High-throughput immunoassay methods for assessing the stages of ovarian aging

Abstract

The presently claimed and described technology provides methods for detecting Anti-Mullerian Hormone (AMH) in a plasma sample using a high-throughput immunoassay analyzer.

Description

HIGH-THROUGHPUT IMMUNOASSAY METHODS FOR ASSESSING THE STAGESOF OVARIAN AGINGRELATED APPLICATIONS

[0001] The present patent application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63 / 610,126, filed December 14, 2023, U.S. Provisional Patent Application Ser. No. 63 / 553,763, filed February 15, 2024, and U.S. Provisional Patent Application Ser. No. 63 / 730,968, filed December 12, 2024, the content of each is hereby incorporated by reference in its entirety into this disclosure.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (67543sequencelisting.xml; Size: 2,240 bytes; and Date of Creation: December 1, 2024) is herein incorporated by reference in its entirety.BACKGROUND

[0003] 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.

[0004] 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. Particularly, criteria is provided for various pre-menopausal (late reproductive stage), peri -menopausal (from aroundwhen menopause begins to 12 months after the FMP), and post-menopausal (the time after 12 months after the FMP) stages. A need exists for an AMH assay that is sensitive and specific enough to distinguish between the stages of ovarian aging.BRIEF SUMMARY

[0005] 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 (LOQ) of the high-throughput immunoassay analyzer for measuring AMH is equal to or less than about 0.003 ng / mL, and wherein the first, second, and third corresponding reference ranges are above the LOQ.

[0006] 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.

[0007] In an aspect, 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.

[0008] 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.

[0009] In an aspect, 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.

[0010] In an aspect, the ovarian response is a response to an ovarian stimulation.

[0011] In an aspect, the degree of ovarian response is selected from low, normal, or high.

[0012] One aspect of the invention is a method for quantitatively assessing ovarian failure 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 failure; and iii) assigning to the subject a degree of ovarian failure 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.

[0013] In an aspect, the degree of ovarian failure is selected from the group consisting of poor ovarian response and normal -to-high ovarian response.

[0014] In an aspect, the method further comprises identifying a subject in need of treatment based on the degree of ovarian failure and administering an effective amount of a pharmaceutical composition to the subject or recommending a therapeutic intervention to the subject.

[0015] In an aspect, the method further comprises detecting the presence or amount of at least one additional biomarker in one or more plasma samples obtained from the subject using the high-throughput immunoassay analyzer and generating a corresponding at least one additional biomarker value.

[0016] In an aspect, the at least one additional biomarker is inhibin B, follicle-stimulating hormone (FSH), or a combination thereof.

[0017] In an aspect, 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.

[0018] In an aspect, 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.

[0019] In an aspect, 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.

[0020] In an aspect, 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.

[0021] In an aspect, 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.

[0022] In an aspect, the plasma sample volume is less than about 10 pL, alternatively between about 2 pL to about 9.9 pL.

[0023] In an aspect, 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

[0024] In an aspect, the reagent vessels comprise an elastomeric self-sealing membrane. In an aspect, the reagent pack further comprises containment walls arranged between the reagent vessels.In an aspect, the high-throughput immunoassay analyzer further comprises a reagent storage unit, wherein the reagent pack is housed in the reagent storage unit.

[0025] In an aspect, 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.

[0026] In an aspect, the first reagent pipettor, the second reagent pipettor, the third reagent pipettor, and / or fourth reagent pipettor are selectively and / or simultaneously operated.

[0027] In an aspect, 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.

[0028] In an aspect, 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.

[0029] In an aspect, 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.

[0030] In an aspect, the reaction in the detection mixture generates a chemiluminescent signal, and the method comprises correlating the chemiluminescent signal with the presence and / or concentration of AMH in the plasma sample.

[0031] In an aspect, 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, and the substrate formulation is configured to produce chemiluminescence.

[0032] In an aspect, 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.

[0033] In an aspect, 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.

[0034] In an aspect, 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.

[0035] In an aspect, the reaction in the detection mixture generates a chemiluminescent signal, and the method comprises correlating the chemiluminescent signal with the presence and / or concentration of at least one additional biomarker in the plasma sample.

[0036] In an aspect, 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, and the substrate formulation is configured to produce chemiluminescence.

[0037] In an aspect, 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.

[0038] In an aspect, 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.

[0039] In an aspect, the detection molecule comprises the second antibody. In an aspect, the detection molecule comprises an alkaline phosphatase (AP)-conjugated secondary antibody.

[0040] In an aspect, the affinity molecule and / or the detection molecule is conjugated to a magnetic bead or a magnetic particle.

[0041] In an aspect, the first reaction mixture, second reaction mixture, and / or detection mixture is subjected to a magnetic field prior to detection.

[0042] In an aspect, 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.

[0043] In an aspect, 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.

[0044] In an aspect, the substrate formulation is configured to produce chemiluminescence and comprises:

[0045] a chemiluminescent compound of formula I or a salt thereof:

[0047] wherein 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;

[0048] Ri is selected from the group consisting of Cs- aryl, C1-6 alkyl, C1-6 haloalkyl, and C5-14 aralkyl groups;

[0049] 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;

[0050] R15 is C1-6 alkyl;

[0051] 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;

[0052] Z is O or S; and

[0053] n is O, 1, or 2;

[0054] a cationic aromatic compound (CAC);

[0055] a background reducing agent; and

[0056] an ether-linked nonionic surfactant or a hydrophilic polymer.

[0057] In an aspect, the detector arrangement comprises 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.

[0058] In an aspect, the high-throughput immunoassay analyzer comprises an ultrasonic mixing module. In an aspect, the first reaction mixture, second reaction mixture, and / or detection mixture is agitated via the ultrasonic mixing module.

[0059] In an aspect, the high-throughput immunoassay analyzer comprises a washing arrangement, wherein the washing arrangement 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.

[0060] In an aspect, 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.

[0061] In an aspect, 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.

[0062] In an aspect, the instrument functionalities are selected from the group consisting of optical sensors, pressure sensors and thermistors. In an aspect, the assay functionalities are selected from the group consisting of sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring.

[0063] In an aspect, 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.

[0064] In an aspect, time to first result (TTFR) 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.

[0065] These and other advantages, aspects, and novel features of the present disclosure, as well as details of illustrated embodiments thereof, will be more fully understood from the following description and drawings.BRIEF DESCRIPTION OF THE FIGURES

[0066] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures, wherein:

[0067] 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.

[0068] FIG. 2 is a table showing the Staging of Reproductive Aging Workshop (STRAW)+10 staging system for reproductive aging in women.

[0069] FIG. 3 is a graph showing the patient demographics of samples analyzed according to an aspect of the disclosure.

[0070] 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.

[0071] 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.

[0072] 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 .DETAILED DESCRIPTIONI. Introduction

[0073] Anti- Mullerian Hormone (AMH) is a glycoprotein hormone, the expression of which is thought to contribute to sex differentiation during fetal development. In the ovary, AMH is expressed by granulosa cells of growing follicles and has shown to be detectable in circulating serum. In some instances, the sequence of AMH is SEQ ID NO: 1. In some instances, assays herein detect AMH of SEQ ID NO: 1, or variants thereof.

[0074] 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 indicatorof 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.

[0075] However, 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 without the ability to distinguish between the stages.

[0076] 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.II. Definitions

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong. Any reference to standard methods (e.g., ASTM, TAPPI, AATCC, etc.) refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated.

[0078] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0079] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0080] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or morepreferred embodiments or aspects does not imply that other embodiments or aspects are not useful and is not intended to exclude other embodiments or aspects from the scope of the invention.

[0081] The term "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0082] By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0083] The singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). As used herein, the term "or" is generally employed in its usual sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means any one or more of the items in the list joined by "and / or". As an example, "x and / or y" means any element of the three-element set {(x), (y), (x, y)}. In other words, "x and / ory" means "one or both of x and y". As another example, "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)}. In other words, "x, y and / or z" means "one or more of x, y and z".

[0084] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Herein, "up to" a number (for example, up to 50) includes the number (for example, 50). The term "in the range" or "within a range" (and similar statements) includes the endpoints of the stated range.

[0085] Reference throughout this specification to "one aspect," "an aspect," "certain aspects," or"some aspects," "one embodiment," "an embodiment," "certain embodiment," or "some embodiment," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0086] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein in connection with a measured quantity, the term "about" refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is + / -10%. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0087] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0088] The term "exemplary" means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms "e g.," and "for example" set off lists of one or more non-limiting aspects, examples, instances, or illustrations.

[0089] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, "substantially" may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.

[0090] The invention is defined in the claims. However, below is a non-exhaustive listing of nonlimiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.

[0091] One aspect of the invention is a method for quantitatively assessing a degree of ovarian aging in a subject using a high-throughput immunoassay analyzer. The term "immunoassay" may be a laboratory method used to determine the amount of an analyte in a sample. It can be based on the interaction of antibodies with antigens, and because of the degree of selectivity for the analyte (either antigen or antibody), an immunoassay can be used to quantitatively determine very low concentrations of analyte in a test sample. An "immunoassay analyzer" can include an instrument on which immunoassays have been automated. Various immunoassay analyzer are commercially available including the Dxl® system (Beckman Coulter, CA).

[0092] As used herein, the terms "subject", "individual", and "patient" are interchangeable, and relate to vertebrates, preferably mammals. For example, mammals in the context of the disclosure are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., as well as animals in captivity such as animals in zoos. The term "animal" as used herein includes humans. The term "subject" may also include a patient, i.e., an animal, having a disease. In exemplary aspects, a subject, individual, or patient refers to a human (e.g., a man, a woman, or a child).III. Assay Methods For Assessing The Stages Of Ovarian Aging

[0093] AMH is a useful biomarker for the assessment of ovarian aging due to its stability across menstrual cycles; its early decline, providing a more sensitive measure of ovarian aging; and its predictive value, useful in fertility planning. However, most assay methods that assess AMH levels are not sensitive enough to distinguish between the various stages of ovarian aging. The disclosedmethods allow a user to rapidly determine a differentiation between a pre-menopausal, peri- menopausal, and post-menopausal sample.

[0094] In some instances, 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. In some cases, menopause is diagnosed after 12 months of amenorrhea, at an average age of 51 years, when total follicles number approximately 1000.

[0095] In June 2023, the Endocrine Society, published a summary of the current state of research investigating ovarian aging. The Journal of Clinical Endocrinology & Metabolism, Volume 108, Issue 8, August 2023, Pages 1835-1874, https: / / doi.org / 10.1210 / clinem / dgad225. In some cases, the STRAW criteria provides nomenclature and a staging system for ovarian aging including menstrual and qualitative hormonal criteria to define each stage. Particularly, criteria is provided for various pre-menopausal (late reproductive stage), perimenopausal (from around when menopause begins to 12 months after the FMP), and post-menopausal (the time after 12 months after the FMP) stages. See FIG. 2.

[0096] 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. For instance, the guidelines recommend AMH for monitoring the progression of ovarian aging. Low and very low levels of AMH and inhibin B were listed as the supportive criteria for menopause staging, but no specific cutoffs were recommended.

[0097] Assays disclosed herein offer specific cut-off values or reference ranges for distinguishing between each stage. Furthermore, the high sensitivity of the disclosed assays, including low LOQ and LOD, allows for the possibility of differentiation between the low levels of AMH associated with each stage of ovarian aging.

