Assay reader, device and method of measuring HCG

Abstract

Disclosed is a method for determining a quantitative estimate of the length of time since conception in a female mammalian subject, the method comprising: a) providing a liquid sample suspected of containing hCG; b) measuring, by means of an assay or assay device, an analyte measurement signal, whose value is dependent upon the level of hCG; c) comparing the measured signal value to an analyte threshold, wherein said analyte threshold corresponds to a time since conception; d) providing an quantitative estimate of the length of time since conception based upon the comparison in step (c).

Claims

1. An electronic pregnancy test device for calculating a quantitative estimate of the length of time since conception in a female mammalian subject, the device comprising: a) a first assay flow-path, comprising a mobilizable labeled binding reagent for hCG, and a detection zone; b) a second assay flow-path, comprising a mobilizable labeled binding reagent for hCG and a second binding reagent for hCG, wherein the second binding reagent for hCG alters the sensitivity of an assay for hCG, and a detection zone, such that the first assay flow-path detection zone is for measuring hCG in a lower concentration range and the second assay flow-path detection zone is for measuring hCG in a higher concentration range; c) a processor; d) a first stored analyte threshold corresponding to a first time since conception, a second stored analyte threshold corresponding to a second time since conception, which is a longer period of time than that corresponding to the first stored analyte threshold, and a third stored analyte threshold which is a minimum pregnancy threshold, said analyte thresholds being stored in said electronic pregnancy test device and accessible to the processor; e) a shared reference zone, the shared reference zone being located within the first or second assay flow-path, the other of the first or second assay flow path having no reference zone located therein, wherein the value of a signal obtained at the shared reference zone compensates the value of a signal obtained at the detection zone of the other of the first or second assay flow-path; f) a measurement means for measuring a first analyte signal value from said first assay flow-path, and for measuring a second analyte signal value from said second assay flow-path, said first and second analyte signal values corresponding to the level of hCG in a liquid sample obtained from said subject, and comparing said first and/or second analyte signal values to the first, second, or third stored analyte thresholds, wherein a second analyte signal value greater than the second stored threshold indicates the subject is 3+ weeks pregnant; a second analyte signal value less than said second stored threshold but a first analyte signal value greater than the first stored threshold indicates the subject is 2-3 weeks pregnant; a first analyte signal value less than the first stored threshold but greater than the third stored threshold indicates the subject is 1-2 weeks pregnant; and a first analyte signal value less than the third stored threshold indicates that the subject is not pregnant; and g) a display means to display a result of the assay which is the quantitative estimate of the length of time since conception.

2. The test device according to claim 1, which further comprises one or more additional stored analyte thresholds corresponding to one or more times since conception.

3. The test device according to claim 1, wherein the first assay flow-path and the second assay flow-path each comprise a single detection zone.

4. The test device according to claim 1, further comprising a shared control zone located within the first or second assay flow-path, the other of the first or second assay flow-path having no control zone located therein, wherein measurement of a signal at the shared control zone provides a value or indication that the assay has been carried out correctly.

5. The test device according to claim 1, wherein the measurement means comprises a single photodetector to detect light from both detection zones, and four light sources to illuminate respectively the shared reference zone, a shared control zone and the two detection zones.

6. The test device according to claim 1, wherein the shared reference zone is provided downstream of the detection zone for the first or second assay flow-path.

7. The test device according to claim 1, wherein one or both assay flow-paths comprises a lateral flow porous carrier.

8. The test device according to claim 7, wherein the lateral flow porous carrier comprises nitrocellulose.

9. The device of claim 1, wherein the second analyte signal value is diminished compared to the first analyte signal value due to the presence of the second binding reagent for hCG, which diminishes the amount of hCG available to be bound at the detection zone relative to the first assay flow-path.

10. The device of claim 1, wherein the first assay flow-path or the second assay flow-path comprises a control zone downstream of their respective detection zones.

11. The device of claim 10, wherein the control zone is a shared control zone.

12. The device of claim 10, wherein a signal value determined at the control zone is used to validate the result of the first and second assay flow-paths respectively.

