DHEAS assay, reagents therefor, and method for producing and using the same

Abstract

This invention provides novel and improved DHEAS assay reagents and assays, as well as methods for manufacturing and using them to detect DHEAS levels in biological samples. [Solution] An immunoassay reagent containing a dehydroepiandrosterone sulfate-fluorescein (DHEAS-FITC) conjugate or a dehydroepiandrosterone sulfate-carboxymethoxylaminodimethylacridinium ester (DHEAS-CMO-DMAE) conjugate is disclosed. Immunosay kits and devices containing one or more DHEAS conjugates are also disclosed. Methods for producing and using DHEAS conjugates are further disclosed.

Description

Background Art

[0001] Cross-reference to related applications / incorporation by reference Not applicable.

[0002] Description of research and development by federal government funds Not applicable.

[0003] Background Dehydroepiandrosterone Sulphate (DHEAS) is an adrenal steroid having the chemical structure shown in FIG. 1, and abnormal levels of DHEAS are known in the art to be associated with many different disorders and conditions. For example, (but not limited to), measurement of circulating levels of DHEAS is important in the investigation of abnormal hair growth (hirsutism) and baldness (alopecia) in women. Also, since plasma levels of DHEAS gradually increase from around 7 years old and then gradually decrease from the 30s, measurement of DHEAS levels is useful in the evaluation of adrenocortical pubertal signs and delayed puberty. Furthermore, DHEAS is often assayed in combination with free testosterone as an initial screening for androgen excess in hirsutism, and high DHEAS levels are commonly seen in polycystic ovary syndrome (PCOS). Furthermore, levels of DHEAS higher than 700 - 800 μg / dL in women suggest a hormonally secreting adrenal tumor.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Methods for the measurement of DHEAS are desirable to provide a mechanism for the evaluation of DHEAS levels in human patients. However, immunoassays for the detection of DHEAS have faced problems with assay sensitivity, ambient temperature effect (ATE), and susceptibility to biotin interference.

[0005] Therefore, there is a need in this field for new and improved DHEAS assay reagents and assays, as well as methods for manufacturing and using them to detect DHEAS levels in biological samples. [Brief explanation of the drawing]

[0006] [Figure 1] Figure 1 shows the chemical structure of dehydroepiandrosterone sulfate (DHEAS). [Figure 2] Figure 2 shows one non-limiting embodiment of a DHEAS assay format constructed in accordance with this disclosure. [Figure 3] Figure 3 shows another non-limiting embodiment of the DHEAS assay format constructed in accordance with this disclosure. [Figure 4] Figure 4 provides one non-limiting embodiment of an assay reagent that may be used in accordance with this disclosure, a scheme for the synthesis of DHEAS-CMO-EDA-fluorescein. [Figure 5] Figure 5 provides another non-limiting embodiment of an assay reagent that may be used in accordance with this disclosure, a scheme for the synthesis of DHEAS-CMO-PEG3-fluorescein. [Figure 6] Figure 6 provides another non-limiting embodiment of an assay reagent that may be used in accordance with this disclosure, a scheme for the synthesis of DHEAS-BSA-fluorescein. [Figure 7] Figure 7 provides yet another non-limiting embodiment of an assay reagent that may be used in accordance with this disclosure, a scheme for the synthesis of a DHEAS-CMO-EDA-DMAE conjugate. [Figure 8] Figure 8 provides yet another non-limiting embodiment of an assay reagent that may be used in accordance with this disclosure, a scheme for the synthesis of a DHEAS-CMO-Z-NSP-DMAE conjugate. [Figure 9]Figure 9 graphically shows the optimization of the test definition of the binding curve for the DHEAS assay format of Figure 2 using the assay reagent DHEAS-CMO-EDA-fluorescein, in contrast to a commercially available assay (using DHEAS). Test definitions 811-833 cover various sequences and timings of reagent addition using the DHEAS II format, all showing improved binding profiles compared to the commercially available DHEAS assay. [Figure 10] Figure 10 graphically shows the binding curve for the DHEAS assay format in Figure 3 using the DHEAS-CMO-EDA-DMAE conjugate, in comparison to a commercially available assay (using DHEAS). [Modes for carrying out the invention]

[0007] Detailed explanation Before describing in detail at least one embodiment of this disclosure by example terminology and results, it should be noted that this disclosure is not limited in its application to the details of the construction and arrangement of components shown below. This disclosure may take other forms, or it may be implemented or carried out in various ways. Accordingly, the terms used herein are intended to be given the broadest possible scope and meaning; and the embodiments are illustrative and not exhaustive. Also, naturally, the expressions and terms used herein are for illustrative purposes only and should not be considered limiting.

[0008] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall be plural and plural terms shall be singular. The techniques and procedures described above shall generally be performed in accordance with conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. The nomenclature used in relation to analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry, as well as the methods and techniques thereof, are well known and commonly used in the art.

[0009] All patents, published patent applications, and non-patent publications referenced in this specification represent the level of skill of a person skilled in the art to which this disclosure belongs. All patents, published patent applications, and non-patent publications referenced in any part of this application are expressly incorporated herein by reference as a whole to the same extent as each individual patent or publication is specifically and individually indicated to be incorporated by reference.

[0010] All compositions / devices, kits, and / or methods disclosed herein can be manufactured and performed without excessive experimentation by taking this disclosure into consideration. While compositions / devices, kits, and / or methods have been described in relation to specific embodiments, it will be apparent to those skilled in the art that modifications can be applied to the compositions / devices, kits, and / or methods and the steps or sequences of steps of the methods described herein without departing from the concepts, spirit, and scope of this disclosure. All such similar substitutions and modifications that are apparent to those skilled in the art are deemed to be within the spirit, scope, and concepts of this disclosure as defined by the appended claims.

