Surfactant treatment of urine samples for antigen detection

Abstract

Provided herein are methods for detecting a target antigen having a complex chemical nature, such as lipoarabinomannan (LAM), in a urine sample. In certain embodiments, to detect a target antigen having a complex chemical nature, such as LAM, the disclosure provides methods of treating the urine sample with a surfactant. The urine sample so treated, when used in an assay, such as an immunoassay that is based on the binding of the target antigen to a binding agent, provides enhanced detection of the target antigen. Also provided herein are kits for performing the methods disclosed herein.

Description

SURFACTANT TREATMENT OF URINE SAMPLES FOR ANTIGEN DETECTIONCROSS REFERENCE TO APPLICATIONS

[0001] Pursuant to 35 U.S.C. § 119(e), this application claims priority to the filing date of U.S. Provisional Application No. 63 / 682,563, filed August 13, 2024, the disclosure of which is incorporated herein by reference.INTRODUCTION

[0002] Detection is challenging for a target antigen having a complex chemical nature, such as the lipoarabinomannan (LAM) in a urine sample. For target antigens having a complex chemical nature, the complex and highly competitive background nature of urine samples makes the target antigen detection even more challenging. One reason for such difficulty in detection is limited availability of the target antigen in a form that is accessible for binding to a binding agent. No currently available method allows for increasing or enhancing the binding availability of LAM in urine samples for LAM detection.SUMMARY

[0003] In certain aspects, the disclosure provides methods for detecting in a urine sample, a target antigen having a complex chemical nature, such as LAM.

[0004] A significant advantage of the methods disclosed herein is to enhance detection capabilities of downstream assays by making the target antigen more accessible for binding to a binding agent. This is achieved by treating a urine sample with a surfactant. Such treatment may: a) release the bound target antigen, such as LAM that is possibly unavailable for binding to a binding agent, b) reduce the loss of the target antigen, such as LAM, due to non-specific binding to various surfaces and assay components, and / or c) release LAM from vesicles and / or previously bound agents in the sample that then makes it available for detection..

[0005] According to certain embodiments, to detect a target antigen having a complex chemical nature, such as LAM, the disclosure provides methods of treating a urine sample with a surfactant. The urine sample so treated, when used in an assay based on the binding of the target antigen to a binding agent, such as an immunoassay, provides enhanced detection of the target antigen.

[0006] In some cases, the target antigen is LAM, and the surfactant is an ionic surfactant, a nonionic surfactant, a zwitterionic surfactant, or a combination thereof. In specific cases, the target antigen is mycobacterial LAM, and the surfactant is a non-ionic surfactant, a zwitterionic surfactant, or a combination thereof.

[0007] Also provided herein are kits for performing the methods disclosed herein.DETAILED DESCRIPTION

[0008] Certain aspects of the present disclosure provide methods for detecting a target antigen having a complex chemical nature, such as LAM in a urine sample.

[0009] According to certain aspects, to detect a target antigen having complex chemical nature, such as LAM, this disclosure provides methods of treating a urine sample with a surfactant. The urine sample so treated, when used in an assay based on the binding of the target antigen to a binding agent, such as an immunoassay, provides enhanced detection of the target antigen.

[0010] In some cases, the target antigen is LAM, and the surfactant is an ionic surfactant, a nonionic surfactant, a zwitterionic surfactant, or a combination thereof. In specific cases, the target antigen is mycobacterial LAM, and the surfactant is an ionic surfactant, a non-ionic surfactant, a zwitterionic surfactant, or a combination thereof.

[0011] Before the present methods and kits are described in greater detail, it is to be understood that the present disclosure is not limited to particular embodiments described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0013] The present disclosure may be understood more readily by reference to the following detailed description of desired embodiments and the examples included therein. In the following specification and the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings.

[0014] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. The presentdisclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0015] The term “comprising” is used herein as requiring the presence of the named component and allowing the presence of other components. The term “comprising” should be construed to include the term “consisting essentially of’ and “consisting of.” The “consisting essentially of’ allows the presence of the named component(s), along with other components which do not change the function / structure of the named component(s). The “consisting of’ allows the presence of the named component(s), along with any adhesives or other bonding means for attaching the listed component(s).

