SAMPLE ANALYSING DEVICE

Abstract

The present invention relates to a device and a method for analysing a sample comprising from 0.1 pg to 1 μg of analyte, and more specifically to a lateral flow device and a method for testing the presence of very low amounts of drugs or drug meta bolites in a sample. The present invention also relates to a method of dissolving a bodily fluid.

Claims

1.-40. (canceled)

41. A method for analysing a sample comprising from 0.1 pg to 1 μg of analyte, the method comprising: (a) providing a sample, the sample containing or not containing from 0.1 pg to 1 μg of an analyte of interest; (b) dissolving at least a portion of the sample in a buffer to form a dissolved sample solution; (c) contacting at least a portion of the dissolved sample solution with a probe zone comprising a labelled probe to dissolve at least a portion of the labelled probe and allow the labelled probe to bind with the analyte, where present, in the portion of the dissolved sample solution to form a labelled probe-analyte complex; (d) passing the labelled probe and/or labelled probe-analyte complex through a test site comprising a first immobilised capture reagent capable of binding to the labelled probe; (e) determining whether or not the amount of analyte, if any, in the sample exceeds a threshold value by detecting the amount of labelled probe in the test site; wherein the sample comprises finger-sweat and/or palm-sweat and/or toe-sweat; and wherein the sample is dissolved in step (b) such that the sample is contacted with the probe zone at the solvent front in step (c).

42. (canceled)

43. The method of claim 41, wherein the sample comprises finger-sweat.

44. The method of claim 41, wherein the sample is provided in step (a) on a sample receiving portion of a lateral flow device, the device further comprising: the probe zone downstream of the sample receiving portion; the test site, downstream of the probe zone, comprising a first immobilised capture reagent capable of binding to the labelled probe; the device being configured to permit movement of a buffer from the sample receiving portion to the probe zone and from the probe zone to the test site.

45. The method of claim 44, wherein the sample is provided directly to the sample receiving portion.

46. The method of claim 41, wherein the sample provided in step (a) is substantially dry, such that the sample comprises insufficient liquid to move from the sample receiving zone to the probe zone.

47. The method of claim 41, wherein the sample is provided in step (a) as a fingerprint, the fingerprint comprising sweat deposited as an impression of a finger's ridge pattern.

48. The method of any of claim 41, wherein step (b) is carried out by providing the buffer upstream of the sample receiving portion and passing the buffer through the sample receiving portion.

49. The method of claim 41, wherein the buffer is provided in a volume of from 100 to 500 μl.

50. The method of claim 41, wherein the buffer to sample volume ratio is from 50:1 to 1,000 to 1.

51-55. (canceled)

56. The method of claim 41, wherein the sample comprises from 0.1 pg to 5 ng of analyte.

57. (canceled)

58. The method of claim 41, wherein the probe zone comprises from 10 pg to 1,000 ng of labelled probe.

59. The method of claim 41, wherein the probe is selected from the group consisting of an antibody, an aptamer, an affimer, and mixtures thereof, preferably wherein the probe is an antibody.

60. (canceled)

61. (canceled)

62. The method of claim 41, wherein the first immobilised capture reagent comprises an antigen capable of binding to the labelled probe.

63. The method of claim 41, wherein the first immobilised capture reagent comprises two or more different antigens capable of binding to the labelled probe.

64. The method of claim 41, wherein the analyte, if present, comprises a drug metabolite and/or a drug.

65. The method of claim 41, further comprising: (f) passing the labelled probe and/or the labelled probe-analyte complex through a control site comprising a second immobilised capture reagent capable of binding to the labelled probe and to the labelled probe-analyte complex; and (g) determining whether or not the test result is reliable by detecting or not detecting the labelled probe and/or the labelled probe-analyte complex in the control site.

66. The method of claim 41, wherein the labelled probe is detectable in radiation having a wavelength of 400 nm to 1 mm, and wherein step (e) and/or step (g) is carried out by illuminating the test site and/or the first control site with radiation having a wavelength of 400 nm to 1 mm to show the labelled probe and/or labelled probe-analyte complex, if present.

67. The method of claim 41, further comprising: (h) illuminating a normalisation site with radiation having a wavelength of 400 nm to 1 mm, measuring the signal intensity of the normalisation site and comparing said signal intensity to the signal intensity detected at the test site and/or the control site; wherein the normalisation site comprises an immobilised labelled protein incapable of binding to the labelled probe, the analyte, and any labelled probe-analyte complex comprising the labelled probe and the analyte; and wherein the immobilised labelled protein and the labelled probe are labelled with the same label.

68. The method of claim 41, further comprising obtaining a fingerprint pattern on a fingerprint pattern receiving zone, wherein the fingerprint pattern receiving zone is separate to the sample receiving portion but is housed within the same device.

69. The method of claim 68, further comprising scanning and/or recording the fingerprint pattern.

70-76. (canceled)

Description

[0200] These and other aspects of the invention will now be described with reference to the accompanying Figures, in which:

[0201] FIG. 1: is a diagram of the lateral flow device according to one embodiment of the present invention.

[0202] FIG. 2: is a photograph of lateral flow test strips obtained as set out in Example 1.

[0203] FIGS. 3 to 6: are images of lateral flow test strips obtained as set out in Example 2.

[0204] FIGS. 7 to 10: are images of lateral flow test strips obtained as set out in Example 3.

[0205] FIG. 11: is a graph showing the results of a comparison between a nitrocellulose lateral flow assay vs. plate assay.

[0206] FIG. 12: is a diagram showing the steps of a ten minute assay using the device of the present invention.

[0207] FIG. 13: is a graph showing results of the lateral flow tests on fingerprint samples taken from people testing positive for morphine by oral fluid analysis.