[0098] One aspect of the disclosure is a method for quantitatively assessing a degree of ovarian aging in a subject. In an embodiment, 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. The corresponding AMH value is then 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 associatedwith 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 the subject is assigned a degree of ovarian aging based on the comparison. In this aspects, 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, and wherein the first, second, and third corresponding reference ranges are above the LOQ.

[0099] In some aspects, the methods may be further combined with other biomarkers to provide confirmatory information related to ovarian aging. In some aspects, the additional biomarker is part of a panel immunoassay, which is a laboratory technique used to simultaneously measure multiple analytes within a single sample. As disclosed herein, the methods allow for inclusion of several analytes to be assessed in parallel, enabling an simultaneous analysis of AMH and other biomarkers such as inhibin B and follicle-stimulating hormone (FSH). In some aspects, this allows for a comprehensive analysis of ovarian aging in one assay. In some embodiments, the methods further include detecting a presence or amount inhibin B and / or FSH in one or more plasma samples obtained from the subject using a high-throughput immunoassay analyzer and generating a corresponding inhibin B or FSH value. The methods may further include comparing the corresponding inhibin B or FSH 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.

[0100] In some aspects, the methods disclosed herein employ affinity molecules, such as antibodies, capable of binding to AMH. If additional biomarkers are detected, the methods disclosed herein may employ affinity molecules capable of binding to those biomarkers.

[0101] Affinity molecules include, but are not limited to, antibodies (including monoclonal antibodies, polyclonal antibodies, antibody fragments, synthetic antibody mimics, and the like), aptamers, affimers, DARPins, oligonucleotides, peptides, and antigens.

[0102] As used herein, the term “antibody” or “antibodies” refers to a binding protein, immunoglobulin, or glycoprotein that maintains antigen-binding properties. An antibody oftencomprises a variable domain and a constant domain in each of a heavy chain and a light chain. Accordingly, most antibodies have a heavy chain variable domain (VH) and a light chain variable domain (VL) that together form the portion of the antibody that binds to the antigen. Within each variable domain are three complementarity determining regions (CDR) which form loops in the heavy chain variable domain (VH) and light chain variable domain (VL) that contact the surface of the antigen. Antibodies herein also include “antigen binding portion” or fragments of the antibody that are capable of binding to the antigen.

[0103] As used herein, the term “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.

[0104] In an embodiment, 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. In some aspects, the detection reaction is a chemiluminescent reaction, an electrochemiluminescence reaction, an electrogenerated chemiluminescence reaction, a photoluminescence reaction, or a bioluminescence reaction. In some embodiments, 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. In some embodiments, the detector affinity molecule is conjugated to an enzyme, such alkaline phosphatase or horseradish peroxidase. In this embodiment, a substrate for the enzyme is added to the second reaction mixture triggering a chemiluminescent reaction.

[0105] In an aspect, the generated corresponding AMH value from the methods disclosed herein is compared with a corresponding reference range. In certain embodiments, 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. In an embodiment, 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 rangeestablished 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. In certain embodiments, the values of the first corresponding reference range is less than the second corresponding reference range and / or third corresponding reference range. In certain embodiments, the values of the second corresponding reference range is less than the third corresponding reference range.

[0106] In an aspect, 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 equal to or greater than about 0.39 ng / mL, is equal to or greater than about 0.41 ng / mL, is equal to or greater than about 0.43 ng / mL, is equal to or greater than about 0.45 ng / mL, is equal to or greater than about 0.47 ng / mL, is equal to or greater than about 0.49 ng / mL, is equal to or greater than about 0.51 ng / mL, is equal to or greater than about 0.53 ng / mL, is equal to or greater than about 0.55 ng / mL, is equal to or greater than about 0.57 ng / mL, is equal to or greater than about 0.59 ng / mL.

[0107] In an aspect, the second corresponding reference range is 0.014 ng / mL ± 0.02; alternatively is equal to or greater than about 0.006 ng / mL; alternatively is equal to or greater than about 0.008 ng / mL; alternatively is equal to or greater than about 0.010 ng / mL; alternatively is equal to or greater than about 0.012 ng / mL; alternatively is equal to or greater than about 0.014 ng / mL; alternatively is equal to or greater than about 0.016 ng / mL; alternatively is equal to or greater than about 0.018 ng / mL; alternatively is equal to or greater than about 0.020 ng / mL; alternatively is equal to or greater than about 0.022 ng / mL; alternatively is equal to or greater than about 0.024 ng / mL; alternatively is equal to or greater than about 0.026 ng / mL; alternatively is equal to or greater than about 0.028 ng / mL; alternatively is equal to or greater than about 0.030 ng / mL; alternatively is equal to or greater than about 0.032 ng / mL; alternatively is equal to or greater than about 0.034 ng / mL.

[0108] In an aspect, 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 0.0032 ng / mL; alternatively is equal to or greater than about 0.0033 ng / mL; alternatively is equal to or greater than about 0.0034 ng / mL; alternatively is equal to or greater than about 0.0035 ng / mL; alternatively is equal to or greater than about 0.0036 ng / mL; alternatively is equal to or greater than about 0.0037 ng / mL; alternatively is equal to or greater than about 0.0038 ng / mL; alternatively is equal to or greater than about 0.0039 ng / mL; alternatively is equal to or greater than about 0.004.

[0109] Based on the results of the comparison, the subject may be assigned a degree of ovarian aging. In certain embodiments, when an additional biomarker in is detected, the method may also include comparing the corresponding additional biomarker in 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 for the second biomarker. In certain embodiments, the method may include 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 additional biomarker in value, or a combination thereof.

[0110] In some cases, for subjects who are pre-menopausal, peri-menopasual, or post-menopausal the levels of AMH in blood may be in the pmol / L, Milli-intemational Unit (mIU), or ng range. One unexpected advantage of this method is the ability to determine the presence or concentration of small amounts or low levels of AMH. In a non-limiting example, the quantitation detection limit (LOQ) for measuring AMH is equal to or less than about 0.004 ng / mL, alternatively equal to orless 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. In a non-limiting example, 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. In a non-limiting example, 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. In a non-limiting example, 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. In a non-limiting example, 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. In a non-limiting example, 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. In a non-limiting example, 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.

[0111] In another non-limiting example, 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 theLOQ, 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.

[0112] In another non-limiting example, 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.

[0113] In yet another non-limiting example, 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. In yet another non-limiting example, the detected AMH exhibits a coefficient of variation (CV) of 20% or less. In yet another nonlimiting example, the detected AMH exhibits a coefficient of variation (CV) of 4% or less.

[0114] In some aspects, 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.

[0115] In some instances, 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. In some instances, 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. In some instances, a patient is recommended an intervention that that treats or prevents ovarian dysfunction and / or premature decline in ovarian reserve.

[0116] In an aspect, 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. In an embodiment, 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.

[0117] The terms "treat", "treating", or "treatment" refer 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 moreof the symptoms of a condition are ameliorated or otherwise beneficially altered. As used herein, 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. For example, 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.

[0118] An "effective amount" includes a "therapeutically effective amount" and a "prophylactically effective amount." The term "therapeutically effective amount" refers to an amount effective in treating and / or ameliorating a condition in a subject. The term "prophylactically 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.

[0119] In some aspects, 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. However, in some instances, the reproductive ageing may be premature, i.e., occurring before peri-menopause. In these instances, a clinician may administer an effective amount of a pharmaceutical composition to the subject or recommend a therapeutic intervention to the subject.

[0120] In an aspect, 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. In an aspect, 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. In some embodiments, the ovarian stage of aging and / or a risk score is based on the AMH value(s). In someembodiments, 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). In an embodiment, 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.

[0121] In an aspect, 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. Non-limiting examples of fertility treatments include intrauterine insemination (IUI), ovulation induction, IUI with ovulation induction, in vitro fertilization (IVF), and IVF with intra-cytoplasmic sperm injection (ICSI). In an aspect, 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.

[0122] In an embodiment, if the ovarian stage of aging or risk score is used to predict an ovarian response to IVF or other types of fertility treatments, 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. In an aspect, 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.

[0123] In an aspect, the method includes detecting a presence of AMH and generating a corresponding AMH value using the assays described herein. The corresponding AMH value is compared to a reference range and a degree of ovarian aging is assigned. In an aspect, the method includes detecting a presence of an additional biomarker and generating a corresponding additional biomarker value using the assays described herein. The corresponding additional biomarker value may be compared to a reference range and a degree of ovarian aging is assigned. In an embodiment, the degree of ovarian aging may indicate that a subject has entered into premature menopause. In this embodiment, subject then may be administered a pharmaceutical composition, hormone treatment, or similar treatment to slow the progression in menopause. In an embodiment, the degree of ovarian aging may indicate that a subject has entered menopause. In this embodiment,the subject may then be administered a pharmaceutical composition, hormone treatment, or similar treatment to eliminate some of the discomfort caused by the condition.

[0124] In an embodiment, the degree of ovarian aging may indicate that a subject has diminished fertility or is infertile. In this embodiment, the subject may then be administered a fertility treatment or ovulation induction agent. Exemplary fertility treatments include, but are not limited to assistive reproductive technology (ART), non- ART fertility treatments, and fertility preservation technologies (egg, embryo, or ovarian preservation). Exemplary ARTs include, but are not limited to, IVF, zygote intrafallopian transfer (ZIFT), gametic intrafallopian transfer (GIFT), or ICSI paired with one of the methods above. Exemplary non-ART fertility treatments include, but are not limited to, ovulation induction protocols with or without IUI with sperm. Exemplary ovulation induction agents include, but are not limited to, gonadotropins such as LH, FSH, human menopausal gonadotropin (hMG), and human chorionic gonadotropin (hCG); and oral ovulation induction agents. 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.

[0125] Ovarian response refers to the endocrine response of the ovaries to a stimulus. In some cases, ovarian response refers to the growth of one or more follicles in the ovary. In some cases, the ovarian response may culminate in the release of an egg by the ovary. In some cases, the ovarian stimulation is produced by an exogenous compound (e.g., exogenous gonadotropin). In some cases, the ovarian stimulation is administered for an in vitro fertilization therapy or for timed intercourse or insemination. In the case where the ovarian stimulation is administered for an in vitro fertilization therapy, predicting ovarian response may allow a health care provider to inform a patient receiving the therapy whether there is a risk of retrieving a lower number of oocytes in the in vitro fertilization procedure. In some cases, if a patient is determined to have low or poor ovarian response, they may choose a different course of action.

[0126] 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.

[0127] In some instances, in an aspect, the subject may be categorized as a low responder, normal responder, or high responder based on the AMH levels. In certain embodiments, 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.

[0128] In an aspect, the generated corresponding AMH value is compared with a corresponding reference range where the reference ranges are associated with various stages of ovarian response. In an embodiment, the corresponding AMH value is compared with a corresponding reference range established in a reference population and associated with a poor (or low ovarian) response. In certain embodiments, the corresponding AMH value may also be compared with additional reference ranges established in a reference population and associated with normal ovarian response and / or high ovarian response. In certain embodiments, the values of the corresponding reference range associated with a poor ovarian response is less than the corresponding reference ranges for the normal and / or high ovarian response. In certain embodiments, the values of the corresponding reference range for the normal ovarian response is less than the corresponding reference range for the high ovarian response. In an aspect, the corresponding reference ranges for poor, normal and / or high ovarian response may have a lower range value equal to or greater than about 0.004 ng / mL, alternatively equal to or greater than about 0.003 ng / mL, alternatively equal to or greater than about 0.002 ng / mL, equal to or greater than about 0.001 ng / mL, or equal to or greater than about 0.0006 ng / mL. Based on the results of the comparison, the subject may be assigned a degree of ovarian response.