13. The device of claim 1, wherein the reference zone is downstream of the detection zone within the first or second assay flow-path.

14. An electronic pregnancy test device for calculating a quantitative estimate of the length of time since conception in a female mammalian subject, the device comprising: a) a first assay flow-path, comprising a mobilizable labeled binding reagent for hCG, and a detection zone; b) a second assay flow-path, comprising a mobilizable labeled binding reagent for hCG and a second binding reagent for hCG, wherein the second binding reagent for hCG alters the sensitivity of an assay for hCG, and a detection zone, such that the first assay flow-path detection zone is for measuring hCG in a lower concentration range and the second assay flow-path detection zone is for measuring hCG in a higher concentration range; c) a processor; d) a first stored analyte threshold corresponding to a first time since conception, a second stored analyte threshold corresponding to a second time since conception, which is a longer period of time than the first stored analyte threshold, and a third stored analyte threshold which is a minimum pregnancy threshold, said analyte thresholds being stored in said electronic pregnancy test device and accessible to the processor; e) a shared reference zone, the shared reference zone being located within a subsidiary flow-path to the first and second assay flow-paths, the first and second assay flow-paths having no reference zone located therein, wherein the value of a signal obtained at the shared reference zone compensates the value of signals obtained at the detection zones of the first and second assay flow-paths; f) a measurement means for measuring a first analyte signal value from said first assay flow-path, and for measuring a second analyte signal value from said second assay flow-path, said first and second analyte signal values corresponding to the level of hCG in a liquid sample obtained from said subject, and comparing said first and/or second analyte signal values to the first, second, or third stored analyte thresholds, wherein a second analyte signal value greater than the second stored threshold indicates the subject is 3+ weeks pregnant; a second analyte signal value less than said second stored threshold but a first analyte signal value greater than the first stored threshold indicates the subject is 2-3 weeks pregnant; a first analyte signal value less than the first stored threshold but greater than the third stored threshold indicates the subject is 1-2 weeks pregnant; and a first analyte signal value less than the third stored threshold indicates that the subject is not pregnant; and g) a display means to display a result of the assay which is the quantitative estimate of the length of time since conception.

15. The test device according to claim 14, which further comprises one or more additional stored analyte thresholds corresponding to one or more times since conception.

16. The test device according to claim 14, wherein the first assay flow-path and the second assay flow-path each comprise a single detection zone.

17. The test device according to claim 14, further comprising a shared control zone located within the first or second assay flow-path, the other of the first or second assay flow-path having no control zone located therein, wherein measurement of a signal at the shared control zone provides a value or indication that the assay has been carried out correctly.

18. The test device according to claim 14, wherein the measurement means comprises a single photodetector to detect light from both detection zones, and four light sources to illuminate respectively the shared reference zone, a shared control zone and the two detection zones.

19. The test device according to claim 14, wherein the shared reference zone is provided downstream of the detection zone for the first or second assay flow-path.

20. The test device according to claim 14, wherein one or both assay flow-paths comprises a lateral flow porous carrier.

21. The test device according to claim 20, wherein the lateral flow porous carrier comprises nitrocellulose.

22. The test device according to claim 14, wherein the second analyte signal value is diminished compared to the first analyte signal value due to the presence of the second binding reagent for hCG, which diminishes the amount of hCG available to be bound at the detection zone relative to the first assay flow-path.

23. The test device according to claim 14, wherein the first assay flow-path or the second assay flow-path comprises a control zone downstream of their respective detection zones.

24. The test device according to claim 23, wherein the control zone is a shared control zone.

25. The test device according to claim 23, wherein a signal value determined at the control zone is used to validate the result of the first and second assay flow-paths respectively.

26. The test device according to claim 14, wherein the reference zone within the first or second assay flow-path is downstream of the detection zone.

Description

(1) Aspects of the invention are further illustrated by reference to the following figures:

(2) FIG. 1 shows the variation in hormone levels and basal body temperature that occurs during a typical 28 day menstrual cycle.

(3) FIGS. 2 (a) and (b) show an exemplary assay device of the invention.