[0011] To be used in accordance with this disclosure, the following terms shall be understood to have the following meanings unless otherwise indicated: When the terms "a" or "an" are used in connection with the term "comprising" in the claims and / or specification, they may mean "one," but also coincide with the meanings of "one or more," "at least one," and "one or more." Thus, the terms "a," "an," and "the" include multiple references unless the context explicitly indicates otherwise. For example, a reference to "a compound" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or a larger number of compounds.

[0012] The use of the term "at least one" is understood to include any quantity of one or more than one, including, but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" can extend to 100 or 1000 or more, depending on the term it is accompanied by; furthermore, the quantity 100 / 1000 should not be considered limiting, as higher upper bounds may also produce satisfactory results. Furthermore, the use of the term "at least one of X, Y, and Z" is understood to include X only, Y only, and Z only, as well as any combination of X, Y, and Z.

[0013] The use of ordinal terms (i.e., "1st," "2nd," "3rd," "4th," etc.) is solely for the purpose of distinguishing two or more items, and is not intended to indicate any order, rank, or importance of one item relative to another, such as any order of addition, unless otherwise explicitly stated.

[0014] The use of the term “or” in the claims is used to mean an inclusive “and / or” unless it is explicitly indicated that it refers only to the options, or the options are not mutually exclusive. For example, the condition “A or B” is satisfied by any of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0015] Where used herein, any reference to “one embodiment,” “embodiment,” “several embodiments,” “one example,” “for example,” or “example” means that any particular element, feature, structure, or characteristic described in relation to that embodiment is included in at least one embodiment. The appearance of the phrase “in several embodiments” or “one example” in various places in the specification does not necessarily refer to the same embodiment, for example. Furthermore, all references to one or more embodiments or examples should be construed as not limiting the scope of the claims.

[0016] Throughout this application, the term “about” is used to indicate that a value includes inherent variations in error of the composition / apparatus / device, the method used to determine its value, or variations that exist between the test subjects. For example, where the term “about” is used, but not as an limitation, the following variations are reasonable for performing the disclosed method, and as will be understood by those skilled in the art, the specified value may vary by plus or minus 20 percent, or 15 percent, or 12 percent, or 11 percent, or 10 percent, or 9 percent, or 8 percent, or 7 percent, or 6 percent, or 5 percent, or 4 percent, or 3 percent, or 2 percent, or 1 percent from a particular value.

[0017] As used in this specification and in the claims, the word "including" The terms "comprising" (and any form of including such as "comprise" and "comprises"), "having" (and any form of having such as "have" and "has"), "including" (and any form of including such as "includes" and "include"), or "containing" (and any form of containing such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. For example, a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent therein.

[0018] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed before that term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and, where order is important in a particular context, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations containing repetitions of one or more of the items such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. are also expressly included. As will be apparent to those of ordinary skill in the art, typically there is no limit to the number of items in any combination, unless otherwise apparent from the context.

[0019] As used herein, the term "substantially" means that the event or situation described thereafter occurs completely or that the event or situation described thereafter occurs to a great extent or to a significant degree. For example, when related to a particular event or situation, the term "substantially" means that the event or situation described thereafter occurs with a probability of at least 80%, or at least 85%, or at least 90%, or 95%. The term "substantially adjacent" can mean that two items are 100% adjacent to each other, or that two items are very close to each other but not 100% adjacent to each other, or that a part of one of the two items is not 100% adjacent to the other item but is very close to the other item.

[0020] As used herein, the phrase "associated with" includes both direct association of two parts with each other and indirect association of two parts with each other. Non-limiting examples of association include covalent bonding of one part to another part by direct bonding or via a spacer group, non-covalent bonding of one part to another part using specific binding pair members directly or attached to a part, for example incorporating one part into another part by dissolving one part into another part or by synthesis, and coating one part onto another part.

[0021] As used herein, the term "liquid test sample" is understood to include any kind of biological fluid sample that can be utilized in accordance with the present disclosure. Examples of biological samples that can be utilized include, but are not limited to, whole blood or any part thereof (i.e., plasma or serum), saliva, sputum, cerebrospinal fluid (CSF), intestinal fluid, intraperitoneal fluid, cyst fluid, sweat, interstitial fluid, tears, mucus, urine, bladder wash fluid, semen, combinations thereof, and the like. The volume of the liquid test sample utilized in accordance with the present disclosure can be (for example, but not by way of limitation) from about 0.1 μl to about 100 μl.

[0022] As used herein, the term “volume” in relation to liquid test samples used in accordance with this disclosure means approximately 0.1 μl to approximately 100 μl, or approximately 1 μl to approximately 75 μl, or approximately 2 μl to approximately 60 μl, or equal to or less than approximately 50 μl.

[0023] The term “patient” includes both human and veterinary subjects. In certain embodiments, patient is a mammal. In certain other embodiments, patient is a human. For diagnostic / procedural purposes, “mammal” refers to any animal classified as a mammal, including humans, livestock and farm animals, non-human primates, and zoo, sport, or pet animals such as dogs, horses, cats, and cattle.

[0024] Returning here to specific, non-limiting embodiments of the concept of the present invention, the disclosure relates to immunoassay reagents (and moreover, kits and devices containing them) that can be used in a method for determining the concentration of DHEAS in a biological sample. The immunoassay reagents of the disclosure comprise novel DHEAS conjugates, and immunoassays utilizing these novel DHEAS conjugates overcome the problems of assay sensitivity, ambient temperature effect (ATE), and sensitivity to biotin interference observed in currently available DHEAS immunoassays.

