Controls for protein-based assays

Abstract

A method for producing an antigen-based positive control for detecting a pathogen, comprising: providing a culture medium containing a pathogen having a first detectable antigen, wherein the culture medium has a first concentration of the first detectable antigen; and exposing the culture medium to UV electromagnetic radiation for a period of time sufficient to inactivate the pathogen, thereby producing an antigen-based positive control, wherein the antigen-based positive control has a second concentration of the first detectable antigen that differs from the first concentration by no more than about 50% as determined via ELISA. Also described are antigen-based positive controls and kits containing the same.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. patent application Ser. No. 18 / 344,693, filed June 29, 2023, which in turn claims priority to U.S. patent application Ser. No. 63 / 357,415, filed June 30, 2022, U.S. patent application Ser. No. 63 / 378,735, filed October 7, 2022, and U.S. patent application Ser. No. 63 / 490,886, filed March 17, 2023. This application also claims priority to U.S. patent application Ser. No. 63 / 378,735 and U.S. patent application Ser. No. 63 / 490,886. The contents of all priority applications are expressly incorporated herein in their entirety. [Background technology]

[0002] background Due to the rapidly evolving nature of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, molecular and rapid assays have recently been developing at the same speed and intensity for the first time in history. The development of antigen-based assays, in particular, has received considerable attention, but adequate positive controls for such assays remain a challenge. For example, common positive controls for antigen-based assays utilize recombinant proteins. However, while such controls can support the assay's ability to detect free proteins, these controls often fail to provide reliable data regarding the assay's ability to detect whole organisms, where detection of the selected antigen may be complicated by other components of the organism (e.g., other proteins contained and / or produced by the organism).

[0003] On the other hand, the use of whole organisms as positive controls presents its own challenges. For example, many laboratories require non-infectious samples for antigen-based assay development, but current methods for rendering pathogens, e.g., SARS-CoV-2, non-infectious have deleterious effects on the proteins targeted by the assay. Furthermore, traditional pathogen measurements (e.g., TCID 50Quantification of non-infectious organisms is hindered because the concentration of non-infectious organisms (NBIs) is often inconsistent with the actual pathogen concentration. This inconsistency presents a significant challenge in the production of reliable positive controls. Summary of the Invention [Problem to be solved by the invention]

[0004] Thus, there is a need in the art for safe and reliable positive controls for the development, testing and use of antigen-based assays. [Means for solving the problem]

[0005] overview Disclosed herein is a method for producing a positive control for detecting a pathogen. The method utilizes a culture medium containing a target pathogen, such as an infectious virus, having at least one detectable antigen, such as a protein, at a first concentration. The method may further include exposing the culture medium to UV radiation for a period sufficient to inactivate the pathogen, thereby producing a non-infectious positive control. The positive control may contain a second concentration of the detectable antigen. BRIEF DESCRIPTION OF THE DRAWINGS [Brief explanation of the drawings]

[0006] [Figure 1A] FIG. 1 shows an illustrative first step of an ELISA procedure according to an embodiment of the present disclosure. [Figure 1B] FIG. 1 shows an illustrative second step of an ELISA procedure according to an embodiment of the present disclosure. [Figure 1C] FIG. 1 shows an illustrative third step of an ELISA procedure according to an embodiment of the present disclosure. [Figure 1D] FIG. 10 shows an illustrative fourth step of an ELISA procedure according to an embodiment of the present disclosure. [Figure 2] FIG. 1 shows TCID50 measurements as described in Example II. [Figure 3] FIG. 1 shows TCID50 measurements as described in Example III. [Figure 4] FIG. 1 shows SARS-CoV-2 qPCR measurements as described in Example III. [Figure 5] FIG. 1 shows ELISA measurements described in Example III. DETAILED DESCRIPTION OF THE INVENTION

[0007] Detailed Description Disclosed herein is a method for producing a positive control for detecting a pathogen, wherein the pathogen has at least one detectable target antigen, e.g., a protein. The method includes providing a culture medium containing the pathogen, wherein the culture medium has a first concentration of the detectable target antigen, and exposing the culture medium to electromagnetic radiation for a period of time sufficient to inactivate the pathogen, thereby producing the positive control. The positive control may have an acceptable second concentration of the detectable target antigen. According to some embodiments, the first and second detectable target antigen concentrations can be determined via immunoassay, e.g., enzyme-linked immunosorbent assay (ELISA).

[0008] In some examples, the method may include providing a culture medium having a pathogen, wherein the pathogen is a virus and the culture medium has a first concentration of a detectable target antigen, and exposing the culture medium to electromagnetic radiation for a period of time sufficient to inactivate the pathogen.