(4) FIG. 3 shows an exemplary assay device according to an embodiment of the invention

(5) FIGS. 4A-B show an exemplary algorithm for determination of flow rate for an assay device comprising a high sensitive (HS) assay and a low sensitive (LS) assay.

(6) FIGS. 5A-B show an exemplary algorithm for the determination of a time since conception for an assay device comprising a high sensitive (HS) assay and a low sensitive (LS) assay.

(7) FIGS. 6(a)-(c) show typical % A vs. assay time profiles that are obtained for an assay device and method for the detection of hCG

(8) FIG. 7 shows the graph plotted for time since conception vs. log(hCG+0.1) obtained for a cohort of women and a fitted exponential curve according to Example 2.

(9) FIGS. 8(a) and (b) show the fitted and observed relationships for the high and low sensitivity assays prepared according to Example 1.

DETAILED DESCRIPTION OF THE FIGURES

(10) FIG. 2a shows an exemplary assay device of the invention. The device is elongate having a length of about 14 cm and a width of about 25 mm, comprising housing (50), a porous sample receiver (51) and an LCD display (53) for displaying the results of the assay. Also provided within the assay and not shown are the assay flow-paths, optical means, a power source and associated electronic components. The assay device may also have a removable cap (52) to fit over the porous sample receiver.

(11) FIG. 2b shows an exemplary assay device according to the invention which has been opened to show some of the components. The device comprises upper and lower housings (510, 512), a desiccant tablet (513) to maintain low levels of humidity within the device, a battery (516) an optical baffle (514) and computer chip (515).

(12) FIG. 3 shows the layout of the individual assay porous carriers of an assay device according to an exemplary embodiment having a shared control and reference zone. Assay device (60) has a common sample application region (61) which fluidically connects first and second assays (62) and (63). Zones (64) and (65) correspond respectively to a detection and control zone for the assay. Zones (66) and (67) correspond respectively to a detection and reference zone.

(13) FIG. 4 shows an exemplary algorithm for determination of flow rate for an assay device comprising a high sensitivity (HS) assay and a low sensitivity (LS) assay.

(14) Once the calibration is complete, all of the windows are measured and filtered.

(15) The minimum flow detection time (Min FDT) may be defined as the minimum time that flow will reach one or more measurement zones. If flow is detected at times less than the minimum flow detection time, it is assumed that the device has “woken up” late and that any calibration values are likely to have been affected by flow already being present in the window during calibration. These devices may be classed as “late wake-up errors”.

(16) The maximum flow detection time (Max FDT) may be defined as a time by which liquid sample has to reach all of the measurement zones after having been applied to the device or after the device has been activated following application of liquid sample. If flow has not been detected in the all of the measurement zones before this maximum time from wake-up the device is assumed to be under sampled, namely not enough liquid sample has been applied to the assay device. These devices may be classed as being under sampled.

(17) The minimum window transit time (Min WTT) may be defined as the minimum time by which fluid passes between one zone and downstream zone. Flow between the windows corresponding to these zones must occur by a minimum time difference. Flow times less than this time may be rejected as it is likely that the device has been flooded or over sampled.

(18) The following is an example of the flow-rate parameters that may be employed:

(19) TABLE-US-00002 Parameter Value Minimum Flow Detection Time (Min FDT) 3 seconds Maximum Flow Detection Time (Max FDT) 64 seconds  Minimum Window Transit Time (Min WTT) 2 seconds

(20) FIG. 5 shows an exemplary algorithm for the determination of a time since conception for an assay device for the determination of hCG over an extended analyte range comprising a high sensitivity (HS) assay and a low sensitivity (LS) assay, each comprising a test zone.

(21) Timing for the assay is from the point of detection of flow through the high sensitivity test line window. The algorithm provides for an early indication of “pregnant”, earlier than the full assay test time. Indications of the time since conception or “not pregnant” are given at the full assay test time (full assay development time).

(22) Once the flow has been determined to be valid by the flow algorithm parameter, after the minimum development time, if HS.sub.t (% A) exceeds the Early Pregnant Decision Threshold and Ctrl.sub.t (% A) is above the Control Line Threshold then a result of PREGNANT is displayed.