[0025] Certain non-limiting embodiments of this disclosure include immunoassay reagents comprising a dehydroepiandrosterone sulfate-fluorescein (DHEAS-FITC) conjugate. Any DHEAS-FITC conjugate disclosed herein or otherwise intended may be included in immunoassay reagents constructed in accordance with this disclosure. In certain non-limiting embodiments, the DHEAS-FITC conjugate includes DHEAS-CMO-EDA-FITC (DHEAS-carboxymethoxylamino-ethyldiamine-fluorescein), DHEAS-CMO-PEG3-fluorescein (DHEAS-carboxymethoxylamino-(polyethylene glycol)3-fluorescein), or DHEAS-CMO-BSA-fluorescein (DHEAS-carboxymethoxylamino-bovine serum albumin-fluorescein).

[0026] As used in the context of DHEAS conjugates, the term "fluorescein" is understood to refer to fluorescein alone, as well as fluorescein derivatives such as (but not limited to) fluorescein isothiocyanate (FITC).

[0027] Certain non-limiting embodiments of this disclosure relate to immunoassay reagents comprising dehydroepiandrosterone sulfate-carboxymethoxylamino-dimethylacridinium ester (DHEAS-CMO-DMAE) conjugates. Any DHEAS-CMO-DMAE conjugate disclosed herein or otherwise intended may be included in immunoassay reagents constructed in accordance with this disclosure. In certain non-limiting embodiments, the DHEAS-AE conjugate includes DHEAS-CMO-EDA-DMAE (DHEAS-carboxymethoxylamino-ethyldiamine-dimethylacridinium ester) or DHEAS-CMO-Z-NSP-DMAE (DHEAS-carboxymethoxylamino-Zn-sulfopropyl-dimethylacridinium ester).

[0028] Certain non-limiting embodiments of this disclosure relate to kits useful for conveniently performing immunoassays for determining DHEAS concentrations. These kits contain one of the DHEAS conjugates disclosed herein or otherwise intended, either alone or in combination with other assay reagents disclosed herein or otherwise intended. Furthermore, the kits may further contain other components and / or reagents for performing any of the specific assays described herein or otherwise intended. The nature of these further reagents depends on the specific assay format, and their identification is well within the skill of those skilled in the art.

[0029] In certain non-limiting embodiments of this disclosure, the immunoassay kit uses any of the DHEAS-FITC conjugates disclosed herein or otherwise intended, alone. or in combination with anti-fluorescein paramagnetic solid-phase particles and / or acridinium ester-labeled anti-DHEAS monoclonal antibody.

[0030] Any paramagnetic solid particles (PMPs) known in the art or otherwise contemplated herein may be used in accordance with this disclosure. The term “paramagnetic” refers to a material in which slight magnetism can be introduced to produce a weak attraction to either magnetic pole, a state that is lost when removed from a magnetic field. Paramagnetic materials typically have unpaired “d” electrons. Examples of paramagnetic materials include, but are not limited to, metal oxides and metal salts such as metal halides; and, for example, metallic elements. Examples of metals, though not limited to, include, for example, iron, chromium, lithium, sodium, magnesium, aluminum, manganese, strontium, zirconium, molybdenum, ruthenium, rhodium, palladium, tin, samarium, europium, tungsten, platinum, and combinations thereof.

[0031] In certain non-limiting embodiments, paramagnetic particles generally have an average diameter of, for example, about 0.02 to about 100 microns, or about 0.05 to about 100 microns, or about 0.1 to about 100 microns, or about 0.5 to about 100 microns, or about 0.02 to about 50 microns, or about 0.05 to about 50 microns, or about 0.1 to about 50 microns, or about 0.5 to about 50 microns, or about 0.02 to about 20 microns, or about 0.05 to about 20 microns, or about 0.1 to about 20 microns, or about 0.5 to about 20 microns. In some embodiments, particles have an average diameter of, for example, about 0.05 to about 20 microns, or about 0.3 to about 10 microns, or about 0.3 to about 5 microns. In some embodiments, and not limited to, the paramagnetic particles are, for example, iron(II) oxide particles, iron(III) oxide particles, mixtures of iron(II) oxide particles and iron(III) oxide particles, chromium oxide particles, and particles formed from oxides of lithium, sodium, magnesium, aluminum, manganese, strontium, zirconium, molybdenum, ruthenium, rhodium, palladium, tin, samarium, europium, tungsten, or platinum, as well as mixtures of two or more of the above.

[0032] Paramagnetic solid particles may or may not be coated; if a coating is present, the coating may (but not limited to) be an aldehyde coating, a polymer coating, a copolymer coating, etc.

[0033] Anti-fluorescein antibodies are well-known, widely commercialized, and extensively studied in this field. For example (though not limited to), some commercial sources of anti-fluorescein antibodies are available from Abbexa Ltd (Houston, TX); Abcam (Cambridge, UK); Biorbyt Ltd (St. Louis, MO); Jackson Immuno Research Labs, Inc. (West Grove, PA); Lifespan Biosciences, Inc. (Seattle, WA); Novus Biologicals, LLC (Centennial, CO); RayBiotech Life (Peachtree Corners, GA); Roche Diagnostics (Basel, CH); Rockland Immunochemicals, Inc. (Pottstown, PA); Santa Cruz Biotechnology, Inc. (Dallas, TX); Sigma-Aldrich Corp. (St. Louis, MO); and Thermo Fisher Scientific (Waltham, MA). However, this list is not comprehensive, and there are many further commercial sources of anti-fluorescein antibodies available in accordance with this disclosure. Therefore, those skilled in the art can clearly and definitively identify and select the various anti-fluorescein antibodies available in accordance with this disclosure, and thus further description of anti-human Ig antibodies or their characteristics is deemed unnecessary.