[0129] The assessment of ovarian failure also can provide a clinician with information regarding oocyte yield. In certain embodiments, based on AMH levels, a subject may be categorized as having a poor ovarian response or a normal -to-high ovarian response. In certain instances, if a subject is categorized as having a poor ovarian response, the clinician may recommend that the subject not undergo oocyte retrieval and may establish a treatment protocol which includes a therapeutic intervention. If a subject is deemed to have a normal -to-high ovarian response, the clinician may proceed with oocyte retrieval.

[0130] Ovarian failure, also referred to as premature menopause and primary ovarian insufficiency, refers to menopause occurring before age 40. Generally, ovarian failure ischaracterized 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).

[0131] AMH is thought to aid in the prediction of ovarian failure by being able to broadly identify those at increased risk, but current conventional assays do not offer the precision adequate to predict the timing and onset of ovarian failure. Therefore, an automated AMH assay with higher sensitivity and specificity and a lower LOQ allows for identifying clinically relevant ranges below the thresholds of existing AMH assays for predicting ovarian failure.

[0132] In an aspect, the generated corresponding AMH value is compared with a corresponding reference range where the reference ranges are associated with various stages of ovarian failure. In an embodiment, the corresponding AMH value is compared with a corresponding reference range established in a reference population and associated with a likelihood of ovarian failure. In certain embodiments, the corresponding AMH value may also be compared with additional reference ranges established in a reference population and associated with normal-to-high ovarian response. In certain embodiments, the values of the corresponding reference range associated with a likelihood of ovarian failure is less than the corresponding reference ranges for the normal-to- high ovarian response. In an aspect, the corresponding reference ranges for a likelihood of ovarian failure and / or a normal-to-high ovarian response may have a lower range value equal to or greater than about 0.004 ng / mL, alternatively equal to or greater than about 0.003 ng / mL, alternatively equal to or greater than about 0.002 ng / mL, equal to or greater than about 0.001 ng / mL, or equal to or greater than about 0.0006 ng / mL. Based on the results of the comparison, the subject may be assigned a degree of likelihood of ovarian failure.IV. Automated Analyzer and Systems for Detecting AMH

[0133] In an embodiment, 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. In an aspect of the methods disclosed herein, a plasma sample is analyzed for AMH using a high-throughput immunoassay analyzer disclosed herein. In an embodiment, the method includes aspirating a portion of the plasma sample from a samplevessel and dispensing the aspirated sample into a reaction vessel of a high-throughput immunoassay analyzer.

[0134] In an aspect, an immunoassay analyzer disclosed herein includes the following basic structural and functional modules: a sample presentation unit, an analytic unit, an incubator station, a washing station, a read station, and reagent storage. In addition, the immunoassay analyzer may include a pipettor arrangement with at least one sample pipettor and at least one reagent pipettor, and at least one transport device. In an aspect, the transport device includes mechanisms, such as pick-and-place grippers, which are used to transport sample and reaction vessels among the various modules of the immunoassay analyzer.

[0094] 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. Examples of 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.

[0095] The read station may include a detector arrangement. In an aspect, 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. In an embodiment, 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.

[0096] In an aspect, primary sample containers can be placed into an onload section (e.g., individually or on racks) of the sample presentation unit. In certain embodiments, the samplepresentation 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. In certain embodiments, 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. In a specific embodiment, 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.

[0097] In certain embodiments, 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. In one embodiment, 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.

[0098] After being fed into the onload section, the sample containers may be moved into a presentation section of the sample presentation unit. In certain embodiments, the sample presentation unit is maintained at between about 4.5°C to 14°C. The transfer station may receive primary sample containers from the sample presentation unit from a transport device. Primary sample containers delivered to the transfer station from the transport device may be initially processed in different ways. For example, the primary sample container may temporarily remain at the transfer station while the sample provided within is aspirated by the sample pipettor at the transfer station. Following such aspiration, the primary sample container may be expelled from the automated analyzer at the offload station of the sample presentation unit, or may be passed on to the transport device for further processing. The instrument may also contain a sample retentionunit 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. As used herein, “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. In certain embodiments, the vessels are tubes with diameters between about 12mm to about 16mm and / or heights between about 75mm to about 100mm. In certain embodiments, 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.

[0099] In an aspect, the analytic unit is configured to receive and analyze samples. In an aspect, the analytic unit configured to perform an immunoassay. In certain embodiments, the analytic unit includes the pipettor arrangement. The pipettor arrangement may be configured to aliquot, aspirate, and dispense fluidic substances into various vessels, including, but not limited to sample vessels, diluent vessels, reagent vessels, and reaction vessels. Fluidic substances are substances that have fluidic characteristics. In some embodiments, the fluidic substance is a single fluidic substance. In other embodiments, the fluidic substance is a mixture of a plurality of substances.

[0100] The pipettor arrangement may contain at least one, two, three, or four reagent pipettors used to mix reagents with sample aliquots for an assay. The pipettor arrangement may also contain at least one, two, three, or four sample pipettors used to transfer sample aliquots for an assay. In some instances, the pipettor arrangement contains one sample pipettor. In some instances, the pipettor arrangement contains one sample pipettor and four reagent pipettors. In an aspect, the reagent pipettors may be arranged as dual reagent pipetting stations and are independent to each other, each having its own fluid pumps and valves, wash towers, reaction vessel carriages, and pipettor. A sample aliquot may be transferred from a sample retention vessel into a reaction vessel using the sample pipettor in order to mix the sample aliquot with one or more reagents. In an aspect, the at least one reagent pipettor and at least one sample pipettor are configured to aspirate and / or dispense less than about 10 pL. In an embodiment, the at least one reagent pipettor and at least one sample pipettor are configured to aspirate and / or dispense less than about 9.9 pL, alternatively less than about 9.5 pL, alternatively less than about 8.0 pL, alternatively less than about 7.0 pL, alternatively less than about 6.0 pL, alternatively less than about 5.0 pL, alternativelyless than about 4.0 pL, alternatively less than about 3.0 pL, alternatively less than about 2.0 pL, alternatively between than about 9.9 pL and 2.0 pL.

[0101] In an aspect, the first reagent pipettor, the second reagent pipettor, the third reagent pipettor, and / or the fourth reagent pipettor are selectively and / or simultaneously operated. In certain embodiments, the sample pipettor, the first reagent pipettor, the second reagent pipettor, the third reagent pipettor, and / or the fourth reagent pipettor are configured to engage a dispense tip prior to aspiration.

[0102] In an aspect, 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.

[0103] In an embodiment, the reagent pack may be configured to accommodate sufficient volumes of reagents for multiple instances of an assay. In some embodiments, each reagent pack includes reagents for about 20 to about 100 instances of an assay and in some cases about 50 instances.

[0104] In some embodiments a reagent pack may supplied with empty or partially filled reagent vessels, to which reagents are subsequently transferred from bulk containers, such as bottles. Individual reagent vessels may differ in dimension to accommodate the requirements of an assay type. Factors that can determine the size of a reagent vessel include the number of uses desired for the reagent pack type, concentration-dependent stability issues with reagent components, and the need to minimize the volume of the final reaction mixture. As noted above, in some embodiments, each reagent pack can include a large reagent vessel and a plurality of small reagent vessels. 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. In certain embodiments, the reagent pack may be maintained at a temperature of between about 4°C to 10°C.

[0105] A “reagent vessel” may refer to a vessel, unit, fluid container, or the like that is configured to store reagents. In an embodiment, 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. For some embodiments, 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.

[0106] 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. In some embodiments, 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. In some embodiments, 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).

[0107] In methods where multiple samples are analyzed, the elastomeric self-sealing membrane allows for aspiration of the reagents without concern of evaporation. In some embodiments, the reagent pack further includes containment walls arranged between the reagent vessels. In an aspect, 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. In an embodiment, the immunoassay analyzer further includes a reagent storage unit, wherein the reagent pack is housed in the reagent storage unit.

[0108] At operation, a fluidic substance is dispensed to a reaction vessel. Examples of the fluidic substance include a sample, diluent, reagent, substrate, or any combination thereof, as described herein. In some embodiments, 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. In some aspects, the immunoassay analyzer includes an ultrasonic mixing module. For instance, a reagent pipettor maybe 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.

[0109] The transfer unit transfers the reaction vessels to and from the incubator station which includes an incubator. In some embodiments, the transfer unit transfers one or more of the pipetted reaction vessels from the reagent carriage unit to the incubator. Further, 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.

[0110] The incubator is thermally controlled to maintain a predetermined temperature. In some embodiments, the incubator is maintained about 30 °C to 40 °C. In other embodiments, the incubator is maintained about 37 °C to ensure immunological reaction and enzyme reaction, for example. By way of example, the incubator performs assay incubation.

[0111] In some embodiments, 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.

[0112] At operation, a sample and a reagent are dispensed into a reaction vessel and mixed. The mixture is then transferred to the incubator. During the incubation, 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. In a non-limiting example, 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.

[0113] At operation, 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 theincubator. 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. In a non-limiting example, 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.

[0114] The wash station receives and supports reaction vessels thereon such that various aspects of diagnostic process are performed with the immunoassay analyzer. In an embodiment, 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. In certain embodiments, 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. In some embodiments, the wash station is a thermally controlled device to separate bound or free analytes from particles after incubation. In some embodiments, 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.

[0115] 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. At operation, 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. Atoperation, a base number of wash actions performed may be three and the additional number of wash action(s) may be one or two. In this embodiment, 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. According to the principles of the present disclosure, 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).

[0116] In an aspect, the affinity molecule and / or the detection molecule is conjugated to a magnetic bead or a magnetic particle. In some instances, magnetic beads (also known as magnetic particles, paramagnetic particles, or superparamagnetic particles) consist of a polystyrene core surrounded by a thin layer of small iron oxide particles (-20-30 nm), such as magnetite. On the surface, 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. In some embodiments, there is about 2 pg or antibody per mg of magnetic bead. In some embodiments, there is about 3 pg or antibody per mg of magnetic bead. In some embodiments, there is about 4 pg or antibody per mg of magnetic bead. In some embodiments, there is about 5 pg or antibody per mg of magnetic bead. In some embodiments, there is about 6 pg or antibody per mg of magnetic bead. In some embodiments, there is about 7 pg or antibody per mg of magnetic bead. In some embodiments, there is about 8 pg or antibody per mg of magnetic bead. In some embodiments, there is about 9 pg or antibody per mg of magnetic bead. In some embodiments, there is about 10 pg or antibody per mg of magnetic bead. In some embodiments, there is about 12 pg or antibody per mg of magnetic bead. In some embodiments, there is about 13 pg or antibody per mg of magnetic bead. In some embodiments, there is about 14 pg or antibody per mg of magnetic bead. In some embodiments, there is about 15 pg or antibody per mg of magnetic bead.

[0117] In an embodiment, 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 amagnetic 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.

