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WO2025128956A1 - Méthodes de dosage immunologique à haut débit pour évaluer les stades du vieillissement ovarien - Google Patents

Méthodes de dosage immunologique à haut débit pour évaluer les stades du vieillissement ovarien Download PDF

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WO2025128956A1
WO2025128956A1 PCT/US2024/059973 US2024059973W WO2025128956A1 WO 2025128956 A1 WO2025128956 A1 WO 2025128956A1 US 2024059973 W US2024059973 W US 2024059973W WO 2025128956 A1 WO2025128956 A1 WO 2025128956A1
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reagent
alternatively
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minutes
pipettor
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Lindsay SUN
Frederic Jean
Anne Young
Zivjena VUCETIC
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Beckman Coulter Inc
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Beckman Coulter Inc
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    • G01N33/57545—
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H10/00—ICT specially adapted for the handling or processing of patient-related medical or healthcare data
    • G16H10/40—ICT specially adapted for the handling or processing of patient-related medical or healthcare data for data related to laboratory analysis, e.g. patient specimen analysis
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/30—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for calculating health indices; for individual health risk assessment
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • Ovarian aging refers to the gradual decline of ovarian function and egg quality and quantity leading up to menopause. Menopause marks the end of a woman’s reproductive years and is characterized by the permanent cessation of menstrual periods. Menopause is diagnosed after 12 months of amenorrhea, at an average age of 51 years, when total follicles number approximately 1000. The Journal of Clinical Endocrinology & Metabolism, 2023, 00, 1-40, https ://doi . org/ 10.1210/ clinem/ dgad225.
  • the Stages of Reproductive Aging Workshop (STRAW) criteria provides nomenclature and a staging system for ovarian aging including menstrual and qualitative hormonal criteria to define each stage. Harlow SD et al, “STRAW + 10 Collaborative Group. Executive summary of the Stages of Reproductive Aging Workshop + 10: addressing the unfinished agenda of staging reproductive aging” J Clin Endocrinol Metab. 2012 Apr;97(4): 1159-68. doi: 10.1210/jc.2011- 3362. Epub 2012 Feb 16. PMID: 22344196; PMCID: PMC3319184.
  • pre-menopausal late reproductive stage
  • peri -menopausal from around when menopause begins to 12 months after the FMP
  • post-menopausal the time after 12 months after the FMP
  • One aspect of the invention is a method for quantitatively assessing a degree of ovarian aging in a subject in need thereof, the method comprising: i) detecting a presence or amount of Anti -Mullerian Hormone (AMH) in one or more plasma samples obtained from the subject using a high-throughput immunoassay analyzer and generating a corresponding AMH value; ii) comparing the corresponding AMH value with (a) a first corresponding reference range established in a reference population and associated with a pre-menopausal stage of ovarian aging, (b) a second corresponding reference range established in a reference population and associated with a peri -menopausal stage of ovarian aging, and (c) a third corresponding reference range established in a reference population and associated with a post-menopausal stage of ovarian aging; and iii) assigning to the subject a degree of ovarian aging based on the comparison; wherein the quantitative detection limit
  • One aspect of the invention is a method for quantitatively assessing reproductive aging in a subject in need thereof, the method comprising: i) detecting a presence or amount of Anti- Mullerian Hormone (AMH) in one or more plasma samples obtained from the subject using a high- throughput immunoassay analyzer and generating a corresponding AMH value; iijcomparing the corresponding AMH value with a corresponding reference range established in a reference population and associated with reproductive aging; and iii) assigning to the subject a degree of reproductive aging based on the comparison; wherein the quantitative detection limit (LOQ) of the high-throughput immunoassay analyzer for measuring AMH is equal to or less than about 0.003 ng/mL.
  • AMH Anti- Mullerian Hormone
  • the method further comprises identifying a subject in need of treatment based on the degree of reproductive aging and administering an effective amount of a pharmaceutical composition to the subject or recommending a therapeutic intervention to the subject.
  • One aspect of the invention is a method for quantitatively assessing an ovarian response in a subject in need thereof, the method comprising: i) detecting a presence or amount of Anti- Mullerian Hormone (AMH) in one or more plasma samples obtained from the subject using a high- throughput immunoassay analyzer and generating a corresponding AMH value; ii) comparing the corresponding AMH value with a corresponding reference range established in a reference population and associated with ovarian response; and iii) assigning to the subject a degree of ovarian response based on the comparison; wherein the quantitative detection limit (LOQ) of the high-throughput immunoassay analyzer for measuring AMH is equal to or less than about 0.003 ng/mL.
  • LOQ quantitative detection limit
  • the method further comprises identifying a subject in need of treatment based on the degree of ovarian response and administering an effective amount of a pharmaceutical composition to the subject or recommending a therapeutic intervention to the subject.
  • the ovarian response is a response to an ovarian stimulation.
  • the degree of ovarian response is selected from low, normal, or high.
  • the degree of ovarian failure is selected from the group consisting of poor ovarian response and normal -to-high ovarian response.
  • the method further comprises comparing the corresponding at least one additional biomarker value with (a) a first corresponding reference range established in a reference population and associated with a pre-menopausal stage of ovarian aging, (b) a second corresponding reference range established in a reference population and associated with a peri- menopausal stage of ovarian aging, and (c) a third corresponding reference range established in a reference population and associated with a post-menopausal stage of ovarian aging; and assigning to the subject a degree of ovarian aging based on the comparison.
  • the method further comprises determining a degree of ovarian aging score for the subject based upon a risk score, wherein the risk score is calculated using the AMH value, the at least one additional biomarker value, or a combination thereof.
  • the detected AMH and/or the at least one additional biomarker is present in the one more plasma samples in an amount that is at least IX greater than the LOQ, alternatively at least 2X greater than the LOQ, or alternatively at least 3X greater than the LOQ.
  • the LOQ corresponds to a coefficient of variation (CV) of the assay of 20% or less, alternatively 15% or less, alternatively 10% or less, alternatively 5% or less, or alternatively 4% or less.
  • CV coefficient of variation
  • the method further comprises identifying a subject in need of treatment based on the degree of ovarian aging and/or risk score and administering an effective amount of a pharmaceutical composition to the subject or recommending a therapeutic intervention to the subject.
