Compositions, devices, and methods for mitigating lipoprotein interference in in vitro diagnostic assays of hydrophobic analytes.

Abstract

Methods for mitigating lipoprotein interference in in vitro diagnostic assays of target hydrophobic analytes are disclosed, as well as compositions, kits, and devices useful for said methods. [Solution] A pretreatment reagent containing at least one enzyme that digests lipoproteins is used.

Description

Technical Field

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

[0002] Explanation regarding research or development funded by the federal government Not applicable.

Background Art

[0003] In vitro diagnostic assays are used to detect hydrophobic analytes in biological samples. However, lipoproteins present in biological samples (e.g., but not limited to, low - density lipoprotein (LDL), very - low - density lipoprotein (VLDL), intermediate - density lipoprotein (IDL), high - density lipoprotein (HDL), and chylomicrons, etc., which are often quantified by their cholesterol content) are known to interfere with such assays, especially in the case of small hydrophobic analytes (e.g., but not limited to, hydrophobic drugs and hormones). These hydrophobic haptens tend to enter the hydrophobic core of lipoprotein particles when present in hydrophilic blood or serum, and currently, there is no method to eliminate this interference. As a result, a portion of the analyte is "tied up" within the lipoprotein particles, and thus the assay reagents are not available, and negative interference by lipoproteins (cholesterol) is often observed.

[0004] Current attempts to overcome cholesterol or lipoprotein interference involve extraction of the analyte using organic solvents. This approach is to break down the lipoproteins so that the hydrophobic analyte can dissolve in the organic solvent. However, the extraction process needs to be carried out manually and is thus not suitable for reducing interference in assays performed on fully automated analyzers.

[0005] Another approach involves adding an organic solvent or surfactant to the reagent mixture to help solubilize the analyte, partially preventing it from entering the hydrophobic core of the lipoprotein particles. However, this method only provides limited mitigation of lipoprotein interference and cannot completely eliminate it.

[0006] An ELISA assay for the small molecule immunosuppressant tacrolimus (also known as FK506) was previously developed that utilizes proteinase to digest immunophilin FK506-binding protein (FKBP), thereby releasing tacrolimus to which FKBP is bound. However, the impact of using proteinase to digest the protein on cholesterol / lipoprotein interference in the assay is limited. [Overview of the project] [Problems that the invention aims to solve]

[0007] Therefore, there is a need for novel and improved methods to mitigate lipoprotein interference, particularly for use with automated analyzers. [Means for solving the problem]

[0008] This disclosure covers such methods, as well as compositions, kits, and devices used therein. [Modes for carrying out the invention]

[0009] Before describing in detail, by illustrative language and results, at least one embodiment of the concept of the present invention, it should be understood that the application of the concept of the present invention is not limited to the structural and arrangement details of the components described below. Other embodiments of the concept of the present invention are possible, or can be carried out or implemented in various ways. For this reason, the language used herein is intended to be as broad as possible in scope and meaning, and the embodiments are illustrative and not exhaustive. Furthermore, it should be understood that the language and terminology used herein are for illustrative purposes only and should not be considered limiting.

[0010] Unless otherwise defined herein, scientific and technical terms used in this disclosure have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms. The techniques and procedures described herein are generally 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 analytical chemistry, organic synthesis chemistry, and medicinal and pharmaceutical chemistry described herein, as well as their experimental procedures and techniques, are well known and commonly used in the art. Standard techniques are used in chemical synthesis and chemical analysis.

[0011] All patents, patent application publications, and non-patent publications referenced herein represent the level of skill of a person skilled in the art to which this disclosure pertains. All patents, patent application publications, and non-patent publications referenced in any part of this application are expressly incorporated herein by reference in whole to the same extent as if each individual patent or publication were directed to be incorporated by reference specifically and individually.