(23) After the Minimum Development Time and PREGNANT is displayed, subsequently if LS.sub.t (% A) exceeds the Early Pregnant Max Decision Threshold then the conception guide indication for the maximum time (+3 weeks) is displayed.

(24) At Full Assay Development Time

(25) If by this time PREGNANT is already displayed then the control line is not tested. Otherwise if the control line is below the Control Line Threshold then “Device Error” is indicated and the test is complete.

(26) If HS.sub.t (% A) exceeds the Pregnant Mid Decision Threshold then the conception guide indication for the mid time is displayed (2-3 weeks) and the test is complete.

(27) If by this time PREGNANT is already displayed then the conception guide indication for the minimum time (1-2 weeks) is displayed and the test is complete. If HS.sub.t (% A) exceeds the Pregnant Minimum Decision Threshold (PDTmin) then PREGNANT and the conception guide indication for the minimum time is displayed and the test is complete.

(28) Otherwise NOT PREGNANT is displayed and the test is complete.

(29) An example of the particular decision algorithm parameters for an assay device for the detection of hCG that may be employed is as follows:

(30) TABLE-US-00003 Parameter Value Control Line Threshold (CLT) 30% A Minimum Development Time (MDT)  60 seconds Early Pregnant Decision Threshold (EPDT) 14% A Early Pregnant Max Decision Threshold (EPDTmax) 45% A Full Assay Development Time (FDT) 150 seconds Pregnant Mid Decision Threshold (PDTmid) 25% A Pregnant Min Decision Threshold (PDTmin)  9% A

(31) In particular the decision algorithm parameters may be employed in the assay device according to example 1 for the detection of hCG. In particular the decision algorithm parameters may be employed in the assay device comprising a shared reference and control zone as illustrated in FIG. 3.

(32) FIGS. 6 (a)-(c) show a typical signal profile of % A vs. time (s) obtained when testing assay devices according to Example 2 having a shared control and reference line according to FIG. 3 for varying levels of hCG in urine. FIGS. 6 (a)-(c) refer to assay devices tested with buffer solution containing respectively 0, 100 and 2000 mIU hCG.

(33) The invention may be further characterised with reference to the following examples:

EXAMPLE 1

(34) Preparation of an Assay Device Comprising a Low Sensitivity Assay and a High Sensitivity Assay for the Determination of hCG Over an Extended Analyte Range.

(35) The high sensitivity assay was prepared for the determination of hCG analyte comprising a mobilisable labelled binding reagent for hCG on a glass fibre porous carrier provided upstream from a detection zone and a control zone provided on a nitrocellulose porous carrier The detection zone comprised immobilised binding reagent for hCG.

(36) The detection zone was prepared by dispensing a line of anti-β-hCG antibody (in-house clone 3468) at a concentration of 3 mg/ml in PBSA buffer, at a rate of 1 μl/cm on onto bands of nitrocellulose of dimensions 350 mm length×40 mm width (Whatman) having a pore-size of 8 microns and a thickness between 90-100 microns which had been laminated to a 175 micron backing layer. The anti-β-hCG antibody was applied using the Biodot xyz3050 dispensing platform as a line ˜1.2 mm in width and ˜300 mm in length at a position of 10 mm along the length of the nitrocellulose.

(37) The control zone was prepared plotting goat-anti-rabbit antibody (Lampire), 2 mg/ml in PBSA buffer at 1 μl/cm onto nitrocellulose at the 13 mm position, 3 mm downstream of the detection zone, using a Biodot XYZ3050 dispensing platform.

(38) The bands of NC were dried using Hedinair drying oven serial #17494 set at 55° C. and speed 5 (single pass). The NC was then blocked using a blocking buffer comprising a mixture of 5% ethanol (BDH Analar 104766P) plus 150 mM Sodium Chloride (BDH Analar 10241AP) plus 50 mM trizma base from (Sigma T1503) plus Tween 20 (Sigma P1379) and 1% (w/v) polyvinyl alcohol (PVA, Sigma 360627). The blocking buffer was applied at a rate of 1.75 μl/mm to the proximal end of the band. Once the blocking solution had soaked into the membrane a solution of 2% (w/v) sucrose (Sigma S8501 in deionised water) was applied using the same apparatus at a rate of 1.6 μl/mm and allowed to soak into the nitrocellulose membrane for ˜5 minutes). The bands of NC were then dried using a Hedinair drying oven serial #17494 set at 75° C. and speed 5 (single pass).