  • the plasma sample volume is less than about 10 pL, alternatively between about 2 pL to about 9.9 pL.
  • the high-throughput immunoassay analyzer comprises a reagent pack configured to hold a plurality of reagent vessels; a pipettor arrangement comprising at least one reagent pipettor and at least one sample pipettor; and a detector arrangement
  • the reagent vessels comprise an elastomeric self-sealing membrane.
  • the reagent pack further comprises containment walls arranged between the reagent vessels.
  • the high-throughput immunoassay analyzer further comprises a reagent storage unit, wherein the reagent pack is housed in the reagent storage unit.
  • the pipettor arrangement comprises at least a first reagent pipettor, a second reagent pipettor, a third reagent pipettor, a fourth reagent pipettor, and at least one sample pipettor.
  • the first reagent pipettor, the second reagent pipettor, the third reagent pipettor, and/or fourth reagent pipettor are selectively and/or simultaneously operated.
  • the first reagent pipettor, second reagent pipettor, third reagent pipettor, fourth reagent pipettor and/or sample pipettor are configured to engage a dispense tip prior to aspiration.
  • the method is configured to analyze at least about 200 plasma samples/hr. In an aspect, the method is configured to analyze at least about 300 plasma samples/hr. In an aspect, the method is configured to analyze at least about 400 plasma samples/hr.
  • detecting the amount or presence of AMH comprises aspirating a portion of the one or more plasma samples from a sample vessel and dispensing the aspirated plasma sample into a reaction vessel of the high-throughput immunoassay analyzer; aspirating a portion of a first reagent from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel, generating a first reaction mixture; aspirating a portion of a second reagent from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel, generating a second reaction mixture; dispensing a substrate formulation into the reaction vessel, generating a detection mixture; detecting, using the detector arrangement, a reaction in the detection mixture; and wherein the corresponding AMH value is based on the reaction detected in the detection mixture.
  • the reaction in the detection mixture generates a chemiluminescent signal
  • the method comprises correlating the chemiluminescent signal with the presence and/or concentration of AMH in the plasma sample.
  • the first reagent comprises at least one affinity molecule configured to bind to at least one portion AMH
  • the second reagent comprises at least one detection molecule
  • the substrate formulation is configured to produce chemiluminescence.
  • the affinity molecule is selected from the group consisting of an antibody, a monoclonal antibody, a polyclonal antibody, a synthetic antibody mimic, an aptamer, an affimer, DARPins, or oligonucleotides or peptides that bind to at least one epitope of AMH.
  • the affinity molecule is an antibody and the method further comprises exposing the plasma sample to a first antibody which binds to a first AMH epitope and a second antibody which binds to a second AMH epitope.
  • detecting the presence or amount of at least one additional biomarker comprises aspirating a portion of the one or more plasma samples from a sample vessel and dispensing the aspirated plasma sample into a reaction vessel of the high-throughput immunoassay analyzer; aspirating a portion of a first reagent from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel, generating a first reaction mixture; aspirating a portion of a second reagent from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel, generating a second reaction mixture; dispensing a substrate formulation into the reaction vessel, generating a detection mixture; detecting, using the detector arrangement, a reaction in the detection mixture; and wherein the corresponding at least one biomarker value is based on the reaction detected in the detection mixture.
  • the reaction in the detection mixture generates a chemiluminescent signal
  • the method comprises correlating the chemiluminescent signal with the presence and/or concentration of at least one additional biomarker in the plasma sample.
  • the first reagent comprises at least one affinity molecule configured to bind to at least one portion of the at least one additional biomarker
  • the second reagent comprises at least one detection molecule
  • the substrate formulation is configured to produce chemiluminescence.
  • the affinity molecule is selected from the group consisting of an antibody, a monoclonal antibody, a polyclonal antibody, a synthetic antibody mimic, an aptamer, an affimer, DARPins, or oligonucleotides or peptides that bind to at least one epitope of the at least one additional biomarker.
  • the affinity molecule is an antibody and the method further comprises exposing the plasma sample to a first antibody which binds to a first at least one biomarker epitope and a second antibody which binds to a second at least one biomarker epitope.
  • the detection molecule comprises the second antibody.
  • the detection molecule comprises an alkaline phosphatase (AP)-conjugated secondary antibody.
  • the affinity molecule and/or the detection molecule is conjugated to a magnetic bead or a magnetic particle.
  • the first reaction mixture, second reaction mixture, and/or detection mixture is subjected to a magnetic field prior to detection.
  • incubation time of the first reaction mixture is at least about 30 minutes, alternatively at least about 40 minutes, or alternatively at least about 50 minutes.
  • incubation time of the second reaction mixture is at least about 5 minutes, alternatively at least about 8 minutes, or alternatively at least about 10 minutes.
  • the substrate formulation is configured to produce chemiluminescence and comprises:
  • A is Ci-ehaloalkyl, naphthyl, phenyl, substituted phenyl, or heteroaryl, wherein substituted phenyl comprises from 1 to 3 halo, Ci-6 alkyl, Ci-6 alkoxy, Ci-6 haloalkyl, C(O)Ris, CN or NO substituents;
  • Ri is selected from the group consisting of Cs- aryl, C1-6 alkyl, C1-6 haloalkyl, and C5-14 aralkyl groups;
  • R7-R14 are independently H, C1-6 alkoxy, halo, C1-4 alkyl, or R7 or R8-R9 or R9-R10 or R11-R12 or R12-R13 or R13-R14, can be joined together as a carbocyclic or heterocyclic ring system comprising at least one 5 or 6-membered ring;
  • R15 is C1-6 alkyl
  • each M is independently selected from the group consisting of H, an alkali metal, alkaline earth metal, transition metal, ammonium, phosphonium, organic amine salt, and an amino acid salt;
  • Z is O or S
  • n O, 1, or 2;
  • CAC cationic aromatic compound
  • the washing arrangement is configured to wash away at least a portion of the unreacted components in the first reaction mixture, second reaction mixture, and/or detection mixture.
  • the high-throughput immunoassay analyzer comprises a machine vision apparatus comprising an image capture device and an image interpretation device configured to monitor instrument and/or assay functionalities of the high-throughput immunoassay analyzer.
  • the instrument functionalities are selected from the group consisting of optical sensors, pressure sensors and thermistors.