[0016] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0017] It is noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation.

[0018] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both the limits, ranges excluding either or both of those included limits are also included.

[0019] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, thenear or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions belong. Although any methods and compositions similar or equivalent to those described herein can also be used in the practice or testing of the methods and compositions, representative illustrative methods and compositions now described.

[0021] It is noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only,” and the like in connection with the recitation of claim elements or use of a “negative” limitation.

[0022] It is appreciated that certain features of the methods and compositions which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods and compositions, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present methods and compositions and are disclosed herein just as if each such sub-combination was individually and explicitly disclosed herein.

[0023] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present devices and methods. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.METHODS

[0024] As summarized above, certain aspects of the disclosure provide methods for detecting in a urine sample, a target antigen having a complex chemical nature, such as LAM.

[0025] According to certain embodiments, to detect a target antigen having a complex chemical nature, such as LAM, this disclosure provides methods of treating a urine sample with a surfactant. The methods further comprise using the surfactant-treated urine sample in an assay for detecting the target antigen. In certain such methods, the detection of the target antigen is performed using an assay based on binding of the target antigen to a binding agent.

[0026] In some cases, the urine sample is from a subject suspected of having tuberculosis infection and HIV infection.

[0027] The urine sample treated with a surfactant according to the methods disclosed herein, when used in an assay based on the binding of the target antigen to a binding agent, such as an immunoassay, provides enhanced detection of the target antigen.

[0028] The term “enhanced detection of the target antigen” indicates that, compared to a urine sample that is not treated with a surfactant, the treatment of a urine sample with a surfactant as described herein may result in one or more of the following: 1) lowering of the limit of detection of the target antigen, 2) higher signal intensity of the detectable signal, for example, darker colorimetric signal, and 3) quicker development of the detectable signal. Additional aspects of a target antigen detection are well-known in the art and improvements in one or more of such aspects are within the purview of this disclosure.

[0029] The target antigen used in the methods disclosed herein can be any suitable antigen having a complex chemical nature. The term “an antigen having a complex chemical nature” refers to an antigen that comprises a combination of two or more types of biomolecules, such as proteins, carbohydrates, nucleic acids, and lipids. Certain such target antigens include proteoglycans, lipoproteins, nucleoproteins, and glycolipids. A glycolipid can be a lipopolysaccharide. In certain such embodiments, the target antigen is a lipopolysaccharide, such as a complex lipid - carbohydrate chain with or without fragmentation. In a specific embodiment, the target antigen is LAM. In a further specific embodiment, the target antigen is mycobacterial LAM.

[0030] The surfactant that can be used in the methods disclosed herein can be any suitable surfactant, including but not limited to, a non-ionic surfactant, an anionic surfactant, a cationic surfactant, an amphoteric surfactant, or a zwitterionic surfactant.

[0031] A non-ionic surfactant has no ionic charge. Certain non-limiting examples of nonionic surfactants include alcohol ethoxylates, nonylphenoxy polyethylenoxy alcohols, ethylene oxide / propylene oxide block copolymers, polyethylene glycol hexadecyl ether, and polysorbate 20.

[0032] An anionic surfactant has negatively charged hydrophilic end of the molecule. The negatively charged part of an anionic molecule can be sulfonates, sulfates, or carboxylates. Theseare usually neutralized by positively charged metal cations such as sodium or potassium. Nonlimiting examples of anionic surfactants include sodium dodecyl sulfate, sodium alkylbenzene sulfonates, sodium stearate, and potassium alcohol sulfates.

[0033] A cationic surfactant has a positively charged hydrophilic end of the molecule. The positively charged end is typically derived from nitrogen compounds. Non-limiting examples of cationic surfactants include Cs to Cio alkyl hydroxy ethyl dimethylammonium chloride and Cs to Cio alkylamidodimethyl propylamine.

[0034] An amphoteric surfactant can change its charge depending on the pH of the medium. Therefore, an amphoteric surfactant can be a cationic, anionic, or non-ionic surfactant depending on the pH. Certain non-limiting examples of amphoteric surfactants include alkylamidopropylamine N-oxide (APAO), alkyldimethylamine N-oxide (AO), alkylbetaine (Bt), alkylamidopropylbetaine (APB), cocamidopropyl betaine, cocoamphoacetate, and cocoamphodiacetate.

