SYSTEMS AND METHODS FOR TRACKING PROGESTERONE
20200078781 · 2020-03-12
Assignee
Inventors
Cpc classification
B01L2300/025
PERFORMING OPERATIONS; TRANSPORTING
B01L2300/0636
PERFORMING OPERATIONS; TRANSPORTING
B01L2200/148
PERFORMING OPERATIONS; TRANSPORTING
B01L2300/0627
PERFORMING OPERATIONS; TRANSPORTING
B01L2300/027
PERFORMING OPERATIONS; TRANSPORTING
B01L2300/023
PERFORMING OPERATIONS; TRANSPORTING
B01L2300/12
PERFORMING OPERATIONS; TRANSPORTING
International classification
Abstract
Embodiments of the invention comprise a test strip configured to detect one or more metabolites of progesterone in urine, and optionally further detect one or more additional metabolites or hormones in urine within the same test strip. Embodiments of the invention are methods of utilizing such a test strip in association with avoidance of pregnancy, detection of menopause, and in association with fertility planning purposes. Embodiments of the invention are associated with the incorporation of a test strip within a testing kit, optionally further comprising a digital reader.
Claims
1. A test strip, configured to evaluate a sample of urine for the presence of pregnanediol and display an indication of whether the sample of urine contains pregnanediol; wherein the indication consisting of one line indicates a positive result, demonstrating that the sample of urine contains pregnanediol above a pre-defined threshold; and, wherein the indication consisting of two colored lines indicates a negative result, demonstrating that the sample of urine does not contain pregnanediol above a pre-defined threshold.
2. The test strip of claim 1, further comprising: a receiving zone, and a testing zone.
3. The test strip of claim 2, the receiving zone comprising anti-pregnanediol antibodies of the IgG isotype conjugated to a visual dye.
4. The test strip of claim 3, the anti-pregnanediol antibodies of an isotype selected from the group consisting of IgG1, IgG2a, IgG2b, and IgG2c.
5. The test strip of claim 3, wherein the concentration of the anti-pregnanediol antibodies is 1-10 g/ml.
6. The test strip of claim 5, wherein the concentration of the anti-pregnanediol antibodies is 5-9 g/ml.
7. The test strip of claim 3, the anti-pregnanediol antibodies incorporating the ability to bind to other metabolites of progesterone aside from and in addition to PdG.
8. The test strip of claim 7, the other metabolites of progesterone selected from the group consisting of: Pregnanediol Sulfate (PdS), 20(alpha)-hydroxyprogesterone, 20(beta)-hydroxyprogesterone, 5beta-Pregnane-3(alpha), 17(alpha), 20(alpha)-triol pregnanediol diacetate, allopregnanediol, epiallo-pregnanediol, and allo-pregnanediol disulphate.
9. The test strip of claim 2, the testing zone comprising a nitrocellulose membrane configured to comprise two specific reagents bound in sequential order.
10. The test strip of claim 2, the testing zone comprising pregnanediol conjugated to a Globulin carrier protein at a concentration of a value selected from the range of 0.5 g/ml-2 g/ml.
11. The test strip of claim 1, the pre-defined threshold selected from within the range of 3 g/m1-10.
12. The test strip of claim 1, the pre-defined threshold set at 5 g/ml.
13. The test strip of claim 2, further comprising a control line comprising anti-goat antibodies.
14. The test strip of claim 1, the test strip configured for interpretation by a digital reader.
15. A test strip, comprising: a control line; a first testing zone configured to evaluate urine for the presence of PdG; and a second testing zone configured to evaluate urine for the presence of LH.
16. The test strip of claim 15, the first testing zone further configured to evaluate urine for the presence of non-PdG metabolites of progesterone.
17. The test strip of claim 15, further comprising: a third testing zone configured to evaluate urine for the presence of one of the hormones or metabolites selected from the group consisting of E3G, FSH, Testosterone and hCG.
18-24. (canceled)
25. A method of utilizing and interpreting a test strip configured to evaluate urine for the presence of pregnanediol, comprising: applying a urine sample to the test strip, reading the results indicated on the test strip, interpreting the results as either positive or negative, wherein a positive result is indicated by the presence of one colored line on the test strip, and wherein a negative result is indicated by the presence of two colored lines on the test strip, and deciding whether to avoid sexual intercourse based upon the interpretation of the results of the test strip to lessen the risk of pregnancy, and engaging in sexual intercourse following a positive result indicated on the test strip.
26-29. (canceled)
Description
BRIEF DESCRIPTION OF FIGURES
[0048]
[0049]
[0050]
[0051]
[0052]
[0053]
[0054]
[0055]
[0056]
[0057]
[0058]
[0059]
[0060]
[0061]
DETAILED DESCRIPTION
[0062] An embodiment of the invention consists of a test strip 1001 configured to evaluate urine for the presence of pregnanediol (PdG) in a concentration above a pre-defined threshold.
[0063] Aspects of the preferred embodiment of the invention are directed to a pregnanediol (PdG) urine lateral flow assay, also referred to herein as a test strip. Embodiments of the test strip 1001 are characterized by having specific reagent combinations as described further herein, said specific reagent combinations uniquely enabling a strong enough interaction in the testing zone 1002 (also variably referred to as the capture zone) of the test strip 1001 to allow for visual, naked eye inspection of the test results. Embodiments of the test strip 1001 are characterized by having specific reagent combinations as described further herein alternatively described as strong enough interaction in the testing zone 1002 of the test strip 1001 of the test strip 1001 for detection by a reader, said reader optionally forming part of a test kit. In an embodiment, the test kit further comprises a digital reader, optionally consisting of a stand-alone digital reader 2003 as depicted in
[0064] In an embodiment, the test strip 1001 includes a nitrocellulose portion. As the urine sample placed at the distal end of the test strip 1001 opposite the control line 1005 continues along the test strip 1001, the sample next encounters a testing zone 1002. In the example shown in
[0065] After the testing zone 1002, the test strip 1001 may include a control line 1005. The control line 1005 may also generally be referred to as a reference line. When present, the control line 1005 includes anti-mouse antibodies or other proteins that specifically bind the gold conjugated antibody. In embodiments, such configuration may be interpreted to provide a measurement of gold-bound antibody in the fluid that is not specifically bound to the analyte being tested.
[0066] In embodiments of the invention, a digital reader 2001 is configured to measure reflectance measurements from the testing zone 1002. In embodiments, the control line (also variably referred to as the reference line) 1005 may be used separately as a reference to assist the digital reader 2001 to define successful testing and PdG concentrations. In alternative embodiments, the test strip 1001 is configured without a control line, as the digital reader 2001 would have an alternative mechanism to ensure the assay completed correctly. In an embodiment, such alternative mechanism consists of the optical determination by the digital reader 2001 that fluid has passed a predetermined point on the assay indicating completion. In some embodiments, the reflectance of light from the testing zone 1002 may be compared by the digital reader 2001 with the reflectance from the test strip 1001 downstream from the testing zone 1002 in a region where there may or may not be a control line 1005 to define successful testing and PdG concentrations. The present inventor has recognized that a test strip 1001 without a control line or reference line may be advantageous because it eliminates the need for the antibodies at this line, reducing cost of the test strip 1001.
[0067] In the first part of
[0068] In an embodiment, the test strip 1001 comprises a nitrocellulose portion, which may terminate with a second overlapping region. The second overlapping region may serve as a border between the nitrocellulose portion and an absorbent portion of the test strip 1001. The absorbent portion of the test strip 1001 facilitates the uptake of the fluid sample as it arrives at the end of the test strip 1001.
[0069] The configuration of the test strip 1001 in its preferred embodiment comprises at least one testing zone 1002. In an embodiment, the at least one testing zone 1002 is created by striping a PdG-carrier protein conjugate onto the membrane of the test strip 1001. In its preferred embodiment, the test strip 1001 is configured to determine the presence of PdG in urine at a specific pre-defined threshold. The specific pre-defined threshold is implemented to the test strip 1001 by the application, in the proper ratio, of the amount of PdG-carrier protein conjugate to the testing zone 1002 of the test strip 1001, and the anti-PdG-collodial gold conjugate to the receiving zone of the test strip 1001. In the preferred embodiment of the invention, the combination of the components making up the receiving zone and the testing zone 1002 in the proper concentrations as defined herein, enables the test strip 1001 to determine whether a concentration of PdG above a pre-defined threshold exists within a sample of urine applied to the test strip 1001.
[0070] The present inventor has recognized that PdG levels in female urine range from 0.2-30 g/ml throughout the typical menstrual cycle. Prior to ovulation, PdG levels in urine samples range from 0.2-5 g/ml, and however PdG levels in urine samples increase to 5-30 g/ml after ovulation. In an embodiment of the invention, the test strip 1001 is configured with a testing zone 1002 threshold of 5 g/ml, such that when urine containing a concentration of PdG exceeding 5 g/ml is applied to the test strip 1001, the test strip 1001 displays a positive result. In an embodiment, a positive result confirms that ovulation has taken place if the subject of the test is not pregnant. In contrast, in an embodiment, the test strip 1001 is configured such that when urine containing a concentration of PdG below 5 g/ml is applied to the test strip 1001, the test strip 1001 displays a negative result.
