) detecting any detectable signals from the labelled and bound control material and from any labelled and bound pathogen.
In one embodiment, the substrate and label are selected to give off a chemiluminescent signal.
In another embodiment, the substrate and label are selected to give off a fluorescent signal.
A further aspect of the invention provides a method for detecting the presence of a DNA in a sample, comprising the steps of:
i) denaturing the predetermined known DNA materials;
ii) adding a sample, which contains at least one denatured control DNA segment, to a column which has at least one control snare and at least one test snare, one of the control snares having thereon a first control single strand capture DNA segment for binding to the denatured control DNA segment, and one of the test snares having thereon a test single strand capture DNA segment for detecting the denatured test DNA segment for which detection is being sought, so that the denatured control DNA segment binds with the first control single strand capture DNA segment to form a double strand control DNA segment, and any denatured test DNA present binds with the test single strand capture DNA segment to form a double strand test DNA segment;
iii) adding a wash solution to the column to remove any unbound DNA;
iv) adding S1 nuclease to the column to destroy any single strand DNA;
v) adding a wash solution and a denaturing solution to the column to re-form the first control single strand capture DNA segment and the test single strand capture DNA segment;
vi) adding DNA probes to provide detectable labels for the first control single strand capture DNA segment and the test single strand capture DNA segment formed in step v);
vii) adding a wash solution to the column to remove any unbound DNA probe;
viii) adding a substrate which reacts with the labels to give off detectable signals; and
ix) detecting any signals from the control and test snares.
In yet another embodiment the method comprises:
i) preparing a positive control DNA material from a DNA material for which detection is sought, by a process selected from the group consisting of a) inserting a control DNA sequence into the DNA material at a predetermined scission point in the first DNA material and b) removing a small fragment of DNA from the first DNA material at a predetermined scission point;
ii) denaturing a test sample which contains at least th
ACGT Medico Inc.
Jones W. Gary
Li Yi
Siew Jeffrey
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