[0009] In some examples, the method may include providing a culture medium having a pathogen, the culture medium having a first concentration of a detectable target antigen, the detectable target antigen comprising a nucleocapsid protein, and exposing the culture medium to electromagnetic radiation for a period of time sufficient to inactivate the pathogen.

[0010] In some examples, the method may include providing a culture medium having a pathogen, wherein the culture medium has a first concentration of a detectable target antigen, and exposing the culture medium to electromagnetic radiation for a period of time sufficient to inactivate the pathogen, wherein the electromagnetic radiation comprises UV radiation provided by UV-C light.

[0011] In some examples, the method may include providing a culture medium having a pathogen, wherein the culture medium has a first concentration of a detectable target antigen, and exposing the culture medium to electromagnetic radiation for a period of time sufficient to inactivate the pathogen, wherein the period of time is between about 1 minute and 15 minutes.

[0012] Also disclosed herein is a positive control comprising an inactivated pathogen provided with at least two quantification values. The present disclosure also relates to the positive controls provided by the methods described herein, and to methods of using the positive controls described herein, for example, in protein biomarker assays.

[0013] Also disclosed herein is a kit having an antigen-based positive control comprising an inactivated pathogen and at least two quantification values.

[0014] In some instances, the kit includes an antigen-based positive control comprising an inactivated virus and at least two quantification values.

[0015] In some examples, the kit includes an antigen-based positive control comprising an inactivated pathogen, and at least two quantification values ​​including the TCID50 value of the pathogen before inactivation and the concentration of the detectable target antigen.

[0016] In some examples, the kit includes an antigen-based positive control comprising an inactivated pathogen, a protease inhibitor, and at least two quantification values.

[0017] In some examples, the kit includes a kit antigen-based positive control comprising an inactivated pathogen provided in a liquid, and at least two quantification values.

[0018] Also disclosed herein is an inactivated pathogen for use as an antigen-based positive control, wherein the inactivated pathogen is provided in the kit with at least two quantification values.

[0019] As used herein, the term "pathogen" refers to any agent capable of causing infection and / or disease in its natural state. Illustrative pathogens include viruses, bacteria, fungi, protists, and yeast. "Virus," according to the present disclosure, includes enveloped and non-enveloped viruses and those containing RNA and / or DNA as nuclear material. Illustrative viruses useful according to the present disclosure include, but are not limited to, SARS-CoV-2, human immunodeficiency virus (HIV), influenza virus (Flu A and B), respiratory syncytial virus (RSV), cytomegalovirus (CMV), human lymphotrophic virus (HTLV), Epstein-Barr virus (EBV), and herpesvirus (HSV).

[0020] The methods of the present disclosure may include providing a culture medium having a pathogen described herein. The culture medium may be a natural medium, an artificial medium, or a combination thereof. Non-limiting examples of culture medium according to the present disclosure include Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), RPMI medium, IMDM, PBS, DPBS, HBSS, EBSS, and combinations thereof. According to some embodiments, the culture medium may be purified.

[0021] In some non-limiting examples, the pathogen may be isolated from an infected biological fluid, including blood, serum, plasma, defibrinated plasma, stabilized pooled plasma, cerebrospinal fluid, urine, saliva, semen, mucus, and sputum. Additionally or alternatively, the pathogen may be cultured as known in the art.

[0022] As described herein, the pathogen may be provided in a purified medium, and the purified medium may be purified as known in the art. Without limitation, purification may include removing cells and cell debris (e.g., by size and / or mass-based separation techniques, such as filtration and low-speed centrifugation), concentrating the pathogen (e.g., by filtration and / or high-speed centrifugation), and / or performing ultracentrifugation and / or density gradient purification techniques, as known in the art.

[0023] The methods of the present disclosure may include providing a selected concentration of a pathogen in a culture medium described herein. According to embodiments, the concentration of the pathogen contained in and / or provided to the culture medium is determined by the Tissue Culture Infectious Dose (TCID) of the pathogen. 50 In some non-limiting examples, the pathogen may be determined by measuring the 4 Units / mL and 10 8 Units / mL and TCID 50 The culture medium may be provided with the following:

[0024] The pathogen-containing culture medium described herein contains a first concentration of detectable target antigen. It should be understood that the first concentration described herein may correspond to the concentration of detectable target antigen before inactivation of the pathogen. As used herein, the term "target antigen" refers to any antigen contained and / or produced by a pathogen that can be targeted by an antigen biomarker assay. A "detectable target antigen" refers to a target antigen that can be identified and / or quantified by an antigen biomarker assay. As used herein, the term "antigen biomarker assay" refers to an assay designed to detect a target antigen, particularly an assay designed to detect a target antigen for detecting infection by a pathogen and / or diagnosing a disease associated with the pathogen.