[0012] All articles, compositions, kits, and / or methods disclosed herein can be manufactured and performed without any experimentation beyond what is necessary in light of this disclosure. While articles, compositions, kits, and / or methods are described in relation to specific embodiments, it will be apparent to those skilled in the art that modifications can be applied to the articles, compositions, kits, and / or methods, as well as to 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, apparent to those skilled in the art, are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0013] When used in accordance with this disclosure, the following terms shall be understood to have the following meanings unless otherwise indicated:

[0014] The use of the terms “a” or “an,” when used in conjunction with the term “including” in the claims and / or herein, 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 referents unless the content clearly 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 more compounds. The term “multiple” means “two or more.”

[0015] The use of the term "at least one" is understood to include one, as well as any number greater than one, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" is understood to include, if it is added to Depending on the term used, the range may extend to a maximum of 100, 1000, or more; in addition, the quantity 100 / 1000 should not be considered limiting, as it may result in satisfying even greater limits. Furthermore, the use of the term "at least one of X, Y, and Z" is understood to include X alone, Y alone, Z alone, and any combination of X, Y, and Z. The use of ordinal terms (i.e., "first," "second," "third," "fourth," etc.) is merely for distinguishing two or more items and is not intended to imply any arbitrary order, sequence, importance, or any additional order of one item relative to another.

[0016] The use of the term “or” in the claims is used to mean an inclusive “and / or” unless it is explicitly indicated that only substitutes are being referred to, or that the substitutes are mutually exclusive. For example, the condition “A or B” means that any of the following are true: 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).

[0017] In this specification, any reference to “one embodiment,” “an embodiment,” “some embodiments,” “one example,” “for example,” or “one example” means that any particular element, configuration, structure, or feature described in relation to that embodiment is included in at least one embodiment. For example, expressions such as “in some embodiments” or “one example” appearing in various parts of this specification do not necessarily all refer to the same embodiment. Furthermore, all references to one or more embodiments or examples should be construed as not limiting the scope of the claims.

[0018] Throughout this application, the term “approximately” is used to indicate that a certain value includes error variability inherent to a composition / apparatus / device, and that the method is used to determine that value, or the variability present between the subjects of study. For example, but not limited to, when the term “approximately” is used, 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 the specified value, because such variation is appropriate for performing the disclosed method and will be understood by those skilled in the art.

[0019] As used herein and in the claims, the words “comprising” (and any form of “comprising,” 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 non-restrictive and do not exclude any additional and undescribed elements or steps of the method.

[0020] As used herein, the term "or combination thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or combination thereof" includes at least one of A, B, C, AB, AC, BC, or ABC, and where the order is important in a particular context, it may also include BA, CA, CB, CBA, BCA, A This also includes CB, BAC, or CAB. Continuing this example, it clearly includes combinations containing one or more repetitions of items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, and CABABB. A person skilled in the art will understand that, unless it is clearly evident from the context, there is generally no limit to the number of items or terms in any combination.

[0021] As used herein, the term “substantially” means that the event or situation described below will occur completely, or that the event or situation described below will occur to a large degree or extent. For example, in relation to a particular event or situation, the term “substantially” means that the event or situation described below will occur with at least 80% probability, or at least 85% probability, or at least 90% probability, or at least 95% probability. The term “substantially adjacent” may mean that two items are 100% adjacent to each other, or that two items are very close to each other but not 100% adjacent, or that a portion of one item is very close to the other but not 100% adjacent to the other.

[0022] As used herein, the expressions “bonded to” and “connected to” include both direct and indirect bonding / connections between two parts. Non-exclusive examples of bonding / connections include, for example, covalent bonding between one part and another by direct bonding or via spacer groups, non-covalent bonding between one part and another by direct or specific bond pair members bonded to both parts, incorporation of one part into another by dissolving one part into another, and coating one part into another.

[0023] As used herein, the term "sample" is understood to include any type of biological sample that can be utilized in accordance with the present disclosure. Examples of available fluid biological samples include whole blood or any portion thereof (including but not limited to plasma or serum), whole blood cells or lysed blood cells (including but not limited to whole red blood cells or lysed red blood cells), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cyst fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, feces, pleural fluid, nasopharyngeal fluid, combinations thereof, and the like, but are not limited thereto.

[0024] As used herein, the term "hapten" refers to a low molecular weight protein or non-protein antigenic determinant (or "epitope") that can be recognized in an in vitro diagnostic assay (which may include a binding partner specific for a target analyte such as, but not limited to, an antibody).