[0071] In an embodiment of the invention, the test strip 1001 is configured to determine for the presence of PdG in a sample of urine applied to the test strip 1001 in an amount exceeding 5 g/ml. In embodiments, the PdG-carrier protein conjugate is applied to the testing zone 1002 at a concentration level of a value selected from within the range of 0.5 g/ml-2 g/ml. In an embodiment, the PdG-carrier protein conjugate is applied to the testing zone 1002 at concentration level of 0.7 g/ml.
[0072] In embodiments of the invention, the testing zone 1002 is configured to comprise a progesterone metabolite, optionally pregnanediol (PdG), conjugated to a Globulin carrier protein at a concentration of a value selected from the range of 0.5 g/ml-2 g/ml within the testing zone 1002. In an embodiment of the invention, the Globulin carrier protein consists of Bovine Gamma Globulin (BGG). The present inventor has recognized that the application of the PdG-carrier protein conjugate at the concentration levels described above, when applied to the test strip 1001 in conjunction with the application of the anti-PdG of a specified IgG isotype conjugated to a visual label of the specific concentration levels described herein, accomplishes the proper ratio of those specific binding partners to enable the test strip 1001 to detect for the presence of PdG in a sample of urine applied to the test strip 1001 at the pre-defined thresholds described above and further visually indicate that the sample of urine contains PdG above the pre-defined threshold or visually indicate that the sample of urine does not contain PdG above the pre-defined threshold.
[0073] Embodiments of the present invention are configured measure additional analytes of progesterone in addition to PdG in the testing zone 1002. The present inventor has recognized that testing for and tracking multiple progesterone metabolites as opposed to merely testing for and tracking PdG provides a more accurate understanding of progesterone metabolism generally. The present inventor has further recognized that measuring additional analytes of progesterone (in addition to PdG) provides a more accurate representation of the level of serum progesterone on the test strip 1001 in an embodiment. Thus, in embodiments, the test strip 1001 is configured to measure progesterone metabolites other than and in addition to PdG in the testing zone 1001 and therefore more accurately provides an indication of ovulation. The present inventor has specifically noted that configuring a test strip 1001 by placing an anti-PdG antibody of the IgG isotype, optionally the IgG2b isotype, in the receiving zone facilitates the binding of other metabolites of progesterone aside from and in addition to PdG. Such other metabolites of progesterone include those selected from the list consisting of: sulfate conjugates of progesterone (PdS), 20(alpha)-hydroxyprogesterone, 20(beta)-hydroxyprogesterone, 5beta-Pregnane-3(alpha), 17(alpha), 20(alpha)-triol, pregnanediol diacetate, allopregnanediol, epiallo-pregnanediol, and allo-pregnanediol disulphate. The present inventor has discovered that the antigen binding region associated with anti-PdG of the IgG isotype and its subtypes not only binds to PdG but to additional progesterone metabolites. Therefore, in an embodiment of the invention, the test strip 1001 is configured to detect additional metabolites of progesterone in addition to PdG to provide a more accurate representation of progesterone in serum and represents a significant departure from the prior art.
[0074] Due to its ability to detect multiple metabolites of progesterone, embodiments of the test strip 1001 differ from the prior art as embodiments of the test strip present results that are linear, as depicted in
[0075] In an embodiment, the chosen anti-PdG antibodies of the IgG isotype and its subtypes contain conformational epitopes that allow for the antibody to bind not only PdG, but other metabolites of progesterone as well. The present inventor has discovered that when compared to other antibodies that solely bind to PdG, the chosen anti-PdG antibody of the IgG isotype capable of binding to other metabolites of progesterone produced linear and reproducible test results. Linear results, in association with embodiments of the invention, demonstrate high concentrations of PdG by depiction in the testing zone no visually detectible line; and medium concentrations of PdG by depiction in the testing zone 1002 of a faint line; and low or zero concentrations of PdG by depiction in the testing zone 1002 of a dark line. The linear relationship associated with embodiments of the invention is further depicted in
[0076] In the preferred embodiment, a positive result is indicated by the visual display of one colored line on the test strip 1001, indicating the presence of PdG above the pre-defined threshold within the urine test sample applied to the test strip 1001. In the preferred embodiment, a negative result is indicated by the visual display of two colored lines on the test strip 1001, indicating the absence of PdG above the pre-defined threshold within the urine test sample applied to the test strip 1001. In various embodiments, the test strip 1001 is configured to display a positive indication or a negative indication based on a chosen pre-defined threshold configured by correspondingly altering the concentration level of at least one of the specific binding partners within the testing zone 1002, the receiving zone, or both the testing zone 1002 and the receiving zone.
[0077] The present inventor has recognized that such configuration is a novel improvement over other attempts, including the attempt disclosed in United States Patent U.S. Pat. No. 6,924,153 B1, issued to Quidel on Aug. 2, 2005, because the present embodiment enables the anti-PdG antibodies to bind immediately to the PdG within the applied sample of urine, rather than requiring the PdG to travel to the testing zone 1002 prior to binding as described, for instance, in the Quidel reference. Practically speaking, the present embodiment represents an improvement over the configuration disclosed in Quidel and elsewhere because it reduces the opportunity for other potential binding partners to compete with PdG to bind its antibody, thereby potentially increasing the efficacy of the test strip 1001 in the present embodiment as compared with previous attempts.
[0078] In various embodiments of the invention, the test strip 1001 comprises at least one specially configured receiving zone. In an embodiment, the receiving zone serves to receive a bodily fluid sample which may contain the metabolite of interest and to begin the flow of the sample along the test strip 1001. The receiving zone is prepared from a natural or synthetic porous or macroporous material which is capable of conducting lateral flow of the fluid sample. A porous or macroporous material suitable for purposes of this invention generally has a pore size greater than 12 m. Examples of porous materials include, but are not limited to, glass, cotton, cellulose, nitrocellulose, polyester, rayon, nylon, polyethersulfone, and polyethylene. In an embodiment, the test strip 1001 is configured to comprise an anti-PdG antibody of the IgG isotype conjugated to a visual label at a concentration of a value selected from within the range of 1 g/ml-10 g/ml within at least one receiving zone. In an embodiment, the test strip 1001 is configured to comprise an anti-PdG antibody conjugated to a visual label at a concentration of 7 g/ml within at least one receiving zone. In various embodiments, the visual label consists of colloidal gold. In various embodiments, the colloidal gold particles chosen to make up the anti-PdG antibody-collodial gold conjugate are of a size dimension of a value selected from the range of 20-100 nm. In various embodiments, the colloidal gold particles chosen to make up the anti-PdG antibody-collodial gold conjugate are of a concentration of 0.7-1.3 OD. The present inventor has recognized that the application of the anti-PdG conjugated to a visual label at the concentration levels described above, when applied to the receiving zone of the test strip 1001 in conjunction with the application of the PdG-carrier protein conjugate of the specific concentration levels described herein applied to the testing zone 1002 of the test strip 1001, accomplishes the proper ratio of those specific binding partners to enable the test strip 1001 to detect for the presence of PdG in a sample of urine applied to the test strip 1001, and further visually indicate that the sample of urine contains PdG above the pre-defined threshold or visually indicate that the sample of urine does not contain PdG above the pre-defined threshold.
[0079] Therefore, in various embodiments of the invention, the present inventor has recognized that the test strip 1001 configured as described effectively and reproducibly produces a negative test result if the PdG level in tested urine is below approximately 5 g/ml; and the Test strip 1001 configured as described effectively and reproducibly produces a positive test result if the PdG level in tested urine is above approximately 5 mg/ml. The present inventor recognizes that due to variations in the ability of different populations to metabolize progesterone in urine, the 5 g/ml threshold is not always the appropriate threshold. Therefore, various embodiments of the Test strip 1001 are configured to display a positive or negative result based on pre-defined thresholds of a PdG level of a value selected from values within the range of 3 g/ml-20 g/ml. The present inventor has recognized that embodiments of the invention are appropriately used in association with tracking progesterone levels during pregnancy, since as pregnancy progresses, pregnanediol levels also increase. Therefore, having a predetermined threshold of a higher value (e.g. 10 g/ml-20 g/ml) allows the user to monitor pregnanediol levels through the duration of pregnancy. Such monitoring, with strips configured to measure pregnanediol with a pre-defined threshold from within the range of 10 g/ml-20 g/ml, allows the user to confirm that progesterone levels are increasing appropriately throughout pregnancy, and such configuration is incorporated as a teaching of an embodiment of the invention.
[0080] In embodiments of the invention, PdG is conjugated to a specified Globulin as a carrier protein. The present inventor recognizes that non-globulin carrier proteins as utilized in prior art devices, including especially Bovine Serum Albumin (BSA), fail to enable adequate binding to the anti-PdG antibodies of the isotypes described herein, and thus fail to create an effective and reproducible testing mechanism. In embodiments of the invention, a specialized anti-PDG antibody isotype chosen specifically from the class of IgG isotypes more particularly described below, is conjugated to colloidal gold or similar visual label. The present inventor has recognized that only a highly specific subset of anti-PdG isotypes, said specific subset described further below, functions to provide an effective and reproducible urine testing mechanism for PdG. The invention as described herein, therefore, is presented as a solution to this and other persisting problems associated with previously known urine testing strips.