[0118] At operation, the vessel containing the first reaction mixture is moved near one or more magnets. The one or more magnets attract the magnetic bead(s) to one or more sides of the reaction vessel. The reaction vessel is then subject to a wash process in which a cleaning dispense nozzle dispenses a rinsing fluid and a cleaning aspiration nozzle aspirates the unreacted components. The aspiration nozzle may be washed with a probe washer before and / or after the aspirating. The reaction vessel may undergo a series of wash process which may include at least two series of dispensing the rinsing fluid and aspirating the uncollected fluid components, alternatively at least three series, alternatively at least four series, alternatively at least five series. As a result, an unreacted substance or substances in the vessel is removed (e g., rinsed away) by the bound-free cleaning aspiration nozzle.

[0119] At operation, the vessel containing the second reaction mixture is moved near one or more magnets. The one or more magnets attract the magnetic bead(s) or magnetic particle(s) to one or more sides of the reaction vessel. The reaction vessel is then subject to a wash process in which a cleaning dispense nozzle dispenses a rinsing fluid and a cleaning aspiration nozzle aspirates the unreacted components. The aspiration nozzle may be washed with a probe washer before and / or after the aspirating. The reaction vessel may undergo a series of wash process which may include at least two series of dispensing the rinsing fluid and aspirating the uncollected fluid components, alternatively at least three series, alternatively at least four series, alternatively at least five series. As a result, an unreacted substance or substances in the vessel is removed (e.g., rinsed away) by the bound-free cleaning aspiration nozzle.

[0120] At operation, a substrate is dispensed into the second reaction mixture and mixed. After a certain reaction time necessary for the substrate and the second reaction mixture to interact. The reaction vessel is then subject to a wash process in which a cleaning dispense nozzle dispenses a rinsing fluid and a cleaning aspiration nozzle aspirates the unreacted components. The aspiration nozzle may be washed with a probe washer before and / or after the aspirating. The reaction vessel may undergo a series of wash process which may include at least two series of dispensing the rinsing fluid and aspirating the uncollected fluid components, alternatively at least three series, alternatively at least four series, alternatively at least five series. As a result, an unreacted substanceor substances in the vessel is removed (e.g., rinsed away) by the bound-free cleaning aspiration nozzle. The resulting detection mixture is transferred to the detector arrangement.

[0121] Assays including features and / or characteristics described herein may benefit from one or more additional number(s) of wash actions. In some instances, “two-site” or “sandwich” immunoassays employ a first antibody or antibody fragment, which is described as the “capture” antibody, is bound to a solid support, such as magnetic beads or particles disclosed herein, using procedures known in the art. Further, a second antibody or antibody fragment, which is described as the “detection” antibody, is coupled or conjugated with a label, such as the enzymes disclosed herein, using procedures known in the art. The label produces a detectable signal when provided with substrate(s), so that the amount of signal measured corresponds to the amount of detection antibody that is bound to the analyte.

[0122] In certain embodiments, the transport device includes three pick-and-place grippers, where a first pick-and-place gripper may be used to transport sample containers among the onload section, the transfer station, and reagent pipetting stations. A second pick-and-place gripper may be used to transport reaction vessels between the reagent pipetting stations and the incubator station or read station. A third pick-and-place gripper may be used to transport reaction vessels between the incubator station and the wash station or read station. A detailed description of the configurations and functions of one embodiment of the vessel pick-and-place grippers is provided in U.S. Patent No. 7128874 and is incorporated herein in its entirety by reference. However, it should be understood that other pick-and-place mechanism that are capable of transporting sample and reaction vessels among the various modules of the immunoassay analyzer is also contemplated for the purpose of the present invention.

[0123] In an aspect, the immunoassay analyzer includes a machine vision apparatus comprising an image capture device and an image interpretation device configured to monitor instrument and / or assay functionalities of the immunoassay analyzer. In some embodiments, instrument functionalities may include optical sensors, pressure sensors and thermistors. In some embodiments, the assay functionalities may include sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring. The machine vision apparatus operates to evaluate the preparation of samples for subsequent analysis. In some embodiments, the machine vision apparatus utilizes one or more image capture units to determinewhether samples have been appropriately prepared for analysis. As described herein, the machine vision apparatus provides direct and simple measurements of volume or integrity of a sample to determine whether the sample is appropriately prepared so that the analytic unit produces a reliable result. An exemplary machine vision apparatus is described in U.S. Patent No. 11,263,433, which is incorporated by reference herein.

[0124] In some aspects, 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.

[0125] In some aspects, the machine vision apparatus operates to detect any interferents, which can interfere with an analytic procedure and may generate incorrect results in the dispense tip. As described herein, this dispense tip evaluation is configured to determine a quality of a fluidic substance at a dispense tip using a dispense tip image capture unit, and an alignment of the dispense tip with respect to the dispense tip image capture unit.

[0126] In some aspects, the machine vision apparatus operates to determine a particle concentration in a fluidic substance contained in a vessel, such as a reaction vessel, a sample vessel, a dilution vessel, a cuvette, or any suitable type of vessel, which is used throughout the process in the immunoassay analyzer. In some embodiments, this reaction vessel particle concentration check uses the vessel image capture unit.

[0127] The dispense tip image capture unit operates to capture images of dispense tips in one or more locations. In some embodiments, the dispense tip image capture unit is fixed at a particular location in the instrument. In other embodiments, the dispense tip image capture unit is movably disposed in the instrument, which can move either independently from other components of the instrument or together with one or more components of the instrument. Some embodiments of the instrument include a plurality of dispense tip image capture units. As described herein, the dispense tip image capture unit can include a camera unit.

[0128] The vessel image capture unit operates to capture images of vessels in one or more locations. In some embodiments, the vessel image capture unit is fixed at a particular location in the immunoassay analyzer. In other embodiments, the vessel image capture unit is movablydisposed in the immunoassay analyzer, which can move either independently from other components of the immunoassay analyzer or together with one or more components of the immunoassay analyzer. Some embodiments of the immunoassay analyzer include a plurality of vessel image capture units. As described herein, the vessel tip image capture unit includes a camera unit.

[0129] The carriage image capture unit operates to capture images of container carriage devices with or without containers in one or more locations. In some embodiments, the carriage image capture unit is fixed at a particular location in the instrument. In other embodiments, the carriage image capture unit is movably disposed in the instrument, which can move either independently from other components of the instrument or together with one or more components of the instrument. Some embodiments of the instrument include a plurality of carriage image capture units.

[0130] All of the units of the immunoassay analyzer are connected to a controller, which can perform block control of all of the analyzer functions by using, for example, a microcomputer. The controller may contain subunits such as a data processing unit, a communication interface, and others. A controller in accordance with an exemplary embodiment of the present technology may comprise a data processor, a non-transitory computer-readable medium, and a data storage coupled to the data processor. The non-transitory computer-readable medium may comprise code, executable by the data processor, to perform the functions described herein. The data processor may store, for example, data for processing samples, sample data, or data for analyzing sample data.

[0131] The data processor may include any suitable data computation device or combination of such devices. An exemplary data processor may comprise one or more microprocessors working together to accomplish a desired function. The data processor may include a CPU that comprises at least one high-speed data processor adequate to execute program components for executing user and / or system-generated requests. The CPU may be a microprocessor such as AMD’s Athlon, Duron and / or Opteron; IBM and / or Motorola’s PowerPC; IBM’s and Sony’s Cell processor; Intel’s Celeron, Itanium, Pentium, Xeon, and / or XScale; Apple Ml, and / or the like processor(s).

[0132] The computer-readable medium and the data storage may be any suitable device or devices that can store electronic data. Examples of memories may comprise, for example, one or morememory chips, disk drives, etc. Such memories may operate using any suitable electrical, optical, and / or magnetic mode of operation.

[0133] The computer-readable medium may comprise code, executable by the data processor to perform any suitable method. For example, the computer-readable medium may comprise code, executable by the processor, to cause the controller to operate on a pre-determined schedule. In some embodiments of the presently claimed technology, the pre-determined schedule is a constituent test.

[0134] In an aspect the cycle time of an immunoassay analyzer described herein is about 45 seconds or less. The “cycle time” is the time required for all modules and / or functions of an immunoassay analyzer to complete its tasks necessary for generating a result. In certain embodiments, the cycle time is 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. At operation, the sample pipettor can complete its tasks in 8 seconds and the reagent pipettor can complete its tasks in 32 seconds. In this embodiment, to maintain a higher throughput, four reagent pipettors are present in the immunoassay analyzer (32 seconds / 4 = 8 seconds). If the incubation of one of the reaction mixtures is 5 minutes, at least 38 incubation positions are needed to support this incubation time (300 seconds / 8 seconds = 37.5 seconds). In certain embodiments, the immunoassay analyzer has at least 30 incubation positions, alternatively at least 40 incubation positions, alternatively at least 50 incubation positions, alternatively at least 60 incubation positions, alternatively at least 70 incubation positions, alternatively at least 80 incubation positions, alternatively at least 90 incubation positions, alternatively at least 100 incubation positions, alternatively at least 125 incubation positions, alternatively at least 150 incubation positions, alternatively at least 175 incubation positions, or alternatively at least 200 incubation positions.

[0135] In an aspect, the time to first result (TTFR) of an immunoassay analyzer described herein is about 60 minutes or less. The “TTFR” is a measure of time from when the sample is aspirated to when the presence and / or concentration of an analyte is determined. In certain embodiments the TTFR 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 about25 minutes or less, alternatively about 20 minutes or less, alternatively about 15 minutes or less, or alternatively about 10 minutes or less.

[0136] In an embodiment, the reagent pack may be configured to accommodate sufficient volumes of reagents for multiple instances of an assay. In some embodiments, each reagent pack includes reagents for about 20 to about 100 instances of an assay and in some cases about 50 instances. In some embodiments, the reagent pack may include a plurality of reagent vessels, wherein the reagent pack is configured to store a volume of reagent required for at least about 20 instances of an AMH assay. In a non-limiting example, the reagent pack includes at least three reagent vessels, wherein each reagent vessel is independently configured to store a volume of reagent required for at least about 20 instances of an AMH assay. In a non-limiting example, the reagent pack includes at least four reagent vessels, wherein each reagent vessel is independently configured to store a volume of reagent required for at least about 20 instances of an AMH assay. In a non-limiting example, the reagent pack includes at least five reagent vessels, wherein each reagent vessel is independently configured to store a volume of reagent required for at least about 20 instances of an AMH assay.

[0137] In an aspect, the first reagent includes at least one affinity molecule configured to bind to at least one portion AMH. Depending on the assay, the affinity molecule may be 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. In a nonlimiting example 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.

[0138] The “first reaction mixture” is a result of an incubation between the sample and the first reagent. During the incubation, the sample and the first reagent interact. In an embodiment, the affinity molecule of the first reagent binds with the AMH. In a non-limiting example, the 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.

[0139] In an embodiment, the method includes 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. In an aspect, the second reagent includes a detection molecule. Thedetection molecule, may be, for example, an alkaline phosphatase (AP)-conjugated secondary antibody.

[0140] The “second reaction mixture” is a result of an incubation between the first reaction mixture and the second reagent. During the incubation, the first reaction mixture and the second reagent interact. In an embodiment, the detection molecule of the second reagent binds with the affinity molecule of the first reagent (which is bound with the AMH in the plasma sample.) In an aspect, 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.