[0035] A zwitterionic surfactant Zwitterionic surfactants has two distinct and opposite charges on the molecule at either adjacent or non-adjacent sites. The presence of both positive and negative charges renders the molecule overall neutral at neutral pH. Non-limiting examples of zwitterionic surfactants include betaines, such as laurylamidopropyldimethylbetaine; sulfobetaines, such as lauryl hydroxysultaine and myristyl sulfobetaine; amine oxides, such as lauryl dimethylamine oxide; amido betaines; and imidazoline betaines.

[0036] In certain embodiments, the surfactant used in the methods disclosed herein is: sodium dodecyl sulfate and its variants, polyoxyethylenesorbitan monolaurate and its variants, polyethylene glycol hexadecyl ether, alcohol ethoxylate, secondary alcohol ethoxylate and its variants, myristyl sulfobetaine and its variant and the like.

[0037] In a specific embodiment, the surfactant used in the methods disclosed herein is: myristyl sulfobetaine, polyethylene glycol hexadecyl ether, polysorbate 20, secondary alcohol ethoxylate - 9, secondary alcohol ethoxylate - 7, sodium dodecyl sulfate, alcohol ethoxylate - 9, or a combination thereof.

[0038] As used herein, the term “treating a urine sample with a surfactant” refers to mixing the urine sample with a solution comprising the surfactant.

[0039] The urine sample so mixed can be incubated for an appropriate time, ranging, for example, from 1 second to 1 hour or more. For example, a urine sample mixed with a solution comprising a surfactant can be incubated for at least or about: between 1 second and 60 seconds, such as 5 seconds, 15 seconds, 30 seconds, 45 seconds, or 60 seconds. A urine sample mixed with a solution comprising a surfactant can be incubated for at least or about: between 1 minute and 60 minutes,such as 5 minutes, 15 minutes, 30 minutes, 45 minutes, or 60 minutes. In a specific embodiment, the surfactant is incubated with the sample for at least 1 minute. An appropriate incubation time also depends on the temperature of incubation. At colder temperatures, the incubation time needs to be longer as compared to incubation at warmer temperatures.

[0040] The concentration of surfactant in the solution comprising the surfactant and in the final mixture with the urine sample can be selected depending on the surfactant used. For example, a surfactant concentration in the final mixture of a solution comprising the surfactant and a urine sample can be at least or about: between 1 nM and 1 mM, such as 100 nM, 100 pM, 1 mM, or 100 mM.

[0041] A mixture of a urine sample and a solution comprising a surfactant can be incubated at a temperature of: between 4°C and 100°C, such as 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C or 95°C.

[0042] In certain embodiments of the methods disclosed herein, the detection of the target antigen is performed using an assay based on binding of the target antigen to a binding agent. The binding agent can be any agent that specifically binds to the target antigen. Certain non-limiting examples of such binding agents include antibody or antigen-binding fragment thereof, specifically binding protein partners, aptamers, and the like. Additional examples of binding agents are well-known in the art and such embodiments are within the purview of the disclosure.

[0043] Any suitable binding agent-based assay can be used to detect the target antigen according to the methods disclosed herein. Certain non-limiting examples of a binding-agent based assay include lateral flow immunoassay, enzyme-linked immunosorbent assays (ELISA), radioimmunoassay (RIS), real-time immunoquantitative PCR (iqPCR), and the like. Additional examples of binding agent-based assays are well-known in the art and such embodiments are within the purview of the disclosure.Kits

[0044] Certain aspects of the disclosure provide kits for carrying out the methods disclosed herein. Such kits may comprise one or more of the following: means for collecting and / or processing a urine sample, one or more surfactants, buffers, devices and reagents for the assays, instructions for conducting the assays, etc. Additional components that could be included in the kits disclosed herein are well-known to a person of ordinary skill in the art and such embodiments are within the purview of the disclosure.

[0045] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

[0046] Clause 1. A method for detecting in a urine sample a target antigen having a complex chemical nature, the method comprising treating the urine sample with a surfactant.