[0081] Embodiments of the invention comprise a method of creating a reproducible and effective test strip 1001, comprising the following steps. In the first conjugating step, PdG is conjugated to a specified Globulin as a carrier protein, and, separately, a mouse anti-PdG antibody chosen from the group of isotypes including IgG1, IgG1 Kappa, IgG2a, or IgG2c is conjugated to colloidal gold. In the configuring step, to create the Test strip 1001, the PdG-Globulin conjugate is impregnated or striped onto the nitrocellulose membrane in the testing zone 1002 of the test strip 1001. Colloidal gold conjugated anti-PdG antibody is applied or soaked into the receiving zone of the test strip 1001. When aurine sample is applied, the free PDG will bind to the anti-PDG antibody and travel to the testing zone 1002 of the test strip 1001. Any unbound anti-PDG antibody will bind to the testing zone 1002 area and produce a colored line. In this type of competitive assay format, the absence of color in the testing zone 1002 indicates a positive test result for the presence of a progesterone metabolite, and the presence of color in the testing zone 1002 indicates a negative result for the presence of a progesterone metabolite.
[0082] The present inventor has recognized that the use of Bovine Gamma Globulin (BGG), a Globulin, as a carrier protein represents an improvement over the use of other non-Globulin carrier proteins, such as BSA, in other known devices. Thus, embodiments of the present invention utilize BGG as the carrier protein. The present inventor has recognized that BGG has an ability to better bind to colloidal gold and thereby produce a more visually interpretable test result. On the other hand, the present inventor saw a need to create a test that preserves the benefit of BGG without the drawbacks noted in the Echogard et. al. study. Specifically, the inventor notes that the positive characteristics in the context of BGG's ability to not only bind better to colloidal gold, but to also to better present a PdG antigen to an antibody and to have a higher affinity to PdG. The present inventor notes that such advantages exist within the broader category of Globulins as carrier proteins, thus various embodiments of the invention utilize one of the Globulins as a carrier protein. These advantages are especially marked in comparison to the non-Globulin carrier protein BSA.
[0083] The present inventor has discovered that the drawback of utilizing a three zone immunoassay test strip 1001 as disclosed by Quidel for PdG quantification produces inconsistent results and does not meet the current needs of the market. In contrast, the preferred embodiment of the Test strip 1001 described herein, in contrast, is configured as a two zone immunoassay test strip 1001. The present inventor has determined that such a two zone immunoassay test strip 1001 produces more consistent results than strips configured as disclosed in Quidel and are therefore more accurate and reliable. Moreover, the increased simplicity and accuracy associated with the test strip 1001 as described herein as embodiments of the invention provides for increased compatibility and usability with test kits as described herein.
[0084] In the context of assays, the present inventor has recognized that immunoglobulins when used as carrier proteins can potentially bind additional proteins within a sample, creating potentially inconsistent or non-specific assay results for the analyte being evaluted. The present inventor has also recognized that with fewer assay components, the most robust and accurate the test strip 1001 will be. These advantages are viewed in stark contrast to the results observed in the 2013 Ecochard et. al. study, which involved a more complex device as described in the Quidel disclosure. Therefore, the present inventor has devised improvements from the discovered flaws associated with previously utilized prior art which are described in detail as embodiments of the invention disclosed herein.
[0085] The present inventor has discovered that to create a functional and reproducibly reliable urine test strip 1001 able to present clear results to the naked eye, or optionally able to present clear results to an external reader, a suitable anti-PdG antibody able to be conjugated to a visual label such as colloidal gold and/or latex beads, must be incorporated into an embodiment of the invention. The present inventor has discovered that the use of a mouse anti-PdG antibody of a peculiar and specific IgG isotype presents the desired characteristics and therefore is a teaching of the invention. In the preferred embodiment of the invention, the IgG2b isotype of mouse monoclonal anti-PdG antibody is utilized as the binding partner to a visual label. In alternative embodiments, the binding partner to a visual label comprises mouse anti-PdG antibody of the IgG1, IgG1 Kappa, IgG2a or IgG2c isotype. As referred to herein, the IgG2b, IgG1, IgG1 Kappa, IgG2a and IgG2c isotypes are subclasses of the IgG isotype, and are thereby incorporated as teachings of the invention.
[0086] The present inventor has recognized that such a specific combination uniquely allows for colloidal gold to be conjugated to the anti-PdG antibody of one of the specific isotypes mentioned above. Other combinations have been attempted, such as that described in Patent Application WO2016142610A1, which have failed to allow the colloidal gold to reproducibly function to produce the color needed to allow the test results to be viewable visually by the naked and untrained (layperson) eye. The present inventor has noted that the utilization of a Globulin conjugated to PdG allows for anti-PdG antibody of an immunologically active IgG isotype, to bind in such a manner that colloidal gold is carried at a concentration sufficient for naked eye visualization, and is therefore a teaching of embodiments of the invention. The present inventor notes that Globulins evidence the preferable binding ratio of 8-32 PdG antigens per Globulin, which favor presentation of a visual result perceptible to the naked eye or to a reader, and is therefore a teaching of embodiments of the invention. The present inventor has recognized the benefit associated with embodiments of the invention described herein that a PdG test may be producible allowing the results to be visually interpreted with the naked eye.
[0087] Embodiments of the present invention comprise a Test strip 1001 configured to detect the presence of PdG in urine. In embodiments, the Test strip 1001 is optimized for visual detection by a layperson's, or non-expert's, naked eye while utilizing the Test strip 1001 in a non-laboratory context in association with intended purposes as described herein. The present inventor has recognized that in embodiments of the invention, the Test strip 1001 is configured to comprise a combination of a mouse anti-PdG antibody of the IgG1, IgG2a, IgG2b, or the IgG2c isotype conjugated to a visual label, such as colloidal gold and/or latex beads, and PdG conjugated to an Globulin carrier protein, optionally BGG.
[0088] In embodiments of the invention, the carrier protein comprises one of the following human, non-human, or plant globulins: vicilin, legumin, casein, Alpha 1-antichymotypsin, seruam amylid A, Alpha 1-lipoprotein, Haptogolulin, Alphy 2-antiplasmin, Protein C, Angiotensinogen, cortisol binding protein, beta-2 microglobulin, plasminogen, angiostatins, sex-hormone-binding protein, transferrin, fibronectin, microglobuln, gamma globulin, thyroglobulin, 11S globulin family, 7S family of globulins. In various embodiments, the Globulin serving as the carrier protein derives from a plant or animal source, including an animal source such as human, mouse, rat, bovine, equine, goat, or rabbit. The present inventors has recognized that the resulting an embodiment of the invention configured as described herein comprises a visual test strip 1001 readable by the untrained eye in a context outside of a laboratory environment, as depicted in
[0089] The preferred embodiment of the current invention comprises a test strip 1001 comprising at least a testing area. Said testing area comprises a Globulin carrier protein conjugated with PdG, which is combined with an immunologically active mouse anti-PdG antibody of IgG2b isotype as a binding partner conjugated to colloidal gold. The conjugation of the Globulin carrier protein to the immunologically active mouse anti-PdG antibody takes place in association with methods known in the prior art, including methods known in United States Patent U.S. Pat. No. 6,924,153 B1, issued to Quidel on Aug. 2, 2005, which is hereby incorporated by reference in its entirety. The present inventor notes that such configuration preserves the ability associated with Globulins more generally to better present PdG antigen to an antibody, a higher affinity to PdG and better conjugation ratios of PdG to carrierespecially in comparison to the use of BSA as a carrier protein. Specifically, the present inventor has recognized that Globulin carriers more generally demonstrate the favorable conjugation ration of 8-32 antigens per one Globulin.
[0090] Such characteristics of Globulins specifically address the shortcomings identified with relation to the art described in PCT/FR2016/050506 published on Mar. 4, 2016, which discloses Bovine Serum Albumin (BSA) as the carrier protein utilized in association with a test for evaluating urine for PdG. The test disclosed in such does not have the ability to bind to colloidal gold, thereby resulting in a progesterone test that delivers results that are problematically imperceptible to the naked eye. In contrast, the present inventor has discovered that the urine PdG test disclosed herein utilizing a Globulin as the carrier protein also solves another related problem associated with prior art test attempts that utilize BSA, namely, that they are impractical for use with some readers that utilize light interpretation to determine the results for the test, because such tests incorporating BSA as a carrier protein fail to reliably produce enough color intensity to deliver perceptible test results.
[0091] The present inventors have recognized the benefit of a configuration of the preferred embodiment of the invention, embodied as a test strip 1001 configured to simultaneously analyze urine beyond mere analysis for the presence of pregnanediol, by further analyzing up to six (6) analytes and/or hormones in an alternative test strip 1001 configuration featuring a testing zone 1002 configured to evaluate urine for the presence of PdG beyond a specific threshold and optionally additional testing zones. In an embodiment, the test strip 1001 comprises a testing zone 1002 configured to evaluate urine for the presence of PdG beyond a specific threshold, a second testing zone 1003 and a third testing zone 1004. It is a teaching of an embodiment of the present invention for the test strip 1001 to optionally incorporate one or more additional testing zones beyond the testing zone configured to analyze urine for the presence of PdG, each additional testing zone specifically configured to evaluate urine for the presence of an item selected from the group consisting of LH, HCG, FSH, Testosterone and/or Estrogen or analytes thereof, such as E3G.