[0141] In an embodiment, the method includes aspirating a portion of a substrate formulation from at least one reagent vessel and dispensing the aspirated substrate formulation into the reaction vessel, generating a detection mixture. In an aspect, the substrate formulation is configured to produce chemiluminescence. These substrates can produce light and thereby provide detection corresponding to a quantity of analytes captured. The term “chemiluminescent compound” refers to a compound that produces chemiluminescence in the presence of a phosphatase enzyme and oxygen under appropriate conditions as provided herein. In a non-limiting example, the substrate formulation includes a chemiluminescent compound of formula I or a salt thereof:

[0143] wherein

[0144] 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)Ri5, CN or NCh substituents;

[0145] Ri is selected from the group consisting of Cs-uaryl, Ci-6 alkyl, Ci-6 haloalkyl, and C5-14 aralkyl groups;

[0146] R7-R14 are independently H, C1-6 alkoxy, halo, C1-4 alkyl, or R7 or R8-R9 or R9-R10 or RH- R12 or Rn-Ri3 or R13-R14, can be joined together as a carbocyclic or heterocyclic ring system comprising at least one 5 or 6-membered ring;

[0147] R15 is Ci-6 alkyl;

[0148] 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;

[0149] Z is O or S; and

[0150] n is 0, 1, or 2;

[0151] a cationic aromatic compound (CAC);

[0152] a background reducing agent; and

[0153] an ether-linked nonionic surfactant or a hydrophilic polymer.

[0154] Chemiluminescent compounds useful in the present formulations are capable of generating chemiluminescence when contacted with an alkaline phosphatase. Such compounds can be synthesized as described in U.S. Pat. Nos. 6,45,727, 6,90,571, 6,139,782, 6,218,137, 6,270,695, 6,296,787, and 10,703,971 each of which is incorporated by reference herein.

[0155] At operation, the chemiluminescent substrate is added to the vessel with the second reaction mixture and light generated by the reaction is measured with a luminometer. The amount of analyte in the sample is then determined from a stored, multi-point calibration curve. In an embodiment, the detector can generate an output signal that can be processed to generate a relative light unit (“RLU”) value (i.e., an output response) indicating a result of the assay. For example, a larger RLU value indicates more light, which indicates a larger amount of the analyte in the biological sample than a smaller RLU value indicates.

[0156] In an exemplary method of producing light from the reaction of the chemiluminescent substrate with a phosphatase enzyme (e.g., detection antibody), the reaction is performed at a temperature between 5° C and 50° C, preferably between 20° C and 40° C in an aqueous buffersolution at a pH between 7 and 12, 8 and 11 , or preferably between 8.5 and 10. The enzyme is preferably an alkaline phosphatase or an alkaline phosphatase conjugate.

[0157] In one aspect, the substrate formulation comprises 0.01 mM-50 mM compound I, 0.01-200 pM cationic aromatic compound, 1 pM -10 mM background reducing agent, 0.05-20 g / L ether- linked non-ionic surfactant or hydrophilic polymer, 0.01-10 g / L anionic surfactant, and an amine buffer at from 0.025M to 0.65M and at pH 7-12.

[0158] In one aspect, the substrate formulation comprises 0.05 mM-10 mM compound I, 0.05-50 pM cationic aromatic compound, 10 uM- 1000 pM background reducing agent, 0.1 to 10 g / L ether- linked non-ionic surfactant or hydrophilic polymer, 0.1 to 5 g / L anionic surfactant, and an amine buffer at from 0.05M to 0.5M and at pH 8-11.

[0159] In one aspect, the substrate formulation comprises 0.1 mM-5 mM compound I, 0.1-25 pM cationic aromatic compound, 50 to 500 pM background reducing agent, 0.2 to 5 g / L ether-linked non-ionic surfactant or hydrophilic polymer, 0.1 to 5 g / L anionic surfactant, and an amine buffer at from 0.1M-0.4M and at pH 8-11.

[0160] In one aspect, the compound I has the formula

[0162] In an aspect, the immunoassay analyzer includes a detector arrangement. In an aspect, 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. In an embodiment, 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. 1 1,604,146, which is incorporated by reference in its entirety herein, discloses nonlimiting examples of a detector that may be used in an aspect of the invention.

[0163] In an embodiment, the method further includes detecting, using a detector arrangement, a presence of a reaction in the detection mixture and determining a presence and / or concentration of AMH in the plasma sample based on the presence of the reaction in the detection mixture. In an aspect, the reaction in the detection mixture generates a chemiluminescent signal, wherein the chemiluminescent signal corresponds to the presence and / or concentration of AMH in the plasma sample. In an embodiment, the detector can generate an output signal that can be processed to generate a relative light unit (“RLU”) value (i.e., an output response) indicating a result of the assay. For example, a larger RLU value indicates more light, which indicates a larger amount of the analyte in the plasma sample than a smaller RLU value indicates.

[0164] The claimed configuration allows for the simultaneous performance of at least two assays for a plurality of plasma samples, alternatively at least three assays for a plurality of plasma samples, or at least four assays for a plurality of plasma samples. Depending on the analysis desired the plasma samples may be from the same subject or from multiple subjects. In some aspects, the method further comprises a plurality of sample vessels. In some embodiments, the method comprises at least one sample vessel, at least two sample vessels, at least three sample vessels, at least four sample vessels, at least five sample vessels, at least ten sample vessels, at least twenty sample vessels, at least fifty sample vessels, at least one hundred sample vessels, at least two hundred sample vessels, at least three hundred sample vessels, at least four hundred sample vessels, or at least five hundred sample vessels. In some aspects, the sample vessels are housed within the immunoassay analyzer.

[0165] In certain embodiments, at least one of the assays is AMH assay, alternatively at least two of the assays are AMH assays, alternatively at least three of the assays are AMH assays, or alternatively at least four of the assays are AMH assays.

[0166] In certain embodiments, the method may include detecting at least one additional biomarker associated with ovarian aging. In some embodiments, at least one of the assays is AMH assay and at least one of the assays is a second analyte assay, wherein the second analyte is inhibin B or follicle-stimulating hormone (FSH).

[0167] In a non-limiting example, if multiple assays for multiple biomarkers are being performed, the first pipettor may be configured to aspirate a reagent from a reagent vessel comprising a first affinity molecule (e.g., a AMH antibody) and dispense into a first reaction vessel, while a second pipettor may be configured to simultaneously aspirate a reagent from a reagent vessel comprising a second affinity molecule (e.g., second analyte antibody) and dispense into a second reaction vessel. Depending on the analysis desired, the third and fourth pipettor may also be configured to simultaneously aspirate a reagent from a reagent vessel comprising a third and fourth affinity molecule and dispense into a third and fourth reaction vessel.

[0168] The disclosed simultaneous and / or selective operation of the pipettors allows for a high- throughput analysis. In some embodiments, the method is configured to analyze at least about 200 plasma samples / hr., alternatively at least about 300 plasma samples / hr., alternatively at least about 400 plasma samples / hr, alternatively at least about 440 plasma samples / hr. or alternatively at least 500 plasma samples / hr.

[0169] In an aspect of the methods disclosed herein, a biological sample is analyzed for AMH using a high-throughput immunoassay analyzer disclosed herein. In an embodiment, the method includes aspirating, using a sample pipettor, a portion of the biological sample from a sample vessel and dispensing the aspirated sample into a reaction vessel of an immunoassay analyzer. In an aspect, the biological sample is serum, whole blood, and / or plasma.

[0170] In an embodiment, the method includes aspirating, using a reagent pipettor, a portion of a first reagent from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel. In some embodiments, the reagent pack may include a plurality of reagent vessels, wherein the reagent pack is configured to store a volume of reagent required for at least about 20 instances of an AMH assay. In a non-limiting example, the reagent pack includes at least three reagent vessels, wherein each reagent vessel is independently configured to store a volume of reagent required for at least about 20 instances of an AMH assay. In a non-limiting example, the reagent pack includes at least four reagent vessels, wherein each reagent vessel is independently configured to store a volume of reagent required for at least about 20 instances of an AMH assay. In a non-limiting example, the reagent pack includes at least five reagent vessels, wherein each reagent vessel is independently configured to store a volume of reagent required for at least about 20 instances of an AMH assay.

[0171] In an aspect, the reagent pack is configured to store a volume of reagent required for at least about 20 instances of an additional biomarker assay. In a non-limiting example, the reagent pack includes at least three reagent vessels, wherein each reagent vessel is independently configured to store a volume of reagent required for at least about 20 instances of an additional biomarker assay. In a non-limiting example, the reagent pack includes at least four reagent vessels, wherein each reagent vessel is independently configured to store a volume of reagent required for at least about 20 instances of an additional biomarker assay. In a non-limiting example, the reagent pack includes at least five reagent vessels, wherein each reagent vessel is independently configured to store a volume of reagent required for at least about 20 instances of an additional biomarker assay.

[0172] In an aspect, a first reagent includes at least one affinity molecule configured to bind to at least one portion of AMH. In an aspect, a first reagent includes at least one affinity molecule configured to bind to at least one portion of an additional biomarker.

[0173] In a non-limiting example, the 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, alternatively at least about 55 minutes, or alternatively at least about 60 minutes.

[0174] In a non-limiting example, the affinity molecule is an antibody and the method further comprises exposing the biological sample to a first antibody which binds to a first AMH epitope and a second antibody which binds to a second AMH epitope.

[0175] In a non-limiting example, the affinity molecule is an antibody and the method further comprises exposing the biological sample to a first antibody which binds to a first additional biomarker epitope and a second antibody which binds to a second additional biomarker epitope.

[0176] In certain embodiments, the first antibody is a capture antibody and the second antibody is a detection antibody. In other embodiments, both antibodies are capture antibodies.

[0177] In an embodiment, an AMH antibody is bound to magnetic bead. In an embodiment, an additional biomarker antibody is bound to magnetic bead.

[0178] Magnetic beads comprising said antibody or antibodies are added to the sample in a reaction vessel along. At least one buffer or other reagents may be added as required by the assay. The mixture is incubated for a period of time necessary for the antibody to bind with the targeted epitope forming a first reaction mixture. A magnetic field is applied to the reaction vessel and theisolated, bead-bound proteins are washed using the washing arrangement. A base number (three) of wash actions is performed with optional one or two additional wash actions.

[0179] In an embodiment, the method includes, aspirating, using the reagent pipettor, a portion of a second reagent from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel. In an aspect, a second reagent includes a detection molecule. The detection molecule, may be, for example, a detection antibody, such as alkaline phosphatase (AP)- conjugated secondary antibody. In an embodiment, the detection molecule of the second reagent binds with the affinity molecule of the first reagent (which is bound with the AMH in the biological sample) or binds to a different AMH epitope. In an embodiment, the detection molecule of the second reagent binds with the affinity molecule of the first reagent (which is bound with the additional biomarker in the biological sample) or binds to a different additional biomarker epitope. In an aspect, 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.

[0180] In an embodiment, an AMH antibody conjugated to alkaline phosphatase is added to the first reaction mixture in a reaction vessel. The mixture in incubated for a period of time necessary for the antibody to bind with a second AMH epitope, forming a second reaction mixture. In an embodiment, an additional biomarker antibody conjugated to alkaline phosphatase is added to the first reaction mixture in a reaction vessel. The mixture in incubated for a period of time necessary for the antibody to bind with a second additional biomarker epitope, forming a second reaction mixture. A magnetic field is applied to the reaction vessel and the isolated, bead-bound proteins are washed using the washing arrangement. A base number (three) of wash actions is performed with optional one or two additional wash actions.