  • the assay functionalities are selected from the group consisting of sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring.
  • cycle time is about 45 seconds or less, alternatively about 40 seconds or less, alternatively about 35 seconds or less, alternatively about 30 seconds or less, alternatively about 25 seconds or less, alternatively about 20 seconds or less, or alternatively about 15 seconds.
  • time to first result is about 60 minutes or less, alternatively about 55 minutes or less, alternatively about 50 minutes or less, alternatively about 45 minutes or less, alternatively about 40 minutes or less, alternatively about 35 minutes or less, alternatively about 30 minutes or less, alternatively about 25 minutes or less, alternatively about 20 minutes or less, alternatively about 15 minutes or less, or alternatively about 10 minutes or less.
  • FIG. 1A is a LOQ plot for AMH detected using a first high-throughput immunoassay analyzer according to an embodiment of the disclosure.
  • FIG. IB is a LOQ plot for AMH detected using a second high-throughput immunoassay analyzer according to an embodiment of the disclosure.
  • FIG. 1C is a LOQ plot for AMH detected using a third high-throughput immunoassay analyzer according to an embodiment of the disclosure.
  • FIG. 3 is a graph showing the patient demographics of samples analyzed according to an aspect of the disclosure.
  • FIGs. 4A and 4B are graphs depicting patient samples analyzed using an AMH assay according to an aspect of the disclosure.
  • FIG. 4A is a box and whisker plot depicting the ability of the AMH assay to differentiate between patient samples identified as premenopausal, perimenopausal, or postmenopausal.
  • FIG. 4B is graph plotting the age of each patient sample against the measured AMH concentration.
  • FIGs. 5A and 5B are graphs depicting patient samples analyzed using a FSH assay according to an aspect of the disclosure.
  • FIG. 5A is a box and whisker plot depicting the ability of the FSH assay to differentiate between patient samples identified as premenopausal, perimenopausal, or postmenopausal.
  • FIG. 5B is graph plotting the age of each patient sample against the measured FSH concentration.
  • FIGs. 6A and 6B are graphs depicting patient samples analyzed using an Inhibin B assay according to an aspect of the disclosure.
  • FIG. 6A is a box and whisker plot depicting the ability of the Inhibin B assay to differentiate between patient samples identified as premenopausal, perimenopausal, or postmenopausal and
  • FIG. 6B is graph plotting the age of each patient sample against the measured Inhibin B concentration .
  • Anti- Mullerian Hormone is a glycoprotein hormone, the expression of which is thought to contribute to sex differentiation during fetal development.
  • AMH is expressed by granulosa cells of growing follicles and has shown to be detectable in circulating serum.
  • the sequence of AMH is SEQ ID NO: 1.
  • assays herein detect AMH of SEQ ID NO: 1, or variants thereof.
  • Biomarkers such as AMH have been found helpful in monitoring the stages of ovarian aging. Secreted by granulosa cells of ovarian follicles, AMH provides a stable and reliable measure of ovarian health, remaining relatively constant throughout the menstrual cycle. AMH begins to decline in the late 20s to early 30s and continues decreasing with age, making it an early indicator of diminished ovarian reserve and reflects the continuous decline of the oocyte/follicle pool with age and, accordingly, ovarian aging. Freeman EW, et al. Contribution of the rate of change of antimullerian hormone in estimating time to menopause for late reproductive-age women. Fertil Steril. 2012; 98(5): 1254-9.
  • AMH levels may also be used to predict ovarian aging and guide clinical decisions in fertility planning. As menopause approaches, AMH levels drop significantly, often becoming undetectable years before menstruation ceases. This makes AMH a valuable tool not only for assessing reproductive potential but also for predicting the onset of menopause 5-10 years in advance. In assisted reproductive technologies like in vitro fertilization (IVF), AMH levels guide ovarian stimulation protocols and predict treatment outcomes.
  • IVF in vitro fertilization
  • Disclosed herein is an objective, standardized immunoassay method that employs an automated analyzer for detecting low levels of AMH in a plasma sample and offers specific cutoff values or reference ranges for distinguishing between each stage of ovarian aging.
  • the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
  • x, y, and/or z means any element of the seven-element set ⁇ (x), (y), (z), (x, y), (x, z), (y, z), (x, y, z) ⁇ .
  • x, y and/or z means "one or more of x, y and z”.
  • One aspect of the disclosure is a method for quantitatively assessing a degree of ovarian aging in a subject.
  • the method includes detecting a presence or amount of Anti- Mullerian Hormone (AMH) in one or more plasma samples obtained from the subject using a high- throughput immunoassay analyzer and generating a corresponding AMH value.
  • AMH Anti- Mullerian Hormone
  • epitope refers to a binding site recognized by an antibody.
  • Epitopes may include any molecule or grouping thereof, including, but not limited to, amino acid side chains, sugars, and lipids, and can have a specific three-dimensional structure or conformation.
  • a portion of at least one plasma sample from a subject is exposed to a capture antibody capable of binding AMH or capable of recognizing an epitope of AMH, and a first detector affinity molecule specific to AMH, generating a first reaction mixture.
  • a detection reaction is conducted in the first reaction mixture wherein the first detector affinity molecule generates a first detection signal, which is then recorded.
  • the detection reaction is a chemiluminescent reaction, an electrochemiluminescence reaction, an electrogenerated chemiluminescence reaction, a photoluminescence reaction, or a bioluminescence reaction.
  • the chemiluminescent reaction is a dioxetane-based reaction, a luminol-based reaction, a acridinium ester-based reaction, a peroxyoxalate reaction, a luciferin-luciferase reaction, a metal-catalyzed reaction, a halogen-based reaction, or a hydrazine-based reaction.
  • the detector affinity molecule is conjugated to an enzyme, such alkaline phosphatase or horseradish peroxidase.
  • a substrate for the enzyme is added to the second reaction mixture triggering a chemiluminescent reaction.
  • the generated corresponding AMH value from the methods disclosed herein is compared with a corresponding reference range.
  • the reference ranges are associated with various stages of the STRAW staging system. Current guidance merely offers that each of perimenopause, post-menopause, and the later stages of pre-menopause are being associated with low levels of AMH.
