WO2022147178A1 - Procédés, réactifs et kits améliorés pour l'inactivation à base de détergent de bêta-coronavirus avant et/ou pendant l'évaluation d'un échantillon biologique pour un antigène ou un anticorps du sars-cov-2 - Google Patents

Procédés, réactifs et kits améliorés pour l'inactivation à base de détergent de bêta-coronavirus avant et/ou pendant l'évaluation d'un échantillon biologique pour un antigène ou un anticorps du sars-cov-2 Download PDF

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WO2022147178A1
WO2022147178A1 PCT/US2021/065581 US2021065581W WO2022147178A1 WO 2022147178 A1 WO2022147178 A1 WO 2022147178A1 US 2021065581 W US2021065581 W US 2021065581W WO 2022147178 A1 WO2022147178 A1 WO 2022147178A1
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cov
sars
sample
ionic surfactant
antibody
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PCT/US2021/065581
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Philip M. HEMKEN
Eitan Israeli
A. Scott Muerhoff
Michael G. BERG
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Abbott Laboratories
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus

Definitions

  • the present disclosure relates to methods of inactivating ⁇ -coronaviruses (e.g., SARS- CoV or SARS-CoV-2) in a biological sample prior to, while (i.e., concurrent with), or both prior to and while testing the sample for the presence of a ⁇ -coronavirus (e.g., SARS-CoV or SARS- CoV-2) antigen or antibody.
  • ⁇ -coronaviruses e.g., SARS- CoV or SARS-CoV-2
  • Viruses of the family Coronaviridae possess a single-strand, positive-sense RNA genome ranging from 26 to 32 kilobases in length (reviewed by Lu et al., The Lancet, 395:565- 574 (February 22, 2020)).
  • the Coronaviridae are further subdivided (initially based on serology but now based on phylogenetic clustering) into four groups, the alpha, beta, gamma and delta coronaviruses.
  • Coronaviruses have been identified in several avian hosts, as well as in various mammals, including camels, bats, masked palm civets, mice, dogs, and cats.
  • SARS coronavirus Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is a novel betacoronavirus ( ⁇ -coronavirus) that emerged in Guangdong, southern China, in November 2002 and resulted in more than 8000 human infections and 774 deaths in 37 countries in 2002-03.
  • Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) was first detected in Saudi Arabia in 2012 and was responsible for 2494 laboratory-confirmed cases of infection and 858 deaths from 2012-20.
  • Coronavirus virions are spherical with diameters of approximately 125 nanometers, as demonstrated by cryo-electron tomography and cryo-electron microscopy.
  • a prominent feature of coronaviruses is the club-shape spike projections emanating from the surface of the virion, giving the virion the appearance of a solar corona and resulting in the name, coronaviruses.
  • Within the envelope of the coronavirus virion is the helically symmetrical nucleocapsid, which binds to and creates a shell around the coronavirus RNA genome.
  • the spike (S) and nucleocapsid (N) proteins are the main immunogens of the coronavirus.
  • the other two main structural proteins of the coronavirus particles are the membrane (M) and envelope (E) proteins. All four proteins are encoded within the 3’ end of the viral genome.
  • the S protein ( ⁇ 150 kDa) is heavily N-linked glycosylated and utilizes an N-terminal signal sequence to gain access to the endoplasmic reticulum (ER). Homotrimers of the virus- encoding S protein make up the distinctive spike structure on the surface of the virus. In many, but not all, coronaviruses, the S protein is cleaved by a host cell furin-like protease into two separate polypeptides known as S1 and S2. S1 makes up the large receptor-binding domain of the S protein while S2 forms the stalk of the spike molecule. The trimeric S glycoprotein mediates attachment of the coronavirus virion to the host cell by interactions between the S protein and its receptor.
  • angiotensin-converting enzyme 2 (ACE2) is the receptor for SARS-CoV.
  • RBD receptor binding domains
  • S1 S1 region of a coronavirus S protein
  • Some viruses have the RBD at the N-terminus of S1 (e.g., murine hepatitis virus) and others have the RBD at the C-terminus of S1 (e.g., SARS-CoV).
  • S-protein/receptor interaction is the primary determinant for the coronavirus to infect a host species and also governs the tissue tropism of the virus.
  • the M protein is the most abundant structural protein in the virion. It is a small ( ⁇ 25-
  • E protein ( ⁇ 8-12 kDa) protein with 3 transmembrane domains and is believed to give the virion its shape. It has a small N-terminal glycosylated ectodomain and a much larger C-terminal endodomain that extends 6-8 nm into the viral particle.
  • the E protein ( ⁇ 8-12 kDa) is found in small quantities within the virion. E proteins in coronaviruses are highly divergent but have a common architecture. Data suggests that the E protein is a transmembrane protein with an N-terminal ectodomain and a C-terminal endodomain that has ion channel activity. Recombinant viruses lacking the E protein are not always lethal - although this is virus-type dependent. The E protein facilitates assembly and release of the virus, but also has other functions (e.g., ion channel activity in SARS-CoV E protein is not required for viral replication but is required for pathogenesis).
  • the N protein is the only protein present in the nucleocapsid. It is composed of two separate domains, an N-terminal domain (NTD) and a C-terminal domain (CTD), both capable of binding RNA in vitro using different mechanisms, which may suggest that optimal RNA binding requires contributions from both domains.
  • NTD N-terminal domain
  • CCD C-terminal domain
  • the N protein is heavily phosphorylated, and phosphorylation has been suggested to trigger a structural change enhancing the affinity for viral versus non-viral RNA.
  • the N protein binds the viral genome in a beads-on-a-string type conformation. Two specific RNA substrates have been identified for N protein; the transcriptional regulatory sequences and the genomic packaging signal.
  • the genomic packaging signal has been found to bind specifically to the second, or C-terminal RNA binding domain.
  • the N protein also binds nsp3, a key component of the replicase complex, and the M protein. These protein interactions likely help tether the viral genome to the replicase-transcriptase complex, and subsequently package the encapsidated genome into viral particles.
  • Lu et al. reported obtaining complete and partial SARS-CoV-2 genome sequences using next-generation sequencing of bronchoalveolar lavage fluid samples and cultured isolates from nine patients from Wuhan diagnosed with viral pneumonia but negative for common respiratory pathogens. Lu et al., The Lancet, 395: 565-574 (February 22, 2020). Based on their analysis, Lu et al.
  • SARS-CoV-2 was closely related (with 88% identity) to two bat-derived severe acute respiratory syndrome (SARS)-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21, collected in eastern China in 2018, but was more distant from SARS-CoV (about 79%) and MERS-CoV (about 50%). Additionally, Zhou et al. confirmed that SARS-CoV-2 uses the same cellular entry receptor, ACE2, as SARS- CoV. Zhou et al., Nature, 579:270-273 (March 2020).
  • SARS-CoV-2 primarily spreads through the respiratory tract, by droplets, respiratory secretions, and direct contact. Additionally, SARS-CoV-2 has been found in fecal swabs and blood, indicating the possibility of multiple routes of transmission. Zhang et al., Microbes 9(1):386-9 (2020). SARS-CoV-2 is highly transmissible in humans, especially in the elderly and people with underlying diseases. Symptoms can appear 2 to 14 days after exposure. Patients present with symptoms such as fever, malaise, cough, and/or shortness of breath. Most adults or children with SARS-CoV-2 infection present with mild flu-like symptoms, however, critical patients rapidly develop acute respiratory distress syndrome, respiratory failure, multiple organ failure and even death.