[0181] In an embodiment, the method includes dispensing a substrate formulation into the reaction vessel, generating a detection mixture. In certain embodiments, the substrate formulation comprises compound I, a cationic aromatic compound, a background reducing agent, an ether- linked non-ionic surfactant or hydrophilic polymer, an anionic surfactant, and an amine buffer. A magnetic field is applied to the reaction vessel and the isolated, bead-bound proteins are washed using the washing arrangement. A base number (three) of wash actions is performed with optional one or two additional wash actions.

[0182] In an embodiment, the method further includes detecting, using a detector arrangement, a presence of a reaction in the detection mixture and determining a presence and / or concentration ofAMH in the biological sample based on the presence of the reaction in the detection mixture. In an aspect, the reaction in the detection mixture generates a chemiluminescent signal, wherein the chemiluminescent signal corresponds to the presence and / or concentration of AMH in the biological sample.

[0183] In an embodiment, the method further includes detecting, using a detector arrangement, a presence of a reaction in the detection mixture and determining a presence and / or concentration of an additional biomarker in the biological sample based on the presence of the reaction in the detection mixture. In an aspect, the reaction in the detection mixture generates a chemiluminescent signal, wherein the chemiluminescent signal corresponds to the presence and / or concentration of an additional biomarker in the biological sample.

[0184] Both the immunochemical assays and immunoassays may be “two-site” or “sandwich” assays which employ a first antibody or antibody fragment, which is described as the “capture” antibody, that is bound to a solid support, such as magnetic beads or particles disclosed herein, using procedures known in the art. Further, a second antibody or antibody fragment, which is described as the “detection” antibody, is coupled or conjugated with a label, such as the enzymes disclosed herein, using procedures known in the art. The label produces a detectable signal when it interacts with a substrate or substrates, so that the amount of signal measured corresponds to the amount of detection antibody that is bound to the analyte. Other types of immunochemical assays and immunoassays include competitive assays and antibody detection assays.

[0185] In an embodiment, the high-throughput immunoassay analyzer further includes an ultrasonic mixing module. In certain embodiments, the first reaction mixture, second reaction mixture, and / or detection mixture is agitated via the ultrasonic mixing module.

[0186] In an aspect, the immunoassay analyzer further comprises a washing arrangement. In an embodiment, the washing arrangement is configured to wash away at least some of the unreacted components from the sample, first reagent, second reagent, or substrate formulation in the first reaction mixture, second reaction mixture, and / or detection mixture. Unreacted components may include unreacted reagents (e.g., free antigens, antibodies, unbound reactants, particles, and / or fluid, etc.) and unreacted sample. The washing arrangement may be configured to perform a set number of wash actions depending on the assay. The washing arrangement may also be configured to perform a set number of washes within a predetermined sequence. In certain embodiments, the washing arrangement is configured to perform at least one wash action to wash away at least aportion 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.

[0187] In an aspect, the affinity molecule and / or the detection molecule is conjugated to a magnetic bead or a magnetic particle. In an embodiment, the first reaction mixture, second reaction mixture, and / or detection mixture is subjected to a magnetic field prior to detection. Magnetization may be used to retain desired components within a reaction vessel. In certain embodiments, the reaction vessel is moved near one or more magnets after the introduction of the first reagent or second reagent. The one or more magnets attract the magnetic bead(s) or magnetic particle(s) to one or more sides of the reaction vessel. The washing arrangement is used to wash the reaction vessel for a predetermined number to times. While washing the magnet(s) retain the magnetic bead(s) or magnetic particle(s) while the unreacted components are washed away. In certain embodiments, increasing the numbers of wash actions may result in a better signal to noise ratio and increase the sensitivity of the assay.

[0188] In an aspect the cycle time is about 45 seconds or less. The “cycle time” is the time required for all modules and / or functions of the high-throughput immunoassay analyzer to complete its tasks necessary for generating a result. In certain embodiments, the cycle time is 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.

[0189] In an aspect, the time to first result (TTFR) is about 60 minutes or less. The “TTFR” is a measure of time from when the sample is aspirated to when the presence and / or concentration of AMH is determined. In certain embodiments the TTFR is 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.

[0190] Additional examples are provided below.

[0191] EXAMPLES

[0192] Example 1 ; AMH Detection Capability

[0193] Limit of Detection (LOD), and Limit of Quantitation (LOQ) were conducted on an exemplary high-throughput immunoassay analyzer according to an aspect of the disclosure. LODis the lowest detectable amount of analyte in a sample. LOQ is the lowest amount of analyte in a sample that can be quantitatively determined.

[0194] Exemplary Limit of Detection Study

[0195] The LOD study included a total of 45 replicates from nine low-level AMH samples using multiple reagent packs on exemplary high-throughput immunoassay analyzers. The exemplary immunoassay analyzers had (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) ultrasonic mixing; (iv) and the capability to perform over 5 wash cycles per reaction vessel.

[0196] The data generated for each sample is summarized below in Tables 1 - 3. As can be seen, for each sample, a mean concentration of forty-five replicates was determined, as was the standard deviation and % CV.

[0197] Table 1: LOD Study Data Summary for High Throughput Immunoassay Analyzer 1

[0198] Table 2: LOD Study Data Summary for High Throughput Immunoassay Analyzer 2

[0199] Table 3: LOD Study Data Summary for High Throughput Immunoassay Analyzer 3

[0200] The regression models were used to calculate the LOD for each immunoassay analyzer, the results of which are summarized in the table below. As can be seen in Table 4 below, the study determined the LOQ to be 0.002 ng / mL (0.01 pmol / L).

[0201] Table 4: LOQ Analysis Summary

[0202] Exemplary Limit of Quantitation Study

[0203] The LOQ study included a total of 45 replicates from eleven low-level AMH samples using multiple reagent packs on exemplary high-throughput immunoassay analyzers. The exemplary immunoassay analyzers had (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) ultrasonic mixing; (iv) and the capability to perform over 5 wash cycles per reaction vessel.

[0204] The data generated for each sample is summarized below in Tables 5-7. As can be seen, for each sample, a mean concentration of forty-five replicates was determined, as was the standard deviation and % CV. A log-log regression model was fitted to the data of Tables 5 - 7, as is shown in FIGs 1A - 1CTable 5: LOQ study data summary for high-throughput immunoassay analyzer 1Table 6: LOQ study data summary for high-throughput immunoassay analyzer 2Table 7: LOQ study data summary for high-throughput immunoassay analyzer 3

[0205] The log-log regression models were used to calculate the LOQ for each immunoassay analyzer, the results of which are summarized in the table below. As can be seen in Table 8 below, the study determined the LOQ to be 0.002 ng / mL (0.02 pmol / L).Table 8: LOQ Analysis Summary

[0206] Example 2: Exemplary AMH assay

[0207] 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..

[0208] Table 9: Exemplary AMH assay

[0209] 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 mixtureis 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.

[0210] 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.

[0211] Example 3: AMH Assay Patient Testing

[0212] 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.

[0213] 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.

[0214] Inclusion

[0215] Able to provide written informed consent; Willing to provide subject health questionnaire information; > 40 years of age; Both ovaries present.

[0216] Exclusion

[0217] Evidence of polycystic ovarian syndrome (PCOS);Previous history of 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.

[0218] The patient ages and corresponding self-reported menopausal status are depicted in FIG. 3. Table 10 depicts the range of patients reporting in each menopausal group, and corresponding reported clinical features, including the average number of symptoms experienced at each of the following frequencies: none, little, quite, and extremely for each patient group

[0219] Table 10: Patient Demographics

[0220] The AMH 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 AMH capture antibodies conjugated to paramagnetic particle, a second reagent vessel containing AMH 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.

[0221] Step 1 : The paramagnetic particle conjugated with capture antibodies from the first reagent vessel (AMH capture antibodies) were pipetted into a reaction vessel using one of the four reagent pipettors along with a buffer. A first sample aliquot was pipetted into the reaction vessel using a sample pipettor. Then the reaction vessel was mixed using the ultrasonic mixer and incubated. A magnetic field was applied to the reaction vessel and the first incubated mixture was washed using a wash buffer to remove any unreacted components.

[0222] Step 2: A secondary antibody capable of binding AMH conjugated to ALP was added to the reaction vessel containing the incubated mixture. The reaction vessel was mixed ultrasonically and incubated, generating a reaction mixture. A magnetic field was applied to the reaction vessel and the second reaction mixture was washed five times using a wash buffer to remove any unreacted components. A substrate was added to the reaction vessel, allowed to incubate, and the signal generated from the resulting reaction was read using a luminometer.

[0223] All samples were analyzed in duplicate.

[0135] For each sample, a concentration of AMH was calculated. The calculated concentration are plotted in FIGs. 4A and 4B. 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, including degree of variation. FIG. 4B is graph plotting the age of each patient sample against the measured AMH concentration. Table 11 lists the median concentrations measure for each group and the 95% confidence interval. As can been seen in FIG. 4A and Table 11, the separation between the sample populations is well-defined, indicating the ability of the assay to stage a subject to a degree of ovarian aging based on AMH levels.

[0224] Table 11

[0225] Example 4 ; FSH and Inhibin B Assay Patient Testing

[0226] The samples analyzed in Example 3, were further analyzed for follicle-stimulating hormone (FSH) and an inhibin B.

[0227] The FSH assay was performed 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 FSH capture antibodies conjugated to paramagnetic particle, a second reagent vessel containing FSH 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.

[0228] Step 1 : The paramagnetic particle conjugated with capture antibodies from the first reagent vessel (FSH capture antibodies) were pipetted into a reaction vessel using one of the four reagent pipettors along with a buffer. A first sample aliquot was pipetted into the reaction vessel using a sample pipettor. Then the reaction vessel was mixed using the ultrasonic mixer and incubated. Amagnetic field was applied to the reaction vessel and the first incubated mixture was washed using a wash buffer to remove any unreacted components.

[0229] Step 2: A secondary antibody capable of binding FSH conjugated to ALP was added to the reaction vessel containing the incubated mixture. The reaction vessel was mixed ultrasonically and incubated, generating a reaction mixture. A magnetic field was applied to the reaction vessel and the second reaction mixture was washed five times using a wash buffer to remove any unreacted components. A substrate was added to the reaction vessel, allowed to incubate, and the signal generated from the resulting reaction was read using a luminometer.

[0230] All samples were analyzed in duplicate.

[0231] For each sample, a concentration of FSH was calculated. The calculated concentration are plotted in FIGs. 5A and 5B. 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.

[0232] Table 12

[0233] 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.

[0234] Step 1 : The paramagnetic particle conjugated with capture antibodies from the first reagent vessel (Inhibin B capture antibodies) were pipetted into a reaction vessel using one of the four reagent pipettors along with a buffer. A first sample aliquot was pipetted into the reaction vessel using a sample pipettor. Then the reaction vessel was mixed using the ultrasonic mixer and incubated. A magnetic field was applied to the reaction vessel and the first incubated mixture was washed using a wash buffer to remove any unreacted components.