[0034] Furthermore, the method for binding antibodies to paramagnetic solid particles is widely known in the field and falls well within the scope of the usual art in that field. Therefore, further explanation is considered unnecessary.

[0035] Anti-DHEAS monoclonal antibodies are well-known, widely commercialized, and extensively studied in the field. For example (but not limited to), some commercial sources of anti-DHEAS monoclonal antibodies are available from Antibodies Online, Inc. (Limerick, PA); Creative Diagnostics (Shirley, NY); Lifespan Biosciences, Inc. (Seattle, WA); and MyBioSource, Inc. (San Diego, CA). However, this list is not exhaustive, and there are many further commercial sources of anti-DHEAS monoclonal antibodies available pursuant to this disclosure. Therefore, those skilled in the art can clearly and definitively identify and select the various anti-DHEAS monoclonal antibodies available pursuant to this disclosure, and thus further description of anti-human Ig antibodies or their characteristics is deemed unnecessary.

[0036] Furthermore, the method of labeling monoclonal antibodies with aclidinium esters is widely known in the art and falls well within the scope of the skills of those skilled in the art. Therefore, further explanation is deemed unnecessary.

[0037] In another, non-limiting embodiment, the immunoassay kit may include any of the DHEAS-CMO-DMAE conjugates disclosed herein or otherwise intended, either alone or in combination with paramagnetic solid-phase particles labeled with a fluoresceinated anti-DHEAS monoclonal antibody.

[0038] Paramagnetic solid-phase particles and anti-DHEAS monoclonal antibodies may be either disclosed herein or otherwise intended. Furthermore, methods for labeling monoclonal antibodies with fluorescein are well known in the art and well within the scope of those skilled in the art; therefore, further explanation is deemed unnecessary.

[0039] Furthermore, the kits of this disclosure may further contain one or more other components or reagents for performing biological sample collection and / or diagnostic application in accordance with this disclosure. For example (but not limited to), a kit may include one or more biological sample collection devices, one or more assay reagents, one or more calibration reagents, one or more quality control reagents, one or more washing reagents, etc. The nature of these further components / reagents depends (but not limited to) various factors such as the type of biological sample and the diagnostic assay format, and their identification is well within the scope of the art for those skilled in the art; therefore, further explanation is deemed unnecessary.

[0040] Furthermore, the various components / reagents present in the kit may be in separate containers / compartments, or they may be mixed in one or more containers / compartments, depending on their cross-reactivity and stability. The kit may also contain other separately packaged reagents for performing the assay. In addition, the kit may contain a microfluidic device to which the components / reagents are applied.

[0041] The relative amounts of the various components / reagents present in the kit can vary widely to provide component / reagent concentrations that substantially optimize the reactions that need to occur during the assay method, and further substantially optimize the sensitivity of the assay. Under appropriate circumstances, one or more of the components / reagents in the kit may be supplied as dry powders, such as lyophilized powders, and The kit may further contain excipients for dissolving the dried reagents; in this way, reagent solutions having concentrations suitable for performing the method or assay according to this disclosure can be obtained from these components. Positive and / or negative controls may be included in the kit. The kit may further include a set of written instructions describing how to use the kit. A kit of this nature can be used in any of the methods described herein or otherwise intended.

[0042] The reagents of the compositions / kits / methods may be provided in any form that enables them to function in accordance with the concepts of the present invention disclosed and claimed herein. For example, but not limited to, the reagents may be provided in the form of single aliquot lyophilized reagents. The use of lyophilized reagents in microfluidic devices is described in detail in U.S. Patent No. 9,244,085 (the entirety of which is expressly incorporated herein by reference).

[0043] Certain non-limiting embodiments of this disclosure relate to a method for determining the concentration of DHEAS in a biological sample. The method includes: (a) a step of simultaneously, partially or whole, sequentially mixing (1) a biological sample suspected to contain DHEAS; (2) any of the dehydroepiandrosterone sulfate-fluorescein (DHEAS-FITC) conjugates disclosed herein or otherwise intended; (3) any of the anti-fluorescein paramagnetic solid particles disclosed herein or otherwise intended; and (4) any acridinium ester-labeled anti-DHEAS monoclonal antibody disclosed herein or otherwise intended, to form a mixture; and (b) a step of enabling the binding of (2), (3), and (4) to each other and the binding of (4) to DHEAS present in the biological sample in the mixture formed in (a). The method may further include (c) measuring the fluorescence signal generated in the mixture; and / or (d) determining the concentration of DHEAS present in the biological sample based on the decrease in the observed fluorescence signal compared to the fluorescence signal observed in the absence of the biological sample. That is, the method utilizes a competitive format in which the amount of fluorescence is inversely proportional to the amount of DHEAS present in the sample.

[0044] In certain (but non-limiting) embodiments, in step (d) of the above method, the concentration of DHEAS present in the biological sample is determined by comparing the fluorescence signal with a calibration curve.