[0092] In an embodiment, the test strip 1001 is configured to simultaneously indicate, in a testing zone 1002, a positive or negative result for the presence of PdG above a pre-set threshold in a sample of urine applied to the test strip 1001, in addition to indicating, in a second testing zone 1003, a positive or negative result for the presence of at least one additional analyte and/or hormone, and, optionally, indicating in an additional third testing zone 1004, a positive or negative result for the presence of at least one additional analyte and/or hormone, all contained within a single test strip 1001. In an embodiment of the invention, the at least one additional analyte and/or hormone to be tested within the second testing zone 1003 and the at least one additional analyte and/or hormone to be tested within the third testing zone 1004 is selected from the following group: (1), Estradiol (E2) at concentrations 25-250 pg/ml in a competitive assay format; (2), Follicle Stimulating Hormone (FSH) at concentrations 3-20 mlU/ml in a sandwich assay format; (3), Luteinizing Hormone (LH) at concentrations 0-25 mlU/ml in a sandwich assay format; (4), Progesterone (P4) at concentrations of 0-40 ng/ml in a competitive or sandwich assay format; (5) human chorionic gonadotropin, (hCG) at concentrations of 0-10,000 mlU/ml; and Testosterone, at concentrations of 0 to 50 g, in a sandwich assay format. In an embodiment, the analyte and/or hormone that the test strip 1001 will simultaneously measure in at least a second testing zone 1003 and optionally a third testing zone are selected from the group consisting of E2, FSH, LH, P4, Testosterone and HcG. In an embodiment, the second testing zone 1003 is configured to detect for the presence of a hormone or analyte differing from the hormone or analyte detected by the third testing zone. In an embodiment, a digital reader 2001 is configured to evaluate the second testing zone 1003 and optionally the third testing zone 1004 in association with the methods further described herein. In an embodiment, the test strip 1001 further incorporates, in addition to a testing zone 1002, a second testing zone 1003, and a third testing zone 1004, a fourth testing zone configured to similarly detect for the presence of a hormone or analyte differing from the hormone or analyte detected by the other (first, second and third) testing zones within the test strip 1001. The methods of evaluation and further configurations optionally applied to the test strip 1001 associated with the testing of analytes and/or hormones beyond and in addition to pregnanediol are further described in U.S. patent application Ser. No. 15/974,229, filed on May 25, 2018, which is hereby incorporated by reference in its entirety.
[0093] In an embodiment of the invention, labels (such as colloidal gold) are varied, with a separate and distinct label configured to attach to a separate hormone. In such embodiment, the present inventor has recognized the advantage that the test strip 1001 is configured to provide a different color for each hormone analyte indicating either the presence or absence of each hormone analyte following application of urine to the test strip 1001. In an embodiment, the test strip 1001 is configured to comprise a conjugate pad (the conjugate pad also referred to as the receiving zone herein) comprising anti-PdG antibody-collodial gold conjugate, and at least one other conjugate. In an embodiment, the at least one other conjugate comprises anti-LH antibody-conjugated with a different label, optionally differently colored latex beads.
[0094] The present inventor has recognized that LH and HCG commonly exhibit cross-reactivity, specifically due to the fact that HCG can bind to LH antibodies. Therefore, having different colors corresponding to the presence of different hormones provides a benefit by allowing an observer to determine whether cross-reactivity has taken place. In other words, if an area designated to test for the presence of LH displayed the coloration of the label for HCG, one skilled in the art would understand such a read to indicate that cross-reactivity has been demonstrated and that the test therefore is invalid. Alternatively, the present inventor has noted that due to the similarities in structure between estrogen analytes and progesterone analytes (in at least one example, said progesterone analytes consisting of PdG), cross reactivity could take place between those two hormone metabolites specifically. Thus, it is beneficial to have different hormones or hormone metabolites labelled with different colors. Such labelling is accomplished in an embodiment by binding to colloidal gold and/or one or more differently colored latex beads, each testing zone within the strip featuring a differently colored label, and is therefore a teaching of an embodiment of the invention.
[0095] The present inventor has discovered that because PdG is a small hormone metabolite, in order to strongly bind to the surface of a membrane, PdG requires a strong carrier protein, which is a teaching of an embodiment of the invention. However, the present inventor has discovered that, for the preferred embodiment of the invention to function as intended, not only does the strong carrier protein need to bind the nitrocellulose membrane of the Test strip 1001, but the strong carrier protein also needs to bind the PdG and present it to the anti-PdG antibody, which is a teaching of an embodiment of the invention. Such teachings as disclosed herein, solve the challenges associated with suboptimal prior art teachings, which lacked the ideal combination of a strong carrier protein able to bind the PdG and present it to the anti-PdG antibody.
[0096] In accord with teachings of the invention, the test strip 1001 relies on the certain reagents being able to interact with other reagents to produce color in the testing zone 1002 of the membrane. Specifically, in the absence of PdG hormone in the urine sample, the following reagents must interact in order for the test results to be useful. First, in the preferred embodiment, colloidal gold must be conjugated to the immunologically active anti-PdG antibody of one of the specific IgG isotypes described elsewhere herein. In alternative embodiments, as a replacement for colloidial gold in other embodiments described herein, an alternative visual dye such as latex beads may be utilized to a similar effect. Further, in embodiments of the invention, the colloidal gold conjugated anti-PdG antibody must interact with the PdG-Globulin conjugate. Moreover, the PdG-Globulin must bind a nitrocellulose membrane. The present inventor has recognized that for these embodiments to function as intended, these interactions between and among the colloidal gold conjugated anti-PdG antibody and the PdG-Globulin conjugate, must be strong enough and stable enough to form and stay bound during urine sample application and lateral flow of urine across the reaction zone to solve the problems faced by the suboptimal prior art mechanisms described elsewhere herein. The disclosures in this paragraph constitute teachings of an embodiment of the invention.
[0097] In association with teachings of the invention, the test strip 1001 is configured to comprise a conjugate of Globulin with PdG. Such PdG-Globulin conjugate is combined with a mouse anti-PdG antibody of one the class of the IgG isotypes in an embodiment. The class of Ig isotypes includes IgG1, IgG2b, IgG1 Kappa, IgG2a or IgG2c isotype as contemplated in association with embodiments of the invention. The conjugation of a selection from within of a class of Globulins to PdG, and the combination of the PdG-conjugated Globulin with a mouse anti-PdG antibody of Ig isotype is accomplished in accord with general conjugation procedures as well-known by those skilled in the art.
[0098] It is a further teaching of the invention that in order for the preferred embodiment of the invention to function as intended, the specifically chosen anti-PdG antibody needs to be monoclonal, due to the nature of the PdG antigen presentation on the PdG-Globulin conjugate. In order for the embodiments of the invention to function as intended, the specifically chosen anti-PdG antibody must incorporate one of the following isotypes: IgG1, IgG2a, IgG2b, or IgG2c. The present inventor has discovered that isotypes other than IgG1, IgG2a, IgG2b, or IgG2c, including but not limited to IgM, IgS, and IgE anti-PdG antibody isotypes, remain unable to effectively bind the colloidal gold (or other visual label) and produce a strong enough color signal on the reaction zone due to their size and structure and are therefore excluded from the preferred embodiment of the invention. Since the colloidal gold must bind the Ig region of the anti-PdG antibody, the present inventor has discovered that the IgG1, IgG2a, IgG2b, and IgG2c isotypes of the anti-PdG antibody sufficiently bind colloidal gold and are therefore incorporated into embodiments of the invention. As a result, the IgG1, IgG2a, IgG2b and IgG2c isotypes of the anti-PdG antibody therefore produce the strongest color. In the preferred embodiment of the invention, the IgG2b isotype is included in the invention, as the present inventor has recognized that the IgG2b isotype performs slightly better when producing color. Therefore, the preferred embodiment of the invention incorporates the IgG2b isotype of the anti-PdG antibody. Alternative embodiments of the invention incorporate the IgG2a, IgG2c or IgG1 isotypes of the anti-PdG antibody.
[0099] The present inventor has recognized that the utilization of a Globulin within a specific combination uniquely allows for colloidal gold to be conjugated to the immunologically active anti-PdG antibody of one of the class of the IgG isotypes. In an embodiment, the combination enables the colloidal gold conjugated anti-PdG antibody to interact with the PdG-Globulin conjugate. In embodiments of the invention, therefore, the conjugate striped on the membrane in the testing area is PdG-Globulin and the anti-PdG antibody must be a monoclonal anti-PdG antibody of one of the following isotypes: IgG1, IgG2a, IgG2b, or IgG2c.
[0100] The present inventor recognizes that embodiments of the invention differ from other combinations that have been attempted in the prior art. Specifically, the other combinations have failed to allow the colloidal gold to function to produce the color needed to allow the test results to be viewable visually by the naked and untrained (layperson) eye. The present inventor has recognized that the novel utilization of a Globulin conjugated to PdG as described herein allows for anti-PdG antibody specifically of the Ig isotype, in accordance with the specific concentration levels described herein in embodiments of the invention, to bind in such a manner that colloidal gold is carried at a concentration sufficient for naked eye visualization. The present inventor has recognized the benefit associated with embodiments of the invention that a PdG test may be producible allowing the results to be visually interpreted with the naked eye and/or an external reader affordable to a typical consumer.
[0101] In embodiments of the invention, one carrier protein is conjugated to eight or more PdG molecules. In the preferred embodiment, the one carrier protein is conjugated to no more than thirty two PdG molecules. The present inventor has discovered that such a ratio allows for the colloidal gold conjugated anti-PdG antibody to bind with both enough affinity and avidity to produce a bright enough color in the test reaction zone for typical users to distinguish visually. The present inventor has discovered the specific property of Globulin enabling such combination. In embodiments of the invention, as Globulin exhibits the optimal number of active sites optimally spaced, the inclusion of Globulin results in a lesser amount of steric hindrance, and therefore embodiments of the invention are enabled to receive and bind PdG at sufficient ratios. Therefore, Globulin is essential for the preferred embodiment of the invention to function as intended. In the preferred embodiment of the invention, therefore, PdG is conjugated to a Globulin.