[0047] Clause 2. The method of clause 1 , further comprising using the surfactant-treated urine sample in an assay for detecting the target antigen.

[0048] Clause 3. The method of clause 2, wherein the assay is based on the binding of the target antigen to a binding agent.

[0049] Clause 4. The method of clause 3, wherein the assay based on the binding of the target antigen to the binding agent is: lateral flow immunoassay, enzyme- linked immunosorbent assays (ELISA), radioimmunoassay (RIS), or real-time immunoquantitative PCR (iqPCR).

[0050] Clause 5. The method of any one of the preceding clauses, wherein the target antigen having the complex chemical nature comprises a combination of two or more types of biomolecules.

[0051] Clause 6. The method of clause 5, wherein the two or more types of biomolecules are selected from proteins, carbohydrates, nucleic acids, and lipids.

[0052] Clause 7. The method of any one of the preceding clauses, wherein the target antigen is a proteoglycan, lipoprotein, nucleoprotein, or glycolipid.

[0053] Clause 8. The method of clause 7, wherein the target antigen is a glycolipid.

[0054] Clause 9. The method of clause 8, wherein the target antigen is lipoarabinomannan(LAM).

[0055] Clause 10. The method of any one of the preceding clauses, wherein the urine sample is from a subject suspected of having a tuberculosis infection.

[0056] Clause 11. The method of clause 10, wherein the subject is also further suspected of having an HIV infection.

[0057] Clause 12. The method of any one of the preceding clauses, wherein the surfactant is a non-ionic surfactant, an anionic surfactant, a cationic surfactant, an amphoteric surfactant, or a zwitterionic surfactant.

[0058] Clause 13. The method of clause 12, wherein the surfactant is the non-ionic surfactant.

[0059] Clause 14. The method of clause 13, wherein the non-ionic surfactant is polyethylene glycol hexadecyl ether, polysorbate 20, secondary alcohol ethoxylate - 9, secondary alcohol ethoxylate - 7, alcohol ethoxylate - 9, or a combination thereof.

[0060] Clause 15. The method of clause 12, wherein the surfactant is a zwitterionic surfactant.

[0061] Clause 16. The method of clause 15, wherein the zwitterionic surfactant is myristyl sulfobetaine.

[0062] Clause 17. The method of clause 12, wherein the surfactant is the ionic surfactant.

[0063] Clause 18. The method of clause 17, wherein the ionic surfactant is sodium dodecyl sulfate.

[0064] Clause 19. A method for detecting lipoarabinomannan (LAM) in a urine sample, the method comprising treating the urine sample with a surfactant.

[0065] Clause 20. The method of clause 19, further comprising using the surfactant-treated urine sample in an assay for detecting LAM.

[0066] Clause 21. The method of clause 20, wherein the assay is based on the binding of LAM to a binding agent.

[0067] Clause 22. The method of clause 21, wherein the assay based on the binding of LAM to the binding agent is: lateral flow immunoassay, enzyme-linked immunosorbent assays (ELISA), radioimmunoassay (RIS), real-time immunoquantitative PCR (iqPCR).

[0068] Clause 23. The method of any one of clauses 19 to 22, wherein the surfactant is a nonionic surfactant, an anionic surfactant, a cationic surfactant, an amphoteric surfactant, or a zwitterionic surfactant.

[0069] Clause 24. The method of clause 23, wherein the surfactant is a non-ionic surfactant.

[0070] Clause 25. The method of clause 24, wherein the non-ionic surfactant is polyethylene glycol hexadecyl ether, polysorbate 20, secondary alcohol ethoxylate - 9, secondary alcohol ethoxylate - 7, alcohol ethoxylate - 9, or a combination thereof.

[0071] Clause 26. The method of clause 23, wherein the surfactant is a zwitterionic surfactant.

[0072] Clause 27. The method of clause 26, wherein the zwitterionic surfactant is myristyl sulfobetaine.

[0073] Clause 28. The method of clause 23, wherein the surfactant is the ionic surfactant.

[0074] Clause 29. The method of clause 29, wherein the ionic surfactant is sodium dodecyl sulfate.