[0025] The term "analyte" refers to a molecule that can be recognized in an in vitro diagnostic assay (which may include a binding partner specific for a target analyte such as, but not limited to, an antibody). When the assay is an immunoassay, the analyte includes at least one antigenic determinant or "epitope" that is the region of the analyte that binds to a binding partner (i.e., an antibody) specific for the target analyte. When the analyte is a hapten, the entire hapten molecule typically forms an epitope.

[0026] As used herein, the term "binding partner specific for a target analyte" is understood to refer to any molecule that can specifically bind to the target analyte. For example, without limitation, the binding partner can be an antibody, a receptor, a ligand, an aptamer, a molecular imprint polymer (i.e., an inorganic matrix), a combination or derivative thereof, and any other molecule capable of specifically binding to the target analyte.

[0027] The term "antibody" is used herein in the broadest sense and refers to, for example, intact monoclonal and polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments and conjugates thereof that exhibit the desired biological activity of analyte binding (including, but not limited to, Fab, Fab’, F(ab’)2, Fv, scFv, Fd, diabody, single-chain antibodies, and other antibody fragments and conjugates thereof that retain at least a portion of the variable region of an intact antibody), antibody surrogate proteins or peptides (i.e., modified binding proteins / peptides), and combinations or derivatives thereof. Antibodies can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0028] As used herein, the term "microfluidic device" includes any device capable of performing at least one diagnostic assay described herein. Microfluidic devices are typically inserted into a system that automates the performance of diagnostic assays. In one non-limiting embodiment, a microfluidic device is constructed for use in an automated diagnostic assay performed by, for example, but not limited to, one of the DIMENSION® integrated chemistry systems commercially available from Siemens Healthcare Diagnostics, Inc. (Newark, DE). However, it is understood that microfluidic devices can be any commercially available product described herein or otherwise contemplated that can perform one or more diagnostic assays in accordance with the present disclosure. Additionally, a microfluidic device can include multiple compartments that integrate multiple assays mounted on a single microfluidic device for use with a clinical chemistry system, whereby a single volume of a biological sample is inserted into the microfluidic device and then delivered to the multiple assay compartments.

[0029] Turning to the concept of the present invention, some non-limiting embodiments of this disclosure generally relate to compositions, kits, devices, and methods for improving the performance and reliability of in vitro diagnostic assays of hydrophobic analytes. In particular, some embodiments of this disclosure relate to compositions, kits, devices, and methods for mitigating lipoprotein interference in in vitro diagnostic assays of hydrophobic analytes.

[0030] Some non-limiting embodiments of this disclosure relate to methods for detecting the presence and / or concentration of a target hydrophobic analyte in a biological sample. In some specific (but non-limiting) embodiments, the method is further defined as a method for minimizing lipoprotein interference in an in vitro diagnostic assay of a hydrophobic analyte.

[0031] The method comprises combining, simultaneously, or entirely or partially sequentially: (1) a sample suspected of containing a target hydrophobic analyte; (2) a pretreatment reagent containing at least one enzyme that digests lipoproteins (e.g., but not limited to lipase and / or at least one other digestive enzyme (e.g., but not limited to gastric enzymes, e.g. (but not limited to) pepsin and pancreatic enzymes, e.g. (but not limited to) amylase and protease)); and (3) at least one assay reagent capable of detecting the hydrophobic analyte. The method further comprises performing one or more detection assays based on (3) to determine the concentration of the target hydrophobic analyte present in the sample.

[0032] When (1) and (2) are incubated together, the enzymes present in the pretreatment reagent digest the lipids and proteins in the lipoprotein. This releases the target analyte from the hydrophobic core of the lipoprotein particles, thereby releasing the target analyte into the assay reagent. In other words, it becomes possible to access antibodies that specifically recognize the target analyte (though not limited to these). As a result, the target analyte is detected with higher efficiency compared to assays that do not utilize pretreatment reagents, thereby resulting in an assay for the target analyte that is less susceptible to such influences.

[0033] One or more steps of the method may be performed manually; or the steps of the method may be fully automated on a clinical chemistry analysis system as described herein.