[0025] Antigen biomarker assays according to the present disclosure are not particularly limited. For example, antigen biomarker assays can be qualitative and / or quantitative. In some non-limiting examples, antigen biomarker assays can be used in research and / or medical settings. Types of antigen biomarker assays can include, but are not limited to, lateral flow assays and ELISA assays.

[0026] A detectable target antigen according to the present disclosure can be any antigen that can be targeted by the antigen biomarker assays described herein. For example, a detectable target antigen can include a protein, such as a viral protein. Illustrative viral proteins include, but are not limited to, structural proteins, nonstructural proteins, regulatory proteins, accessory proteins, or combinations thereof. In a non-limiting example where the pathogen is SARS-CoV-2, a detectable target antigen can include a nucleocapsid protein (N protein) and / or a spike protein (S protein). In some non-limiting examples, a target antigen can include a conserved region of a pathogen protein, such as a viral protein. As used herein, a "conserved region" of a pathogen protein refers to a sequence of amino acids in the pathogen protein that does not vary between strains of the pathogen.

[0027] Methods according to the present disclosure may include exposing a culture medium described herein to electromagnetic radiation for a period of time sufficient to inactivate the pathogen, also referred to herein as an inactivated pathogen. The term "inactivated pathogen," as used herein, refers to a pathogen that has been modified so that it is unable to cause infection and / or disease in humans. In some non-limiting examples, an inactivated pathogen may include a pathogen that has been modified so that it is unable to replicate meaningfully, i.e., unable to replicate sufficiently to cause infection and / or disease in humans.

[0028] According to some embodiments, pathogen inactivation is achieved by administering a TCID 50Inactivation can be determined using a cytopathic effect assay or infectivity assay. As known in the art, such assays involve subjecting serially diluted virus cultures or inactivated pathogen samples to cells in a 96-well plate format. In some non-limiting examples, the assay may include one initial dilution followed by additional serial dilutions across the remainder of the 96-well plate. According to the infectivity assay, inactivation corresponds to the absence of cytopathic effect (CPE) observed in any well across the 96-well plate.

[0029] According to some embodiments, the electromagnetic radiation may include UV radiation, gamma radiation, microwave radiation, IR radiation, X-ray radiation, or a combination thereof.

[0030] As described herein, UV radiation can be provided to the culture medium via a UV source, including, but not limited to, a UV lamp. It should be understood that the level of UV radiation provided to the culture medium can depend, for example, on the characteristics of the UV source (e.g., the wattage of the UV lamp), the distance between the UV source and the culture medium, the UV radiation wavelength, the volume of the culture medium exposed to UV radiation, or a combination thereof.

[0031] As described herein, UV sources can include UV lamps, as known in the art. Some non-limiting examples of UV lamps include the Model G30T8 Ultraviolet UV-C Lamp.

[0032] According to some embodiments, the distance between the culture medium and the UV source (e.g., UV bulb) during UV irradiation can be between about 1 inch and 50 inches, optionally between about 10 inches and 50 inches, optionally between about 20 inches and 40 inches. However, it should be understood that the present disclosure is not necessarily limited to this method. In particular, the distance between the culture medium and the UV source can vary based on the UV source, the culture volume, or a combination thereof.

[0033] In some embodiments, the culture medium may be exposed to UVA light, UVB light, UVC light, or a combination thereof. It should be understood that UVA light has a wavelength between about 315 nm and 400 nm, UVB light has a wavelength between about 280 nm and 315 nm, and UVC light has a wavelength between about 100 nm and 280 nm. In some embodiments, the culture medium may be exposed to UV light having a wavelength between about 200 nm and 300 nm, optionally between about 225 nm and 275 nm, and optionally about 254 nm.

[0034] In some non-limiting examples, UV exposure can be provided to a volume of culture medium between about 1 mL and 500 mL, optionally between about 50 mL and 250 mL, optionally between about 100 mL and 200 mL, optionally about 125 mL.

[0035] Methods according to the present disclosure may include exposing the culture medium to electromagnetic radiation for a period of time sufficient to inactivate the pathogens, as described herein. It should be understood that the period of time sufficient to inactivate the pathogens may depend, at least in part, on the level of electromagnetic radiation provided to the culture medium, as described above.