  • the corresponding AMH value is compared with (a) a first corresponding reference range established in a reference population and associated with a pre-menopausal stage of ovarian aging, (b) a second corresponding reference range established in a reference population and associated with a peri -menopausal stage of ovarian aging, and (c) a third corresponding reference range established in a reference population and associated with a post-menopausal stage of ovarian aging.
  • the values of the first corresponding reference range is less than the second corresponding reference range and/or third corresponding reference range.
  • the values of the second corresponding reference range is less than the third corresponding reference range.
  • the first corresponding reference range is 0.19 ng/mL ⁇ 0.4; alternatively is equal to or greater than about 0.09 ng/mL, is equal to or greater than about 0.11 ng/mL, is equal to or greater than about 0.13 ng/mL, is equal to or greater than about 0.15 ng/mL, is equal to or greater than about 0.17 ng/mL, is equal to or greater than about 0.19 ng/mL, is equal to or greater than about 0.21 ng/mL, is equal to or greater than about 0.23 ng/mL, is equal to or greater than about 0.25 ng/mL, is equal to or greater than about 0.27 ng/mL, is equal to or greater than about 0.29 ng/mL, is equal to or greater than about 0.31 ng/mL, is equal to or greater than about 0.33 ng/mL, is equal to or greater than about 0.35 ng/mL, is equal to or greater than about 0.37 ng/mL, is
  • third corresponding reference range 0.003 ng/mL ⁇ 0.001 alternatively is equal to or greater than about 0.002 ng/mL; alternatively is equal to or greater than about 0.0021 ng/mL; alternatively is equal to or greater than about 0.0022 ng/mL; alternatively is equal to or greater than about 0.0023 ng/mL; alternatively is equal to or greater than about 0.0024 ng/mL; alternatively is equal to or greater than about 0.0025 ng/mL; alternatively is equal to or greater than about 0.0026 ng/mL; alternatively is equal to or greater than about 0.0027 ng/mL; alternatively is equal to or greater than about 0.0028 ng/mL; alternatively is equal to or greater than about 0.0029 ng/mL; alternatively is equal to or greater than about 0.003 ng/mL; alternatively is equal to or greater than about 0.0031 ng/mL; alternatively is equal to or greater than about 0.00
  • the levels of AMH in blood may be in the pmol/L, Milli-intemational Unit (mIU), or ng range.
  • mIU Milli-intemational Unit
  • the quantitation detection limit (LOQ) for measuring AMH is equal to or less than about 0.004 ng/mL, alternatively equal to or less than about 0.003 ng/mL, alternatively equal to or less than about 0.002 ng/mL, equal to or less than about 0.001 ng/mL, equal to or less than about 0.0006 ng/mL and wherein the detected AMH has a concentration above the LOQ.
  • the LOQ for measuring AMH is equal to or less than about 0.10 ng/mL, alternatively equal to or less than about 0.090 ng/mL, alternatively equal to or less than about 0.080 ng/mL, alternatively equal to or less than about 0.070 ng/mL, alternatively equal to or less than about 0.060 ng/mL, alternatively equal to or less than about 0.50 ng/mL, alternatively equal to or less than about 0.40 ng/mL, alternatively equal to or less than about 0.30 ng/mL, alternatively equal to or less than about 0.020 ng/mL, or alternatively equal to or less than about 0.010 ng/mL.
  • the LOQ for measuring AMH is equal to or less than about less than about 100 pg/mL, less than about 90 pg/mL, less than about 80 pg/mL, less than about 70 pg/mL, less than about 60 pg/mL, less than about 50 pg/mL, less than about 40 pg/mL, less than about 30 pg/mL, less than about 20 pg/mL, or less than about 10 pg/mL.
  • the LOQ for measuring AMH is equal to or less than about 10 pg/mL, less than about 9 pg/mL, less than about 8 pg/mL, less than about 7 pg/mL, less than about 6 pg/mL, less than about 5 pg/mL, less than about 4 pg/mL, less than about 3 pg/mL, less than about 2 pg/mL, or less than about 1 pg/mL.
  • the LOQ for measuring AMH is equal to or less than about Ipg/mL, less than about 0.9pg/mL, less than about 0.8pg/mL, less than about 0.7pg/mL, less than about 0.6pg/mL, less than about 0.5pg/mL, less than about 0.4pg/mL, less than about 0.3pg/mL, less than about 0.2pg/mL, or less than about O.lpg/mL.
  • the LOQ for measuring AMH is equal to or less than about 100 fg/mL, less than about 90 fg/mL, less than about 80 fg/mL, less than about 70 fg/mL, less than about 60 fg/mL, less than about 50 fg/mL, less than about 40 fg/mL, less than about 30 fg/mL, less than about 20 fg/mL, or less than about 10 fg/mL.
  • the LOQ for measuring AMH is equal to or less than about 10 fg/mL, less than about 9 fg/mL, less than about 8 fg/mL, less than about 7 fg/mL, less than about 6 fg/mL, less than about 5 fg/mL, less than about 4 fg/mL, less than about 3 fg/mL, less than about 2 fg/mL, or less than about 1 fg/mL.
  • the detected AMH has a concentration that is at least 0.5X greater than the LOQ
  • the detected AMH has a concentration that is at least IX greater than the LOQ, alternatively at least 2X greater than the LOQ, alternatively at least 3X greater than the LOQ, alternatively at least 4X greater than the LOQ, alternatively at least 5X greater than the LOQ, alternatively at least 10X greater than the LOQ, alternatively at least 1OOX greater than the LOQ, or alternatively at least 1000X greater than the LOQ.
  • the detected AMH has a concentration that is at least 0.5X lower than the LOQ
  • the detected AMH has a concentration that is at least IX lower than the LOQ, alternatively at least 2X lower than the LOQ, alternatively at least 3X lower than the LOQ, alternatively at least 4X lower than the LOQ, alternatively at least 5X lower than the LOQ, alternatively at least 10X lower than the LOQ.
  • the detected AMH exhibits a coefficient of variation (CV) of 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less.
  • the detected AMH exhibits a coefficient of variation (CV) of 20% or less.
  • the detected AMH exhibits a coefficient of variation (CV) of 4% or less.
  • clinical factors may be used in combination with the disclosed assay to assess the progression of ovarian aging.