[0045] Certain non-limiting embodiments of this disclosure relate to a method for determining the concentration of DHEAS in a biological sample. The method includes: (a) a step of simultaneously, partially or whole, sequentially mixing (1) a biological sample suspected to contain DHEAS; (2) any paramagnetic solid particles labeled with a fluoresceinized anti-DHEAS monoclonal antibody disclosed herein or otherwise intended; and (3) any dehydroepiandrosterone sulfate-carboxymethoxylaminodimethylacridinium ester (DHEAS-CMO-DMAE) conjugate disclosed herein or otherwise intended, to form a mixture; (b) a step of enabling the binding of (3) to (2) or the binding of DHEAS present in the biological sample to (2) in the mixture formed in (a); (c) a step of measuring the fluorescence signal generated in the mixture; and / or (d) a step of determining the concentration of DHEAS present in the biological sample based on the amount of decrease in the observed fluorescence signal compared to the fluorescence signal observed in the absence of the biological sample. In other words, this method utilizes a competitive format in which the amount of fluorescence is inversely proportional to the amount of DHEAS present in the sample.

[0046] In certain (but non-limiting) embodiments, in step (d) of the method, the concentration of DHEAS present in the biological sample is determined by comparing the fluorescence signal with a calibration curve.

[0047] Certain non-limiting embodiments of this disclosure relate to a microfluidic device for determining the concentration of DHEAS in a sample. The microfluidic device comprises a compartment capable of containing a sample suspected to contain DHEAS, and one or more combinations of assay reagents described herein or otherwise intended. Not limiting, for example, the microfluidic device may comprise: (i) a first compartment capable of containing a sample suspected to contain DHEAS; (ii) any of the dehydroepiandrosterone sulfate-fluorescein (DHEAS-FITC) conjugates disclosed herein or otherwise intended; (iii) any of the anti-fluorescein paramagnetic solid particles disclosed herein or otherwise intended; and (iv) one or more of the acridinium ester-labeled anti-DHEAS monoclonal antibodies disclosed herein or otherwise intended. In another non-limiting example, a microfluidic device may comprise: (i) a first compartment capable of containing a sample suspected to contain DHEAS; (ii) paramagnetic solid particles labeled with a fluoresceinated anti-DHEAS monoclonal antibody; and (iii) one or both of a dehydroepiandrosterone sulfate-carboxymethoxylaminodimethylacridinium ester (DHEAS-CMO-DMAE) conjugate.

[0048] Any of reagents (ii) to (iv) may be placed in any part of a microfluidic device that enables the device to function in accordance with the disclosure. For example (but not limited to), at least one of (ii) to (iv) may be placed in a first compartment. Or (and / or in addition to them), the microfluidic device may include at least a second compartment that is in fluid communication with the first compartment, and one or more of (ii) to (iv) may be placed in the second compartment. Furthermore, the microfluidic device may include other additional compartments, and reagents (ii) to (iv) may be divided among three different compartments, if desired.

[0049] The microfluidic devices of this disclosure may include sample application chambers to which samples can be applied, and inlet channels that are in fluid communication with them and also in fluid communication with one or more compartments containing one or more of the reagents described herein. The devices may have any number of compartments, any arrangement of compartments, and any distribution of components between them, insofar as the device can function in accordance with this disclosure.

[0050] Any compartment of a microfluidic device may be sealed to maintain the reagents applied within them in a substantially airtight environment until use; for example, a compartment containing lyophilized reagents may be sealed to prevent any unintended reconstitution of the reagents. An inlet channel and a compartment, and two further compartments, may be described as "capable of fluid communication" with one another; this phrase indicates that the compartments may still be sealed, but if a hole is made in the seal formed in or between the compartments, the two compartments may have fluid flow between them.

[0051] The microfluidic devices of this disclosure may have any other desired features known in the art or otherwise contemplated herein. For example, the microfluidic device may further include a reading chamber; the reading chamber may be a compartment containing paramagnetic solid particles, or the reading chamber may be in fluid communication with the compartment. The microfluidic device may also include other solutions such as cleaning solutions, diluents, excipients, interference solutions, positive controls, negative controls, quality control solutions, etc. The microfluidic device may further include one or more compartments for containing a cleaning solution, and these compartments may be able to fluidize to any other compartments of the device. In another example, the microfluidic device may further include one or more compartments for containing at least one excipient for dissolving one or more dry reagents, and these compartments may be able to fluidize to any other compartments of the device. Furthermore, the microfluidic device may further include one or more compartments for containing a dilution solution, and these compartments may be able to fluidize to any other compartments of the device.

[0052] Furthermore, any kit / microfluidic device or any other device contemplated herein may include multiplexed multiplex assays within a single kit / device. [Examples]

[0053] Examples are provided herein and thereafter. However, it should be understood that this disclosure is not limited in its application to the specific experiments, results, and test procedures disclosed herein and thereafter. Rather, the examples are provided simply as one of many embodiments and are intended to be illustrative rather than comprehensive.

[0054] DHEAS (Figure 1) is an adrenal steroid, and abnormal levels of DHEAS are known in the field to be associated with a wide variety of disorders and conditions, as outlined in the background section above. Methods for measuring DHEAS are desired to provide a mechanism for evaluating DHEAS levels in human patients. However, immunoassays for detecting DHEAS have faced problems associated with assay sensitivity, ambient temperature effect (ATE), and sensitivity to biotin interference.

[0055] In response to the disadvantages and shortcomings of currently available DHEAS assays, a series of novel DHEAS derivatives and conjugates were designed and synthesized for use in an acridinium ester (AE) technology-based immunoassay platform (Natrajan et al. (2010); Natrajan et al. (2011); and Natrajan et al. (2012)). These novel DHEAS derivatives and conjugates included dehydroepiandrosterone sulfate-fluorescein (DHEAS-FITC) and dehydroepiandrosterone sulfate-acridinium ester (DHEAS-AE) conjugates. These DHEAS-FITC and DHEAS-AE conjugates were evaluated using a competitive assay format on an Atellica / ADVIA Centaur immunoassay analyzer (Siemens Healthcare Diagnostics Inc., Tarrytown, NY). The DHEAS-CMO-EDA-FITC(8) compound (DHEAS-carboxymethoxylaminoethyldiamine-fluorescein paired with anti-FITC labeled paramagnetic solid particles (anti-FITC-PMP)) has demonstrated superior assay performance compared to assay requirements and has been selected as one of the leading candidate reagents for the DHEAS II immunoassay.

