Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
2000-05-12
2003-03-18
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C422S051000, C422S051000, C422S067000, C422S067000, C435S004000, C435S007100, C435S007940, C435S287100, C435S287200, C435S287300, C435S287700, C435S287900, C435S805000, C435S810000, C435S970000, C436S514000, C436S810000
Reexamination Certificate
active
06534324
ABSTRACT:
BACKGROUND OF INVENTION
1. Technical Field
The present invention relates in general to a device and method for rapidly analyzing liquids for the presence of certain target chemicals. The invention more particularly relates to an apparatus and method which provides a portable, inexpensive, rapid, and yet highly accurate test for the presence of certain chemicals at predetermined threshold levels.
2. Background Art
In a variety of instances, persons wish to test liquids, and particularly biological samples, for the presence of particular chemicals. For example, water samples may be tested for the presence of particular toxins or pesticides. Similarly, such tests may be used to determine whether or not deceased or sick wildlife have been exposed to such environmental toxins. In addition, urine samples taken from individuals are tested for the presence of certain drugs, or their metabolic byproducts.
The prior-known most accurate method to test such fluids is to preserve a sample of the fluid for analysis by gas chromatography with mass spectroscopy detection (“GC/MS”). However GC/MS, although highly accurate and reliable, is extremely expensive and the large machines which perform GC/MS are not portable and require skilled labor to operate and interpret. The utilization of GC/MS therefore necessitates preservation of a biological sample taken in the field, and unwanted and undesirable delays during transportation and analysis can result in some applications.
Accordingly, portable assay strips were developed which could be used for rapid screening of biological samples in the field. Such assay strips were useful as presumptive tests for the presence of certain compounds. Samples not showing a positive result could be eliminated from further analysis, and samples showing a positive result could be readily preserved for more accurate testing by GC/MS or other methods.
In order to be useful, it was necessary that the certain threshold levels be established for the target chemical, such that an amount of the chemical below such levels did not show a positive result. As an example, the Substance Abuse and Mental Health Services Administration (SAMHSA) has developed the following guidelines for the threshold or cutoff levels of certain classes of chemicals in urine:
Drug/
Threshold
Substance
(in ng/mL)
Canabinnoids metabolites
50
Cocaine metabolites
300
Opiates (morphine, codeine)
2,000
Amphetamines
1,000
Phencyclidine
25
Ideally, a strip assay would always show a positive result for a drug (or drug metabolite) concentration at or above the cutoff value, and always show a negative result below the cutoff value. In reality, strip assay devices are subject to false positive and false negative results. A false positive occurs when the analyte is not present in an amount equal to or greater than the cutoff value, but the test shows a positive result. False negatives results occur when the analyte is present at a level equal to or above the cutoff, but the test shows a negative result. False positives are undesirable for a variety of reasons. For example, because all positive results are retained for additional laboratory testing (for confirmation), false positives reduce the accuracy of screening, and increase time delay and costs. False negatives are undesirable for several reasons, and in particular that they decrease the accuracy of the results.
The strip assay devices currently developed for such applications do not have sufficient accuracy to achieve the ideal, or even close to the ideal. For example, the College of American Pathologists has recommended that tests be accurate for samples within 25% of the cutoff range (i.e., that a test show no false positives at a level 25% below the cutoff, and no false negatives at a level 25% above the cutoff). However, conventional strip assays do not achieve this level of precision. Indeed, conventional assay strips are often unable to achieve accuracy within 100% of the cutoff range.
Conventional assay strips for screening for the presence of certain drug and/or drug metabolites in urine are generally constructed as competitive assay tests. These strips function by collecting an amount of urine to be analyzed. Specifically, one end of the assay strip is designed to be dipped downwardly into the urine sample and comprises a sample pad which absorbs the urine. The urine migrates along the pad. The urine is then mixed with an amount of competitor, which is generally made by conjugating the target compound with a carrier molecule (often Bovine Serum Albumin, or BSA) which has dye or other indicia means attached. Specifically, the urine flows from the sample pad into the conjugate pad, onto which an amount of competitor has been dried. Also dried onto the conjugate pad is an amount of control protein conjugated with the dye or other indicia, which provides a reference and control. The mixture is then exposed to antibodies specific to the particular analyte.
In practice, the mixture flows along a strip of nitrocellulose to which antibodies have been adhered in two rows. In the first row, antibodies specific to the particular analyte are affixed. In the second row, antibodies specific to the control protein are affixed to serve as a reference and control (showing a level of development of dye). As the mixture flows over the first row of analyte-specific antibodies, any analyte in the urine competes with the competitor for binding with the antibodies. If the level of analyte in the urine is insufficient to displace a specific amount of competitor, the dye (attached to the carrier-analyte conjugate) is seen, and the assay is read as negative. If the level of analyte in the urine is sufficient to displace the competitor to a significant degree, no dye is seen and the test is read as being a positive.
As the mixture continues to flow along the nitrocellulose strip, the second reference row is developed, and the dye is seen as the antibodies specific to the control protein bind the control protein-dye conjugate, as a reference. The assay strips also incorporate an absorbent pad at the terminus of the nitrocellulose, to absorb excess mixture.
In an alternative conventional methodology, the assay operates as above initially, except that the conjugate pad referenced is treated with antibodies specific to the analyte, conjugated with dye or other indicia. In this method, the analyte in the urine is bound by the anti-analyte antibodies in the conjugate pad, and the mixture is then allowed to flow along a nitrocellulose strip on which the first row is composed of analyte (conjugated to a carrier protein such as BSA). If the level of analyte in the urine is insufficient to saturate the anti-analyte antibodies, then the anti-analyte antibodies will bind to the immobilised analyte-BSA conjugate on the strip and the result will be read as negative. If, however, the level of analyte in the urine is sufficient to saturate the anti-analyte antibodies, these antibodies will flow over the immobilised analyte-BSA conjugate on the membrane, and the result will be read as positive. As in the first method, the mixture continues to flow along the nitrocellulose membrane, encountering the second row of antibodies, which in this method bind the antibody-dye conjugate.
Using assay strips, a urine sample can be screened within minutes after it is obtained. In practice, multiple strips (each specific for a particular analyte) are dipped into a sample, and the results obtained. In such a manner, small, relatively inexpensive, and portable assay strips can be provided to persons which enable them to screen a particular urine sample in the field for a variety of compounds. However, prior known assay strips do not achieve a level of precision which is desirable.
As discussed above, the result of an assay is determined by examining the color intensity (or other indicia) at the test line (the first row). Ideally, this line would be very intense at values below the cutoff, visible at the cutoff, and invisible for values above the cutoff. In pract
Foley & Lardner
Kleinke Bernard L.
Mizuho USA, Inc.
Nguyen Bao-Thuy L.
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