  • Clinical factors include, but are not limited to, age, basal antral follicle count (BAFC), menstrual cycle, race, ethnicity, current medications, BMI, onset of vasomotor symptoms, fertility treatment history, and clinical diagnoses.
  • determination of ovarian aging as disclosed herein can be used to aid in fertility assessment, reproductive treatment planning, menopause prediction, and menopause related risk assessment and management.
  • a patient in response to the results of an assay disclosed herein, a patient may be recommended a fertility treatment, hormonal therapy, or an immune-modulating therapy.
  • a patient is recommended an intervention that that treats or prevents ovarian dysfunction and/or premature decline in ovarian reserve.
  • the method includes identifying a subject in need of treatment based on the degree of ovarian aging and/or risk score and administering an effective amount of a pharmaceutical composition to the subject.
  • the method includes assigning a stage of ovarian aging and/or risk score to the subject and identifying a course of treatment (or treatment protocol) based on the assigned stage and/or risk score.
  • treat refers to administering to a subject a compound or pharmaceutical composition to partially or completely alleviate, inhibit, ameliorate, or relieve the condition from which the subject is suffering. This means any manner in which one or more of the symptoms of a condition are ameliorated or otherwise beneficially altered.
  • amelioration of the symptoms of a particular condition refers to any lessening, whether permanent or temporary, lasting or transient, that can be attributed to or associated with treatment by the compounds, compositions, and methods of the present disclosure.
  • treating a subject can mean eliminating or reducing the clinical signs of a condition in the subject; arrest, inhibit, or slow the progression of the condition in the subject; and/or decrease the number, frequency, or severity of clinical symptoms of the condition in the subject.
  • a “treatment protocol” is a protocol or regime developed regarding specific therapies (including pharmaceuticals or therapeutic interventions) for treatment.
  • a “therapeutic intervention” refers to a clinical intervention intended to manage a disease, condition, disorder or injury and avoid further clinical interventions.
  • an "effective amount” includes a “therapeutically effective amount” and a “prophylactically effective amount.”
  • therapeutically effective amount refers to an amount effective in treating and/or ameliorating a condition in a subject.
  • prolactically effective amount refers to an amount effective in preventing and/or substantially lessening the chances of a condition in a subject.
  • the effective amount of the pharmaceutical composition may be administered orally or via intravenous injection. The exact amount required to achieve a therapeutically effective outcome will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition, the particular composition, its mode of administration, its mode of activity, and the like.
  • the method incudes using the AMH measurements to assess reproductive aging, ovarian response, and/or ovarian failure.
  • Reproductive or ovarian aging may refer to the progressive decline in oocyte numbers and/or poor oocyte quality. This decline generally occurs during the fourth decade of a human subject’s life.
  • the reproductive ageing may be premature, i.e., occurring before peri-menopause.
  • a clinician may administer an effective amount of a pharmaceutical composition to the subject or recommend a therapeutic intervention to the subject.
  • the generated corresponding AMH value is compared with a corresponding reference range and the subject is assigned an ovarian stage of aging and/or a risk score.
  • the generated corresponding additional biomarker value is compared with a corresponding reference range and the subject is assigned an ovarian stage of aging and/or a risk score.
  • the ovarian stage of aging and/or a risk score is based on the AMH value(s).
  • the ovarian stage of aging and/or a risk score is based on a combination of the AMH value(s) and the additional biomarker value(s).
  • the related ovarian stage of aging or risk score may assist a medical professional with providing assessment, treatment, and/or other clinically relevant information to the subject.
  • the treatment and/or assessment will vary from subject to subject, but non-limiting examples includes administering an effective amount of a pharmaceutical composition to alleviate or ameliorate side effects from a condition, achieve a desired clinical effect, or to treat or prevent a disease.
  • the related ovarian stage of aging or risk score may be used to provide guidance or an assessment related to the initiation of a fertility treatment.
  • fertility treatments include intrauterine insemination (IUI), ovulation induction, IUI with ovulation induction, in vitro fertilization (IVF), and IVF with intra-cytoplasmic sperm injection (ICSI).
  • IUI intrauterine insemination
  • IVF in vitro fertilization
  • ICSI intra-cytoplasmic sperm injection
  • the related ovarian stage of aging or risk score may be used to provide guidance or an assessment related to infertility, including, but not limited to diminished ovarian reserve (DOR), natural aging, premature ovarian aging, and gonadotoxic treatment.
  • DOR diminished ovarian reserve
  • DOR diminished ovarian reserve
  • natural aging premature ovarian aging
  • gonadotoxic treatment gonadotoxic treatment.
  • a medical professional may then establish a treatment protocol for the subject.
  • This protocol may include administering an effective amount of a pharmaceutical composition to, for example, effect follicular development in the subject.
  • the assigning of an ovarian stage of aging or risk score may assist a medical professional with developing a protocol comprising the administration of an effective amount of a pharmaceutical composition to ameliorate a certain condition or achieve a desired clinical effect.
  • Exemplary oral ovulation induction agents include, but are not limited to, clomiphene citrate, aromatase inhibitors, such as letrozole and anastrozole; insulin sensitizing drugs, such as metformin, rosiglitazone, and pioglitazone; bromocriptine; cabergoline; GnRH; and GnRH analogs, such as leuprolide acetate, nafarelin acetate, goserelin acetate, ganirelix, and cetrorelix acetate and combinations thereof.
  • AMH levels are known in the art to be correlated with patient response to ovarian stimulation. Exemplary AMH assays described herein may offer higher sensitivity and specificity and a lower LOQ than a conventional AMH assays and may therefore allow for the assessment of AMH levels relevant to ovarian stimulation below the thresholds of existing AMH assays.
  • the subject may be categorized as a low responder, normal responder, or high responder based on the AMH levels.
  • a clinician may determine that high or normal responders may continue an IVF cycle, where poor responders be recommended for additional intervention, such as adjuvant therapy, prior to continuing IVF. Additionally for subjects with high ovarian response, the clinician may choose a treatment protocol which will minimize and/or reduce the risk of ovarian hyperstimulation syndrome.
  • Ovarian failure also referred to as premature menopause and primary ovarian insufficiency, refers to menopause occurring before age 40.
