Patent ID: 12259385

The following non-limiting examples further illustrate the present invention.

EXAMPLES

The present invention will now be described in relation to several examples.

Example 1: Homogeneously Distributed Dried Drug Over the Sample Receiving Portion is Concentrated at the Solvent Front when Solvent is Applied Upstream of the Deposited Sample

Solution set 1 (Set 1): Morphine was dissolved in methanol at 0 to 900 pg per 100 μl of solvent.

Solution set 2 (Set 2): Morphine was dissolved in fingerprint solubilisation buffer at 0 to 900 pg per 100 μl of solvent.

A series of lateral flow strips of the present invention were set up having two test sites and no control sites. The volume of the sample receiving portion is around 110 μl.

Each test line (each test site) is BSA-Morphine (40 μg/ml) conjugate applied at 0.25 μl/cm.

The labelled probe zone is provided with 50 ng labelled antibody/zone in loading buffer (100 mM Sucrose in

10 mM PB pH 7.42, 1% (w/v) Tween-80).

100 μl of solution from Set 1 were applied to the sample receiving portion of a series of lateral flow strips and then dried.

100 μl of solutions from Set 2 were applied to the sample receiving portion of a second series of lateral flow strips and while still wet, fresh fingerprint solubilisation buffer was used to chase the liquid through the lateral flow strip by applying the buffer to the bottom edge of the pad i.e. upstream of the sample receiving portion. A similar application of fingerprint solubilisation buffer to the pads containing dried Morphine was performed in parallel.

Set 2 represents what would happen if solvent were applied directly to the fingerprint, similar to sample application

to sample pad areas of conventional test strips. Set 1 shows what happens when buffer is applied upstream of the print. Here buffer slowly wets the fingerprint area, solubilising the drug/drug metabolites as it advances downstream the lateral flow strip, incrementally concentrating the drug material at the buffer solvent front.

The assay results are shown inFIG.2. The data clearly demonstrates that the drug in the membranes from Set 1 has been concentrated with respect to similar levels found in Set 2.

The method used by set 2 (similar to a typical lateral flow approach) is not sensitive enough and is unable to detect the lowest levels of drug found on the fingerprint which would indicate a person is just above the legal cut-off. The present invention clearly detects the drug/metabolite at 150 pg per sample.

Testing using the set 2 format only appears to detect the presence of drug/metabolite between 750 to 900 pg which is well above the cut-off and of no use for routine testing, as the value is too high.

Example 2

Fingerprints were collected from three patients on a variety of sample receiving portion materials on lateral flow strips of the present invention having two test sites and no control sites.

The saliva opiate levels of the patients were confirmed by LGC as follows:Donor 04745007: NegativeDonor 04745008: >1700 ng/ml of opiates—Very high levelsDonor 04745009: >10 ng/ml of 6-acetyl morphine (opiate heroin marker)—level 2.5× Cut-off value

Lateral flow assays were run by applying the solubilisation buffer of Example 1. Images of the resulting test lines were captured by illumination of the samples for 5 seconds each.

The results are shown inFIGS.3to6.

For each of the four substrates tested, clear inhibition (opiate detection) is seen for sample 04745008. Partial

inhibition is observed for sample 04745009 which is in line with low levels of metabolite. Use of a calibration curve would allow quantitation of this value.

The results show that the sample receiving portion may comprise any material that does not bind the analyte (drug or drug metabolite) being investigated and allows buffer to migrate through it slowly in order to collect and concentrate the metabolite at the solvent front.

Example 3: Evidence for Varying the Assay Sensitivity Range

Samples having 0 pg, 150 pg, 300 pg and 450 pg of morphine were provided on sample receiving portion of lateral flow strips of the present invention having two test sites and no control sites.

Assays were run, varying the amount of labelled antibody and/or amount of antigen in the test sites.

FIGS.7to10show the results obtained.

Cut-off values for opiates may be around 90 pg per print. Similar studies have been done for the cocaine metabolite BZE, the cut-off value for which is around 68 pg per print.

The results show that the lower the amount of antibody, the more sensitive the assay is, which is useful for detecting low levels of drugs and/or drug metabolites. Also, the lower the amount of immobilised antigen in the test sites, the more sensitive the assay is.

Example 4: Comparison Between a Nitrocellulose Lateral Flow Assay Vs. Plate Assay