(39) Preparation of the Labelled Binding Reagent for hCG.

(40) Labelled binding reagent was prepared according to the following protocol:

(41) Coating Latex Particles with Anti-α hCG

(42) 1. Dilute blue latex particles from Duke Scientific (400 nm in diameter, DB1040CB at 10% solids (w/v)) to 2% solids (w/v) with 100 mM di-sodium tetra borate buffer pH 8.5 (BDH AnalaR 102676G) (DTB). 2. Wash the diluted latex by centrifuging a volume of (2 mls) of diluted latex in two Eppendorf centrifuge tubes at 17000 rpm (25,848 rcf) for 10 minutes on an Heraeus Biofuge 17RS centrifuge. Remove and discard the supernatant and re-suspend the pellets in 100 mM DTB to give 4% solids (w/v) in a total volume of 1 ml. 3. Prepare a mixture of ethanol and sodium acetate (95% Ethanol BDH AnalaR 104766P with 5% w/v Sodium Acetate Sigma S-2889). 4. Add 100 μls ethanol-sodium acetate solution to the washed latex in step 2 (this is 10% of the volume of latex). 5. Dilute the stock antibody (in-house clone 3299) to give ˜1200 μg/ml antibody in DTB. 6. Heat a volume of 1 ml of the diluted antibody from step 5 in a water bath set at 41.5° C. for ˜2 minutes. Also heat the washed latex plus ethanol-sodium acetate from step 4 in the same water bath for 2 minutes. 7. Add the diluted antibody to the latex plus ethanol-acetate, mix well and incubate for 1 hour in the water bath set at 41.5° C. whilst mixing using a magnetic stirrer and a magnetic flea placed in the mixture. 8. Prepare 40 mg/ml Bovine Serum Albumin (BSA) Solution (Intergen W22903 in de-ionised water). Block the latex by adding an equal volume of 40 mg/ml BSA to the mixture of latex/antibody/ethanol-acetate and incubate in the water bath at 41.5° C. for 30 minutes with continued stirring. 9. Centrifuge the mixture at 17000 rpm for 10 minutes as in step 2, (split the volume into 1 ml lots between Eppendorf tubes). Remove and discard the supernatant and re-suspend the pellet in 100 mM DTB. Repeat the centrifugation as in step 2, remove and discard the supernatant and re-suspend in pellet in Air Brushing Buffer (20% (w/v) Sucrose Sigma S8501, 10% BSA (w/v) in 100 mM Trizma Base Sigma T1503 pH to 9). Add Air Brushing Buffer to give 4% solids (w/v) latex.

(43) The conjugated latex was and sprayed in a mixture of BSA and sucrose onto a glass-fibre porous carrier (F529-09, Whatman) at a rate of 50 g/hr and 110 mm/s and dried using a Hedinar Conveyor Oven Serial number 17494 set at 65° C. and speed 5 (single pass).

(44) Labelled binding reagent for the control zone was also deposited onto the same region of the porous carrier as the labelled binding reagent for the analyte as follows: Rabbit IgG (Dako) was conjugated to 400 nm blue latex polystyrene latex (Duke Scientific) in BSA/sucrose to give a final % blue latex of 0.7% solids and sprayed at 65 g/hr onto glass fibre.

(45) The glass fibre material with sprayed labelled binding reagent was attached to the nitrocellulose membrane using a clear adhesive coated laminate film (Ferrisgate, 38 mm wide) arranged such that the labelled reagent was uppermost and the glass fibre overlapped the surface of the nitrocellulose by ˜2 mm along the length (350 mm) of the band of nitrocellulose membrane. The glass fibre was attached to the end of the nitrocellulose such that it was upstream of the detection zone.