  • ovarian failure is characterized by oligomenorrhea or amenorrhea, symptoms of estrogen deficiency, and gonadotropin levels in the menopausal range before age 40 years.
  • causes of ovarian failure may include genetic causes, autoimmune disorders, ovarian damages, and others.
  • Clinical treatments for ovarian failure include estrogen hormone replacement therapy.
  • Experimental therapies include vitro activation, mitochondrial activation, stem cell and exosomes therapy, biomaterials strategies and intra-ovarian infusion of platelet-rich plasma (PRP).
  • PRP platelet-rich plasma
  • the method includes detecting a presence of AMH in one or more plasma samples obtained from the subject using a high-throughput immunoassay analyzer and generating a corresponding AMH value.
  • a plasma sample is analyzed for AMH using a high-throughput immunoassay analyzer disclosed herein.
  • the method includes aspirating a portion of the plasma sample from a sample vessel and dispensing the aspirated sample into a reaction vessel of a high-throughput immunoassay analyzer.
  • the immunoassay analyzer may include a container carriage device which is configured to hold and carry the containers at various locations in the instrument so that the analytic unit, incubator station, wash station, and read station can use the containers in various manners.
  • container carriage devices include vessel racks (e.g., a sample rack, a reagent rack, and a diluent rack), the sample presentation unit, vessel carriage units (e g., a sample carriage unit, a reaction vessel carriage unit, and a reagent carriage unit), vessel transfer units (e.g., a sample transfer unit, a reagent transfer unit, an incubator transfer unit, and an reaction vessel transfer unit), and vessel holding plates or wheels (e.g., a sample wheel, an incubator, and a wash wheel), which are described herein.
  • vessel racks e.g., a sample rack, a reagent rack, and a diluent rack
  • the sample presentation unit e.g., a sample carriage unit, a reaction vessel carriage unit, and
  • the read station may include a detector arrangement.
  • the detector arrangement may include a detector that is configured to detect light or luminescence, for example chemiluminescence.
  • the detector may be a luminescence detector, a chemiluminescence detector, a luminometer, or a photomultiplier-based detection instrument.
  • the detector includes a light detector configured to sense photons emitted from assay reactions over a period of time, an analog circuit configured to provide an analog signal based on the photons emitted from the assay reactions over the period of time, and a counter circuit configured to provide a photon count based on the photons emitted from the assay reactions over the period of time.
  • U.S. Patent No. 11,604,146 which is incorporated by reference in its entirety herein, discloses non-limiting examples of a detector that may be used in an aspect of the invention.
  • primary sample containers can be placed into an onload section (e.g., individually or on racks) of the sample presentation unit.
  • the sample presentation unit has at least one sample rack, alternatively at least two sample racks, alternatively at least three sample racks, alternatively at least four sample racks, alternatively at least five sample racks, alternatively at least six sample racks, alternatively at least seven sample racks, alternatively at least eight sample racks, alternatively at least nine sample racks, alternatively at least ten sample racks, alternatively at least 11 sample racks, alternatively at least 12 sample racks, alternatively at least 13 sample racks, alternatively at least 14 sample racks, alternatively at least 15 sample racks, alternatively at least 16 sample racks, alternatively at least 17 sample racks, alternatively at least 18 sample racks, alternatively at least 19 sample racks, or alternatively at least 20 sample racks.
  • each sample rack can hold at least one sample vessel, alternatively at least two sample vessels, alternatively at least three sample vessels, alternatively at least four sample vessels, alternatively at least five sample vessels, alternatively at least six sample vessels, or alternatively at least seven sample vessels.
  • the sample presentation unit holds about 140 sample vessels. In a specific embodiment, the sample presentation unit has 20 sample racks with each rack holding seven sample vessels.
  • the sample vessels include a barcode label that uniquely identifies the sample vessel in the immunoassay analyzer.
  • the barcode label also may include alphanumeric characters that correspond to the barcode identification information.
  • the immunoassay analyzer may include at least optical reader, such as a barcode scanner.
  • the optical readers are area scan cameras that provide a two-dimensional image of the barcode and/or sample vessel. In another embodiment, optical readers are area scan cameras that provide a three- dimensional image of the barcode and/or sample vessel.
  • the instrument may also contain a sample retention unit configured to receive sample retention vessels and store, analyze, or otherwise process samples retained within the sample retention vessels.
  • a “primary sample container” loaded into the automated analyzer with a sample may also be considered a “sample retention vessel” when placed in a sample retention unit, such as a storage unit.
  • sample retention vessel such as a storage unit.
  • containers or “vessels” are analogous, and can be of various types, such as specimen tubes (also referred to herein as sample tubes) and pipettor tips, such as micro or disposable tips.
  • the vessels are tubes with diameters between about 12mm to about 16mm and/or heights between about 75mm to about 100mm.
  • the vessels are cups with volumes of about 0.5mL, alternatively about l.OmL, alternatively about 1.5mL, alternatively about 2.0mL, alternatively about 2.5mL, or alternatively about 3.0mL.
  • the immunoassay analyzer includes a reagent pack configured to hold a plurality of reagent vessels.
  • a “reagent pack” may include any suitable container that can store a reagent.
  • An example of a reagent pack can include a generally rectangular elongated body formed to include multiple reagent vessels including one or more large reagent vessels, and one or more relatively smaller reagent vessels, as well as features to facilitate handling and automation.
  • US Patent No. 9,519,000 which is incorporated by reference in its entirety herein, discloses nonlimiting examples of a reagent pack that may be used in an aspect of the invention.
  • the reagent pack may be configured to accommodate sufficient volumes of reagents for multiple instances of an assay.
  • each reagent pack includes reagents for about 20 to about 100 instances of an assay and in some cases about 50 instances.
  • Each reagent vessel may be large enough to accommodate a microtip or disposable tip (i.e., dispo- tip) of a reagent pipettor used to remove a volume of reagent for use in an assay.
  • the reagent pack may be maintained at a temperature of between about 4°C to 10°C.
  • a “reagent vessel” may refer to a vessel, unit, fluid container, or the like that is configured to store reagents.
  • the reagent vessels include an elastomeric self-sealing membrane.
  • An elastomeric self-sealing membrane may be a polymer, such as polypropylene, which is able to regain its original shape when pierced.