[0056] Reagent Design: The Atellica / ADVIA Centaur DHEAS reagent included a solid phase, auxiliary reagent, and Wright reagent. Current commercially available assays use DHEAS-NSP-AE (Wright reagent) paired with a biotinylated mouse monoclonal antibody (mAb, auxiliary reagent) and a streptavidin-coated solid phase. In the assay of this disclosure, anti-FITC-PMP particles were selected for the solid phase instead of streptavidin-coated particles to prevent biotin interference. Two assay formats were investigated for this immunoassay (Figures 2-3). In Format 1 (Figure 2), the solid phase reagent contained magnetic particles labeled with fluoresceinated DHEAS, and the Wright reagent contained an mAb-AE conjugate. The Wright reagent antibody binds to DHEAS-FITC on the solid phase. Endogenous DHEAS competes with the DHEAS-FITC-bound solid phase for the mAb-AE in the Wright reagent, resulting in an inverse relationship between the RLU signal intensity and the endogenous analyte concentration when measured against a calibration curve.

[0057] To support Assay Format 1 (Figure 2), both DHEAS-FITC and mAb-AE reagents needed to be newly developed and evaluated. AE-labeled monoclonal antibodies were prepared by methods known and characterized in the art; however, the DHEAS-FITC conjugate and its synthesis method were not known prior to this disclosure, and therefore their performance in DHEAS II immunoassays had not been investigated prior to this disclosure.

[0058] A second DHEAS II format was also investigated – this assay format is called Format 2 and is shown in Figure 3. In Format 2, the solid-phase reagent contains magnetic particles labeled with fluoresceinated mAbs. The Wright reagent contains DHEAS-AE tracers. Format 2 operates using the same competitive main component as Format 1. Adding endogenous DHEAS derived from patient samples to the reaction interferes with the binding of DHEAS-AE tracers to the solid phase, resulting in reduced signal generation. In Format 2, design and development focused on the synthesis of various DHEAS-AE tracers paired with fluoresceinated mAb-coated PMP solid phases to improve assay performance while eliminating biotin interference.

[0059] For Format 1 (Figure 2), DHEAS-CMO-EDA-fluorescein (5) was designed for initial assay studies (Figure 4) and synthesized by reacting DHEAS-CMO with EDA-fluorescein (Figure 4). Early data showed that this compound yielded promising functional data and significantly reduced ambient temperature effects (ATE) compared to commercially available assays. DHEAS-CMO-PEG3-fluorescein (8) was also fabricated to evaluate whether an alternative hydrophilic linker structure would provide further benefits to assay performance (Figure 5). Furthermore, a DHEAS-BSA-fluorescein conjugate was fabricated using BSA as a protein linker that could provide multistoichiometry between DHEAS and the fluorescein moiety on BSA (Figure 6). The detailed assay performance of these three reagents is discussed below.

[0060] For assay format 2 (Figure 3), DHEAS-CMO was selected for conjugation to DMAE acridinium ester for initial characterization studies. Early data demonstrated that using this conjugate, DHEAS-CMO-EDA-DMAE(15) produced a promising standard curve shape and significantly reduced ambient temperature bias. Given this initial performance, conjugates with Z-NSP-DMAE at the same ligation site were also fabricated to determine whether different AE structures would provide any further ATE benefits to the assay. The performance of both DHEAS-AEs(15, 18) is discussed below. The synthesis of both DHEAS-AEs is shown in the schemes in Figures 7-8, respectively.

[0061] Both DHEAS-FITC and DHEAS-AE conjugates were evaluated using the ADVIA Centaur family of immunoassay analyzers at Siemens Healthcare Diagnostics Inc., Tarrytown, NY.

[0062] Functional performance (bonding curve shape, accuracy estimation, and ambient temperature bias (ATE)) of fluorescein-conjugates DHEAS-CMO-EDA-FITC, DHEAS-CMO-PEG3-FITC, and DHEAS-BSA-FITC ((5), (8), and (13) respectively). The conjugates were evaluated using DHEAS II immunoassay format 1. The conjugates DHEAS-CMO-PEG3-FITC and DHEAS-BSA-FITC showed minimal improvement compared to currently available commercial assays (data not shown). The DHEAS-CMO-EDA-FITC conjugate (5) showed an improved binding profile with comparable or better sensitivity compared to currently available commercial assays (Figure 9). More importantly, as shown in Tables 1-2, assay format 1 using conjugate (5) minimized ATE to ≤10%, significantly improving assay performance compared to commercial assays (%ATE approximately 29%).

[0063] Two DHEAS-AE conjugates (15, 18) were also evaluated using assay format 2 with the prototype ADVIA Centaur DHEAS II immunoassay. Both AE conjugates showed promising assay performance compared to the commercially available assay (Figure 10). ATE estimation for both AEs was better than that of the commercially available assay (Table 3).

[0064] Based on overall preliminary assay performance, conjugate (5) exhibits the best ATE and assay sensitivity among the reagents tested.