[0034] Any biological sample known in the art for use in the in vitro diagnostic assays described herein may be used in accordance with this disclosure. Examples of usable biological samples include, but are not limited to, urine, whole blood or any part thereof (including, but not limited to, plasma or serum), whole blood cells (i.e., substantially unlysed) or lysed blood cells (including, but not limited to, whole red blood cells or lysed red blood cells), saliva, sputum, cerebrospinal fluid (CSF), intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, tears, mucus, bladder lavage fluid, semen, combinations, etc. Any hydrophobic analytes detectable by assay methods disclosed herein or otherwise intended may be detected by the methods of this disclosure. Examples of target analytes include small hydrophobic molecules, such as (but not limited to) vitamin D, tacrolimus, sirolimus, everolimus, estrogens, estrone, estradiol, estriol, alfatradiol (estradiol, estrone, and estriol), cyclosporine, ethinylestradiol, esterified estrogens, moxestrol, kinestrol, progestins, progesterone, androgens, such as testosterone and dihydrotestosterone. These include (DHT), dehydroepiandrosterone (DHEA) and DHEA sulfate (DEHE-S), androstenedione, aldosterone, other steroid hormones, cortisol, catecholamines, 25-hydroxyvitamin D2 (25-OH vitamin D2), 25-hydroxyvitamin D3 (25-OH vitamin D3), 1,25-dihydroxyvitamin D2 (1,25-OH vitamin D2), and 1,25-dihydroxyvitamin D3 (1,25-OH vitamin D3), among others.

[0035] The pretreatment reagent comprises at least one enzyme that digests lipids on lipoproteins into more water-soluble and smaller compounds; the enzyme may be a lipase and / or at least one other digestive enzyme (e.g., gastric enzymes, e.g., pepsin, e.g., pepsin, e.g., amylase and protease, e.g., amylase and protease, e.g., pepsin, e.g., pepsin, e.g., amylase and protease

[0036] The enzyme that digests the lipoprotein must be supplied at a sufficient concentration to cause the hydrophobic core of the lipoprotein particle to release the target analyte, thereby making it accessible to the assay reagent, and thus enabling highly efficient detection of the target analyte. The enzyme concentration will depend on the amount of analyte present and the duration of the pretreatment reaction.

[0037] The enzyme that digests lipoproteins is present in the pretreatment reagent and may be present at any concentration that allows the enzyme to function as described herein. Examples of specific concentrations include: approximately 0.01 mg / mL, approximately 0.05 mg / mL, approximately 0.1 mg / mL, and approximately 0.2 mg / mL. L, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1 mg / mL, about 1.5 mg / mL, Approximately 2 mg / mL, approximately 2.5 mg / mL, approximately 3 mg / mL, approximately 3.5 mg / mL, approximately 4 mg / mL, approximately 4.5 mg / mL, approximately 5 mg / mL, approximately 5.5 mg / mL, approximately 6 mg / mL, approximately 6.5 mg / mL, Approximately 7mg / mL, approximately 7.5mg / mL, approximately 8mg / mL, approximately 8.5mg / mL, approximately 9mg / mL, approximately 9.5mg / mL, approximately 10mg / mL, approximately 11mg / mL, approximately 12mg / mL, approximately 13mg / mL, Approximately 14mg / mL, approximately 15mg / mL, approximately 16mg / mL, approximately 17mg / mL, approximately 18mg / mL, approximately 19mg / mL, approximately 20mg / mL, approximately 21mg / mL, approximately 22mg / mL, approximately 23mg / mL, Approx. 24 mg / mL, approx. 25 mg / mL, approx. 26 mg / mL, approx. 27 mg / mL, approx. 28 mg / mL, approx. 29 mg / mL, approx. 30 mg / mL, approx. 31 mg / mL, approx. 32 mg / mL, approx. 33 mg / mL, Approx. 34 mg / mL, approx. 35 mg / mL, approx. 36 mg / mL, approx. 37 mg / mL, approx. 38 mg / mL, approx. 39 mg / mL, approx. 40 mg / mL, approx. 41 mg / mL, approx. 42 mg / mL, approx. 43 mg / mL, The concentrations include, but are not limited to, approximately 44 mg / mL, 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, or higher. In certain (but not limited) embodiments, the enzyme concentration in the pretreatment reagent is defined as being within any two of the above ranges, for example (but not limited to) the range of approximately 0.01 mg / mL to approximately 100 mg / mL, the range of approximately 0.05 mg / mL to approximately 50 mg / mL, the range of approximately 0.1 mg / mL to approximately 20 mg / mL, the range of approximately 0.5 mg / mL to approximately 10 mg / mL, etc.However, the specific concentration ranges described above are for illustrative purposes only and should not be considered limiting; it should be understood that any concentration range having one lower limit and another upper limit is expressly included within the scope of this disclosure.