[0102] In association with teachings of the preferred embodiment of the present invention, a testing system to detect the presence of PdG is optimized for visual detection by a layperson's, or non-expert's, naked eye utilizing the embodiment in a context other than a professional laboratory environment. The present inventor has recognized that in embodiments of the invention, the combination of mouse anti-PdG antibody of an Ig isotype conjugated to a visual label, such as colloidal gold and/or latex beads, and PdG conjugated Globulin as a carrier protein create sufficient binding partners to enable a test strip 1001 capable of visual inspection, or inspection by a reader as further described herein. Resultantly, the preferred embodiment of the invention comprises a visual test readable by the untrained eye in a context outside of a laboratory environment, as depicted in
[0103] In an embodiment of the invention, the method of creating the test strip 1001 comprises the following steps:
[0104] Selecting an immunologically active PdG antibody of a specific isotype. In the preferred embodiment, the specific PdG antibody chosen is anti-PdG antibody of the IgG2b isotype. In alternative embodiments, the specific anti-PdG antibody chosen is one of the anti-PdG antibodies of either the IgG1, IgG2a, or IgG2c isotypes.
[0105] Conjugating the selected anti-PdG antibody to visual dye. Such step transforms the chosen immunologically active anti-PdG antibody into a visually labeled anti-PdG antibody. In the preferred embodiment, the visual dye consists of colloidal gold of a size value selected from within the range of 20-100 nm in diameter. In alternative embodiments, the visual dye comprises colloidal gold or colored latex beads.
[0106] Saturating the conjugate pad with the visually labeled anti-PdG antibody. In embodiments of the invention, the visual labelization takes place as a result of conjugating a anti-PdG antibody to a visual dye, such as colloidal gold.
[0107] Conjugating PdG to a Globulin carrier protein at a ratio of eight (8) or more units of PdG per 1 unit of Globulin. This ratio of units of PdG to units of Globulin constitutes a teaching of embodiments of the invention. In the preferred embodiment, the chosen Globulin carrier protein is BGG. In various embodiments, the Globulin is chosen from the list comprising: human, non-human, or plant globulins: vicilin, legumin, casein, Alpha 1-antichymotypsin, seruam amylid A, Alpha 1-lipoprotein, Haptogolulin, Alphy 2-antiplasmin, Protein C, Angiotensinogen, cortisol binding protein, beta-2 microglobulin, plasminogen, angiostatins, sex-hormone-binding protein, transferrin, fibronectin, microglobulin, gamma globulin, thyroglobulin, 11S globulin family, 7S family of globulins. In varying embodiments of the invention, globulins are chosen from plant or animal sources. Animal sources may include any from the list of: human, mouse, rat, bovine, equine, goat, or rabbit.
[0108] Impregnating PdG conjugated to the Globulin onto a test strip 1001 membrane at a chosen concentration level selected from the range of 0.5-2 mg/ml in a testing zone 1002, and
[0109] Impregnating anti-mouse antibodies into the control line 1005 area.
[0110] In the preferred embodiment, a test strip 1001 results from the above method.
[0111] The present inventor intends for the test strip 1001 to be used in a variety of scenarios and in association with a variety of methods. Such scenarios include utilization of the test strip 1001 as part of a method of quantifying and tracking PdG levels throughout the menstrual cycle. In accord with such method, the user will collect urine samples on different days of her menstrual cycle and use a test strip 1001 with each first morning urine, as depicted in
[0112] Furthermore, the present inventor recognizes that if conception occurs, progesterone and PdG levels remain elevated throughout the duration of the pregnancy in a healthy pregnancy. Therefore, in association with a method of evaluation of whether the progesterone levels are such that a pregnancy may remain healthy, the test strip 1001 is intended to be utilized in association with a method of checking levels of progesterone during the pregnancy.
[0113] The test strip 1001 has other useful hormone monitoring uses. For example, the ovaries produce progesterone in large concentrations after ovulation has taken place. Therefore, lack of progesterone production over several weeks or months could indicate the start of menopause. Low amounts of progesterone could indicate an ovarian dysfunction such as failed ovulation attempt, luteinized unruptured follicle (LUF), or poor ovulation. Low or abnormal progesterone levels could also indicate a luteal phase defect. Additionally, in certain cancers such as breast, ovarian, and uterine, cancer cells are responsive or can secrete progesterone. In other methods of use, a female could utilize a test strip 1001 in association with pregnancy avoidance purposes. Specifically, a test strip 1001 showing a positive result indicates elevated PdG levels in urine, thereby indicating that the subject woman has ovulated and therefore entered her infertile phase.
[0114] In accordance with teachings of the presently contemplated test strip 1001, the present inventor has discovered a configuration including a unique combination of specific elements to allow for the detection of PdG in urine when applied to the test strip 1001 as further described herein. Such configuration allows a user to utilize the test strip 1001 determine whether PdG above a threshold amount is present in a urine sample applied to the test strip 1001. The present inventor has observed that the novel configuration presented herein demonstrates high specificity to PdG. The present inventor has recognized that the high specificity in association with PdG occurs due to the unique chemistry of the test strip 1001 especially in comparison to other attempts. Thus, the preferred embodiment of the test strip 1001s effectively and reproducibly provides an indication of whether a female has produced progesterone above a pre-defined threshold level.
[0115] In embodiments, the invention is characterized as an improvement to test kits known in the art. In such embodiments, the test strip 1001 as described herein is incorporated within a test kit in association a digital reader 2001.
[0116] A variety of test kits are contemplated for the application of the test strip 1001 described herein constituting an improvement thereof, which non-exhaustively may include the Clearblue Easy Fertility Monitor (CBEFM) that incorporates a means to interpret testing results and a digital display, which without a test strip 1001 configured to evaluate urine for pregnanediol (as further described herein) merely provides a method for monitoring the fertility status of an individual using two alternative hormones other than PdG: LH and E3G. Moreover, the test strip 1001 described herein is intended for use in association with a modified version of the Clearblue Digital Ovulation Test (CDOT), which without a test strip 1001 configured to evaluate urine for pregnanediol (as further described herein) merely employs a variable threshold for LH surge determination. Moreover, the test strip 1001 configured to evaluate urine for pregnanediol (as further described herein) is intended for use in association with a modified version of the First Response Advanced Digital Ovulation Test, which otherwise merely predicts the onset of ovulation by measuring LH and displaying a result. An embodiment of the invention comprises the CDOT or the First Response Advanced Digital Ovulation Test configured to comprise a test strip 1001 configured to evaluate urine for progesterone as described herein. In an embodiment, a digital reader 2001, optionally consisting of a stand-alone digital reader 2003, is further configured to read, store and present the results of such a test strip 1001. In an embodiment, a stand-alone digital reader 2003 is configured to wirelessly communicate with a smartphone digital reader 2002, optionally via Bluetooth, to store and present results from a test strip 1001 as collected by the stand-alone digital reader 2001 in association with an app operated by the smartphone digital reader 2002. The First Response Advanced Digital Ovulation Test, the CDOT, CBEFM, and similar devices are referred to as stand-alone digital readers 2003 as referred to herein. A stand-alone digital reader 2003 may comprise an input configured to read a test strip 1001 incorporated into a cartridge (also referred to herein as a test stick 2004). Examples of stand-alone digital readers 2003 are depicted in
[0117] Further, the test strip 1001 configured to evaluate urine for pregnanediol as further described herein is intended for use in association with a variety of other digital readers, including a mobile device based system (such as a smartphone) that utilizes a camera 2005 to interpret the results of a lateral flow assay and which may utilize a variety of apps that track menstrual cycles. Embodiments of an improved test kit incorporating the test strip 1001 configured to evaluate urine for pregnanediol, and optionally other metabolites of progesterone, as further described herein are described below. The present inventor contemplates that additional configuration of test kits incorporating digital readers, aside from mere inclusion of the test strip 1001 configured to evaluate urine for the presence of pregnanediol, optionally and other metabolites of progesterone, is necessary to enable functionality and usefulness, in accordance with the methods and configurations as described herein.
[0118] An embodiment of the invention comprises a method of evaluating a subject's urine for the presence of PdG, such method including the steps of contacting a urine sample collected from a subject, typically a woman, with a test strip 1001. The method of evaluating urine for the presence of PdG includes a detecting the presence of PdG step, comprising determining that PdG of a concentration above a pre-defined threshold is present within the subject woman's urine by visually determining that one colored line is displayed on the test strip 1001, or determining that PdG of a concentration below a pre-defined threshold is present within the subject woman's urine by visually determining that two colored lines are displayed on the test strip 1001. During such method, during the detecting the presence of the PdG step, the visual display of one colored line on the test strip 1001 indicates that progesterone levels in the blood are elevated, and that ovulation has occurred in a non-pregnant woman. During such method, during the detecting the presence of the PdG step, the presence of two colored lines on the testing strip (including one colored line viewable within the testing area of the test strip 1001) indicates that progesterone levels in the blood are not elevated, and therefore indicates that ovulation has not occurred.
[0119] An embodiment of the invention comprises steps for determining whether a woman is in an infertile period by visually inspecting a test strip 1001 with the naked eye following the application of a sample of urine to the test strip 1001. In association with such method, the user may interpret the non-occurrence of ovulation, as indicated by a negative result on the test strip 1001, as a potentially fertile period.