[0065] [Table 1]

[0066] [Table 2]

[0067] [Table 3]

[0068] In summary, a series of DHEAS-FITC and DHEAS-AE conjugates were successfully designed, synthesized, and evaluated for use in DHEAS II immunoassays (Formats 1-2, Figures 2-3, respectively). Based on overall assay performance, the SP phase reagent (Format 1) derived from DHEAS-CMO-EDA-fluorescein (5) (Figure 4) exhibits promising low assay sensitivity and the best ambient temperature effect (ATE) in the assay compared to currently available commercially available assays. Format 1 uses a 2H1-anti-FITC PMP solid phase to minimize the risk of biotin interference. DHEAS-CMO-EDA-fluorescein was selected as an excellent candidate to meet the requirements of the preliminary assay (Tables 2 and 3). This conjugate is a key component in the development of a commercially available ATELLICA / ADVIA Centaur DHEAS II immunoassay.

[0069] Accordingly, in accordance with this disclosure, compositions, devices, and kits, as well as methods for manufacturing and using them, are provided that fully satisfy the purposes and advantages set forth herein. Although this disclosure has been written in conjunction with the specific drawings, experiments, results, and language set forth herein, it is obvious that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to encompass all such alternatives, modifications, and variations that are in the spirit and broad scope of this disclosure.

[0070] References The following references are incorporated herein by reference to the extent that they provide exemplary procedures or other details that supplement those presented herein. Furthermore, the following are not intended to be a disclosure statement; a separate disclosure statement in accordance with 37 CFR § 1.97 will be submitted.

[0071] 1. Haning RV. “Using DHEAS to monitor androgen disorders.” Contemp Ob / Gyn.1981;18(9):117-132. 2.Kasick JM、Bergfeld WF、Steck WD、Gupta MK.「Adrenal androgenic female-pattern alopecia: sex hormones and the balding woman.」 Cleve Clin Q.1983;50(2):111-122. 3.Cohen HN、Wallace AM、Beastall GH、Fogelman I、Thomson JA.「Clinical value of adrenal androgen measurement in the diagnosis of delayed puberty.」 Lancet.1981;1(8222):689-692. 4.Braunstein GD.「Coping with hirsutism - management strategies.」 Contemp Ob / Gyn .1981;17(5):189-193. 5.de Peretti E、Forest MG.「Pattern of plasma dehydroepiandrosterone sulfate levels in humans from birth to adulthood: evidence for testicular production.」 J Clin Endocrinol Metab.1978;47(3):572-577. 6.Zappulla F、Ventura D、Capelli Mら.「Gonadal and adrenal secretion of dehydroepiandrosterone sulfate in prepubertal and pubertal subjects.」 J Endocrinol Invest.1981;4(2):197-202. 7. Vermeulen A. "Androgen secretion by adrenals and gonads." In Mahesh V, Greenblatt RB, eds. Hirsutism and Virilism. Boston: John Wright-PSG; 1983: 17-34. 8. Sciarra F. "Diagnosis of virilizing syndromes: endocrinological parameters." In Molinatti G et al., eds. Androgenization in Women. New York: Raven Press; 1983: 85-113. 9. Haning RV Jr, Carlson IH, Shapiro SS, Nolten WE. "Testosterone free index correlates best with dehydroepiandrosterone sulfate." Fertil Steril. 1981; 36(6): 757-765. 10. Lobo RA, Paul WL, Goebelsmann U. "Serum levels of DHEAS in gynecologic endocrinopathy and infertility." Obstet Gynecol. 1981; 57(5): 607-612. 11. Buvat J, Dewailly D, Marcolin G, Buvat-Herbaut M, Racadot A, Fossati P. "Investigative strategy of hyperandrogenism in women." Horm Res. 1983; 18(1-3): 106-116. 12.Natrajan、A.;Sharpe、D.;Costello、J.;Jiang、Q.P.「Enhanced Immunoassay Sensitivity Using Chemiluminescent Acridinium Esters with Increased Light Output」 Ann Biochem 2010、vol.406、204. 13.Natrajan、A.;Sharpe、D.;Wen、D.「Effect of Surfactants On the Chemiluminescence of Acridinium Dimethylphenyl Ester Labels and Their Conjugates」 Org Biomol Chem 2011、vol.9、5092. 14.Natrajan、A.;Sharpe、D.;Wen、D.「Zwitterionic Reagents for Labeling、Cross-Linking and Improving the Performance of Chemiluminescent Immunoassays」 Org Biomol Chem 2012、vol.10、188。

Claims

1. Dehydroepiandrosterone sulfate-fluorescein (DHEAS-FITC) conjugate Immunoassay reagents, including those mentioned above.

2. The immunoassay reagent according to claim 1, wherein the DHEAS-FITC conjugate comprises DHEAS-CMO-EDA-FITC (DHEAS-carboxymethoxylaminoethyldiamine-fluorescein).

3. The DHEAS-FITC conjugate is DHEAS-CMO-PEG3-fluorescein(DHEAS-carboxymethoxylamino-(polyethylene glycol) ３ The immunoassay reagent according to claim 1, comprising (-fluorescein) or DHEAS-CMO-BSA-fluorescein (DHEAS-carboxymethoxylamino-bovine serum albumin-fluorescein).

4. The immunoassay reagent according to claim 1, wherein fluorescein is fluorescein isothiocyanate (FITC).

5. Dehydroepiandrosterone sulfate-carboxymethoxylaminodimethylacridinium ester (DHEAS-CMO-DMAE) conjugate Immunoassay reagents, including those mentioned above.

6. The immunoassay reagent according to claim 5, wherein the DHEAS-AE conjugate comprises DHEAS-CMO-EDA-DMAE (DHEAS-carboxymethoxylamino-ethyldiamine-dimethylacridinium ester) or DHEAS-CMO-Z-NSP-DMAE (DHEAS-carboxymethoxylamino-Z-N-sulfopropyl-dimethylacridinium ester).