[0038] Similarly, one or more additional substances in the pretreatment reagent can each be present at any concentration that allows the substance to function to enhance enzyme activity. For example, each additional substance (though not limited to these, such as enzyme cofactors like bile acids and / or their salts, surfactants like PLURONIC® block copolymer, or proteases) can be present in the following non-limiting concentrations: approximately 0.001%, approximately 0.005%, approximately 0.01%, approximately 0.02%, approximately 0.03%, approximately 0.04%, approximately 0.05%, approximately 0.06%, approximately 0.07%, approximately 0.08%, approximately 0.09%, approximately 0.1%, approximately 0.15%, approximately 0.2%, approximately 0.25%, approximately 0.3%, approximately 0.35%, approximately 0.4%, approximately 0.45%, approximately 0.5%, approximately 0.55%, approximately 0.6%, approximately 0.65%, approximately 0.7%, approximately 0.75%, approximately 0.8%, approximately 0. 85%, approximately 0.9%, approximately 0.95%, approximately 1%, approximately 1.1%, approximately 1.2%, approximately 1.3%, approximately 1.4%, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8%, approximately 1.9%, approximately 2%, approximately 2.1%, approximately 2.2%, approximately 2.3%, approximately 2.4%, approximately 2.5%, approximately 2.6%, approximately 2.7%, approximately 2.8%, approximately 2.9%, approximately 3%, approximately 3.25%, The solutions can be provided at concentrations selected from approximately 3.5%, 3.75%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, or higher. In certain (but not limited) embodiments, the concentration of each additional substance present in the pretreatment reagent is defined as being within any two ranges of the above values, for example (but not limited to) the range of approximately 0.001% to approximately 25%, the range of approximately 0.005% to approximately 15%, the range of approximately 0.01% to approximately 5%, etc. However, the specific concentration ranges described above are for illustrative purposes only and should not be considered limiting; it should be understood that any concentration range having one lower limit and another upper limit is expressly included within the scope of this disclosure.

[0039] In some non-limiting embodiments, at least one assay reagent capable of detecting a hydrophobic analyte is an immunoassay reagent (i.e., a target analyte-specific binding partner (such as an antibody, but not limited to these)). The at least one target analyte-specific binding partner can then be conjugated to the target analyte or at least one immunoassay reagent.

[0040] The sample preparation and assay / detection steps can be performed in the same compartment of the microfluidic device. Alternatively, the sample preparation step can be performed in the first compartment, and then the prepared sample can be moved to the second compartment for the detection step.

[0041] The biological sample is added to the pretreatment step in its natural form, or the biological sample is dissolved before the pretreatment step. Thus, in certain (but non-limiting) embodiments, the method described herein, or otherwise intended, may further include the step of dissolving the biological sample in a first compartment and then transferring the dissolved biological sample to a second compartment used in the pretreatment step.

[0042] Any of the methods and processes described herein can be performed by a user, for example, but not limited to such methods. However, as used herein, the term “user” is not limited to human use; rather, the term “user” can include (for example, but not limited to) computers, servers, websites, processors, network interfaces, humans, user terminals, virtual computers, and combinations thereof.

[0043] Some non-limiting embodiments of this disclosure relate to reagent kits useful for conveniently carrying out the diagnostic assay methods described herein. The reagent kit comprises one or more pretreatment reagents described herein or otherwise intended, combined with one or more assay reagents capable of detecting hydrophobic analytes, described herein or otherwise intended.