[0208] FIG. 14: is a graph showing results of the lateral flow tests on fingerprint samples taken from people testing negative for morphine by oral fluid analysis.

[0209] FIG. 15: is a graph showing results of the lateral flow tests on fingerprint samples taken from people testing positive for BZE by oral fluid analysis.

[0210] FIG. 16: is a graph showing results of the lateral flow tests on fingerprint samples taken from people testing negative for BZE by oral fluid analysis.

[0211] The following non-limiting examples further illustrate the present invention.

EXAMPLES

[0212] 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

[0213] Solution set 1 (Set 1): Morphine was dissolved in methanol at 0 to 900 pg per 100 μl of solvent.

[0214] Solution set 2 (Set 2): Morphine was dissolved in fingerprint solubilisation buffer at 0 to 900 pg per 100 μl of solvent.

[0215] 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.

[0216] Each test line (each test site) is BSA-Morphine (40 μg/ml) conjugate applied at 0.25 μl/cm.

[0217] 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).

[0218] 100 μl of solution from Set 1 were applied to the sample receiving portion of a series of lateral flow strips and then dried.

[0219] 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.

[0220] 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.

[0221] The assay results are shown in FIG. 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.

[0222] 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.

[0223] 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

[0224] 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.

[0225] The saliva opiate levels of the patients were confirmed by LGC as follows:

Donor 04745007: Negative

[0226] Donor 04745008: >1700 ng/ml of opiates—Very high levels
Donor 04745009: >10 ng/ml of 6-acetyl morphine (opiate heroin marker)—level 2.5× Cut-off value

[0227] 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.

[0228] The results are shown in FIGS. 3 to 6.

[0229] 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.

[0230] 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

[0231] 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.

[0232] Assays were run, varying the amount of labelled antibody and/or amount of antigen in the test sites.

[0233] FIGS. 7 to 10 show the results obtained.

[0234] 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.

[0235] 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

[0236] A Plate was coated with 50 μg/ml BSA-MOR (4.7 mg/ml): at 4° C., overnight: [0237] 5.3 μl stock+495 μl 100 mM bicarbonate buffer pH 9.5 (diluted from 1077RD) [0238] After incubation, the coated wells were washed 3× with 100 μl PBST (from 1402RD, diluted to 1× with H2O) [0239] A series of dilution of MOR was prepared: [0240] Stock solutions: 1 mg/ml [0241] Stock 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) [0242] A 4-fold dilution series was prepared starting with 100 ng/ml: [0243] 25 ng/ml: 50 μl 100 ng/ml+150 μl PBST (from 1402RD, diluted to 1× with H2O) [0244] 6.25 ng/ml: 50 μl 100 ng/ml+150 μl PBST (from 1402RD, diluted to 1× with H2O) [0245] 1.56 ng/ml: 50 μl 100 ng/ml+150 μl PBST (from 1402RD, diluted to 1× with H2O) [0246] 0.39 ng/ml: 50 μl 100 ng/ml+150 μl PBST (from 1402RD, diluted to 1× with H2O) [0247] 0.09 ng/ml: 50 μl 100 ng/ml+150 μl PBST (from 1402RD, diluted to 1× with H2O) [0248] 0.02 ng/ml: 50 μl 100 ng/ml+150 μl PBST (from 1402RD, diluted to 1× with H2O) [0249] 0.006 ng/ml: 50 μl 100 ng/ml+150 μl PBST (from 1402RD, diluted to 1× with H2O) [0250] 0.0015 ng/ml: 50 μl 100 ng/ml+150 μl PBST (from 1402RD, diluted to 1× with H2O) [0251] 0.0004 ng/ml: 50 μl 100 ng/ml+150 μl PBST (from 1402RD, diluted to 1× with H2O) [0252] 0.0001 ng/ml: 50 μl 100 ng/ml+150 μl PBST (from 1402RD, diluted to 1× with H2O) [0253] Control: PBST (from 1402RD, diluted to 1× with H2O) [0254] rabbit anti-MOR-FITC antibody dilution was prepared: [0255] 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) [0256] 18 μl of MOR dilution was incubated with 2 μl of rabbit anti-MOR-FITC and incubated at room temp. for 4 min [0257] 10 μl from these reactions were transferred into the BSA-MOR coated wells and incubated for 4 min at room temperature [0258] After incubation, the wells were washed 3× with 100 μl PBST (from 1402RD, diluted to 1× with H2O) [0259] 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 s [0260] experiment with PBST as solvent: concentration-dependent inhibition; sensitivity: approx. 0.1-0.39 ng/ml

[0261] The results are shown in FIG. 11, a dose response curve which shows the detection of morphine in the 4 pg-225 pg range.

Example 5

[0262] 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

[0263] 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.

[0264] 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).

[0265] The results of the tests done on the fingerprint samples taken from the people who had tested positive by oral fluid analysis are shown in FIG. 13. The results above the horizontal cut off line are false negatives. The results below the horizontal cut off line are true positives.

[0266] The results of the tests done on the fingerprint samples taken from the people who had tested negative by oral fluid analysis are shown in FIG. 14. The results above the horizontal cut off line are true negatives. The results below the horizontal cut off line are false positives.

[0267] 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

[0268] 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

[0269] 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.

[0270] The results of the tests done on the fingerprint samples taken from the people who had tested positive by oral fluid analysis are shown in FIG. 15. The results above the horizontal cut off line are false negatives. The results below the horizontal cut off line are true positives.

[0271] The results of the tests done on the fingerprint samples taken from the people who had tested negative by oral fluid analysis are shown in FIG. 16. The results above the horizontal cut off line are true negatives. The results below the horizontal cut off line are false positives.

[0272] 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%.