  • the present disclosure relates to methods of inactivating any SARS- CoV-2 in a biological sample prior to or while (i.e., concurrent with), or both prior to and while, testing the sample for the presence of a SARS-CoV-2 antigen or antibody.
  • the method involves the steps of maintaining the sample in a medium comprising at least about 0.1% (v/v) of at least one non-ionic surfactant under conditions appropriate for and for an amount of time sufficient for the at least one non-ionic surfactant to inactivate the SARS-CoV-2 in the sample as determined by an inability of the SARS-CoV-2 to replicate in culture, without substantially interfering with testing the sample for the presence of a SARS-CoV-2 antigen or antibody.
  • the at least one non-ionic surfactant is a secondary alcohol ethoxylate, such as tergitol.
  • the at least one non-ionic surfactant is tergitol and polyethylene glycol sorbitan monolaurate.
  • the medium comprises from about 0.1% to about
  • the medium comprises from about 0.1% to about 0.75% (v/v) of the at least one non-ionic surfactant. In further aspects, the medium comprises from about 0.1% to about 0.5% (v/v) of the at least one non-ionic surfactant. In further aspects, the medium comprises about 0.2% (v/v) of at least one non-ionic surfactant Specifically, in some aspects, the medium comprises about 0.2% (v/v) of tergitol. In other aspects, the medium comprises about 0.1 % (v/v) of tergitol and about 0.1% (v/v) of polyethylene glycol sorbitan monolaurate.
  • the at least one non-ionic surfactant is: (a) present in or added to a viral transport medium (VTM) or a universal transport medium (UTM); (b) present in or added to a pretreatment solution; (c) present in or added to an assay-specific reagent; or (d) any combination of (a) to (c).
  • VTM viral transport medium
  • UDM universal transport medium
  • the sample comprises saliva, an oropharyngeal specimen, a nasopharyngeal specimen, an anal swab specimen, or a nasal mucus specimen.
  • CoV-2 antigen or antibody is performed using an immunoassay or a clinical chemistry assay.
  • testing the sample for the presence of a SARS- CoV-2 antigen or antibody is performed using single molecule detection, a lateral flow assay, or point-of-care assay.
  • CoV-2 antigen or antibody is adapted for use in an automated system or a semi-automated system.
  • the present disclosure also relates to an improvement of a method of testing a biological sample for the presence of aSARS-CoV-2 antigen or antibody.
  • the improvement comprises maintaining the sample in a medium comprising at least about 0.1% v/v of at least one non-ionic surfactant prior to or while (i.e., concurrent with), or both prior to and while, testing the sample for the presence of a SARS-CoV-2 antigen or antibody, wherein the sample is maintained under conditions and for an amount of time sufficient for the at least one non-ionic surfactant to inactivate any SARS-CoV-2 present in the sample as determined by an inability of the SARS-CoV-2 to replicate in culture, without substantially interfering with testing the sample for the presence of a SARS-CoV-2 antigen or antibody.
  • the at least one non-ionic surfactant is a secondary alcohol ethoxylate, such as tergitol.
  • the at least one non-ionic surfactant is tergitol and polyethylene glycol sorbitan monolaurate.
  • the medium comprises from about 0.1% to about 1.0% (v/v) of the at least one non-ionic surfactant. In some aspects, the medium comprises from about 0.1% to about 0.75% (v/v) of the at least one non-ionic surfactant. In further aspects, the medium comprises from about 0.1% to about 0.5% (v/v) of the at least one non-ionic surfactant In further aspects, the medium comprises about 0.2% (v/v) of at least one non-ionic surfactant. Specifically, in some aspects, the medium comprises about 0.2% (v/v) of tergitol. In other aspects, the medium comprises about 0.1% (v/v) of tergitol and about 0.1% (v/v) of polyethylene glycol sorbitan monolaurate.
  • the at least one non-ionic surfactant is: (a) present in or added to a viral transport medium (VTM) or a universal transport medium (UTM); (b) present in or added to a pretreatment solution; (c) present in or added to an assay- specific reagent; or (d) any combination of (a) to (c).
  • VTM viral transport medium
  • UDM universal transport medium
  • the sample saliva, an oropharyngeal specimen, a nasopharyngeal specimen, an anal swab specimen, or a nasal mucus specimen is sample saliva, an oropharyngeal specimen, a nasopharyngeal specimen, an anal swab specimen, or a nasal mucus specimen.
  • testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using an immunoassay or a clinical chemistry assay.
  • testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using single molecule detection, a lateral flow assay, or a point-of-care assay.
  • testing the sample for the presence of a SARS-CoV-2 antigen or antibody is adapted for use in an automated system or a semi-automated system.
  • FIG. 1 is graph showing inactivation of SARS-CoV-2 (the causative virus of COVID- 19) in transport culture media to which has been added 0.5%, 0.1%, or 0.01% of the non-ionic surfactant tergitol.
  • the present disclosure is predicated, at least in part, on the discovery that incubating a biological sample with a non-ionic surfactant (e.g., tergitol) inactivates a ⁇ -coronavirus such as SARS-CoV or SARS-CoV-2, if present in the sample.
  • a non-ionic surfactant e.g., tergitol
  • inactivation is achieved by lysis of the virus particles.
  • a ⁇ -coronavirus such as SARS-CoV or SARS-CoV-2
  • any diagnostic testing on a patient sample e.g., an antigen assay, a molecular assay, or an antibody assay.
  • heat inactivation is used, but heat treatment could decrease assay signal.
  • the present disclosure demonstrates that, surprisingly, incubating the biological sample with a non-ionic surfactant such as tergitol can be employed instead of heat inactivation.
  • the disclosure provides a method of inactivating any ⁇ - coronavirus, such as SARS-CoV or SARS-CoV-2, in a biological sample prior to or while (i.e., concurrent with), or both prior to and while, testing the sample for the presence of a ⁇ - coronavirus, such as SARS-CoV or SARS-CoV-2, antigen or antibody, the method comprising maintaining biological sample in a medium containing at least about 0.1% (v/v) of at least one non-ionic surfactant, wherein the at least one non-ionic surfactant inactivates the ⁇ -coronavirus, such as SARS-CoV or SARS-CoV-2, in the biological sample as determined by an inability of the ⁇ -coronavirus, such as SARS-CoV or SARS-CoV-2, to replicate in culture, without substantially interfering with testing the sample for the presence of a ⁇ -coronavirus, such as SARS-CoV or SARS-
  • the biological sample used in the methods of the present disclosure may be obtained from an asymptomatic subject or from a subject exhibiting one or more symptoms of infection with a ⁇ -coronavirus (e.g., SARS-CoV or SARS- CoV-2).
  • a ⁇ -coronavirus e.g., SARS-CoV or SARS- CoV-2.
  • the invention is not limited to any particular mechanism of action and an understanding of the mechanism is not necessary to practice the invention.