[0235] Step 2: A secondary antibody capable of binding Inhibin B conjugated to ALP was added to the reaction vessel containing the incubated mixture. The reaction vessel was mixed ultrasonically and incubated, generating a reaction mixture. A magnetic field was applied to the reaction vessel and the second reaction mixture was washed five times using a wash buffer to remove any unreacted components. A substrate was added to the reaction vessel, allowed to incubate, and the signal generated from the resulting reaction was read using a luminometer.

[0236] All samples were analyzed in duplicate.

[0237] For each sample, a concentration of Inhibin B was calculated. The calculated concentration are plotted in FIGs. 6A and 6B. 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, including degree of variation. FIG. 6B is graph plotting the age of each patient sample against the measured Inhibin B concentration. Table 13 lists the median concentrations measure for each group and the 95% confidence interval. As can be seen in FIG. 6A and Table 13, patients in the postmenopausal group exhibit significantly decreased Inhibin B concentrations as compared to patients in the premenopausal and perimenopausal groups.

[0238] Table 13

[0239] All features disclosed in the specification, including the claims, abstracts, and drawings, and all the steps in any method or process disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in the specification, including the claims, abstract, and drawings, can be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0240] It will be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

CLAIMSWhat is claimed is:

1. 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 (LOQ) of the high-throughput immunoassay analyzer for measuring AMH is equal to or less than about 0.003 ng / mL, and wherein the first, second, and third corresponding reference ranges are above the LOQ.

2. The method of claim 1, wherein the method further comprises detecting the presence or amount of at least one additional biomarker in one or more plasma samples obtained from the subject using the high-throughput immunoassay analyzer and generating a corresponding at least one additional biomarker value.

3. The method of claim 2, wherein the at least one additional biomarker is inhibin B, follicle-stimulating hormone (FSH), or a combination thereof.

4. The method of claim 2 or claim 3, wherein 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 premenopausal 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.

5. The method of any one of claims 1 to 4, wherein 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.

6. The method of any one of claim 1 to 5, wherein 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.

7. The method of any one of claim 1 to 6, wherein 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.

8. The method of any one of claims 1 to 7, wherein 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.

9. The method of any one of claims 1 to 8, wherein the plasma sample volume is less than about 10 pL, alternatively between about 2 pL to about 9.9 pL.

10. The method of any one of claims 1 to 9, wherein 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 arrangement11. The method of claim 10, wherein the reagent vessels comprise an elastomeric self-sealing membrane.

12. The method of claim 10 or claim 11, wherein the reagent pack further comprises containment walls arranged between the reagent vessels.

13. The method of any one of claims 10 to 12, wherein the high-throughput immunoassay analyzer further comprises a reagent storage unit, wherein the reagent pack is housed in the reagent storage unit.

14. The method of any one of claims 10 to 13, wherein 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.

15. The method of claim 14, wherein the first reagent pipettor, the second reagent pipettor, the third reagent pipettor, and / or fourth reagent pipettor are selectively and / or simultaneously operated.

16. The method of claim 14 or claim 15, wherein 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.

17. The method of any one of claims 1 to 16, wherein the method is configured to analyze at least about 200 plasma samples / hr.

18. The method of any one of claims 1 to 16, wherein the method is configured to analyze at least about 300 plasma samples / hr.

19. The method of any one of claims 1 to 16, wherein the method is configured to analyze at least about 400 plasma samples / hr.

20. The method of any one of claims 10 to 19, wherein 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.

21. The method of claim 20, wherein the reaction in the detection mixture generates a chemiluminescent signal, and the method comprises correlating the chemiluminescent signal with the presence and / or concentration of AMH in the plasma sample.

22. The method of claim 20 or claim 21, wherein 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, and the substrate formulation is configured to produce chemiluminescence.

23. The method of claim 22, wherein 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.

24. The method of claim 23, wherein 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.

25. The method of claim 24, wherein the detection molecule comprises the second antibody.

26. The method of any one of claims 20 to 25, wherein the detection molecule comprises an alkaline phosphatase (AP)-conjugated secondary antibody.

27. The method of any one of claims 20 to 26, wherein the affinity molecule and / or the detection molecule is conjugated to a magnetic bead or a magnetic particle.

28. The method of claim 27, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is subjected to a magnetic field prior to detection.

29. The method of any one of claims 10 to 28, wherein 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.

30. The method of claim 29, wherein the reaction in the detection mixture generates a chemiluminescent signal, and the method comprises correlating the chemiluminescent signal with the presence and / or concentration of at least one additional biomarker in the plasma sample.

31. The method of claim 29 or claim 30, wherein 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, and the substrate formulation is configured to produce chemiluminescence.

32. The method of claim 31, wherein 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.

33. The method of claim 32, wherein 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.

34. The method of claim 33, wherein the detection molecule comprises the second antibody.

35. The method of any one of claims 31 to 34, wherein the detection molecule comprises an alkaline phosphatase (AP)-conjugated secondary antibody.

36. The method of any one of claims 31 to 35, wherein the affinity molecule and / or the detection molecule is conjugated to a magnetic bead or a magnetic particle.

37. The method of claim 36, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is subjected to a magnetic field prior to detection.

38. The method of any one of claim 20 to 37, wherein 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.

39. The method of any one of claims 20 to 38, wherein 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.

40. The method of any one of claims 20 to 39, wherein the substrate formulation is configured to produce chemiluminescence and comprises: a chemiluminescent compound of formula I or a salt thereof:whereinA 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 NO2 substituents;Ri is selected from the group consisting of Cs-waryl, C1-6 alkyl, C1-6 haloalkyl, and C5-14 aralkyl groups;R7-R14 are independently H, Ci-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; and n is 0, 1, or 2; a cationic aromatic compound (CAC); a background reducing agent; and an ether-linked nonionic surfactant or a hydrophilic polymer.

41. The method of any one of claims 20 to 40, wherein the detector arrangement comprises 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.

42. The method of any one of claim 1 to 41, wherein the high-throughput immunoassay analyzer comprises an ultrasonic mixing module.

43. The method of claim 42, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is agitated via the ultrasonic mixing module.

44. The method of any one of claim 1 to 43, wherein the high-throughput immunoassay analyzer comprises a washing arrangement, wherein the washing arrangement 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.

45. The method of claim 44, wherein 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.

46. The method of any one of claim 1 to 45, wherein 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.

47. The method of claim 46, wherein the instrument functionalities are selected from the group consisting of optical sensors, pressure sensors and thermistors.

48. The method of claim 46, wherein the assay functionalities are selected from the group consisting of sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring.

49. The method of any one of claim 1 to 48, wherein cycle time is about 45 seconds or less, alternatively about 40 seconds or less, alternatively about 35 seconds or less, alternatively about30 seconds or less, alternatively about 25 seconds or less, alternatively about 20 seconds or less, or alternatively about 15 seconds.

50. The method of any one of claim 1 to 49, wherein time to first result (TTFR) 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.

51. 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; ii) comparing 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.

52. The method of claim 51, wherein 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.

53. The method of claim 51 or 52, wherein the method further comprises detecting the presence or amount of at least one additional biomarker in one or more plasma samples obtained from the subject using the high-throughput immunoassay analyzer and generating a corresponding at least one additional biomarker value.

54. The method of claim 53, wherein the at least one additional biomarker is inhibin B, follicle-stimulating hormone (FSH), or a combination thereof.

55. The method of any one of claim 51 to 54, wherein 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.

56. The method of any one of claim 51 to 55, wherein 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.

57. The method of any one of claims 51 to 56, wherein the plasma sample volume is less than about 10 pL, alternatively between about 2 pL to about 9.9 pL.

58. The method of any one of claims 51 to 57, wherein 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 arrangement59. The method of claim 58, wherein the reagent vessels comprise an elastomeric self-sealing membrane.

60. The method of claim 58 or claim 59, wherein the reagent pack further comprises containment walls arranged between the reagent vessels.

61. The method of any one of claims 58 to 60, wherein the high-throughput immunoassay analyzer further comprises a reagent storage unit, wherein the reagent pack is housed in the reagent storage unit.

62. The method of any one of claims 58 to 61, wherein 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.

63. The method of claim 62, wherein the first reagent pipettor, the second reagent pipettor, the third reagent pipettor, and / or fourth reagent pipettor are selectively and / or simultaneously operated.

64. The method of claim 62 or claim 63, wherein 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.

65. The method of any one of claims 51 to 64, wherein the method is configured to analyze at least about 200 plasma samples / hr.

66. The method of any one of claims 51 to 64, wherein the method is configured to analyze at least about 300 plasma samples / hr.

67. The method of any one of claims 51 to 64, wherein the method is configured to analyze at least about 400 plasma samples / hr.

68. The method of any one of claims 58 to 67, wherein 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.

69. The method of claim 68, wherein the reaction in the detection mixture generates a chemiluminescent signal, and the method comprises correlating the chemiluminescent signal with the presence and / or concentration of AMH in the plasma sample.

70. The method of claim 68 or claim 69, wherein the first reagent comprises at least one affinity molecule configured to bind to at least one portion AMH, the second reagent comprisesat least one detection molecule, and the substrate formulation is configured to produce chemiluminescence.

71. The method of claim 70, wherein 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.

72. The method of claim 71, wherein 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.

73. The method of claim 72, wherein the detection molecule comprises the second antibody.

74. The method of any one of claims 70 to 73, wherein the detection molecule comprises an alkaline phosphatase (AP)-conjugated secondary antibody.

75. The method of any one of claims 70 to 73, wherein the affinity molecule and / or the detection molecule is conjugated to a magnetic bead or a magnetic particle.

76. The method of claim 75, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is subjected to a magnetic field prior to detection.

77. The method of any one of claims 58 to 76, wherein 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.

78. The method of claim 77, wherein the reaction in the detection mixture generates a chemiluminescent signal, and the method comprises correlating the chemiluminescent signal with the presence and / or concentration of at least one additional biomarker in the plasma sample.

79. The method of claim 77 or claim 78, wherein 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, and the substrate formulation is configured to produce chemiluminescence.

80. The method of claim 79, wherein 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.81 . The method of claim 80, wherein 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.

82. The method of claim 81, wherein the detection molecule comprises the second antibody.

83. The method of any one of claims 79 to 82, wherein the detection molecule comprises an alkaline phosphatase (AP)-conjugated secondary antibody.

84. The method of any one of claims 79 to 83, wherein the affinity molecule and / or the detection molecule is conjugated to a magnetic bead or a magnetic particle.

85. The method of claim 84, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is subjected to a magnetic field prior to detection.

86. The method of any one of claim 77 to 85, wherein 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.

87. The method of any one of claims 77 to 86, wherein 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.

88. The method of any one of claims 77 to 87, wherein the substrate formulation is configured to produce chemiluminescence and comprises: a chemiluminescent compound of formula I or a salt thereof:whereinA 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 NO2 substituents;Ri is selected from the group consisting of Cs-uaryl, 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; and n is 0, 1, or 2; a cationic aromatic compound (CAC); a background reducing agent; and an ether-linked nonionic surfactant or a hydrophilic polymer.

89. The method of any one of claims 77 to 88, wherein the detector arrangement comprisesa 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.

90. The method of any one of claim 51 to 89, wherein the high-throughput immunoassay analyzer comprises an ultrasonic mixing module.

91. The method of claim 90, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is agitated via the ultrasonic mixing module.

92. The method of any one of claim 51 to 91, wherein the high-throughput immunoassay analyzer comprises a washing arrangement, wherein the washing arrangement 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.