A Plate was coated with 50 μg/ml BSA-MOR (4.7 mg/ml): at 4° C., overnight:5.3 μl stock+495 μl 100 mM bicarbonate buffer pH 9.5 (diluted from 1077RD)After incubation, the coated wells were washed 3× with 100 μl PBST (from 1402RD, diluted to 1× with H2O)A series of dilution of MOR was prepared:Stock solutions: 1 mg/mlStock was diluted to 10 μg/ml: 10 μl stock+990 μl PBST (from 1402RD, diluted to 1× with H2O) The 10 μg/ml solution was further diluted to 100 ng/ml: 10 μl 10 μg/ml+990 μl PBST (from 1402RD, diluted to 1× with H2O)A 4-fold dilution series was prepared starting with 100 ng/ml:25 ng/ml: 50 μl 100 ng/ml+150 μl PBST (from 1402RD, diluted to 1× with H2O)6.25 ng/ml: 50 μl 100 ng/ml+150 μl PBST (from 1402RD, diluted to 1× with H2O)1.56 ng/ml: 50 μl 100 ng/ml+150 μl PBST (from 1402RD, diluted to 1× with H2O)0.39 ng/ml: 50 μl 100 ng/ml+150 μl PBST (from 1402RD, diluted to 1× with H2O)0.09 ng/ml: 50 μl 100 ng/ml+150 μl PBST (from 1402RD, diluted to 1× with H2O)0.02 ng/ml: 50 μl 100 ng/ml+150 μl PBST (from 1402RD, diluted to 1× with H2O)0.006 ng/ml: 50 μl 100 ng/ml+150 μl PBST (from 1402RD, diluted to 1× with H2O)0.0015 ng/ml: 50 μl 100 ng/ml+150 μl PBST (from 1402RD, diluted to 1× with H2O)0.0004 ng/ml: 50 μl 100 ng/ml+150 μl PBST (from 1402RD, diluted to 1× with H2O)0.0001 ng/ml: 50 μl 100 ng/ml+150 μl PBST (from 1402RD, diluted to 1× with H2O)Control: PBST (from 1402RD, diluted to 1× with H2O)rabbit anti-MOR-FITC antibody dilution was prepared:1 μl stock (mAbF-MOR-004-006; 0.5 mg/ml)+99 μl extraction buffer 1420RD or PBST (from 1402RD, diluted to 1× with H2O)18 μl of MOR dilution was incubated with 2 μl of rabbit anti-MOR-FITC and incubated at room temp. for 4 min10 μl from these reactions were transferred into the BSA-MOR coated wells and incubated for 4 min at room temperatureAfter incubation, the wells were washed 3× with 100 μl PBST (from 1402RD, diluted to 1× with H2O)empty wells were filled with 10 μl PBST (1402RD) and the fluorescence was measured in a plate reader with a 485 nm excitation filter and a 535 nm emission filter for 1 sexperiment with PBST as solvent: concentration-dependent inhibition; sensitivity: approx. 0.1-0.39 ng/ml

The results are shown inFIG.11, a dose response curve which shows the detection of morphine in the 4 pg-225 pg range.

Example 5

Oral fluid was taken from 184 people and the fluid analysed for the presence of morphine and/or metabolites thereof. Positive samples were shown to comprise morphine and/or metabolites thereof. Negative samples were shown to not comprise morphine and/or metabolites thereof. There were 92 positive samples and 92 negative samples

Fingerprint samples were taken from the same people and the lateral flow device as described herein was used to test for the presence of morphine or metabolites thereof. The cut-off point for detecting the presence of morphine in the fingerprint was 180 pg, i.e. the fingerprint samples shown to comprise 180 pg or more of morphine and/or metabolites thereof were deemed positives, whilst the samples shown to comprise less than 180 pg or more of morphine and/or metabolites thereof were deemed negatives.

The results were compared to the results obtained via oral fluid analysis. A test showing the presence of morphine and/or metabolites thereof which agreed with the oral fluid analysis was deemed to be a true positive (TP). A test showing the presence of morphine and/or metabolites thereof which did not agree with the oral fluid analysis was deemed to be a false positive (FP). A test not showing the presence of morphine and/or metabolites thereof which agreed with the oral fluid analysis was deemed to be a true negative (TN). A test not showing the presence of morphine and/or metabolites thereof which did not agree with the oral fluid analysis was deemed to be a false negative (FN).

The results of the tests done on the fingerprint samples taken from the people who had tested positive by oral fluid analysis are shown inFIG.13. The results above the horizontal cut off line are false negatives. The results below the horizontal cut off line are true positives.

The results of the tests done on the fingerprint samples taken from the people who had tested negative by oral fluid analysis are shown inFIG.14. The results above the horizontal cut off line are true negatives. The results below the horizontal cut off line are false positives.

The percentage accuracy, percentage sensitivity and percentage specificity of the lateral flow test for morphine and/or metabolites thereof can thus be calculated as follows:
Accuracy=((Total number of TP+Total number of TN)/Total number of samples)×100
Sensitivity=(Total number of TP/(Total number of TP+Total number of FN))×100
Specificity=(Total number of TN/(Total number of TN+Total number of FP))×100

The results showed that the accuracy of the lateral flow test, which can detect whether a sample has more or less than 180 pg of morphine, was 92.9%. The sensitivity was 85.9%. The specificity was 100%.

Example 6

Example 6 is identical to Example 5 with the exception that the drug metabolite tested was benzoylecgonine (BZE) (the major metabolite of cocaine), there were 100 people's samples taken (50 positive and 50 negative), and the cut-off point for the fingerprint samples was 150 pg of BZE, i.e. the fingerprint samples shown to comprise 150 pg or more of BZE were deemed positives whilst the fingerprint samples shown to comprise less than 150 pg of BZE were deemed negatives.

The results of the tests done on the fingerprint samples taken from the people who had tested positive by oral fluid analysis are shown inFIG.15. The results above the horizontal cut off line are false negatives. The results below the horizontal cut off line are true positives.

The results of the tests done on the fingerprint samples taken from the people who had tested negative by oral fluid analysis are shown inFIG.16. The results above the horizontal cut off line are true negatives. The results below the horizontal cut off line are false positives.

The results showed that the accuracy of the lateral flow test, which can detect whether a fingerprint sample has more or less than 150 pg BZE, was 95%. The sensitivity was 90%. The specificity was 100%.