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
  • Antibody and “antibodies” as used herein refers to monoclonal antibodies, monospecific antibodies (e.g., which can either be monoclonal, or may also be produced by other means than producing them from a common germ cell), multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, a bird (for example, a duck or a goose), a shark, a whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee, etc.), recombinant antibodies, chimeric antibodies, single- chain Fvs (“scFv”),
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an analyte-binding site.
  • Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.
  • an antibody against an analyte is frequently referred to herein as being either an “anti-analyte antibody” or merely an “analyte antibody”.
  • Antibody fragment refers to a portion of an intact antibody comprising the antigen-binding site or variable region. The portion does not include the constant heavy chain domains (i.e., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody.
  • antibody fragments include, but are not limited to, Fab fragments, Fab’ fragments, Fab’-SH fragments, F(ab’) 2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing the three CDRs of the light-chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.
  • Anti-species antibodies refers to an antibody, such as an IgG and/or IgM antibody, that recognize antibodies of another species of interest.
  • anti-human antibodies e.g., anti-human IgG or IgM antibodies
  • Binding protein is used herein to refer to a monomeric or multimeric protein that binds to and forms a complex with a binding partner, such as, for example, a polypeptide, an antigen, a chemical compound or other molecule, or a substrate of any kind.
  • a binding protein specifically binds a binding partner.
  • Binding proteins include antibodies, as well as antigen- binding fragments thereof and other various forms and derivatives thereof as are known in the art and described herein below, and other molecules comprising one or more antigen-binding domains that bind to an antigen molecule or a particular site (epitope) on the antigen molecule.
  • a binding protein includes, but is not limited to, an antibody a tetrameric immunoglobulin, an IgG molecule, an IgG1 molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, an affinity matured antibody, and fragments of any such antibodies that retain the ability to bind to an antigen.
  • coronavirus refers to viruses that belonging to the family Coronaviridae that have a positive-sense, RNA genome ranging from 26 to 32 kilobases in length. Coronaviruses having four main structural proteins: the spike glycoprotein (S protein), the membrane protein (M protein), the envelope protein (E protein) and the nucleocapsid protein (N protein). Coronavirus can be further subdivided into four groups, alpha, beta, gamma and delta coronaviruses. Examples of alpha coronaviruses include HCoV-229E and HCoV-NL63.
  • beta coronaviruses examples include HCoV-OC43, HCoV-HKU1, Middle East Respiratory Syndrome (MERS-CoV), severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) and SARS-CoV-2 (also known as 2019-nCov, COVLD-19, coronavirus disease, and Coronavirus Disease 2019).
  • MERS-CoV Middle East Respiratory Syndrome
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • SARS-CoV-2 also known as 2019-nCov, COVLD-19, coronavirus disease, and Coronavirus Disease 2019.
  • the present disclosure relates to ⁇ -coronaviruses.
  • the ⁇ -coronaviruses are MERS-CoV, SARS-CoV and SARS-CoV-2.
  • the ⁇ -coronaviruses are SARS-CoV and SARC-CoV-2.
  • the ⁇ -coronavirus is SARS-CoV-2.
  • the sequence of SARS-CoV-2 has been described in a variety of publications, such as, for example, Lu et al., Lancet, 395:565-574 (February 2020); NCBI Reference
  • Component refer generally to elements such as, for example, a calibrator, a control, a sensitivity panel, a container, a buffer, a diluent (including an assay specific diluent), a salt, an enzyme, a co-factor for an enzyme, a detection reagent, a pretreatment reagent/solution, a substrate (e.g., as a solution), a stop solution, and the like that can be included in a kit for assay of a test sample, such as a patient saliva, oropharyngeal specimen, nasopharyngeal specimen, nasal mucus specimen, urine, whole blood, serum or plasma sample (e.g., as per “Sample” below), in accordance with the methods described herein and other methods known in the art.
  • Some components can be in solution or lyophilized for reconstitution for use in an assay
  • Controls as used herein generally refers to a reagent whose purpose is to evaluate the performance of a measurement system in order to assure that it continues to produce results within permissible boundaries (e.g., boundaries ranging from measures appropriate for a research use assay on one end to analytic boundaries established by quality specifications for a commercial assay on the other end).
  • permissible boundaries e.g., boundaries ranging from measures appropriate for a research use assay on one end to analytic boundaries established by quality specifications for a commercial assay on the other end.
  • a control should be indicative of patient results and optionally should somehow assess the impact of error on the measurement (e.g., error due to reagent stability, calibrator variability, instrument variability, and the like).
  • control subject relates to a subject or subjects that has not been infected with a coronavirus, such as a ⁇ -coronavirus, or been exposed to any subject that has had a coronavirus, such as a ⁇ -coronavirus.
  • Determined by an assay is used herein to refer to the determination of a reference level by any appropriate assay.
  • the determination of a reference level may, in some embodiments, be achieved by an assay of the same type as the assay that is to be applied to the sample from the subject (for example, by an immunoassay, clinical chemistry assay, a single molecule detection assay, protein immunoprecipitation, immunoelectrophoresis, a point-of-care assay, chemical analysis, SDS-PAGE and Western blot analysis, or protein immunostaining, electrophoresis analysis, a protein assay, a competitive binding assay, or a functional protein assay.
  • a reference level may, in some embodiments, be achieved by an assay of the same type and under the same assay conditions as the assay that is to be applied to the sample from the subject
  • this disclosure provides exemplary reference levels (e.g., calculated by comparing reference levels at different time points). It is well within the ordinary skill of one in the art to adapt the disclosure herein for other assays to obtain assay- specific reference levels for those other assays based on the description provided by this disclosure.
  • a set of training samples comprising samples obtained from subjects known to have been infected by a coronavirus, such as a ⁇ -coronavirus (such as SARS-CoV or SARS-CoV-2), and samples obtained from human subjects known not to have been infected with a coronavirus, such as a ⁇ -coronavirus (such as SARS-CoV or SARS-CoV-2), or been exposed to a subject that has been infected with a coronavirus, such as a ⁇ -coronavirus (such as SARS- CoV or SARS-CoV-2), may be used to obtain assay-specific reference levels.
  • a coronavirus such as a ⁇ -coronavirus (such as SARS-CoV or SARS-CoV-2)
  • samples obtained from human subjects known not to have been infected with a coronavirus such as a ⁇ -coronavirus (such as SARS-CoV or SARS-CoV-2)
  • a reference level “determined by an assay” and having a recited level of “sensitivity” and/or “specificity” is used herein to refer to a reference level which has been determined to provide a method of the recited sensitivity and/or specificity when said reference level is adopted in the methods of the disclosure. It is well within the ordinary skill of one in the art to determine the sensitivity and specificity associated with a given reference level in the methods of the disclosure, for example by repeated statistical analysis of assay data using a plurality of different possible reference levels.
  • lowering a cutoff will improve sensitivity but will worsen specificity (proportion of those without disease who test negative).
  • a coronavirus such as a ⁇ -coronavirus (such as SARS-CoV or SARS-CoV-2), will be readily apparent to those skilled in the art.