7. Dehydroepiandrosterone sulfate-fluorescein (DHEAS-FITC) conjugate; Anti-fluorescein paramagnetic solid phase particles; and Acridinium ester-labeled anti-DHEAS monoclonal antibody An immunoassay kit including...

8. The immunoassay kit according to claim 7, wherein the DHEAS-FITC conjugate comprises DHEAS-CMO-EDA-FITC (DHEAS-carboxymethoxylaminoethyldiamine-fluorescein).

9. The immunoassay kit according to claim 7, wherein the fluorescein in the DHEAS-FITC conjugate is fluorescein isothiocyanate (FITC).

10. The DHEAS-FITC conjugate is DHEAS-CMO-PEG3-fluorescein(DHEAS-carboxymethoxylamino-(polyethylene glycol) ３ The immunoassay kit according to claim 9, comprising (-fluorescein) or DHEAS-CMO-BSA-fluorescein (DHEAS-carboxymethoxylamino-bovine serum albumin-fluorescein).

11. Paramagnetic solid-phase particles labeled with fluoresceinized anti-DHEAS monoclonal antibody; and Dehydroepiandrosterone sulfate-carboxymethoxylaminodimethylacridinium ester (DHEAS-CMO-DMAE) conjugate An immunoassay kit including...

12. The immunoassay kit according to claim 11, wherein the DHEAS-AE conjugate comprises DHEAS-CMO-EDA-DMAE (DHEAS-carboxymethoxylamino-ethyldiamine-dimethylacridinium ester) or DHEAS-CMO-Z-NSP-DMAE (DHEAS-carboxymethoxylamino-Z-N-sulfopropyl-dimethylacridinium ester).

13. A method for determining the concentration of DHEAS in a biological sample, the method being: (a) (1) Biological samples suspected of containing DHEAS; (2) Dehydroepiandrosterone sulfate-fluorescein (DHEAS-FITC) conjugate; (3) Anti-fluorescein paramagnetic solid phase particles; and (4) Acridinium ester-labeled anti-DHEAS monoclonal antibody A process of mixing simultaneously, partially, or entirely in a continuous manner to form a mixture; (b) A step to enable the binding of (2), (3), and (4) to each other and to the binding of (4) to DHEAS present in the biological sample in the mixture formed in (a); and (c) A step of measuring the fluorescence signal generated in the mixture; and (d) A step of determining the concentration of DHEAS present in the biological sample based on the decrease in the observed fluorescence signal compared to the fluorescence signal observed when the biological sample is not present. The above method, including.

14. The method according to claim 13, wherein the DHEAS-FITC conjugate comprises DHEAS-CMO-EDA-FITC (DHEAS-carboxymethoxylaminoethyldiamine-fluorescein).

15. The method according to claim 14, wherein the fluorescein in the DHEAS-FITC conjugate is fluorescein isothiocyanate (FITC).

16. The DHEAS-FITC conjugate is DHEAS-CMO-PEG3-fluorescein(DHEAS-carboxymethoxylamino-(polyethylene glycol) ３ The method according to claim 14, comprising -fluorescein) or DHEAS-CMO-BSA-fluorescein (DHEAS-carboxymethoxylamino-bovine serum albumin-fluorescein).

17. The method according to claim 14, wherein in step (d), the concentration of DHEAS present in the biological sample is determined by comparing the fluorescence signal with a calibration curve.

18. A method for determining the concentration of DHEAS in a biological sample, the method being: (a) (1) Biological samples suspected of containing DHEAS; (2) Paramagnetic solid-phase particles labeled with fluoresceinized anti-DHEAS monoclonal antibody; and (3) Dehydroepiandrosterone sulfate-carboxymethoxylaminodimethylacridinium ester (DHEAS-CMO-DMAE) conjugate; A process of mixing simultaneously, partially, or entirely in a continuous manner to form a mixture; (b) a step in which the mixture formed in (a) enables the binding of (3) to (2) or the binding of DHEAS present in the biological sample to (2); and (c) A step of measuring the fluorescence signal generated in the mixture; and (d) A step of determining the concentration of DHEAS present in the biological sample based on the decrease in the observed fluorescence signal compared to the fluorescence signal observed when the biological sample is not present. The above method, including.

19. The method according to claim 18, wherein the DHEAS-AE conjugate comprises DHEAS-CMO-EDA-DMAE (DHEAS-carboxymethoxylamino-ethyldiamine-dimethylacridinium ester) or DHEAS-CMO-Z-NSP-DMAE (DHEAS-carboxymethoxylamino-Z-N-sulfopropyl-dimethylacridinium ester).

20. The method according to claim 18, wherein in step (d), the concentration of DHEAS present in the biological sample is determined by comparing the fluorescence signal with a calibration curve.

21. A microfluidic device for determining the concentration of DHEAS in a sample, wherein the microfluidic device is: (i) A first compartment capable of containing a sample suspected of containing DHEAS; (ii) Dehydroepiandrosterone sulfate-fluorescein (DHEAS-FITC) conjugate; (iii) Anti-fluorescein paramagnetic solid phase particles; and (iv) Acridinium ester-labeled anti-DHEAS monoclonal antibody The above microfluidic devices include the above.

22. The microfluidic device according to claim 21, wherein at least one of (ii) to (iv) is arranged in the first compartment.

23. The microfluidic device according to claim 21, further defined as including at least a second compartment capable of fluid communication with a first compartment, wherein at least one of (ii) to (iv) is located in the second compartment.

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