[0044] Some other non-limiting embodiments of this disclosure relate to an assay device (such as a microfluidic device) comprising any of the at least one pretreatment reagents described herein or otherwise intended, and any at least one assay reagent described herein or otherwise intended, wherein the assay device is intended for use in any of the in vitro diagnostic assay methods described herein above.

[0045] For example, a microfluidic device may include at least one compartment capable of receiving a sample suspected to contain a target hydrophobic analyte, wherein the at least one compartment contains at least one pretreatment reagent as described in detail herein. The compartment may further include one or more assay reagents as described herein or otherwise intended. Alternatively, the microfluidic device may include at least a second compartment capable of being in fluid communication with the first compartment containing the pretreatment reagent, wherein the assay reagent may be contained in the second compartment. The second compartment may be a reading chamber on which a detection assay for determining the concentration of the target hydrophobic analyte present in the sample is performed; or the microfluidic device may further include a reading chamber capable of being in fluid communication with the second compartment.

[0046] In addition, the reagent kits and / or microfluidic devices of this disclosure may further include other components and / or reagents for performing any of the specific diagnostic assays described herein or otherwise intended. The nature of these additional components / reagents will depend on the specific assay format, and their identification will be well within the scope of the art of the art. Examples of additional reagents / components that may be present in the reagent kits and / or microfluidic devices of this disclosure include, but are not limited to, diluents, solvents (for lysing red blood cells), washing solutions (including, but not limited to, isotonic solutions), positive controls, negative controls, quality controls, and / or activators, as well as any combination thereof.

[0047] The relative amounts of various components / reagents in a kit and / or microfluidic device can vary significantly to arrive at 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.

[0048] The reagent kits of this disclosure may further include a set of written instructions describing how to use the kit. Kits of this nature can be used with any of the microfluidic devices and / or in any of the methods described herein or otherwise intended.

[0049] A microfluidic device may have one or more associated manual functions (i.e., in this case, pipetting is required for the addition of one or more reagents and / or the transfer of mixtures between two compartments); or, during the device's construction, the necessary reagents / components may be arranged in various compartments (the various compartments are in continuous fluid communication (or can be made to be)), so that after the sample is added to the microfluidic device, no manual handling of the sample and / or reagents is required to perform the assay.

[0050] As described herein, a microfluidic device comprises one or more compartments containing the components / reagents described herein. However, it should be understood that a microfluidic device may have any number of compartments, any arrangement of compartments, and any distribution of components / reagents therein, as long as the device can function in accordance with this disclosure. Where multiple compartments are provided, the compartments may be completely isolated from each other, or one or more compartments may be in fluid communication with each other. Various structures of microfluidic devices that can be used in accordance with this disclosure are well known in the art and therefore do not require further explanation.

[0051] In some embodiments, the microfluidic device includes at least first and second compartments. The first compartment is capable of receiving a biological sample and, if desired (but not limited to), includes a mechanism for lysing red blood cells, or otherwise for pretreatment and preparation of the sample for assay. The separation mechanism is well known in the art of microfluidic devices and therefore no further explanation is deemed necessary. The second compartment is capable of fluid communication with the first compartment and includes at least one pretreatment reagent. The second compartment may further include at least one assay reagent; or the microfluidic device may include a third compartment for storing at least one assay reagent, and at least one assay reagent may be transferred from the third compartment to the second compartment. The sample is then transferred to a container and incubated with pretreatment reagents in the pretreatment step.

[0052] The microfluidic device may also include a reading chamber (including, but not limited to, an optical reading chamber) that can be investigated by a clinical chemistry system (e.g., optically investigated by a spectrometer, but not limited to, these). The reading chamber may be connected to any of the compartments described herein, or it may be connected to a compartment separate from those described herein.

[0053] The inlet channel and compartment, as well as the two compartments, may be described as "capable of fluid communication" with one another; this expression indicates that the compartments are still sealed, but the two compartments are capable of having fluid flow between them if the seals formed in or between them are punctured.