  • a coronavirus such as a ⁇ -coronavirus (such as SARS-CoV or SARS-CoV-2)
  • a coronavirus such as a ⁇ -coronavirus (such as SARS-CoV or SARS-CoV-2)
  • ⁇ -coronavirus such as SARS-CoV or SARS-CoV-2
  • SARS-CoV or SARS-CoV-2 the higher the cutoff, specificity improves as more true negatives (i.e., subjects not having been infected by a coronavirus, such as ⁇ -coronavirus (such as SARS-CoV or SARS-CoV-2)) are distinguished from those having been infected by a coronavirus, such as a ⁇ -coronavirus (such as SARS-CoV or SARS-CoV-2).
  • sensitivity improves as more true positives (i.e., subjects having been infected with a coronavirus, such as a ⁇ -coronavirus (such as SARS-CoV or SARS-CoV-2)) are distinguished from those who have not been infected (e.g., do not have) with a coronavirus, such as a ⁇ -coronavirus (such as SARS-CoV or SARS-CoV-2).
  • lowering the cutoff increases the number of cases identified as positive overall, as well as the number of false positives, so the specificity must decrease.
  • a high sensitivity value helps one of ordinary skill rule out disease or condition (such as infection with a coronavirus, such as a ⁇ -coronavirus (such as SARS-CoV or SARS-CoV-2)), and a high specificity value helps one of skill rule in disease or condition.
  • a coronavirus such as a ⁇ -coronavirus (such as SARS-CoV or SARS-CoV-2)
  • a high specificity value helps one of skill rule in disease or condition.
  • Whether one of ordinary skill desires to rule out or rule in disease depends on what the consequences are for the patient for each type of error. Accordingly, one cannot know or predict the precise balancing employed to derive a test cutoff without full disclosure of the underlying information on how the value was selected. The balancing of sensitivity against specificity and other factors will differ on a case-by-case basis.
  • Epitope refers to a site(s) on any molecule that is recognized and can bind to a complementary site(s) on its specific binding partner.
  • the molecule and specific binding partner are part of a specific binding pair.
  • an epitope can be on a polypeptide, a protein, a hapten, a carbohydrate antigen (such as, but not limited to, glycolipids, glycoproteins or lipopolysaccharides), or a polysaccharide.
  • Its specific binding partner can be, but is not limited to, an antibody.
  • “Identical” or “identity,” as used herein in the context of two or more polypeptide or polynucleotide sequences, can mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of the single sequence are included in the denominator but not the numerator of the calculation.
  • “Monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • Monoclonal antibodies may include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological properties.
  • “Nucleocapsid protein” or “N” protein may be used interchangeably herein to refer to one of four main structural proteins of a coronavirus. The N protein is the only protein present in the nucleocapsid. It is composed of two separate domains, an N -terminal domain (NTD) and a C- terminal domain (CTD), both capable of binding RN A in vitro using different mechanisms, which may suggest that optimal RNA binding requires contributions from both domains.
  • Point-of-care device refers to a device used to provide medical diagnostic testing at or near the point-of-care (namely, outside of a laboratory), at the time and place of patient care (such as in a hospital, physician’s office, urgent or other medical care facility, a patient’s home, a nursing home and/or a long-term care and/or hospice facility).
  • point-of-care devices include those produced by Abbott Laboratories (Abbott Park, IL) (e.g., i-STAT and i- STAT Alinity, Universal Biosensors (Rowville, Australia); see US 2006/0134713), Axis-Shield PoC AS (Oslo, Norway) and Clinical Lab Products (Los Angeles, USA).
  • “Quality control reagents” in the context of immunoassays and kits described herein, include, but are not limited to, calibrators, controls, and sensitivity panels.
  • a “calibrator” or “standard” typically is used (e.g., one or more, such as a plurality) in order to establish calibration (standard) curves for interpolation of the concentration of an analyte, such as an antibody or an analyte.
  • a single calibrator which is near a reference level or control level (e.g., “low”, “medium”, or “high” levels), can be used.
  • Multiple calibrators i.e., more than one calibrator or a varying amount of calibrators) can be used in conjunction to comprise a “sensitivity panel.”
  • Reference level refers to an assay cutoff value (or level) that is used to assess diagnostic, prognostic, or therapeutic efficacy and that has been linked or is associated herein with various clinical parameters (e.g., presence of disease, stage of disease, severity of disease, progression, non-progression, or improvement of disease, etc.).
  • cutoff refers to a limit (e.g., such as a number) above which there is a certain or specific clinical outcome and below which there is a different certain or specific clinical outcome.
  • sample may be a sample of blood, such as whole blood (including for example, capillary blood, venous blood, dried blood spot, etc.), tissue, urine, serum, plasma, nasal mucus, amniotic fluid, lower respiratory specimens such as, but not limited to, sputum, saliva, endotracheal aspirate or bronchoalveolar lavage, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes.
  • whole blood including for example, capillary blood, venous blood, dried blood spot, etc.
  • tissue including for example, capillary blood, venous blood, dried blood spot, etc.
  • tissue including for example, capillary blood, venous blood, dried blood spot, etc.
  • tissue including for example, capillary blood, venous blood, dried blood spot, etc.
  • tissue including for example, capillary blood, venous blood, dried blood spot, etc.
  • urine including for example, capillary blood, venous blood
  • the sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.
  • the sample can be a nasopharyngeal or oropharyngeal sample obtained using one or more swabs that, once obtained, is placed in a sterile tube containing a virus transport media (VTM) or universal transport media (UTM), for testing.
  • VTM virus transport media
  • UTM universal transport media
  • the sample can be obtained using a dry swab, in which case a VTM or UTM is not required.
  • the sample may comprise saliva or a nasal mucus specimen.
  • a variety of cell types, tissue, or bodily fluid may be utilized to obtain a sample.
  • Such cell types, tissues, and fluid may include sections of tissues such as biopsy and autopsy samples, oropharyngeal specimens, nasopharyngeal specimens, an anal swab specimen, frozen sections taken for histologic purposes, blood (such as whole blood, dried blood spots, etc.), plasma, serum, red blood cells, platelets, interstitial fluid, cerebral spinal fluid, etc.
  • Cell types and tissues may also include lymph fluid, cerebrospinal fluid, or any fluid collected by aspiration.
  • a tissue or cell type may be provided by removing a sample of cells from a human and a non-human animal, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose). Archival tissues, such as those having treatment or outcome history, may also be used. Protein or nucleotide isolation and/or purification may not be necessary.
  • the sample is a whole blood sample.
  • the sample is a capillary blood sample.
  • the sample is a dried blood spot.
  • the sample is a serum sample.
  • the sample is a plasma sample.
  • the sample is an orophaiyngeal specimen.
  • the sample is a nasopharyngeal specimen. In other embodiments, the sample is sputum. In other embodiments, the sample is endotracheal aspirate. In still yet other embodiments, the sample is bronchoalveolar lavage. In still yet other embodiments, the sample is an anal swab specimen.
  • a biological sample may be diluted or undiluted either prior to or during the performance of the method. In some aspects, the dilution occurs prior to performing the method. In other aspects, the dilution occurs during the performance of the method.