(46) The zone chosen as the reference zone was at a distance of 13 mm along the nitrocellulose, namely downstream of the detection zone.

(47) The laminated sheet was subsequently cut into test-strips of 6 mm width.

(48) The low sensitivity assay for the determination of hCG was prepared comprising a mobilisable labelled binding reagent for hCG and a mobilisable second binding reagent for hCG on a glass fibre porous carrier provided upstream from a detection zone and a control zone provided on a nitrocellulose porous carrier. The detection zone comprised immobilised binding reagent for hCG.

(49) The detection zone was prepared according to that of the high sensitivity assay.

(50) The control zone was prepared plotting goat-anti-rabbit antibody (Lampire), 2 mg/ml in PBSA buffer at 1 μl/cm onto nitrocellulose at the 13 mm position, 3 mm downstream of the detection zone, using a Biodot XYZ3050 dispensing platform.

(51) Preparation of the Mobilisable Labelled and Second Binding Reagents

(52) Mouse-anti-human α-hCG mAb (clone 3299) conjugated to 400 nm blue polystyrene latex (Duke Scientific) was mixed with scavenger antibody mAb mouse anti-human β-hCG (in-house clone 3468) at 3 mg/ml to give a final % blue latex of 3%, a final 3468 concentration of 0.075 mg/ml and 0.06 mg/ml concentration of the free anti-β hCG antibody. The resulting mixture was airbrushed onto Whatman glass fibre (F529 25 mm wide reels) using the BIODOT XYZS (serial number 1673) at 90 g/hr sprayed at 2.02 μg/cm onto F529-09 glass fibre.

(53) Labelled binding reagent for the control zone was also deposited onto the same region of the porous carrier as the labelled binding reagent for the analyte as follows: Rabbit IgG (Dako) was conjugated to 400 nm blue latex polystyrene latex (Duke Scientific) in BSA/sucrose to give a final % blue latex of 0.7% solids and sprayed at 65 g/hr onto glass fibre.

(54) The glass fibre was dried using a Hedinar Conveyor Oven Serial number 17494 set at 65° C. and speed 5 (single pass). A second pass of latex was deposited onto the glass fibre by repeating the above however at an offset of ˜0.8 mm from the original position of spray (further downstream of the glass fibre). The glass fibre was subsequently dried using a Hedinar Conveyor Oven Serial number 17494 set at 65° C. and speed 5 (single pass).

(55) The glass fibre material with sprayed labelled binding reagent was attached to the nitrocellulose membrane using a clear adhesive coated laminate film (Ferrisgate, 38 mm wide) arranged such that the labelled reagent was uppermost and the glass fibre overlapped the surface of the nitrocellulose by ˜2 mm along the length (350 mm) of the band of nitrocellulose membrane. The glass fibre was attached to the end of the nitrocellulose such that it was upstream of the detection zone.

(56) The laminated sheet was subsequently cut into test-strips of 6 mm width.

(57) A common porous sample receiver (505521, Filtrona Fibertech) of 45 mm length, 12 mm width and a thickness of approximately 2.5 mm was provided upstream from and overlapping the first and second assays by approximately 3 mm.

(58) The high and low sensitivity assays were housed in an assay housing with the porous sample partially extending from the housing.

(59) Signals were measured from the respective detection zones and control zone and were measured with respect to the signal measured from the shared reference zone.

(60) As an alternative to providing shared control and reference zones, the high and low sensitivity assay may each comprise a reference and/or control zone.

EXAMPLE 2

(61) The Determination of Threshold Values to be Stored in an Assay Device or Reader in Order to Determine a Date Since Conception.