[0054] The kits / microfluidic devices of this disclosure comprise any other desirable configurations known in the art or otherwise contemplated herein. For example, but not limited to, the kits / microfluidic devices of this disclosure may further comprise one or more additional compartments containing, but not limited to, solvents (for lysing red blood cells), diluents, washing solutions, labeling agents, interference solutions, other solutions such as positive controls, negative controls, quality controls, and / or actuators, and any combination thereof. [Examples]

[0055] Examples are described below. However, it should be understood that this disclosure is not limited in its application to the specific experiments, results, and experimental procedures disclosed herein. Rather, these examples are provided simply as one of many embodiments, and are not exhaustive but illustrative.

[0056] In this example, the effect of using a pretreatment reagent on cholesterol interference in an everolimus (EVRO) assay using the DIMENSION® integrated chemistry system (Siemens Healthcare Diagnostics, Inc., Newark, DE) was tested. Various amounts of lipase, bile salts (as lipase cofactors), and PLURONIC® block copolymer (BASF Corporation, Ludwigshafen, Germany) were added to the pretreatment reagent, incubated with a sample containing a known amount of cholesterol, and then the everolimus (EVRO) assay of the treated sample was performed using the DIMENSION® integrated chemistry system (Siemens Healthcare Diagnostics, Inc., Newark, DE).

[0057] As shown in Table 1, without the pretreatment step, the amount of cholesterol interference observed in the EVRO assay was -7.6%, -12%, and -19% for cholesterol concentrations of 300 mg / mL, 350 mg / mL, and 400 mg / mL, respectively. Adding a pretreatment step with a reagent containing lipase, its cofactor bile acid / salt (for enzyme activity enhancement), and PLURONIC® as a surfactant reduced the amount of cholesterol interference to 1% (from -7.6%), -0.4% (from -12%), and -3% (from -19%) for cholesterol concentrations of 300 mg / mL, 350 mg / mL, and 400 mg / mL, respectively. Therefore, cholesterol interference was substantially eliminated at 400 mg / mL cholesterol using the compositions and methods of this disclosure.

[0058] [Table 1]

[0059] Thus, in accordance with this disclosure, compositions, kits, and devices, as well as methods for manufacturing and using them, have been provided that fully satisfy the above-mentioned objectives and advantages. While this disclosure has been described with the above-mentioned specific drawings, experiments, results, and words, it is obvious that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, all such alternatives, modifications, and variations that fall within the spirit and broad scope of the concepts of the invention disclosed herein shall be encompassed.

Claims

1. A microfluidic device for use with clinical chemistry instruments: It includes at least one compartment capable of receiving a sample suspected to contain a target hydrophobic analyte, wherein the at least one compartment is: A pretreatment reagent comprising at least one enzyme for digesting lipoproteins; and A assay reagent capable of detecting a hydrophobic analyte for determining the concentration of the target hydrophobic analyte present in the sample. The microfluidic device, including the above.

2. The microfluidic device according to claim 1, further defined as comprising at least two compartments, wherein a pretreatment reagent is located in a first compartment capable of receiving a sample, and at least one assay reagent is located in a second compartment capable of being in fluid communication with the first compartment, wherein the first or second compartment is a reading chamber in which a detection assay for determining the concentration of a target hydrophobic analyte present in the sample is performed.

3. The microfluidic device according to claim 1, wherein at least one enzyme for digesting lipoproteins comprises a lipase and / or at least one digestive enzyme.

4. The microfluidic device according to claim 1, wherein the enzyme is present in the pretreatment reagent at a concentration in the range of approximately 0.1 mg / mL to approximately 20 mg / mL.

5. The microfluidic device according to claim 1, wherein the pretreatment reagent further comprises at least one additional substance selected from the group consisting of enzyme cofactors, surfactants, proteases, and combinations thereof.

6. The microfluidic device according to claim 5, wherein at least one enzyme that digests lipoproteins is a lipase, and the pretreatment reagent comprises a bile acid and / or a salt thereof as a cofactor for the lipase.