93. The method of claim 92, wherein 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.

94. The method of any one of claim 51 to 93, wherein 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.

95. The method of claim 94, wherein the instrument functionalities are selected from the group consisting of optical sensors, pressure sensors and thermistors.

96. The method of claim 94, wherein the assay functionalities are selected from the group consisting of sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring.

97. The method of any one of claim 51 to 96, wherein 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.

98. The method of any one of claim 51 to 97, wherein time to first result (TTFR) 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.

99. 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.

100. The method of claim 99, wherein 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.

101. The method of claim 99 or claim 100, wherein the ovarian response is a response to an ovarian stimulation.

102. The method of any one of claims 99 to 101, wherein the degree of ovarian response is selected from low, normal, or high.

103. The method of any one of claims 99 to 102, wherein the method further comprises detecting the presence or amount of at least one additional biomarker in one or more plasma samples obtained from the subject using the high-throughput immunoassay analyzer and generating a corresponding at least one additional biomarker value.

104. The method of claim 103, wherein the at least one additional biomarker is inhibin B, follicle-stimulating hormone (FSH), or a combination thereof.

105. The method of any one of claim 99 to 104, wherein 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.

106. The method of any one of claim 99 to 105, wherein 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.

107. The method of any one of claims 99 to 106, wherein 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.

108. The method of any one of claims 99 to 107, wherein the plasma sample volume is less than about 10 pL, alternatively between about 2 pL to about 9.9 pL.

109. The method of any one of claims 99 to 108, wherein 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 arrangement110. The method of claim 109, wherein the reagent vessels comprise an elastomeric selfsealing membrane.

111. The method of claim 109 or claim 110, wherein the reagent pack further comprises containment walls arranged between the reagent vessels.

112. The method of any one of claims 109 to 111, wherein the high-throughput immunoassay analyzer further comprises a reagent storage unit, wherein the reagent pack is housed in the reagent storage unit.

113. The method of any one of claims 109 to 112, wherein 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.

114. The method of claim 113, wherein the first reagent pipettor, the second reagent pipettor, the third reagent pipettor, and / or fourth reagent pipettor are selectively and / or simultaneously operated.

115. The method of claim 113 or claim 114, wherein 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.

116. The method of any one of claims 109 to 115, wherein the method is configured to analyze at least about 200 plasma samples / hr.

117. The method of any one of claims 109 to 115, wherein the method is configured to analyze at least about 300 plasma samples / hr.

118. The method of any one of claims 109 to 1 15, wherein the method is configured to analyze at least about 400 plasma samples / hr.

119. The method of any one of claims 109 to 118, wherein 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.

120. The method of claim 119, wherein the reaction in the detection mixture generates a chemiluminescent signal, and the method comprises correlating the chemiluminescent signal with the presence and / or concentration of AMH in the plasma sample.

121. The method of claim 119 or claim 120, wherein 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, and the substrate formulation is configured to produce chemiluminescence.

122. The method of claim 121 , wherein 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.

123. The method of claim 122, wherein 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.

124. The method of claim 123, wherein the detection molecule comprises the second antibody.

125. The method of any one of claims 119 to 124, wherein the detection molecule comprises an alkaline phosphatase (AP)-conjugated secondary antibody.

126. The method of any one of claims 119 to 125, wherein the affinity molecule and / or the detection molecule is conjugated to a magnetic bead or a magnetic particle.

127. The method of claim 126, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is subjected to a magnetic field prior to detection.

128. The method of any one of claims 109 to 127, wherein 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.

129. The method of claim 128, wherein the reaction in the detection mixture generates a chemiluminescent signal, and the method comprises correlating the chemiluminescent signal with the presence and / or concentration of at least one additional biomarker in the plasma sample.

130. The method of claim 128 or claim 129, wherein 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, and the substrate formulation is configured to produce chemiluminescence.

131. The method of claim 130, wherein 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.

132. The method of claim 131, wherein 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.

133. The method of claim 132, wherein the detection molecule comprises the second antibody.

134. The method of any one of claims 130 to 133, wherein the detection molecule comprises an alkaline phosphatase (AP)-conjugated secondary antibody.

135. The method of any one of claims 130 to 134, wherein the affinity molecule and / or the detection molecule is conjugated to a magnetic bead or a magnetic particle.

136. The method of claim 135, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is subjected to a magnetic field prior to detection.

137. The method of any one of claim 119 to 136, wherein 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.

138. The method of any one of claims 119 to 137, wherein 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.

139. The method of any one of claims 119 to 138, wherein the substrate formulation is configured to produce chemiluminescence and comprises: a chemiluminescent compound of formula I or a salt thereof:whereinA 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 NO2 substituents;Ri is selected from the group consisting of Cs-uaryl, 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; and n is 0, 1, or 2; a cationic aromatic compound (CAC); a background reducing agent; and an ether-linked nonionic surfactant or a hydrophilic polymer.

140. The method of any one of claims 119 to 139, wherein the detector arrangement comprisesa 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.

141. The method of any one of claim 99 to 140, wherein the high-throughput immunoassay analyzer comprises an ultrasonic mixing module.

142. The method of claim 141, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is agitated via the ultrasonic mixing module.

143. The method of any one of claim 99 to 142, wherein the high-throughput immunoassay analyzer comprises a washing arrangement, wherein the washing arrangement 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.

144. The method of claim 143, wherein 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.

145. The method of any one of claim 99 to 144, wherein 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.

146. The method of claim 145, wherein the instrument functionalities are selected from the group consisting of optical sensors, pressure sensors and thermistors.

147. The method of claim 145, wherein the assay functionalities are selected from the group consisting of sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring.

148. The method of any one of claim 99 to 147, wherein 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.

149. The method of any one of claim 99 to 148, wherein time to first result (TTFR) 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.

150. A method for quantitatively assessing ovarian failure 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 failure; and iii) assigning to the subject a degree of ovarian failure 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.

151. The method of claim 150, wherein the degree of ovarian failure is selected from the group consisting of poor ovarian response and normal -to-high ovarian response.

152. The method of claim 150 or 151, wherein the method further comprises identifying a subject in need of treatment based on the degree of ovarian failure and administering an effective amount of a pharmaceutical composition to the subject or recommending a therapeutic intervention to the subject.

153. The method of any one of claims 150 to 152, wherein the method further comprises detecting the presence or amount of at least one additional biomarker in one or more plasma samples obtained from the subject using the high-throughput immunoassay analyzer and generating a corresponding at least one additional biomarker value.

154. The method of claim 153, wherein the at least one additional biomarker is inhibin B, follicle-stimulating hormone (FSH), or a combination thereof.

155. The method of any one of claim 150 to 154, wherein 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.

156. The method of any one of claim 150 to 155, wherein 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.

157. The method of any one of claims 150 to 156, wherein 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.

158. The method of any one of claims 150 to 157, wherein the plasma sample volume is less than about 10 pL, alternatively between about 2 pL to about 9.9 pL.

159. The method of any one of claims 150 to 158, wherein 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 arrangement160. The method of claim 159, wherein the reagent vessels comprise an elastomeric selfsealing membrane.

161. The method of claim 159 or claim 160, wherein the reagent pack further comprises containment walls arranged between the reagent vessels.

162. The method of any one of claims 159 to 161, wherein the high-throughput immunoassay analyzer further comprises a reagent storage unit, wherein the reagent pack is housed in the reagent storage unit.

163. The method of any one of claims 159 to 162, wherein 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.

164. The method of claim 163, wherein the first reagent pipettor, the second reagent pipettor, the third reagent pipettor, and / or fourth reagent pipettor are selectively and / or simultaneously operated.

165. The method of claim 163 or claim 164, wherein 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.

166. The method of any one of claims 150 to 165, wherein the method is configured to analyze at least about 200 plasma samples / hr.

167. The method of any one of claims 150 to 165, wherein the method is configured to analyze at least about 300 plasma samples / hr.

168. The method of any one of claims 150 to 165, wherein the method is configured to analyze at least about 400 plasma samples / hr.

169. The method of any one of claims 159 to 168, wherein 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.

170. The method of claim 169, wherein the reaction in the detection mixture generates a chemiluminescent signal, and the method comprises correlating the chemiluminescent signal with the presence and / or concentration of AMH in the plasma sample.

171. The method of claim 169 or claim 170, wherein 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, and the substrate formulation is configured to produce chemiluminescence.

172. The method of claim 171, wherein the affinity molecule is selected from the group consisting of an antibody, a monoclonal antibody, a polyclonal antibody, a synthetic antibodymimic, an aptamer, an affimer, DARPins, or oligonucleotides or peptides that bind to at least one epitope of AMH.

173. The method of claim 172, wherein 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.

174. The method of claim 173, wherein the detection molecule comprises the second antibody.

175. The method of any one of claims 169 to 174, wherein the detection molecule comprises an alkaline phosphatase (AP)-conjugated secondary antibody.

176. The method of any one of claims 169 to 175, wherein the affinity molecule and / or the detection molecule is conjugated to a magnetic bead or a magnetic particle.

177. The method of claim 176, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is subjected to a magnetic field prior to detection.

178. The method of any one of claims 159 to 177, wherein 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.

179. The method of claim 178, wherein the reaction in the detection mixture generates a chemiluminescent signal, and the method comprises correlating the chemiluminescent signal with the presence and / or concentration of at least one additional biomarker in the plasma sample.

180. The method of claim 178 or claim 179, wherein 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, and the substrate formulation is configured to produce chemiluminescence.

181. The method of claim 180, wherein 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.

182. The method of claim 181, wherein 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.

183. The method of claim 182, wherein the detection molecule comprises the second antibody.

184. The method of any one of claims 180 to 183, wherein the detection molecule comprises an alkaline phosphatase (AP)-conjugated secondary antibody.

185. The method of any one of claims 180 to 184, wherein the affinity molecule and / or the detection molecule is conjugated to a magnetic bead or a magnetic particle.

186. The method of claim 185, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is subjected to a magnetic field prior to detection.

187. The method of any one of claim 169 to 186, wherein 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.

188. The method of any one of claims 169 to 187, wherein 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.

189. The method of any one of claims 169 to 188, wherein the substrate formulation is configured to produce chemiluminescence and comprises: a chemiluminescent compound of formula I or a salt thereofwhereinA 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 NO2 substituents;Ri is selected from the group consisting of Cs-uaryl, 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; and n is 0, 1, or 2; a cationic aromatic compound (CAC); a background reducing agent; and an ether-linked nonionic surfactant or a hydrophilic polymer.

190. The method of any one of claims 169 to 189, wherein the detector arrangement comprisesa 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.

191. The method of any one of claim 150 to 190, wherein the high-throughput immunoassay analyzer comprises an ultrasonic mixing module.

192. The method of claim 191, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is agitated via the ultrasonic mixing module.

193. The method of any one of claim 150 to 192, wherein the high-throughput immunoassay analyzer comprises a washing arrangement, wherein the washing arrangement 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.

194. The method of claim 193, wherein 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.

195. The method of any one of claim 150 to 194, wherein 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.

196. The method of claim 195, wherein the instrument functionalities are selected from the group consisting of optical sensors, pressure sensors and thermistors.

197. The method of claim 195, wherein the assay functionalities are selected from the group consisting of sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring.

198. The method of any one of claim 150 to 197, wherein 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.

199. The method of any one of claim 150 to 198, wherein time to first result (TTFR) 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.

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