[0075] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the embodiments, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used.Example 1 - Detection of LAM in a urine sample

[0076] LAM is present in cell walls of Mycobacteria and related Actinomyces. LAM is a heat stable lipopolysaccharide with a variable chemical structure and a molecular weight of 19 ± 8.5 kD. Detection of LAM in urine provides a quick diagnosis for tuberculosis infection. Therefore, increasing sensitivity of LAM detection in urine would greatly improve tuberculosis diagnosis.

[0077] This examples describes methods of detecting LAM in urine samples. The methods comprise treating the urine sample with a surfactant. Such treatment with a surfactant may allow LAM in urine samples to be more accessible for binding, and hence, readily detectable in a downstream assay, e.g., an immunoassay. In this Example, a urine sample is treated with a surfactant for a minimum of 1 minute followed by downstream further processing or detection in an assay.

[0078] Without being bound to a specific theory for the operation of the disclosed methods, certain actions of the surfactant may facilitate enhanced detection of LAM. The surfactant treatment may allow for the LAM to stay in solution in a form to be detectable by a downstream assay. Additionally, the surfactant treatment may reduce the surface binding of LAM to other components thereby increasing LAM available for binding to a binding agent.

[0079] For example, the hydrophobic component of the surfactant chains (C-chains typically nonpolar ends) of a surfactant can blend with the lipid (also typically hydrophobic) chain end of LAM molecules thereby allowing for the LAM to stay in solution. The efficacy of may depend on the chain length of the surfactant because it directly influences the degree of hydrophobic interactions. Also, other parts of the surfactant, for example, the hydrophilic end, may also influence the efficacy due to various factors, such as stearic hindrance, micellar behavior, residual charge - for example, when ionic or zwitterionic surfactants are used.

[0080] The above interaction can also allow for the LAM molecule to stay in solution by reduction of the lipid interactions with surfaces within the collection, processing or detection devices thereby reducing losses to non-specific binding events.

[0081] Moreover, some tuberculosis patient urine samples contain extracellular vesicles that can also encapsulate LAM target within the vesicle. The surfactant treatment step allows for release of encapsulated LAM from these vesicles. This can act as a further enhancement for LAM detection due to additional LAM availability released from the vesicles.

[0082] Different surfactants have varying degree of efficacy on the increasing LAM availability. Non-ionic and zwitterionic surfactants show more enhancement of LAM detection in downstream assays, indicating the influence of the sample matrix (urine) on this step.

[0083] According to this Example, a urine sample is obtained from a patient and may be preserved appropriate before the LAM detection assays is conducted. Once ready for the assay, the urine sample is treated with a surfactant, for example: myristyl sulfobetaine (3-(N,N- Dimethyltetradecylammonio)propanesulfonate), polyethylene glycol hexadecyl ether, polysorbate 20, secondary alcohol ethoxylate - 9, secondary alcohol ethoxylate - 7, sodium dodecyl sulfate, alcohol ethoxylate - 9, or a combination thereof.

[0084] After appropriate treatment of a urine sample with a surfactant, for example, the appropriate surfactant, as a suitable concentration for a suitable time, the surfactant-treated urine sample is used in an assay to detect LAM or any further post-processing required before continuing to a detection assay downstream.

[0085] The assay to detect LAM can be a lateral flow immunoassay based on LAM specific antibodies.

[0086] Treatment of a urine sample with a surfactant would result in one or more of the following: 1) lowering of the limit of detection of LAM thereby allowing detection of lower concentrations of LAM, 2) higher signal intensity of the detectable signal, for example, darker colorimetric signal, thereby simplifying the detection of LAM, and 3) quicker development of the detectable signal thereby saving time of the assay.Example 2 - Effects of detergents on detection

[0087] This Example describes the effects of various detergents on detection of LAM via a lateral flow immunoassay.

[0088] Clinical Urine samples with known LAM target concentration (20 pg / ml) were tested with a sample pre-processing step that involved a surfactant treatment step and a concentration step. Detection of LAM in such processed sample was performed in a lateral flow immunoassay. Various tested surfactants and corresponding detection results are provided in Table 1 below. All surfactants are initially tested at 1 mM final concentration in the sample.

[0089] The strength of the signal (1 being the weakest signal) is an indication of the performance of the lateral flow assay where a pair of antibodies capture the LAM target.