7. The microfluidic device according to claim 1, wherein the target hydrophobic analyte is selected from the group consisting of vitamin D, tacrolimus, sirolimus, everolimus, estrogen, estrone, estradiol, estriol, alpha-estradiol, cyclosporine, ethinylestradiol, esterified estrogen, moxestrol, kinestrol, progestin, progesterone, androgen, testosterone, dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), DHEA sulfate, androstenedione, aldosterone, cortisol, catecholamine, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D2, and 1,25-dihydroxyvitamin D3.

8. The microfluidic device according to claim 1, wherein at least one assay reagent is further defined as an immunoassay reagent.

9. A method for detecting the presence and / or concentration of a target hydrophobic analyte in a biological sample, comprising: Simultaneously, or entirely or partially consecutively: (1) A sample suspected of containing the target hydrophobic analyte; (2) A pretreatment reagent comprising at least one enzyme for digesting lipoproteins; and (3) At least one assay reagent capable of detecting hydrophobic analytes. The process of combining, and A step of performing one or more detection assays based on (3) to determine the concentration of the target hydrophobic analyte present in the sample. The method, including the method described above.

10. The method according to claim 9, wherein at least one enzyme that digests lipoproteins comprises a lipase and / or at least one digestive enzyme.

11. The method according to claim 9, wherein the enzyme is present in the pretreatment reagent at a concentration in the range of about 0.1 mg / mL to about 20 mg / mL.

12. The method according to claim 9, wherein the pretreatment reagent further comprises at least one additional substance selected from the group consisting of enzyme cofactors, surfactants, proteases, and combinations thereof.

13. The method according to claim 12, wherein at least one enzyme that digests lipoproteins is a lipase, and the pretreatment reagent comprises a bile acid and / or a salt thereof as a cofactor for the lipase.

14. The method according to claim 9, wherein the target hydrophobic analyte is selected from the group consisting of vitamin D, tacrolimus, sirolimus, everolimus, estrogen, estrone, estradiol, estriol, alpha-estradiol, cyclosporine, ethinylestradiol, esterified estrogen, moxestrol, kinestrol, progestin, progesterone, androgen, testosterone, dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), DHEA sulfate, androstenedione, aldosterone, cortisol, catecholamine, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D2, and 1,25-dihydroxyvitamin D3.

15. The method according to claim 9, wherein at least one assay reagent is further defined as an immunoassay reagent.

16. The method according to claim 9, wherein the sample is a biological sample selected from the group consisting of urine, whole blood or any part thereof, lysed blood or any part thereof, saliva, sputum, cerebrospinal fluid (CSF), intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, tears, mucus, bladder lavage fluid, semen, and any combination thereof.

17. A kit for use in a method for detecting the presence and / or concentration of a target hydrophobic analyte in a biological sample: A pretreatment reagent comprising at least one enzyme for digesting lipoproteins; and A assay reagent capable of detecting a hydrophobic analyte for determining the concentration of the target hydrophobic analyte present in the sample. The kit includes the above.

18. The kit according to claim 17, wherein at least one enzyme for digesting lipoproteins comprises a lipase and / or at least one digestive enzyme, wherein the enzyme is present in the pretreatment reagent at a concentration in the range of about 0.1 mg / mL to about 20 mg / mL.

19. The kit according to claim 17, wherein the pretreatment reagent further comprises at least one additional substance selected from the group consisting of enzyme cofactors, surfactants, proteases, and combinations thereof.

20. The kit according to claim 19, wherein at least one enzyme that digests lipoproteins is a lipase, and the pretreatment reagent comprises a bile acid and / or a salt thereof as a cofactor for the lipase.

21. The kit according to claim 19, wherein the target hydrophobic analyte is selected from the group consisting of vitamin D, tacrolimus, sirolimus, everolimus, estrogen, estrone, estradiol, estriol, alpha-estradiol, cyclosporine, ethinylestradiol, esterified estrogen, moxestrol, kinestrol, progestin, progesterone, androgen, testosterone, dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), DHEA sulfate, androstenedione, aldosterone, cortisol, catecholamine, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D2, and 1,25-dihydroxyvitamin D3.

22. The kit according to claim 19, wherein at least one assay reagent is further defined as an immunoassay reagent.

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