[0036] Non-limiting examples of irradiation durations useful in accordance with the present disclosure include between about 1 and 20 minutes, optionally between about 1 and 15 minutes, optionally between about 5 and 15 minutes, and optionally about 10 minutes. Other non-limiting examples of irradiation durations useful in accordance with the present disclosure include about 1 minute, optionally about 2 minutes, optionally about 3 minutes, optionally about 4 minutes, optionally about 5 minutes, optionally about 6 minutes, optionally about 7 minutes, optionally about 8 minutes, optionally about 9 minutes, optionally about 10 minutes, optionally about 11 minutes, optionally about 12 minutes, optionally about 13 minutes, optionally about 14 minutes, and optionally about 15 minutes.

[0037] According to some embodiments, the culture medium may be agitated (e.g., swirled and / or shaken) for all or a portion of the irradiation period. Additionally or alternatively, the culture medium may remain unagitated for all or a portion of the irradiation period. Furthermore, the culture medium may or may not be placed on ice or maintained at a low temperature during electromagnetic irradiation.

[0038] It should be understood that the irradiation periods described herein can be continuous periods. Alternatively, the irradiation periods of the present disclosure can include two, three, four, or more portions, each portion separated by one or more steps of the method described herein. For example, the method can include exposing the culture medium to electromagnetic radiation for a first portion of the irradiation period, agitating the culture medium, and exposing the culture medium to electromagnetic radiation for a second portion of the irradiation period. The separate portions can be pooled into one volume to increase batch size.

[0039] The disclosed method advantageously provides a positive control having an acceptable second concentration of detectable target antigen. It should be understood that the second concentration described herein may correspond to the concentration of detectable target antigen after inactivation of a pathogen as described herein. As used herein, "acceptable concentration" refers to a concentration of detectable target antigen that is detectable by an antigen biomarker assay described herein and sufficient for use as a positive control in such an antigen biomarker assay. It should be understood that the positive control may refer to a culture medium containing an inactivated pathogen as described herein. Alternatively, if the inactivated pathogen is separated from the culture medium, the positive control may refer to the inactivated pathogen.

[0040] According to some embodiments, the acceptable second concentration of the detectable target antigen may be within a certain range of the first concentration of the detectable target antigen described herein. For example, the second concentration may differ from the first concentration by no more than about 50%, optionally by no more than about 40%, optionally by no more than about 30%, optionally by no more than about 20%, or optionally by no more than about 10%.In some non-limiting examples, the second concentration can differ from the first concentration by about 50% or less, optionally by about 49% or less, optionally by about 48% or less, optionally by about 47% or less, optionally by about 46% or less, optionally by about 45% or less, optionally by about 44% or less, optionally by about 43% or less, optionally by about 42% or less, optionally by about 41% or less, optionally by about 40% or less, optionally by about 39% or less. may differ by about 38% or less, optionally may differ by about 37% or less, optionally may differ by about 36% or less, optionally may differ by about 35% or less, optionally may differ by about 34% or less, optionally may differ by about 33% or less, optionally may differ by about 32% or less, optionally may differ by about 31% or less, optionally may differ by about 30% or less, optionally may differ by about 29% or less from the first concentration, optionally may differ by about 28% or less, optionally may differ by about 27% or less, may differ by about 26% or less, optionally may differ by about 25% or less, optionally may differ by about 24% or less, optionally may differ by about 23% or less, optionally may differ by about 22% or less, optionally may differ by about 21% or less, optionally may differ by about 20% or less, optionally may differ by about 19% or less from the first concentration, optionally may differ by about 18% or less, optionally may differ by about 17% or less, optionally may differ by about 16% or less, optionally may differ by about 15% or less, optionally may differ by about 14% or less may vary by less than about 13%, may vary by less than about 12%, may vary by less than about 11%, may vary by less than about 10%, may vary by less than about 9%, may vary by less than about 8%, may vary by less than about 7%, may vary by less than about 6%, may vary by less than about 5%, may vary by less than about 4%, may vary by less than about 3%, may vary by less than about 2%, or may vary by less than about 1%.According to some embodiments, the first concentration and / or the second concentration may be determinable by ELISA.