  • the elastomeric membrane can be a thermoplastic elastomer with hardness of 30-40 durometer (Shore) A. In other embodiments, the hardness can be 20-50 (Shore) A, or about 30 (Shore) A. Elastomers deform sufficiently to form a tight seal with the vessel base. Thermoplastic elastomers are advantageous because of their compatibility with plastics injection molding processes.
  • the elastomeric membrane can be large enough to provide adequate compression without bottoming on the sealing portion of the vessel.
  • the hardness and dimensions can cooperate to allow the elastomeric membrane to the sealing portion with reasonable sealing force.
  • the elastomeric membrane diameter is small enough so that, when compressed by engagement of the pipettor tip, it conforms to the sealing portion without contacting the wall of the pipettor tip. This advantageously concentrates sealing force to the sealing portion of vessel and distributes sealing force evenly to prevent leaks.
  • the sealing force is about 44 newtons (about 9.9 lbs.) and produces a pressure on the sealing surface of about 300 (about 43.5 pounds per square inch) to about 1000 kPa (145.0 pounds per square inch).
  • the elastomeric self-sealing membrane allows for aspiration of the reagents without concern of evaporation.
  • the reagent pack further includes containment walls arranged between the reagent vessels.
  • the reagent pack may include at least one reagent vessel, alternatively at least two reagent vessels, alternatively at least three reagent vessels, alternatively at least four reagent vessels, alternatively at least five reagent vessels, or alternatively at least ten reagent vessels.
  • the immunoassay analyzer further includes a reagent storage unit, wherein the reagent pack is housed in the reagent storage unit.
  • a fluidic substance is dispensed to a reaction vessel.
  • the fluidic substance include a sample, diluent, reagent, substrate, or any combination thereof, as described herein.
  • the reaction vessel already contains other fluidic substances, such as a sample, and after a fluidic substance is dispensed to a reaction vessel, the fluidic substance is mixed with the other fluidic substances in the reaction vessel.
  • the mixing can be performed with a stirrer in direct contact with the fluidic substances, an ultrasonic probe in direct or indirect contact with the fluidic substances, or any other suitable mixing apparatus.
  • the immunoassay analyzer includes an ultrasonic mixing module.
  • a reagent pipettor may be outfitted with a tip that allows it to perform ultrasonic mixing of a reagent in a reagent pack before aspirating it for transport to a reaction vessel, thereby ensuring that the aspirated reagent would not be impacted by any settling that may have taken place in the reagent pack.
  • Sample pipettors may similarly be specialized.
  • the transfer unit transfers the reaction vessels to and from the incubator station which includes an incubator.
  • the transfer unit transfers one or more of the pipetted reaction vessels from the reagent carriage unit to the incubator.
  • the transfer unit can transfer one or more reaction vessels from the incubator to the reagent carriage unit.
  • the transfer unit can also remove from the reaction vessels that have been read or completed the incubator.
  • the incubator is thermally controlled to maintain a predetermined temperature.
  • the incubator is maintained about 30 °C to 40 °C.
  • the incubator is maintained about 37 °C to ensure immunological reaction and enzyme reaction, for example.
  • the incubator performs assay incubation.
  • the transfer unit transfers incubated reaction vessels from the incubator to the wash unit, transfers assay reaction vessels from the wash unit to the incubator, transfers reaction vessels containing substrate from the wash unit to the incubator for substrate incubation or enzyme reaction, transfers washed reaction vessels from the incubator to detector arrangement after substrate incubation, and transfers the reaction vessels that have been read or completed from the detector arrangement to the incubator.
  • the used reaction vessels can be delivered to a waste location.
  • a sample and a reagent are dispensed into a reaction vessel and mixed.
  • the mixture is then transferred to the incubator.
  • the sample and the reagent interact.
  • the resulting “first reaction mixture” is a result of the incubation between the sample and the reagent.
  • the reagent may include a specific-binding reagent, such as an affinity molecule specific to the analyte being analyzed by the immunoassay analyzer.
  • the incubation time of the first reaction mixture is at least about 30 minutes, alternatively at least about 40 minutes, alternatively at least about 50 minutes, alternatively at least about 55 minutes, or alternatively at least about 60 minutes.
  • a reagent which is different from the reagent used in the first reaction mixture, is added to the first reaction mixture and mixed. The mixture is then transferred to the incubator. During incubation, the first reaction mixture and the reagent interact. The resulting “second reaction mixture” is a result of an incubation between the first reaction mixture and the reagent.
  • the reagent may include a detection molecule, such as an alkaline phosphatase (AP)- conjugated secondary antibody, or a labeled antibody.
  • the incubation time of the second reaction mixture is at least about 5 minutes, alternatively at least about 8 minutes, or alternatively at least about 10 minutes.
  • the wash station receives and supports reaction vessels thereon such that various aspects of diagnostic process are performed with the immunoassay analyzer.
  • the wash station is configured to wash away at least some of the unreacted components. Unreacted components may include unreacted reagents (e.g., free antigens, antibodies, unbound reactants, particles, and/or fluid, etc.) and unreacted sample.
  • the wash station may be configured to perform a set number of wash actions depending on the assay.
  • the wash station may also be configured to perform a set number of washes within a predetermined sequence.
  • the wash station is configured to perform at least one wash action to wash away at least a portion of unreacted components, alternatively configured to perform at least two wash actions, alternatively configured to perform at least three wash actions, alternatively configured to perform at least four wash actions, or alternatively configured to perform at least five wash actions.
  • the wash station is a thermally controlled device to separate bound or free analytes from particles after incubation.
  • the wash unit is maintained about 30 °C to 40 °C. In other embodiments, the wash unit is maintained about 37 °C to ensure enzyme reaction, for example.
  • U.S. Patent Publication No. 2022/0357352 which is incorporated by reference in its entirety herein, discloses configurable wash processes according to an aspect of the invention.
  • the wash station may comprise a washing arrangement which may be configured to provide a base number of wash series (or wash actions) for each reaction vessel and optionally provide an additional number(s) of wash actions.
  • the additional number(s) of wash actions may include one, a plurality, or all of a potential number of wash actions.
  • An additional number of wash actions beyond the base number of wash actions may be specified for certain assays in an assay protocol file.
  • the washing arrangement may include cleaning dispense nozzle (or probe) which dispenses a rinsing fluid and a cleaning aspiration nozzle (or probe) which aspirates the unreacted components.