[0120] An embodiment of the invention comprises steps for determining whether a pregnant woman has produced enough progesterone to sustain pregnancy. Said method comprises the steps of contacting a urine sample of women with a Test strip 1001 configured as further described herein, and detecting the presence of PdG by the absence of color in the testing area and the presence of color on a control line. During such method, during the detecting the presence of the PdG step, the absence of color in the testing area indicates that progesterone levels in the blood are elevated, and that a sufficient amount of progesterone has been produced to sustain a pregnancy. During such method, during the detecting the presence of the PdG step, the presence of color in the testing area indicates that progesterone levels in the blood are not elevated, and therefore indicates that a sufficient amount of progesterone may not have been produced to sustain a pregnancy. Following a result demonstrating that progesterone levels in the blood are not elevated, the subject of the test may pursue a mitigating step, whereby during the step of mitigating the subject of the test engages in the step of additional blood (serum) testing to determine with precision the specific level of progesterone in her blood, and/or a supplementing step to ingest, inject or otherwise add progesterone to her bloodstream in accordance with mechanisms known by those skilled in the art.
[0121] In an embodiment, the test strip 1001 is preferably utilized in accordance with the following steps: First, applying a urine sample 3001 to the test strip 1001. In association with the present method as depicted by
[0122] In an embodiment of the invention, a positive result indicated on the test strip 1001, following the application of a sample of urine collected from a woman, determinable by the presence of only one visually perceptible colored line indicates that the woman whose urine was applied to the test strip 1001 was within her infertile period at the time the urine sample was applied to the test strip 1001. The present inventor intends for such information intended to be used in association with pregnancy avoidance purposes in a method of avoiding pregnancy while still optionally engaging in sexual intercourse, as depicted in
[0123] An embodiment of the invention comprises steps for determining whether menopause has occurred, as depicted in
[0124] An embodiment of the invention comprises steps for determining whether progesterone supplementation is effective, as depicted in
[0125] An alternative embodiment of the invention comprises a system comprising a test strip 1001 and an external reader. In varying embodiments, such a system may be described as or otherwise form a portion of a test kit.
[0126] In an embodiment, the external reader is configured to evaluate a test strip 1001. Following evaluation, the external reader is configured to display a positive result after evaluation of the test strip 1001 upon its determination that the testing zone of the test strip 1001 either does not contain any line of any luminance or color value, or otherwise contains a line perceptible only below a specified color value or luminance threshold as interpreted by an external reader.
[0127] In association with teachings of the invention, the test strip 1001 described herein may be utilized as part of a test kit incorporating a digital reader 2001. In an associated method, a first step of collecting urine takes place, wherein the user engages in a collecting step to collect at least one urine sample, or optionally a plurality of urine samples, each collected on different days of her menstrual cycle. For each sample collected, the user then engages in an applying step, where the user applies urine from the collected urine sample to a test strip 1001. The user then engages in an imaging step, wherein the user utilizes a digital reader 2001, optionally comprising a tablet computer or smartphone incorporating a built-in camera, to optically evaluate the at least one testing zone of the test strip 1001. The digital reader 2001 is configured, optionally by a software application, to perform a step of interpreting the results of either positive or negative by evaluating the presence of a line within the testing zone, or alternatively the intensity of the color or luminescence of the testing zone. Optionally, the digital reader 2001 is configured to compare the results evaluated from within the testing zone to the control line and/or the naked membrane, each of which optionally serves as a background measurement. During the interpretation of the results, the digital reader 2001 optionally generates a signal representative of the pregnanediol level indicated on the at least one test strip 1001.
[0128] In associated methods, a plurality of test strips 1001 are utilized during steps of quantifying and tracking pregnanediol levels throughout the menstrual cycle. In such methods, when such quantifying and tracking steps are performed in association with an external digital reader, each result may be compared to the other to track trends and map the user's menstrual cycle in association with steps and applications well known to those skilled in the art. In such way, the digital reader 2001 is configured to store a predetermined threshold associated with the level of pregnanediol, as previously collected pregnanediol levels remain in storage on the digital reader. In an embodiment, the associated methods include the methods disclosed in U.S. Patent Application 62/611,467 filed on Dec. 28, 2017, which is hereby incorporated by reference in its entirety.
[0129] Optionally, the comparing step is performed by the digital reader, whereby the color intensities of the detection area are compared to other readings taken over time, and the software application is configured to determine significant increases or decreases in PdG concentrations. In an embodiment, the digital reader 2001 comprises a circuit configured to store at least one predetermined pregnanediol concentration threshold, generate a signal representative of the pregnanediol level indicated on at least one test strip 1001, compare the signal to a threshold and display a message indicating the result, as described further herein.
[0130] In an embodiment, a quantifying step follows, wherein the software application quantitates the concentration of PdG in the sample by comparing the intensity of the testing zone with the intensity of a known standard curve of PdG concentrations and line intensity. A displaying step then follows, where the software application presents a display of the test results on a screen incorporated into the digital reader, which may optionally include the word ovulation in association with a positive test and no ovulation in association with a negative test, on a screen perceptible to the user as the message indicating the result. As an alternative to the word ovulation, the word infertile, the phrase safe to have sex, or a similar phrase may be displayed as the message indicating the result.
[0131] In most cases, diagnostic devices known in the art when evaluating hormones other than progesterone rely on the individual's hormone level to be either high or low relative to a fixed threshold value. In embodiments of the diagnostic devices described herein making use of the test strip 1001, an associated test kit comprises a test strip 1001 specially configured to be capable of evaluating urine for at least the presence of pregnanediol at or above a threshold level. When pregnanediol is determined as present in excess of the threshold level, it is a teaching of the invention to display a result related to high, which optionally may include the word ovulation and/or positive as the message indicating the result. When progesterone is determined to be below this level, it is a teaching of the invention to display a result related to low, which optionally may include the words no ovulation and/or negative display a message indicating the result. In an embodiment of the invention, the test strip 1001 is configured to evaluate urine for at least the presence of PdG at 5 g/mL or greater. The configuration is so determined by the ratio of PdG-Globilun embedded into the testing zone to labeled anti-PdG antibody embedding into the test receiving zone. A positive or high progesterone reading will occur when color in the test receiving zone lighter than a certain threshold or no color exists. It has been found that many individual subjects do not conform to the average in terms of basal circulating hormone levels, cycle length and/or the duration of the cycle. Furthermore, variations can extend from one cycle to another in the same individual, making the use of a fixed threshold of PdG concentration as indicitave of a positive result for the entire population problematic. Thus, it is a teaching of the invention to modify the pregnanediol concentration threshold to accommodate populations for particular uses, or to store more than one pre-determined thresholds linked to specific populations on an associated digital reader, in accordance with the disclosures elsewhere herein.
[0132] In the CBEFM device described herein, which constitutes an example of a stand-alone digital reader 2003 in association with the disclosures herein, the user will monitor their urine sample over multiple menstrual cycles. The monitor stores the data, compares readings day to day, and identifies the days of maximum fertility. However, the CBEFM devices described above does not have the capability evaluate urine for the presence of PdG. These drawbacks are resolved in the inventive embodiments described herein, and it is a teaching of the present invention to apply the test strip 1001s to the CBEFM with modifications readily apparent to those skilled in the art, and utilize the CBEFM as a device for evaluation of the strips and the presentation of the results of the strips on a display associated with the CBEFM.
[0133] It is a teaching of the invention to incorporate a test strip 1001 into a test kit. A variety of examples for the implementation of the test kit comprising the specially configured single rest strip 1001 capable of evaluating urine for at least the presence of PdG are described herein. In one implementation, a test kit may include two units, a stand-alone digital reader 2003 and a package that contains multiple disposable test sticks. Because the test method can be effective in a single cycle, the kit may include only enough disposable test sticks 2004 for a single cycle of tests, for example, about 20. The reader may be activated mechanically, more preferably, activation is achieved by the change of the light reflectance of the background with or without the test stick 2004. The reader may also be designed to be activated by inserting a single-use disposable test stick 2004 and to measure the color development at the detection area of the test strip 1001 after a urine sample is applied. At the completion of the test, the test result may be converted to an electronic or digital output, viewable on a display 2010. The disposable test stick design 2004 may be based on lateral flow technology and contain the specially configured Test strip 1001 to evaluate urine for the presence of PdG.
[0134]
[0135]
[0136] An embodiment of the stand-alone digital reader 2003 may incorporate a printed circuit board. The printed circuit board includes one or more sensors. In an embodiment, the printed circuit board includes two optical sensors, configured to serve identical functions as a camera 2005 as described elsewhere herein. In this implementation, the sensors may be phototransistors. In other implementations, the sensors may be one or more photodiodes, electroactive sensors or radioactivity sensors. The sensors may be of the same or different types. The sensors are coupled with the processor chip.
[0137] The printed circuit board may include an emitter. In an implementation including photoelectric sensors, the emitter may be a light source such as a light emitting diode (LED). In an implementation including photoelectric sensors, the light source may be located equidistant between the photoelectric sensors. The light source may be coupled with the processor chip. The light source may illuminate according to a configurable pattern. In an implementation where the light source is coupled with the processor chip, the illumination pattern may be controlled by the processor chip. In an implementation where the light source is not coupled with the processor chip, the illumination pattern may be controlled by a separate timing circuit. In such embodiment, the light source allows the photoelectric sensors to read a test strip 1001 contained within a test stick 2004, and thereby perform identical functions to those performed by the camera 2005 as described elsewhere herein.