[0041] 1A-1D show an illustrative schematic of one ELISA protocol useful according to the present disclosure. In this example, as shown in FIG. 1A, a well plate 101 can be pre-coated with an antibody 102 specific for a detectable target antigen, e.g., SARS-CoV-2 N protein, thereby immobilizing the antibody 102. As shown in FIG. 1A, a positive control sample containing a detectable target antigen 103 can be added to the well plate 101, which can then bind to the immobilized antibody 102, as shown in FIG. 1B. As shown in FIG. 1C, after washing the well plate 101, a signal-generating antibody 104, e.g., a horseradish peroxidase-conjugated anti-SARS-CoV-2 N protein antibody, can then be added to the well plate 101. The well plate 101 can then be washed to remove any unbound signal-generating antibodies 104, leaving behind only the antibody-antigen-antibody sandwich complexes 105, as shown in FIG. 1D. It should be understood that each sandwich complex 105 includes an immobilized antibody 102, a detectable target antigen 103, and a signal-generating antibody 104. The sandwich complexes 105 can be quantified by loading a substrate solution (e.g., TMB substrate solution) into the well plate 101 and allowing color to develop. The intensity of the color is proportional to the number of immobilized sandwich complexes 105 in the well plate 101 (and thus the number of immobilized detectable target antigens 103 in the well plate 101).

[0042] However, it should be understood that the present disclosure is not limited to this example. That is, the first concentration of detectable target antigen and / or the second concentration of detectable target antigen can be determined using tools in addition to or instead of ELISA. According to some embodiments, the first concentration of detectable target antigen and / or the second concentration of detectable target antigen can be determined using any acceptable immunoassay, as known in the art. Non-limiting examples of immunoassays according to the present disclosure include Western blot techniques, immunofluorescence assays (IFAs), and lateral flow tests (LFTs). Additionally or alternatively, the first concentration of detectable target antigen and / or the second concentration of detectable target antigen can be determined by measuring infectivity prior to inactivation via polymerase chain reaction (PCR) techniques, via chromatography (e.g., high-performance liquid chromatography), via mass spectrometry, via total protein assays (e.g., colorimetric or spectrophotometric), or a combination thereof.

[0043] The disclosed methods may include one or more steps that provide a second concentration of detectable target antigen that is closer to the first concentration than that provided by the same method without such a step. In one non-limiting example, the method may include combining the positive control with an agent that reduces and / or eliminates protein degradation, including degradation of the detectable target antigen described herein. Illustrative agents include, but are not limited to, protease inhibitors. Non-limiting examples of protease inhibitors include aprotinin, bestatin, calpain I, calpain II, chymostatin, E-64, leupeptin, alpha-marcoglobulin, pefabloc SC, pepstatin, TLCK-HCL, trypsin inhibitors, and combinations thereof. In some non-limiting examples, the protease inhibitor includes one or more agents that at least partially inhibit the activity of pancreatic extract, pronase, thermolysin, chymotrypsin, trypsin, papain, or combinations thereof. In some non-limiting examples, the agent may be provided with a positive control such that the second concentration may differ from the first concentration by no more than about 50%, optionally by no more than about 40%, optionally by no more than about 30%, optionally by no more than about 20%, and optionally by no more than about 10%.

[0044] According to some embodiments, a protease inhibitor may be combined with the culture medium prior to and / or during the electromagnetic irradiation described herein. Additionally or alternatively, a protease inhibitor may be combined with a positive control.

[0045] According to some aspects, the methods of the present disclosure may include partially or completely removing protease inhibitors before, during, and / or after electromagnetic irradiation as described herein. Additionally or alternatively, all or a portion of the protease inhibitors may serve as components of a positive control as described herein.

[0046] The disclosed methods may include one or more additional processing steps, including pooling and / or aliquoting samples of culture medium as described herein. Additionally or alternatively, the method may include providing a positive control, for example, during packaging in a sterile vial or tube. In some non-limiting examples, the method may include separating the inactivated pathogen from the culture medium and / or providing the positive control as part of a positive control device, e.g., as part of a positive control swab.