  • a base number of wash actions performed may be one, two, three, four, or five and the additional number of wash action(s) may be one, two, three, four or five.
  • a base number of wash actions performed may be three and the additional number of wash action(s) may be one or two.
  • the base number of wash actions may be performed if three probes dispense buffer solution once per vessel and the three probes aspirate the at least some of the unreacted components some of the buffer solution, and/or the at least some of the unreacted reagents once per vessel.
  • certain probe(s) may be selectively used to dispense clean buffer solution into the vessel and aspirate the at least some of the unreacted components of the sample, some of the buffer solution, and/or the at least some of the unreacted reagents from the vessel to perform the additional wash action(s).
  • the affinity molecule and/or the detection molecule is conjugated to a magnetic bead or a magnetic particle.
  • magnetic beads also known as magnetic particles, paramagnetic particles, or superparamagnetic particles
  • the magnetic beads consist of a polystyrene core surrounded by a thin layer of small iron oxide particles (-20-30 nm), such as magnetite.
  • the magnetic beads are encapsulated by, for example, a polymer, protein A, protein G, protein L, a secondary antibody, or an epoxy. Surface modification of the coating minimizes any non-specific protein binding.
  • Antibodies targeting the analyte of interest can be covalently coupled to the surface of the magnetic bead.
  • the first reaction mixture or second reaction mixture is subjected to a magnetic field.
  • the magnetic beads do not exhibit bead-to-bead attraction, only migrating when a magnetic field is applies. Captured analytes or targets are separated from the mixture and magnetization may be used to retain desired components within a reaction vessel.
  • the machine vision apparatus operates to detect a volume of a fluidic substance in a container and determine whether the volume held in the container is appropriate as targeted. As described herein, this volume detection is configured to detect a volume at a dispense tip using the dispense tip image capture unit, and a volume at a vessel using a vessel image capture unit.
  • An exemplary assay protocol (Table 9) is used to detect the presence of AMH in a plasma sample from a subject.
  • the assay was performed using an exemplary high-throughput immunoassay analyzer according to an aspect of the disclosure having (i) four reagent pipettors and one sample pipettor configured to aspirate and dispense less than about 10 pL; (ii) reagent packs configured to store volumes of reagents for at least 20 instances of assays; (iii) an ultrasonic mixer; and (iv) the capability to perform over 5 wash cycles per reaction vessel..
  • Paramagnetic particle conjugated with AMH antibodies capable of binding to an epitope AMH were pipetted into a reaction vessel using one of four reagent pipettors. A sample aliquot is pipetted into the reaction vessel using a sample pipettor. Then the reaction vessel is mixed using the ultrasonic mixer and incubated, generating a first reaction mixture. A magnetic field is applied to the reaction vessel and the first reaction mixture is washed using a wash buffer to remove any unreacted components. A secondary antibody capable of binding a different epitope of AMH conjugated to alkaline phosphatase (“ALP”) is added to the reaction vessel containing the first reaction mixture. The reaction vessel is mixed ultrasonically and incubated, generating a second reaction mixture. A magnetic field is applied to the reaction vessel and the second reaction mixture is washed using a wash buffer to remove any unreacted components. A substrate according to the present disclosure is added to the reaction vessel, and the signal generated from the resulting reaction is read using a luminometer.
  • ALP alkaline
  • a single calibration is generated (duplicate replicates of each level) and a calibration curve is fit to the corresponding signal output.
  • the assay is then used to measure AMH levels in the plasma samples.
  • the resulting AMH levels are compared to reference ranges associated with various stages of the STRAW staging system.
  • the subject is then assigned a degree of ovarian aging based on the comparison with the reference values.
  • Example 3 AMH Assay Patient Testing
  • An exemplary AMH Assay was assessed for the ability to establish AMH reference ranges for pre/peri/post-menopausal stages of ovarian aging and stage a subject to a degree of ovarian aging based on the AMH reference ranges.
  • the AMH Assay was used to screen a panel of samples for patients who were stratified into menopausal stages - Premenopause (group 1), Perimenopause (group 2), or Postmenopause (group 3) - based on the results of a questionnaire in which the patients self-reported their menopausal status and other relevant clinical criteria. Fifteen samples per group, which were subject totheThe following inclusion/exclusion criteria, were analyzed.
  • PCOS polycystic ovarian syndrome
  • ovarian surgery Exposure to cytotoxic drugs or pelvic radiation therapy; Hormonal therapy, or use of oral contraceptive or hormone eluting contraceptive device in the 6 months before entering the study; Medical history of thyroid disease, pituitary hormone disorders, hypercortisolemia, or eating disorders; Currently pregnant.
  • Example 3 The samples analyzed in Example 3, were further analyzed for follicle-stimulating hormone (FSH) and an inhibin B.
  • FSH follicle-stimulating hormone
  • FIG. 5A is a box and whisker plot depicting the ability of the FSH assay to differentiate between patient samples identified as premenopausal, perimenopausal, or postmenopausal, including degree of variation.
  • FIG. 5B is graph plotting the age of each patient sample against the measured FSH concentration. Table 12 lists the median concentrations measure for each group and the 95% confidence interval.
  • the Inhibin B assay was used to screen the patient samples on Beckman Coulter’s Dxl 9000 analyzer having (i) four reagent pipettors and one sample pipettor configured to aspirate and dispense less than about 10 pL; (ii) a reagent pack with a first reagent vessel containing Inhibin B capture antibodies conjugated to paramagnetic particle, a second reagent vessel containing Inhibin B monoclonal antibodies conjugated to ALP and a third reagent vessel containing an assay buffer where each reagent vessel is configured to store volumes of reagents for at least 50 instances of assays; (iii) an ultrasonic mixer; and (iv) the capability to perform over 5 wash cycles per reaction vessel.

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Abstract

La présente invention concerne des méthodes de détection de l'hormone anti-mullérienne (AMH) dans un échantillon de plasma au moyen d'un analyseur de dosage immunologique à haut débit.
PCT/US2024/059973 2023-12-14 2024-12-13 Méthodes de dosage immunologique à haut débit pour évaluer les stades du vieillissement ovarien Pending WO2025128956A1 (fr)

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