  • the sample can be from about 1 to about 25 microliters, about 1 to about 24 microliters, about 1 to about 23 microliters, about 1 to about 22 microliters, about 1 to about 21 microliters, about 1 to about 20 microliters, about 1 to about 18 microliters, about 1 to about 17 microliters, about 1 to about 16 microliters, about 15 microliters or about 1 microliter, about 2 microliters, about 3 microliters, about 4 microliters, about 5 microliters, about 6 microliters, about 7 microliters, about 8 microliters, about 9 microliters, about 10 microliters, about 11 microliters, about 12 microliters, about 13 microliters, about 14 microliters, about 15 microliters, about 16 microliters, about 17 microliters, about 18 microliters, about 19 microliters, about 20 microliters, about 21 microliters, about 22 microliters, about 23 microliters, about 24 microliters or about 25 microliters.
  • the sample is from about 1 to about 150 microliters or less or from about 1 to about 25 microliters or less.
  • the biological sample can be diluted about 1-fold, about 2-fold, about 3-fold, about 4- fold, about 5-fold, about 6-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, about 20-fold, about 21 -fold, about 22-fold, about 23-fold, about 24-fold, about 25-fold, about 26-fold, about 27-fold, about 28-fold, about 29-fold, about 30-fold, about 31 -fold, about 32-fold, about 33-fold, about 34-fold, about 35-fold, about 36-fold, about 37-fold, about 38-fold, about 39-fold, about 40-fold, about 41-fold, about 42-fold, about 43-fold, about 44-fold, about 45-fold, about 46-fold, about 47-fold, about 48
  • “Sensitivity” of an assay as used herein refers to the proportion of subjects for whom the outcome is positive that are correctly identified as positive (e.g., correctly identifying those subjects with a disease or medical condition for which they are being tested). For example, this might include correctly identifying subjects as having been infected with a coronavirus, such as a ⁇ -coronavirus, from those who have not been infected with a coronavirus, such as a ⁇ - corona virus.
  • a coronavirus such as a ⁇ -coronavirus
  • Specificity of an assay as used herein refers to the proportion of subjects for whom the outcome is negative that are correctly identified as negative (e.g., correctly identifying those subjects who do not have a disease or medical condition for which they are being tested). For example, this might include correctly identifying subjects not infected with a coronavirus, such as a ⁇ -coronavirus, from those who have not been infected with a coronavirus, such as a ⁇ - coronavirus.
  • a coronavirus such as a ⁇ -coronavirus
  • single molecule detection refers to the detection and/or measurement of a single molecule of an analyte in a test sample at very low levels of concentration (such as pg/mL or femtogram/mL levels).
  • concentration such as pg/mL or femtogram/mL levels.
  • single molecule analyzers or devices include nanopore and nanowell devices. Examples of nanopore devices are described in PCT International Application WO 2016/161402, which is hereby incorporated by reference in its entirety. Examples of nanowell device are described in PCT International Application WO 2016/161400, which is hereby incorporated by reference in its entirety.
  • “Specific binding” or “specifically binding” as used herein may refer to the interaction of an antibody, a protein, or a peptide with a second chemical species, wherein the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.
  • a particular structure e.g., an antigenic determinant or epitope
  • Specific binding partner or “Specific binding member,” may be used interchangeable herein to refer to a member of a specific binding pair.
  • a specific binding pan- comprises two different molecules, which specifically bind to each other through chemical or physical means. Therefore, in addition to antigen and antibody specific binding pairs of common immunoassays, other specific binding pairs can include biotin and avidin (or streptavidin), carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzymes and enzyme inhibitors, and the like.
  • specific binding pairs can include members that are analogs of the original specific binding members, for example, an analyte-analog.
  • Immunoreactive specific binding members include antigens, antigen fragments, and antibodies, including monoclonal and polyclonal antibodies as well as complexes and fragments thereof, whether isolated or recombinantly produced.
  • spike protein or “S” protein may be used interchangeably herein to refer to one of four main structural proteins of a coronavirus.
  • the spike protein is heavily N-linked glycosylated and utilizes an N-terminal signal sequence to gain access to the endoplasmic reticulum (ER).
  • Homotrimers of the virus-encoding S protein make up the distinctive spike structure on the surface of the virus.
  • the S protein is cleaved by a host cell furin-like protease into two separate polypeptides noted S1 and S2.
  • S1 makes up the large receptor-binding domain (RBD) of the S protein while S2 forms the stalk of the spike molecule.
  • RBD receptor-binding domain
  • SARS-CoV-2 RBD is disclosed in M. Yuan et al., Science
  • ACE2 angiotensin-converting enzyme 2
  • Statistically significant refers to the likelihood that a relationship between two or more variables is caused by something other than random chance.
  • Statistical hypothesis testing is used to determine whether the result of a data set is statistically significant In statistical hypothesis testing, a statistically significant result is attained whenever the observed p-value of a test statistic is less than the significance level defined of the study. The p-value is the probability of obtaining results at least as extreme as those observed, given that the null hypothesis is true. Examples of statistical hypothesis analysis include Wilcoxon signed-rank test, t-test, Chi-Square or Fisher’s exact test. “Significant ” as used herein refers to a change that has not been determined to be statistically significant (e.g., it may not have been subject to statistical hypothesis testing).
  • a mammal e.g., a bear, cow, cattle, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, tiger, lion, cheetah, jaguar, bobcat, mountain lion, dog, wolf, coy
  • the subject may be a human, a non-human primate or a cat In some embodiments, the subject is a human. The subject or patient may be undergoing other forms of treatment. In some embodiments, the subject is a human that may be undergoing other forms of treatment. In some embodiments, the subject is suspected to have, have had or has been exposed to a subject that has had or tested positive for infection with a coronavirus, such as a ⁇ -coronavirus (such as SARS- CoV or SARS-CoV-2).
  • a coronavirus such as a ⁇ -coronavirus (such as SARS- CoV or SARS-CoV-2).
  • the subject is completely asymptomatic and does not exhibit any symptoms of a coronavirus, such as a ⁇ -coronavirus (such as SARS-CoV or SARS-CoV-2), and may or may not have been exposed to a subject that has or has been exposed or infected with a coronavirus, such as a ⁇ -coronavirus.
  • a coronavirus such as a ⁇ -coronavirus (such as SARS-CoV or SARS-CoV-2)
  • surface active agent refers to amphipathic molecules that consist of a non-polar hydrophobic portion, usually a straight or branched hydrocarbon or fluorocarbon chain containing 8-18 carbon atoms, attached to a polar or ionic hydrophilic portion.
  • the hydrophilic portion can be nonionic, ionic, or zwitterionic.
  • the hydrocarbon chain interacts weakly with the water molecules in an aqueous environment, whereas the polar or ionic head group interacts strongly with water molecules via dipole or ion-dipole interactions.
  • surfactants are classified into anionic, cationic, zwitterionic, non-ionic, and polymeric surfactants.
  • a “system” refers to a plurality of real and/or abstract elements operating together for a common purpose.
  • a “system” is an integrated assemblage of hardware and/or software elements.
  • each element of the system interacts with one or more other elements and/or is related to one or more other elements.
  • a system refers to a combination of elements and software for controlling and directing methods.