[0047] The present disclosure also relates to a positive control that can be obtained by the methods described herein. The positive control can include a culture medium described herein having an inactivated pathogen and a detectable target antigen at a concentration that is within about 50%, optionally within about 40%, optionally within about 30%, and optionally within about 20% of the detectable target antigen concentration in the same culture medium before the pathogen is inactivated. Additionally or alternatively, the positive control can include an inactivated pathogen described herein having a detectable target antigen concentration that is within about 50%, optionally within about 40%, optionally within about 30%, optionally within about 20%, and optionally within about 10% of the detectable target antigen concentration in the same pathogen before it is inactivated.In some non-limiting examples, the positive control differs from the first concentration by about 50%, optionally within about 49%, optionally within about 48%, optionally within about 47%, optionally within about 46%, optionally within about 45%, optionally within about 44%, optionally within about 43%, optionally within about 42%, optionally within about 41%, or optionally within about 40%, of the concentration of detectable target antigen in the same pathogen before inactivation; optionally within about 39%, optionally within about 38%, optionally within about 37%, optionally within about 36%, optionally within about 35%, optionally within about 34%, optionally within about 33%, optionally within about 32%, optionally within about 31%, optionally within about 30%, optionally within about 29%, optionally within about 28%, optionally within about 27%, or optionally within about 2 The inactivated pathogen may comprise an inactivated pathogen described herein having a concentration of detectable target antigen that is within 6%, optionally within about 25%, optionally within about 24%, optionally within about 23%, optionally within about 22%, optionally within about 21%, optionally within about 20% different from the first concentration, optionally within about 19%, optionally within about 18%, optionally within about 17%, optionally within about 16%, optionally within about 15%, optionally within about 14%, optionally within about 13%, optionally within about 12%, optionally within about 11%, optionally within about 10% different from the first concentration, optionally within about 9%, optionally within about 8%, optionally within about 7%, optionally within about 6%, optionally within about 5%, optionally within about 4%, optionally within about 3%, optionally within about 2%, optionally within about 1%. In some non-limiting examples, the pathogen includes SARS-CoV-2 and the detectable target antigen includes the N protein and / or the S protein.

[0048] According to some embodiments, the positive control of the present disclosure may be provided with at least two quantification values, which may be the TCID of the pathogen in the culture medium before inactivation. 50 The positive control may be selected from a value, a molecular concentration of the pathogen in the culture medium, a first concentration of the detectable target antigen, and a second concentration of the detectable target antigen. For example, the positive control may be provided in or with a container on which or with at least two quantification values ​​are provided. The present disclosure also relates to kits containing a positive control and instructions having at least two quantification values ​​described herein.

[0049] In some embodiments, the positive control may be storable at non-freezing temperatures, for example, 2-8° C. In some embodiments, the positive control may be storable at room temperature, for example, 20-22° C. In some embodiments, the positive control may be storable at freezing temperatures, for example, below −65° C.

[0050] The present disclosure also relates to methods of using the positive controls described herein in antigen biomarker assays.

[0051] While the embodiments described herein have been described in conjunction with the illustrative embodiments outlined above, various alternatives, modifications, variations, improvements, and / or substantial equivalents, whether known, currently foreseen, or currently foreseeable, may become apparent to those of ordinary skill in the art. Accordingly, the illustrative embodiments as set forth above are intended to be illustrative and not limiting. Various changes may be made without departing from the spirit and scope of the present disclosure. Accordingly, the present disclosure is intended to embrace all known or later-developed alternatives, modifications, variations, improvements, and / or substantial equivalents.

[0052] Accordingly, the claims are not intended to be limited to the embodiments set forth herein, but are to be accorded the full scope consistent with the language of the claims, wherein reference to an element in the singular is not intended to mean "one and only one," unless specifically so stated, but rather "one or more." All structural and functional equivalents to the elements of the various embodiments described throughout this disclosure that are known or later become known to those of ordinary skill in the art are expressly incorporated herein by reference and are intended to be encompassed by the claims. Moreover, none of the subject matter disclosed herein is intended to be made available to the public, regardless of whether such disclosure is expressly recited in the claims. A claim element should not be construed as a means-plus-function unless the element is expressly recited using the phrase "means for."

[0053] The recitation herein of numerical ranges by endpoints (e.g., between about 50:1 and 1:1, between about 100°C and 500°C, between about 1 minute and 60 minutes) includes all numbers subsumed within that range. For example, between about 1 minute and 60 minutes includes 21 minutes, 22 minutes, 23 minutes, and 24 minutes as endpoints within the specified range. Thus, for example, ranges 22-36, 25-32, 23-29, etc., are also ranges with endpoints subsumed within the range of 1-60, depending on the starting materials used, the temperature, the specific application, the specific embodiment, or, where appropriate, the limitations of the claims. Because varying one or more reaction parameters may result in different synthetic products, the examples and methods disclosed herein demonstrate that the recited ranges encompass all points within that range. Furthermore, the examples and methods disclosed herein describe various aspects of the disclosed ranges and effects when those ranges are varied individually or in combination with other recited ranges.