(62) A number of women aged between 18-45 were recruited into a study and selected on the basis that they were having regular menstrual cycles between 21-42 days over the last 6 months, were not breastfeeding, had no known history of infertility, not suffering from polycystic ovarian syndrome, not suffering from chronic renal or liver disease, and either not to have been using hormonal contraceptives during the past 3 months with at least three cycles since discontinuing. The women provided daily samples of urine over a minimum of six cycles and carried out daily measurements of their urinary LH levels using a Unipath FAM™ monitor to monitor and record the date of their LH surge as well as a diary to record the first day of their last menstrual period. Of the 61 women who became pregnant during the study, their, their urinary levels of hCG were measured on a daily basis up to 90 days post the date of their LH surge using an Perkin Elmer AutoDelphia laboratory analyser. Knowing the date of the LH surge for those pregnant individuals, a data set was generated of the measured level of hCG for each individual woman as a function of the date since conception, wherein the date of conception was defined as the date of the LH surge+1 day.

(63) An exponential curve was used to model log(hCG+0.1) over time of pregnancy,

(64) Wherein:
log(hCG+0.1)=A+B*Rx
A (Max)
B (Diff between intercept and Max)
R (rate of increase)
x=time of pregnancy

(65) The raw data and fitted exponential curve is shown in FIG. 7.

(66) 31 urine samples from non-pregnant women were selected and spiked with hCG at 20 concentrations ranging from 10-250,000 mIU/ml hCG. Each spiked sample was applied to six assay devices chosen from three batches according to Example 1. Analyte signal values (% A) for the high and low sensitivity assays were measured as a function of hCG concentration at an FDT of 150 s.

(67) A four parameter logistic curve was used to model % A for both the high sensitivity assay and the low sensitivity assay over the log(hCG+0.1) concentrations.

(68) Where:

(69) $\%A=A+\frac{C}{\left(1+e^{-B^{*}\left(\mathrm{Log}\left(\mathrm{hCG}+0.1\right)-M\right)}\right)}$
And wherein
B=slope
M=log(hCG+0.1) level that results in a response half way between the minimum and maximum
C=difference between maximum and minimum
A=minimum

(70) The fitted and observed relationships for the high and low sensitivity assays are shown in FIGS. 8(a) and (b).

(71) A simulation study was designed to optimise the PDTmin, PDTmid and EPDTmax thresholds in order to maximize the classification of pregnancies into three groups, <=2 wks since conception, 2-3 wks since conception and >3 wks since conception and to quantify the accuracy of the classification using these thresholds.

(72) The thresholds were assessed against the results of real samples from five pregnant subjects in order to determine their accuracy.

(73) The simulation was performed in three steps:

(74) Step 1

(75) Perform the simulation at a broad range of thresholds. The initial thresholds were chosen using prior knowledge of the device and the boundaries between groups as predicted from the exponential model from the first data set.

(76) PDTmin (%)—5, 7.5, 10

(77) PDTmid (%)—30, 32.5, 35

(78) EPDTmax (%)—43, 45, 47

(79) The choice of 3 values for each threshold provided 27 threshold combinations to be simulated.

(80) Step 2

(81) Pick the best combination of thresholds in Step 1 and look at narrower range of thresholds around the best combination.

(82) Step 3

(83) Choose the best combination of thresholds in Step 2 as the optimal set and do a more refined quantification.

(84) The exponential model from the first data set and the two four parameter logistic models from the urine study (FDT=150 secs) in the second data set were used in the simulation process.

(85) 10,000 post-concept time points were generated from a uniform distribution for each of the following intervals:

(86) 7 to 10 (7 to 9 days)

(87) 10 to 15 (10 to 14 days)

(88) 15 to 22 (2-3 weeks)

(89) 22 to 43 (3+ weeks (up to 42 days));

(90) wherein time is continuous and boundaries are points in time.

(91) For each generated time point, log(hCG+0.1) was simulated. For each simulated log(hCG+0.1) value, a value for the lowtest and hightest % A values were calculated and for each pair of % A values the predicted conception guide outcome was calculated using a set of thresholds.

(92) At step 3 a more refined simulation was performed with 10,000 post-concept time points generated from a uniform distribution for each day from day 7 to day 42. Each sampling interval went from midnight to midnight e.g. day 7 generated data were real numbers between 7 and 8.

(93) Based upon the study, threshold values in % A were calculated for PDTmin, PDTmax and EPDTmax. These threshold values may be stored in the assay device or reader in order to calculate a time since conception.