  • the present disclosure relates to methods for inactivating any ⁇ -coronavirus (e.g., SARS-CoV or SARS-CoV-2) in a biological sample (e.g., a sample obtained from a subject who may or may not exhibit signs and/or symptoms of infection and suspected of having a ⁇ - coronavirus (e.g., SARS-CoV or SARS-CoV-2)) prior to and/or while testing the sample for the presence of a ⁇ -coronavirus (e.g., SARS-CoV or SARS-CoV-2) antigen or antibody.
  • the method comprises maintaining the biological sample in a medium containing at least one non-ionic surfactant.
  • medium refers to any solution (e.g., buffer or diluent) that is capable of inactivating a ⁇ - coronavirus (e.g., SARS-CoV or SARS-CoV-2).
  • the medium is particularly useful as an assay component and/or as a diluent of assay components (as defined above).
  • the medium desirably comprises a non-ionic surfactant that can inactivate ⁇ - coronavirus (e.g., SARS-CoV or SARS-CoV-2).
  • Non-ionic surfactants contain uncharged, hydrophilic head groups that consist of either polyoxyethylene moieties, or glycosidic groups, as in octyl glucoside and dodecyl maltoside. Since non-ionic surfactants break lipid-lipid and lipid- protein, but not protein-protein interactions, they are considered non-denaturing. Thus, they are widely used in the isolation of membrane proteins in their biologically active form. Unlike ionic detergents, salts have minimal effect on the micellar size of non-ionic detergents (le Maire et al., Biochim Biophys Acta., 1508(1-2): 86-111(2000)).
  • Nonionic surfactants include, but are not limited to, an ethoxylated surfactant, an alcohol ethoxylated, a secondary alcohol ethoxylate, an alkyl phenol ethoxylated, a fatty acid ethoxylated, a monoalkaolamide ethoxylated, a sorbitan ester ethoxylated, a fatty amino ethoxylated, an ethylene oxide-propylene oxide copolymer, Bis(polyethylene glycol bis(imidazoyl carbonyl)), nonoxynol-9, Bis(polyethylene glycol bis[imidazoyl carbonyl]), BRIJ 35, BRIJ 56, BRIJ 72, BRIJ 76, BRIJ 92V, BRIJ 97, BRIJ 58P, CREMOPHOR, EL, decaethylene glycol monododecyl ether, N-decanoyl-N-methylglucamine,
  • the non-ionic surfactant is a secondary alcohol ethoxylate, such as tergitol.
  • Tergitol surfactants like Tergitol-type NP-40 and other varieties, are nonionic and nonylphenol-ethoxylate based. They have a pH value of six (pH ⁇ 6.0) and are commercially available as detergent coupling agents.
  • Tergitol is an emulsifier and stabilizer, and several varieties having slightly different properties and uses are commercially available.
  • tergitol varieties include, but are not limited to, tergitol 15-S-12, tergitol 15-S-30, tergitol 15-S-5, tergitol type 15-S-7, tergitol 15-S-9, tergitol 15-S-40, tergitol 15-S-40 (70%), tergitol NP-10, tergitol NP-4, tergitol NP-40, tergitol NP-40 (70%), tergitol NP-7, tergitol NP-9, tergitol TMN- 10, tergitol TMN-6, and tergitol TMN-100X.
  • Tergitol surfactants can be purchased from commercial sources such as Sigma- Aldrich (St. Louis, MO) and Dow Chemical Co. (Midland, MI).
  • the non-ionic surfactant is a secondary alcohol ethoxylate, such as tergitol, and polyethylene glycol sorbitan monolaurate, polyoxyethylenesorbitan monolaurate, polyoxyethylenesorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene (4) sorbitan monostearate, polyoxyethylenesorbitan tristearate, polyoxyethylenesorbitan monooleate, polyoxyethylenesorbitan trioleate and any combinations thereof.
  • the non-ionic surfactant is a secondary alcohol ethoxylate, such as tergitol, and polyethylene glycol sorbitan monolaurate (Tween 20).
  • the medium may contain any suitable amount of non-ionic surfactant, so long as the amount of non-ionic surfactant is sufficient to inactivate any ⁇ -coronavirus (e.g., SARS-CoV or SARS-CoV-2) present in the sample without substantially interfering with testing the sample for the presence of a ⁇ -coronavirus (e.g., SARS-CoV or SARS-CoV-2) antigen or antibody.
  • any suitable amount of non-ionic surfactant so long as the amount of non-ionic surfactant is sufficient to inactivate any ⁇ -coronavirus (e.g., SARS-CoV or SARS-CoV-2) present in the sample without substantially interfering with testing the sample for the presence of a ⁇ -coronavirus (e.g., SARS-CoV or SARS-CoV-2) antigen or antibody.
  • the medium comprises from about 0.1 % to about 1.0% (v/v) of the at least one non-ionic surfactant (e.g., about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, or about 0.9% (v/v)).
  • the medium comprises from about 0.1% to about 0.75% (v/v) of the at least one non-ionic surfactant (e.g., about 0.15%, about 0.25%, about 0.35%, about 0.45%, about 0.55%, or about 0.65% (v/v)).
  • the medium comprises from about 0.1% to about 0.5% (v/v) of the at least one non-ionic surfactant (e.g., about 0.125%, about 0.225%, about 0.325%, about 0.425%, about 0.475%, or about 0.499% (v/v)).
  • the medium comprises about 0.2% (v/v) of at least one non-ionic surfactant
  • the medium comprises about 0.2 (v/v) of at least tergitol.
  • the medium comprises about 0.1% (v/v) of tergitol and about 0.1% (v/v) of: polyethylene glycol sorbitan monolaurate, polyoxyethylenesorbitan monolaurate, polyoxyethylenesorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene (4) sorbitan monostearate, polyoxyethylenesorbitan tristearate, polyoxyethylenesorbitan monooleate, or polyoxyethylenesorbitan trioleate.
  • the medium comprises about 0.1% (v/v) of tergitol and about 0.1% (v/v) of polyethylene glycol sorbitan monolaurate.
  • contacting and/or maintaining the sample in a medium comprising at least one non- ionic surfactant desirably is performed under conditions appropriate for, and for an amount of time sufficient for, the at least one non-ionic surfactant to inactivate any ⁇ -coronavirus (e.g., SARS-CoV or SARS-CoV-2) in the sample.
  • ⁇ -coronavirus e.g., SARS-CoV or SARS-CoV-2
  • the “appropriate conditions” and “amount of time” sufficient to inactivate a virus are well understood by one of ordinary skill in the art For example, an amount of time sufficient for virus inactivation may be at least about 10 seconds (e.g., where the medium is changed or transferred), up to years (e.g., where the medium is used for transport or storage of a sample).
  • an amount of time sufficient for virus inactivation may be at least about 1 minute, at least about 30 minutes, at least about one hour, at least about two hours, at least about 5 hours, at least about 10 hours, at least about 12 hours, or at least about 24 hours. In other embodiments, the amount of time sufficient for virus inactivation may be 1, 2, 3, 4, 5, or more days (e.g., 7 days, 14 days, 30 days, etc.), about 6 months, or a year or more (e.g., 2, 3, 4, or 5 years).