[0054] Furthermore, the word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any aspect described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other aspects. Unless otherwise specifically stated, the term "some" refers to one or more. Combinations, e.g., "at least one of A, B, or C," "at least one of A, B, and C," and "A, B, C, or any combination thereof," include any combination of A, B, and / or C, and may include multiple As, multiple Bs, or multiple Cs. Specifically, combinations, e.g., "at least one of A, B, or C," "at least one of A, B, and C," and "A, B, C, or any combination thereof," may be A only, B only, C only, A and B, A and C, B and C, or A and B and C, where any such combination may contain one or more members of A, B, or C. None of the material disclosed herein is intended to be made available to the public, regardless of whether such disclosure is expressly recited in the claims.

[0055] As used herein, the terms "about" and "approximately" are defined as close as would be understood by one of ordinary skill in the art. In one non-limiting embodiment, the terms "about" and "approximately" are defined as within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%.

[0056] The examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, dimensions, etc.), but some experimental error and deviation should be accounted for. [Example]

[0057] Example Example I Preparation of a positive control using SARS-CoV-2 First, frozen cell culture stocks were thawed to provide a medium containing SARS-CoV-2, and the N protein concentration of the medium was determined via ELISA. The frozen cell culture contained the target viral pathogen (i.e., SARS-CoV-2), the host cells used to propagate the viral pathogen, and the culture medium used for growing the viral pathogen. To thaw the frozen cell culture, the tubes containing the frozen cell culture were removed from the freezer (in this example, an ultra-low temperature freezer at or below -65°C) and thawed in a water bath for 30 minutes or less. The medium was then clarified to remove cell debris by centrifugation at 1500 rpm or 500 × g for 10 minutes. The supernatant was collected and pooled. Aliquots of the medium were then placed into sterile Petri dishes. The Petri dishes were each 245 mm x 26 mm and contained 25–45 mL of clarified medium, although other Petri dish sizes and sample volumes are acceptable. Table 1 below shows the maximum volume capacity of various Petri dishes by size.

[0058] [Table 1]

[0059] Each aliquot was then exposed to UV irradiation using a 45 Model G30T8 Ultraviolet UV-C Lamp at a wavelength of 254 nm for approximately 5 minutes. Each aliquot was vortexed and then re-exposed to UV irradiation using a 45 Model G30T8 Ultraviolet UV-C Lamp at a wavelength of 254 nm for approximately 5 minutes to provide a positive control aliquot. The aliquots were then pooled, and pathogen inactivation was determined using an infectivity assay. The N protein concentration of the positive control was determined via ELISA.

[0060] Example II Preparation of a positive control using influenza A H1N1pdm virus Three independent lots of culture medium containing influenza A H1N1pdm virus (strain Gaungdong-Maonan-SWL 1536 / 19) were divided into aliquots. Each aliquot was then subjected to the inactivation conditions shown in Table 2. One aliquot was not subjected to the inactivation conditions to serve as a control.

[0061] [Table 2]

[0062] Next, TCID 50 Measurements were performed on each sample. The results of these measurements are shown in Figure 2. UV exposure for 10 or 15 minutes increased the recoverable TCID 50 It was determined that this was sufficient to inactivate the pathogen, as evidenced by the complete absence of units.

[0063] Example III Preparation of a positive control using SARS-COV2 virus Three independent lots of culture medium containing SARS-COV2 virus (USA-WA 1 / 2020 strain) were divided into aliquots. Each aliquot was then subjected to the inactivation conditions shown in Table 3. One aliquot was not subjected to the inactivation conditions to serve as a control.

[0064] [Table 3]

[0065] Next, TCID 50 Measurements were performed on each sample. The results of these measurements are shown in Figure 3. UV exposure for 10 or 15 minutes increased the recoverable TCID 50 It was determined that this was sufficient to inactivate the pathogen, as evidenced by the complete absence of units.

[0066] SARS-CoV-2 qPCR was then performed to determine the effect of inactivation conditions on nucleic acid integrity. Figure 4 shows the results of this analysis. As shown, qPCR signals across all samples were consistent. Therefore, it was concluded that UV inactivation did not have a detrimental effect on the viral nucleic acid targeted in this study.

[0067] Finally, the N protein concentration of each sample after inactivation was determined via ELISA. To determine the N protein concentration, a standard curve was first prepared using the 200 ng / mL SARS-CoV-2 NP standard shown in Table 4.

[0068] [Table 4]

[0069] Each sample was then diluted in triplicate with assay diluent. The SARS-CoV-2 anti-NP microplate was then pre-washed three times with 350 μL of plate wash buffer, with aspirating between washes. The microplate was then tapped onto an absorbent towel until no droplets remained in the wells, but the wells were not allowed to dry completely. 200 μL of either the SARS-CoV-2 NP standard or sample was then added to each well, and the microplate was incubated at 37°C for 1.5 hours.