[0138] In an embodiment, as the emitter illuminates the test strip 1001, the sensor may detect a response from the illumination. For example, in an implementation where the emitter is a light source, the photoelectric sensor will detect the amount of light reflected by the test strip 1001. The test strip 1001 is configured to provide a positive result for at least the presence of PdG in urine by displaying the absence of a dark line in the testing zone 1002 that does not bind to colloidal gold in the preferred embodiment. Therefore, a positive reading (indicating the presence of PdG) will result in the absence of color or a reading below a certain color threshold reading off of the test strip 1001 by the photoelectric sensor. The photoelectric sensor therefore is configured to read a positive result upon determining that the reflection of light indicates a color level below a determined threshold. An example method of detection will be discussed in more detail below.
[0139] The emitter and sensor may be used to detect the insertion of a test stick 2004. When the stand-alone digital reader 2003 is not assembled with a test stick 2004, the emitter in the stand-alone digital reader 2003 can turn on periodically, for example, every two seconds. Detection of the presence of a test stick 2004 may be achieved by detecting a large difference in sensor response depending on whether the emitter is on or off due to the presence of the nearby reflective surface of the test stick 2004. For example, in an implementation including two photodiode sensors, four readings may be captured: (1) first sensor output with emitter on, (2) first sensor output with emitter off, (3) second sensor output with emitter on, and (4) second sensor output with emitter off. In this example, very low and approximately equal readings for all four indicate that the stand-alone digital reader 2003 is still in the packaging or sitting on the counter waiting for the next test to be performed. Readings indicating a high light intensity at the photodetector for tests 1 and 3 and a low light intensity at the photodetector for tests 2 and 4 indicate the presence of a test stick 2004. The stand-alone digital reader 2003 may use this information to alter operation mode (e.g., from low power stand-by mode in the packaging to higher power test mode when a strip is inserted).
[0140] In an embodiment, the sensor is positioned substantially over the testing zone 1002 of the test strip 1001 contained within a test stick 2004. A sensor may be positioned over a blank region downstream of the testing zone 1002 on the test strip 1001 in an embodiment. In this embodiment, no control/reference line is present. In an embodiment, reflectance measurements are made for these two regions for a time period after a fluid sample is applied to one end of the strip, optionally to determine presence of a sample.
[0141] The circuit includes a light emitter. The light emitter may be an LED. The light emitter is connected a processing/control circuit, optionally comprising a smartphone or tablet computer, that may be in the integrated circuit. The at least one photodetectors and are also each coupled to the processing/control circuit 806 to control initiation of the photodetector operation. In an embodiment, the at least one photodetector comprises a camera 2005, configured to obtain color data of at least one test strip 1001 configured to evaluate urine for the presence of pregnanediol, and a processor, configured to evaluate the color data obtained by the camera 2005. The processor is configured to evaluate a concentration value for each successive test strip 1001 based on the color data collected by the camera 2005 and to output a signal onto a display 2010.
[0142] It may be desirable to align the test strip 1001 when inserted into the device 100 such that the nitrocellulose region is substantially located under the sensor 430. A first sensor 430 may be located directly over the testing zone 1002. A second sensor may be located directly over a second region of the strip that may or may not contain a control line. Further details of one embodiment of a sensor 430, emitter 440, and Test strip 1001 alignment are discussed below. Measurements of the reflectivities provide a measure of PdG concentration. In an embodiment of the invention, the test strip 1001 is placed into a test stick comprising a rigid material, the test stick incorporating an opening such that the sensor can read the test strip 1001 through the test stick, and the test stick 2004 further configured to correspond to the dimensions of a port 2020 configured to accept the test stick 2004.
[0143] Turning now to PdG change detection methods which may be used by a digital reader 2001, optionally a stand-alone digital reader 2003, an implementation of an embodiment of the invention comprises the method for detecting a variation in a PdG level in a urine sample from an individual. At a step, a series of samples may be collected. Preferably, the series of samples is collected over a single biological cycle of the user, for example, over a single menstrual cycle. This allows a baseline and threshold to be developed in the same cycle. At a step, a baseline may be determined from a plurality of samples of the series collected. The baseline samples may be initial samples collected over an initial plurality of days of the cycle. For example, samples collected on the first three days after a user's menses may be used to determine a baseline. In an implementation, the samples may be collected on any one or more of the third through the tenth day from the onset of menses of a menstrual cycle of the individual.
[0144] At a step, a threshold may be associated with the PdG level for the individual based at least in part on the determined baseline. The threshold represents a personalized value for the specific individual monitoring the PdG level for a current biological status rather than relying on previously measured cycle information or a fixed threshold for all individuals. An example of one possible determination of a baseline and threshold will be described in further detail below. In an embodiment of the invention, the threshold value is the pre-determined threshold value, configured by the ratio of the specific binding partners of the test strip 1001.
[0145] At a step, a signal may be generated representative of the PdG level in one or more samples. For example, the signal may be based at least in part on an amount of light reflected by the testing zone 1002 on the test strip 1001, or lack of light reflected.
[0146] At a step, the signal may be compared to the threshold. The comparison may include detection of a difference between the values and/or statistical or probability analyses of the values. At a step, an output may be generated based at least in part on the comparing. Where the signal value drops below the threshold value, the amount of PdG may be higher in the current sample under test than the threshold value. In an embodiment of the invention, when the signal value drops below the threshold value, a positive result is indicated. This condition may correspond with an elevated level of PdG for the individual thereby confirming the occurrence of ovulation.
[0147] Embodiments of the digital reader 2001 comprise a camera 2005 and a processor. The camera may comprise a CMOS sensor or a CCD sensor able to sense photons from the electromagnetic spectrum, or colors displayed upon at least one test strip 1001. The processor performs an analysis of the coloration by processing the color information from the color space (RGB, sRGB, HUV, LAB), then transforming this information mathematically, for example, but not limited to, matrix multiplication, rotation, transposition normalization, etc., resulting in rotation, scaling and skewing of the values, to fulfil standard color spaces notation, or to non-conventional color spaces resulting from said mathematical transformations. The transformation may include a step for fixing the standard illuminant or adjusting the standard illuminant of said resulting color space. The transformation may result in a color space on which a coloration or set of colorations are represented as a larger fraction of the resulting color space. The analysis of the coloration of at least one test strip 1001 by the processor is used to assign hormonal concentration values to the colors detected by the camera. Then, a set of color values in the defined color space (for example R,G,B) is assigned a concentration value. In this way, concentration values are mapped to the analysed color space, thus leading to a high level of accuracy regarding different concentration values of PdG, and optionally other metabolites of progesterone, collected from a photographed test strip 1001. As the color values mapped to concentration values indicate the concentration of PdG within urine, the digital reader 2001 may then present a result of the test strip 1001 by displaying the progesterone concentration value corresponding to the color value presented to the user. The present inventor has discovered the unique attributes of the test strip 1001 as described herein that present a linear correlation between concentrations of PdG, and optionally other additional metabolites of progesterone, and color, such linear correlation necessary for the functioning of the digital reader 2001 to interpret the results of the test strip 1001.
[0148] According to the present embodiment, a pre-calibration step may be performed by the digital reader 2001 prior to reporting hormonal values. In this calibration step, the processor of the digital reader 2001 obtains color data from a set of test strip 1001 exposed to standard concentration values before a measurement cycle. At least two concentration values can be used as minimum, for example one that generates a dark line within the testing zone 1002 of the test strip 1001 indicating a negative result, and one that generates the maximum change in coloration within said testing zone 1002 with a faint line or no line, indicating a positive result. Preferably, a minimum of five concentration values is used. In order to decrease the concentration difference between the calibrated values and increase the accuracy of the measurement, the processor may be calibrated by using as many test strips 1001 as possible exposed to as many standard concentrations within the exposure range of the test strip 1001. In such way, the digital reader 2001 may update the mapping of the coloration value to the progesterone concentration value as indicated by the test strip 1001. In embodiments of the test strip 1001 containing a plurality of testing zones, the similar variations of the above process may be repeated to provide an accurate result for each testing zone.
[0149] The term concentration value also encompasses percentage values, or mathematical transformations of said values to any other numerical values, which mapped onto the color space may comprise further transformations, for example but not limited to logarithmic series, power series, prime series, or arbitrary number series for which one real concentration value corresponds to one mapped numerical value.
[0150] According to an embodiment, coloration and not intensity is measured by the digital reader. Measuring coloration of the test strips 1001 allows for the measurement to be performed under normal ambient conditions, for example, but not limited to, incandescent light, florescent light, day light, LED light, candle light, etc. The intensity of the light source under which the device performs a measurement can be expressed in terms of illuminance ranging from, but not limited to 4 1 lux to 4 1000 lux.
[0151] The digital reader 2001 then measures test strip 1001 values from strips exposed to urine containing hormones at different concentration values. The color information from the test strip 1001s is mapped to the color space as described above, and associated to a corresponding concentration from the calibration with standard concentrations. Then, the processor of the digital reader 2001 interprets the mapped color and associated concentrations to determine the PdG values indicated by the photographed test strip 1001s, optionally in association with an application used in association with the digital reader.
[0152] The digital reader 2001 then measures hormonal values over time and stores them within a circuit configured to store at least one predetermined pregnanediol concentration threshold, thereby allowing the digital reader 2001 to present positive or negative results. The values are collected from the test strip 1001 or from multiple test strips 1001 at time intervals, for example but not limited to, daily or weekly measurements. These measurements are collected for example, but not limited to, first morning urine.