  • Appropriate conditions for virus inactivation typically include standard laboratory or hospital conditions for testing a sample, and also include conventional storage conditions (e.g., where the medium is used for transport or storage of a sample). In one embodiment, appropriate conditions include room temperature. In another embodiment, appropriate conditions include incubation at 37 °C with 5% CO 2 .
  • a virus is “inactivated” when infectivity of the virus is destroyed but immunogenicity is maintained.
  • ⁇ -coronavirus infectivity may be destroyed or disrupted at any state of the life cycle of the virus.
  • the coronavirus lifecycle includes (1) virus binding and entry into cells, (2) release of viral genome, (3) translation of viral polymerase protein, (4) RNA genome replication, (5) translation of viral structural proteins, (6) viral particle (virion) assembly, and (7) exocytosis.
  • the amount of non-ionic surfactant that is added to or present in the medium desirably inactivates ⁇ -coronavirus (e.g., SARS-CoV or SARS-CoV-2) without denaturing viral proteins or secondary structures that bind to assay reagents (e.g., antibodies) for detecting ⁇ -coronavirus (e.g., SARS-CoV or SARS-CoV-2).
  • ⁇ -coronavirus e.g., SARS-CoV or SARS-CoV-2
  • inactivation of ⁇ -coronavirus may be determined by an inability of the ⁇ -coronavirus (e.g., SARS-CoV or SARS-CoV-2) to replicate in culture.
  • CPE viral-induced cytopathic effects
  • cytopathogenic effect may be used interchangeably herein to refer to structural changes in host cells that are caused by viral infection.
  • an infecting virus may cause lysis of a host cell or cell death without lysis due to an inability to reproduce.
  • CPE may also cause changes in cell morphology, cell physiology, and biosynthetic events.
  • CPE may be detected using a variety of techniques, including, but not limited to, qualitative methods such as light microscopy, electron microscopy, and fluorescence microscopy.
  • CPE may also be detected using quantitative methods such as real-time cell analysis (see, e.g., Teng et al., J. Virol. Methods, 193(2); 364-370 (2013)), plaque assays (e.g., focus forming assays (FFAs)), and cell viability assays (e.g., colorimetric tetrazolium reduction assays, resazurin reduction assays, protease viability marker assays, and the luminogenic ATP assay).
  • FFAs focus forming assays
  • cell viability assays e.g., colorimetric tetrazolium reduction assays, resazurin reduction assays, protease viability marker assays, and the luminogenic ATP assay.
  • Methods for measuring virus infectivity and replication are further described in, for example, Storch, G.A., Clinical Infectious Diseases, Volume 31, Issue 3, pp. 739-751, September
  • the medium provided by the invention may be used to collect, transport and/or dilute the biological sample prior to performing the assay itself, or, it can be used to dilute assay components when performing the assay, such as when serial dilutions of test sample or other assay components are performed according to a particular assay method.
  • the medium desirably is designed to preserve the viability of microorganisms (e.g., virus or bacteria) during transport without allowing their multiplication.
  • virus infections such as ⁇ -coronavirus (e.g., SARS-CoV or SARS-CoV-2)
  • the at least one non-ionic surfactant is present in or added to a viral transport medium (VTM) or a universal transport medium (UTM).
  • VTM viral transport medium
  • UDM universal transport medium
  • viral transport medium refers to a liquid medium for the transport of virus specimens to a laboratory for analysis.
  • VTM allows for the safe transfer of viruses, chlamydia, and mycoplasma for further research, including conventional cell culture methods, diagnostic tests, and molecular biology techniques.
  • VTM typically contain components designed to provide an isotonic solution (e.g., protective protein), antibiotics to control microbial contamination, and one or more buffers to control the pH.
  • viral transport media may be used for transporting collection swabs or materials released into the medium from a collection swab. Liquid media may be added to other specimens when inactivation of the viral agent is likely and when the resultant dilution is acceptable.
  • VTM The U.S. Centers for Disease Control and Prevention (CDC) has published a standard operating procedure (SOP) for producing VTM, and recommends that VTM contain the following components: Hanks Balanced Salt Solution (HBSS) 1X with calcium and magnesium ions, no phenol red, 500mL bottle; sterile, heat-inactivated fetal bovine serum (FBS); gentamicin sulfate (50mg/mL) (or similar antibiotic at an appropriate concentration to prevent bacterial contamination and growth); amphotericin B (250 ⁇ g/mL) (Fungizone) (or similar antifungal at an appropriate concentration to prevent fungal contamination and growth); blood agar plate; and disinfectant (e.g., 70% ethanol) (Centers for Disease Control and Prevention, Preparation of Viral Transport Medium, SOP DSR-052-05; cdc.gov/coronavirus/2019-ncov/downloads/Viral- Transport-Medium.pdf).
  • HBSS Hanks Balanced Salt Solution
  • the medium may be a universal transport media (UTM), which is a room temperature stable viral transport medium for collection, transport, maintenance, and long-term freeze storage of viruses, such as Influenza H1N1, Chlamydia, Mycoplasma and Ureaplasma specimens.
  • UTM is used in the art for rapid antigen testing, direct fluorescent antibody (DFA) testing, viral culture, and for molecular-based assays.
  • UTMs Commercially available UTMs that may be used in connection with the inventive methods include, for example, UTM® (COPAN Diagnostics, Murrieta, CA), UNITRANZ-RTTM (Puritan Medical Products, Guilford, ME), BD universal viral transport (UVT) system (Becton, Dickinson and Company, Franklin Lakes, NJ), and Hardy Diagnostics Universal Transport MediumTM (Hardy Diagnostics, Santa Maria, CA).
  • UTM® COPAN Diagnostics, Murrieta, CA
  • UNITRANZ-RTTM Purtan Medical Products, Guilford, ME
  • UVT UVT
  • UVT Ultravioleton, Dickinson and Company, Franklin Lakes, NJ
  • Hardy Diagnostics Universal Transport MediumTM Hardy Diagnostics, Santa Maria, CA
  • Other commercially available virus transport media that may be used in the methods described herein include, but are not limited to, VIROCULT® and (Medical Wire & Equipment, Wiltshire, England), Eagle Minimum Essential Medium (E-MEM) (Sigma
  • VTM marketed by Rhino Diagnostics
  • Rhino Diagnostics see also, e.g., Hubbard et al., International Journal of Microbiology, Volume 2011, Article ID 46309 (2011) and Johnson, F.B., Clinical Microbiology Reviews, 3(2): 120-131 (1990)).
  • Methods for producing viral transport media are described in, e.g., Smith et al., J. Clinical Microbiology, 58(8): e00913-20 (2020); DOI: 10.1128/JCM.00913-20.
  • the VTM used or generated desirably conforms to the Enforcement Policy for Piral Transport Media During the Coronavirus Disease 2019 (COVID19) Public Health Emergency. Guidance for Commercial Manufacturers, Clinical Laboratories, and Food and Drug Administration Staff (FDA-2020-D-1138).
  • the at least one non-ionic surfactant is present in or added to a pretreatment solution or reagent (i.e., the medium is a pretreatment solution).
  • a pretreatment solution or reagent i.e., the medium is a pretreatment solution.