[0070] The wells were then aspirated and washed six times with 350 μL of plate wash buffer, with aspirating between washes. The microplate was then tapped onto an absorbent towel until no droplets remained in the wells, but the wells were not allowed to dry completely.

[0071] 100 μL of SARS-CoV-2 NP detection antibody was then added to each well, and the microplate was incubated for 1 hour at 37°C. The wells were then aspirated and washed six times with 350 μL of plate wash buffer, with aspirating between washes. The microplate was then tapped onto an absorbent towel until no droplets remained in the wells, but the wells were not allowed to dry completely.

[0072] Then, 100 μL of high-sensitivity TMB substrate was added to each well, and the microplate was incubated at room temperature (20-25°C) for 30 minutes. Then, 100 μL of stop solution was added to each well, and the microplate was read at 450 nm on a plate reader.

[0073] The results of this analysis are shown in Figure 5. As shown in Figure 5, the N protein concentration after heat inactivation is significantly lower than the N protein concentration of the control (i.e., the sample that was not inactivated). However, Figure 5 shows that UV inactivation provided minimal changes in N protein concentration compared to the control. [Explanation of symbols]

[0074] 101-well plate 102 Antibodies 103 Detectable Target Antigens 104 Signal-Generating Antibodies 105 Sandwich Complex

Claims

1. 1. A method for producing an antigen-based positive control for detecting a pathogen, comprising: providing a culture medium containing a pathogen having a first detectable antigen, the culture medium having a first concentration of the first detectable antigen; and exposing the culture medium to electromagnetic radiation for a period of time sufficient to inactivate the pathogen, thereby producing an antigen-based positive control. Including, The method, wherein the antigen-based positive control comprises the first detectable antigen at a second concentration that differs from the first concentration by no more than about 50% as determined via ELISA.

2. The method of claim 1 , wherein the pathogen comprises a virus.

3. 3. The method of claim 2, wherein the virus comprises SARS-CoV-2, RSV, Flu A, Flu B, or a combination thereof.

4. The method of claim 3, wherein the detectable target antigen comprises a nucleocapsid protein.

5. The method of claim 1 , wherein the electromagnetic radiation comprises UV radiation provided by UV-C light.

6. The method of claim 1, further comprising combining the culture medium with a protease inhibitor.

7. 7. The method of claim 6, wherein the antigen-based positive control comprises the protease inhibitor.

8. 10. The method of claim 1, further comprising combining the antigen-based positive control with a protease inhibitor.

9. 10. The method of claim 1, wherein the period is between about 1 minute and 15 minutes.

10. an antigen-based positive control, wherein the antigen-based positive control comprises an inactivated pathogen; and At least two quantification values Kit including:

11. The kit of claim 10 , wherein the pathogen comprises a virus.

12. 12. The kit of claim 11, wherein the virus comprises SARS-CoV-2, RSV, Flu A, Flu B, or a combination thereof.

13. The at least two quantification values ​​are the TCID of the pathogen before inactivation. 50 11. The kit of claim 10, comprising a detectable concentration of the target antigen.

14. 11. The kit of claim 10, wherein the virus comprises SARS-CoV-2 and the detectable target antigen is a nucleocapsid protein.

15. 12. The kit of claim 11, further comprising a protease inhibitor.

16. 12. The kit of claim 11, wherein the inactivated pathogen is provided in a liquid.

17. 1. An antigen-based positive control for detecting a pathogen, said antigen-based positive control comprising: providing a culture medium containing a pathogen, the culture medium having a first concentration of a detectable target antigen; and exposing said culture medium to electromagnetic radiation for a period of time sufficient to inactivate said pathogen, thereby producing said antigen-based positive control. and providing a method comprising: the antigen-based positive control comprises the detectable target antigen at a second concentration that differs from the first concentration by no more than about 50%; An antigen-based positive control, wherein the first concentration and the second concentration are determinable via ELISA.

18. 18. The antigen-based positive control of claim 17, wherein the pathogen comprises a virus.

19. 19. The antigen-based positive control of claim 18, wherein the virus comprises SARS-CoV-2, RSV, Flu A, Flu B, or a combination thereof.

20. 20. The antigen-based positive control of claim 19, wherein the detectable target antigen comprises a nucleocapsid protein.

21. 1. An inactivated pathogen for use as an antigen-based positive control, wherein the inactivated pathogen is provided in a kit with at least two quantification values.

Images