[0090] The untreated reference sample is subjected to the same process steps as the test sample samples with the surfactants and LAM was detected using the same lateral flow immunoassay.

[0091] Results shows varying increases in the signal of the surfactant treated samples as compared to the untreated sample. The increases in the signal were higher as the concentration of the target in the sample increases, noting that 20 pg / ml is an extremely low concentration of LAM, lower than LAM concentration typically found in urines of subjects infected with tuberculosis.

[0092] The preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

Claims

CLAIMSWE CLAIM:

1. A method for detecting in a urine sample a target antigen having a complex chemical nature, the method comprising treating the urine sample with a surfactant.

2. The method of claim 1, further comprising using the surfactant-treated urine sample in an assay for detecting the target antigen.

3. The method of claim 2, wherein the assay is based on the binding of the target antigen to a binding agent.

4. The method of claim 3, wherein the assay based on the binding of the target antigen to the binding agent is: lateral flow immunoassay, enzyme-linked immunosorbent assays (ELISA), radioimmunoassay (RIA), or real-time immunoquantitative PCR (iqPCR).

5. The method of any one of the preceding claims, wherein the target antigen having the complex chemical nature comprises a combination of two or more types of biomolecules.

6. The method of claim 5, wherein the two or more types of biomolecules are selected from proteins, carbohydrates, nucleic acids, and lipids.

7. The method of any one of the preceding claims, wherein the target antigen is a proteoglycan, lipoprotein, nucleoprotein, or glycolipid.

8. The method of claim 7, wherein the target antigen is a glycolipid.

9. The method of claim 8, wherein the target antigen is lipoarabinomannan (LAM).

10. The method of any one of the preceding claims, wherein the urine sample is from a subject suspected of having a tuberculosis infection.

11. The method of clause 10, wherein the subject is also further suspected of having an HIV infection.

12. The method of any one of the preceding claims, wherein the surfactant is a non-ionic surfactant, an anionic surfactant, a cationic surfactant, an amphoteric surfactant, or a zwitterionic surfactant.

13. The method of claim 12, wherein the surfactant is the non-ionic surfactant.

14. The method of claim 13, wherein the non-ionic surfactant is polyethylene glycol hexadecyl ether, polysorbate 20, secondary alcohol ethoxylate - 9, secondary alcohol ethoxylate - 7, alcohol ethoxylate - 9, or a combination thereof.

15. The method of claim 12, wherein the surfactant is a zwitterionic surfactant.

16. The method of claim 15, wherein the zwitterionic surfactant is myristyl sulfobetaine.

17. The method of claim 12, wherein the surfactant is the ionic surfactant.

18. The method of claim 17, wherein the ionic surfactant is sodium dodecyl sulfate.

19. A method for detecting lipoarabinomannan (LAM) in a urine sample, the method comprising treating the urine sample with a surfactant.

20. The method of claim 19, further comprising using the surfactant-treated urine sample in an assay for detecting LAM.

21. The method of claim 20, wherein the assay is based on the binding of LAM to a binding agent.

22. The method of claim 21, wherein the assay based on the binding of LAM to the binding agent is: lateral flow immunoassay, enzyme-linked immunosorbent assays (ELISA), radioimmunoassay (RIS), real-time immunoquantitative PCR (iqPCR).

23. The method of any one of claims 19 to 22, wherein the surfactant is a non-ionic surfactant, an anionic surfactant, a cationic surfactant, an amphoteric surfactant, or a zwitterionic surfactant.

24. The method of claim 23, wherein the surfactant is a non- ionic surfactant.

25. The method of claim 24, wherein the non- ionic surfactant is polyethylene glycol hexadecyl ether, polysorbate 20, secondary alcohol ethoxylate - 9, secondary alcohol ethoxylate - 7, alcohol ethoxylate - 9, or a combination thereof.

26. The method of claim 23, wherein the surfactant is a zwitterionic surfactant.

27. The method of claim 26, wherein the zwitterionic surfactant is myristyl sulfobetaine.

28. The method of claim 23, wherein the surfactant is the ionic surfactant.

29. The method of claim 28, wherein the ionic surfactant is sodium dodecyl sulfate.

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