[0153] The first upward PdG trend to be observed indicating a PdG concentration of 5 g/ml after multiple measurements indicates that a woman has ovulated. The present inventor has discovered that a test strip 1001 in an embodiment of the invention configured to incorporate a testing zone able to detect metabolites of progesterone other than PdG provides a greater accuracy for depicting measurements of PdG, and therefore correspondingly, progesterone. The processor then may confirm that a first upward trend of the PdG has occurred by interpreting the successive test strip 1001 measurements, though subsequent negative readings may indicate the subsequent decline of progesterone following the initial production of progesterone following ovulation. Sustained levels of elevated progesterone readings, on the other hand, may indicate pregnancy. A single test indicating a PdG concentration at or above 5 g/ml of PdG confirms ovulation.
[0154] Progesterone hormonal data obtained by the device may include test strip 1001 variability noise, or instrument noise, or ambient conditions variability noise. Noise reduction algorithms as well understood by those skilled in the art may be utilized to enhance the quality of data.
[0155] The digital reader 2001 may further comprise a display, configured to display the result of the test strip 1001. The user may be instructed on the display how to position the camera to obtain the color data of the test strip 1001. Thus, the user may obtain color data of the test strip 1001 with immediate feedback whether the color data suffices the requirements of the digital reader. Also, the results of the estimation may be displayed to the user immediately.
[0156] Progesterone and/or PdG data obtained by the device may include test strip 1001 variability noise, or instrument noise, or ambient conditions variability noise. Noise reduction algorithms, known to those skilled in the art, may be utilized to enhance the quality of data.
[0157] The test strip 1001 optionally additionally comprises a blank zone onto which no test samples are attached to, in addition to the testing zone and receiving zone, and wherein the processor may be configured to calibrate the device based on the obtained camera data of one or more of these zones. The test strip 1001s may comprise multiple testing zones, multiple blank zones, and multiple control line 1005s, wherein the processor may be configured to evaluate the camera data of these zones. The detection, control, and blank zones may vary in color or have the same color. The device may analyze these colors together or separately as described above.
[0158] In more detail, the control line 1005 is a zone onto which the applied test sample permeates, and which gives a clear indication whether the application of the test sample was successful. The blank zone is typically a white portion of the test strip 1001 which acquires a background coloration that may differ to its original color after exposure to the testing fluid. The testing zone is a zone onto which a coloration change is observed depending on the concentration value of pregnanediol in the test problem sample. A concentration value associated with the sensitivity of the test strip 1001 corresponds in coloration to the control line 1005.
[0159] In an embodiment, the digital reader 2001 utilizes the coloration of the testing zone to be mapped to concentrations from the standard calibration described above, and indicate a pregnanedial concentration value, or an alternative message indicating the result, to a user. Furthermore, in an embodiment the color mapping to concentration can also be applied to the control line 1005 and blank zone and the device may utilize the control line 1005 and the blank zone to perform a local calibration on which the color information is utilized to evaluate the quality of the test strip 1001, the correct usage of the test strip 1001, or the illuminating conditions.
[0160] In an embodiment, the digital reader 2001 may be configured to calibrate the device based upon data stored from at least one previous measurement cycle of the device.
[0161] The present inventor recognizes that the progesterone levels of each woman and each cycle differ. In an embodiment, by evaluating the absolute progesterone levels of previous measurement cycles, the digital reader 2001 may estimate the base progesterone levels of the woman. Further, the digital reader 2001 may utilize the previous measurement cycles to estimate peaks of the progesterone levels. Depending on the estimated progesterone levels, the digital reader 2001 may determine a positive test indicating of progesterone and/or PdG levels above a threshold with higher accuracy. In such way, the digital reader 2001 confirms ovulation based on progesterone and/or PdG levels from each individual cycle and only uses previous cycle information to better calibrate for the detection of PdG based upon previous readings. In an embodiment, the digital reader 2001 also may be configured to read certain changes color levels that are not of a lacking intensity sufficient to confirm ovulation in the absolute, but such changes with a confirmatory blood test indicating progesterone production taken shortly before or after the urine progesterone test reading can allow the reader to calibrate to the color of the test strip 1001 at the time of the positive progesterone blood test to indicate a positive reading from a test strip 1001 in subsequent cycles.
[0162] The digital reader 2001 may be configured as a smartphone, as depicted in
[0163] Smartphones comprise cameras, processors, sensors and displays. Furthermore, smartphones are widely used today and carried by most people on a daily basis. By using a smartphone, the accuracy of high quality cameras for evaluation of the test strip 1001s may be utilized. Also, there is no need to provide an additional analyzation device. Smartphones also comprise a power supply such as a battery.
[0164] The digital reader 2001 may use multiple connectivity means, for example wifi, bluetooth, LTE, 3G, 4G, 5G to send and transmit data obtained or generated by the device elsewhere. In an embodiment depicted by
[0165] The present inventor has recognized that women use menstrual cycle apps, so called period-trackers to help them gain insights into their menstrual cycles. Most of these apps are based off standard calendar methods, assuming women have a consistent luteal phase and ovulation date is often calculated as average menstrual cycle length14 days. However, this is only true for less than 20% of women, thus making the rhythm method-based ovulation dating highly inaccurate. Therefore, some of these apps incorporate additional fertility indicators such as cervical mucus observations, basal body temperature (BBT) recordings, ovulation predictor kits results (LH, FSH, E3G measurements), and cervical position. Since a urine PdG level of at least 5 g/ml, utilized as the pre-determined threshold in an embodiment of the invention, is an indicator of ovulation, incorporating results of the test strip 1001 into period tracking apps known in the art will increase the accuracy of such at predicting ovulation. It is, therefore, a teaching of the invention for a digital reader 2001 to be utilized to transmit the results of the test strip 1001 into such period tracking apps in accordance with mechanisms known by those skilled in the art.
[0166] For confirmation of ovulation, the digital reader 2001 may be configured to output a signal onto a display 2010, if the processor determines that the test strip 1001 indicates the presence of PdG in urine above a pre-determined threshold. The signal may comprise a signal representative of the pregnanediol level indicated on the at least one test strip 1001. Alternatively, the signal may comprise a message. The message may state phrases such as positive, ovulation has occurred, Ovulation has been confirmed, or ovulation to the user upon detecting a positive reading. On the contrary the signal indicated on the display 2010, for simplicity, may simply display negative, ovulation has NOT occurred, or no ovulation to the user upon detecting a negative reading.
[0167] Preferably, a wider color spectrum is obtained by the camera 2005 than by using conventional nearly monochromatic light sources and corresponding light receiving elements. Since the color indicating the amount of pregnanediol in the testing zone 1002 varies over the color spectrum, using a wider color spectrum enables that small changes in the amount of pregnanediol present in the testing zone 1002 can be determined more accurately than just a threshold intensity. Similar techniques are applicable to additional testing zones in embodiments of the invention. Thus, the concentration values in the sample to which a test strip 1001 is exposed can be determined more accurately, and therefore, a hormonal profile can be built using the digital reader 2001.
[0168] After calibration of the digital reader 2001, the color of the testing zone 1002 exposed to unknown concentration urine sample of the test strip 1001 is determined, and a pregnanediol value is assigned to the test strip 1001 depending upon the color in the testing zone 1002 and optionally translated into a signal, and the pregnanediol value is compared to the pregnanediol concentration threshold. The digital reader 2001 is then optionally configured to present a message onto a display 2010 indicating a result.
[0169] In an intended method of use, for monitoring the progesterone level, daily urine samples are applied to a series of test strip 1001, starting shortly after menstruation. Color data from each test strip 1001 in the series is obtained by the camera 2005 of the smartphone or other optical reading means of a digital reader 2001. In an embodiment, during the process of collecting color data of the test strip 1001, the camera 2005 of the smartphone is positioned a few centimeters and central over the test strip 1001 and a picture of the test strip 1001 is taken by the user or the color data or the test strip 1001 is processed by the smartphone 22 to compare the signal representative of the pregnanediol level indicated on the test strip 1001 to the predetermined pregnanediol concentration threshold. The smartphone may optionally be configured in association with methods understood by those skilled in the art to then present a message indicating the result on a display 2010.
[0170] In order to correct for different conditions such as different illumination etc., each color data of the test strip 1001 is subjected to a further calibration. For calibration, in an embodiment, the color of the blank zone of the test strip 1001, the blank zone optionally a portion of the receiving zone exclusive of the area of the testing zone 1002 and other testing zones, is compared with the color of the control line 1005 of the test strip 1001, which has maximum color change, induced by the urine sample. The first color (the dark line of the control line) is set to denote a minimum PdG value, while the second color (no line or a faint line) is set to denote a maximum PdG value.
[0171] The methods disclosed herein comprise one or more steps or actions for achieving the described method. The method steps and/or actions may be interchanged with one another without departing from the scope of the claims. The order and/or use of specific steps and/or actions may be modified without departing from the scope of the claims.
[0172] It is to be understood that the claims are not limited to the precise configuration and components illustrated above. Various modifications, changes and variations may be made in the arrangement, operation and details of the methods and apparatus described above without departing from the scope of the disclosure.
[0173] While the foregoing is directed to aspects of the present disclosure, other and further aspects of the disclosure may be devised without departing from the basic scope thereof.