  • pretreatment solution and “pretreatment regimen,” may be used interchangeably to refer to a solution that may lyse, solubilize, and/or precipitate an analyte present in a sample prior to testing the sample for the presence of the analyte. Pretreatment is not necessary for all samples, however.
  • solubilizing the analyte entails release of the analyte from any endogenous binding proteins present in the sample.
  • a pretreatment solution may be homogeneous (not requiring a separation step) or heterogeneous (requiring a separation step). With use of a heterogeneous pretreatment solution there is removal of any precipitated analyte binding proteins from the test sample prior to proceeding to the next step of the assay.
  • the pretreatment solution or reagent optionally may comprise: (a) one or more solvents and salt, (b) one or more solvents, salt, and detergent, (c) detergent, (d) detergent and salt, or (e) any reagent or combination of reagents appropriate for cell lysis and/or solubilization of analyte.
  • the at least one non-ionic surfactant is present in or added to an assay-specific reagent (i.e., the medium is an assay-specific reagent).
  • an assay-specific reagent i.e., the medium is an assay-specific reagent.
  • assay-specific reagent and “analyte-specific reagent,” may be used interchangeably herein to refer to a class of biological molecules which can be used to identify and measure the amount of an individual chemical substance in biological specimens.
  • assay-specific reagents may include monoclonal or polyclonal antibodies, receptors, target ligands, peptides, and other molecules or compounds that have the potential to recognize specific sequences or structural features that are unique to the analyte. Assay-specific reagents contribute to the sensitivity and selectivity of the assay.
  • the at least one non-ionic surfactant may be added to the medium prior to addition of a biological sample to the medium.
  • the at least one non-ionic surfactant may be added to the medium after the biological sample is added to the medium.
  • the medium may comprise a plurality of different non-ionic surfactants.
  • the medium may comprise 2, 3, 4, 5, or more different non-ionic surfactants, such as any of those described above or known in the art
  • at least one of the plurality of non- ionic surfactants is tergitol.
  • the method of inactivating ⁇ -coronavirus (e.g., SARS-CoV or SARS-CoV-2) in a biological sample may be used in conjunction with one or more methods for diagnosing a ⁇ - coronavirus (e.g., SARS-CoV or SARS-CoV-2) infection, such as testing the biological sample for the presence of a ⁇ -coronavirus (e.g., SARS-CoV or SARS-CoV-2) antigen or antibody.
  • a ⁇ -coronavirus e.g., SARS-CoV or SARS-CoV-2
  • the methods described herein can be used in conjunction with clinical presentation and other laboratory tests to aid in the diagnosis of ⁇ -coronavirus (e.g., SARS-CoV or SARS-CoV-2) infection in a subject (e.g., who may or may not exhibit signs and/or symptoms of infection and suspected of having ⁇ -coronavirus (e.g., SARS-CoV or SARS- CoV-2)).
  • laboratory tests include, for example, PCR or other nucleic acid amplification- based assays (also referred to as “molecular” tests), serology or antibody assays, and antigen assays.
  • Molecular and antigen tests are used to diagnose an active coronavirus infection, while antibody tests are used to assess a potential past coronavirus infection.
  • Molecular tests detect the genetic material or nucleic acid present inside a virus particle.
  • Most molecular tests employ PCR-based methods (e.g., RT-PCR), which are also referred to as nucleic acid amplification tests (NAAT).
  • NAAT nucleic acid amplification tests
  • Antigen tests detect one or more specific proteins from a virus particle. Antigen tests tend to be highly specific but are typically less sensitive than molecular tests.
  • Serology/antibody tests detect antibodies produced by a subject in response to a viral infection. With respect to SARS-CoV-2 , serology/antibody tests typically detect antibodies (e.g., IgM and/or IgG antibodies) to the spike (S) protein or nucleocapsid (N) protein.
  • the FDA has authorized molecular tests for SARS-CoV-2 for use in clinical laboratory settings and authorized some for use in a point-of-care (POC) setting, including, for example, ID NOWTM COVID-19, REALTIMETM SARS-CoV-2 EUA, and ALINITYTM m SARS-CoV-2 assay (all marketed by Abbott Laboratories, Abbott Park, IL). All antigen tests currently authorized by the FDA are POC tests and provide results in less than an hour.
  • An exemplary SARS-CoV-2 antigen test is BINAXNOWTM (Abbott Laboratories, Abbott Park, IL).
  • ELISA enzyme- linked immunosorbent assays
  • CLIA chemiluminescent immunoassays
  • SARS-CoV-2 antibody tests may be performed on the ARCHITECT® and/or ALINITY automated analyzers (Abbott Laboratories, Abbott Park, IL).
  • Nanopore devices are described in PCT International Application WO 2016/161402 and examples of nanowell devices are described in PCT International Application WO 2016/161400, both of which are hereby incorporated by reference herein.
  • Other devices and methods appropriate for single molecule detection also can be employed.
  • a medium comprising at least about 0.1% (v/v) of at least one non-ionic surfactant as described herein may be provided in a kit, along with components which may be used in methods for assaying or assessing a biological sample for ⁇ -coronavirus (e.g., SARS-CoV or SARS-CoV-2) infection.
  • the kit comprises at least one component for assaying the biological sample for the presence of ⁇ -coronavirus (e.g., SARS-CoV or SARS-CoV-2) (e.g., components for a molecular assay, an antigen assay, or an antibody assay as described above), and instructions for assaying the sample.
  • kit can be affixed to packaging material, can be included as a package insert, or can be viewed or downloaded from a particular website that is recited as part of the kit packaging or inserted materials. While the instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” can include the address of an internet site that provides the instructions.
  • the kit can comprise a calibrator or control, e.g., purified, and optionally frozen or lyophilized, and/or at least one container (e.g., tube, microtiter plates or strips) for conducting the assay, and/or a buffer, such as an assay buffer or a wash buffer, either one of which can be provided as a concentrated solution, a substrate solution for the detectable label (e.g., an enzymatic label), or a stop solution.
  • the kit comprises all components, i.e., reagents, standards, buffers, diluents, etc., which are necessary to perform the assay.
  • the instructions also can include instructions for generating a standard curve.
  • the kit may further comprise reference standards for quantifying ⁇ -coronavirus (e.g., SARS-CoV or SARS-CoV-2).
  • the reference standards may be employed to establish standard curves for interpolation and/or extrapolation of ⁇ -coronavirus (e.g., SARS-CoV or SARS-CoV- 2) concentration.
  • the kit includes quality control components (for example, sensitivity panels, calibrators, and positive controls).
  • quality control components for example, sensitivity panels, calibrators, and positive controls.
  • Preparation of quality control reagents is well-known in the art and is described on insert sheets for a variety of immunodiagnostic products.
  • Sensitivity panel members optionally are used to establish assay performance characteristics, and further optionally are useful indicators of the integrity of the immunoassay kit reagents, and the standardization of assays.
  • the kit can also optionally include other reagents required to conduct a diagnostic assay or facilitate quality control evaluations, such as buffers, salts, enzymes, enzyme co-factors, substrates, detection reagents, and the like.
  • Other components such as buffers and solutions for the isolation and/or treatment of a test sample (e.g., pretreatment reagents), also can be included in the kit.
  • One or more of the components of the kit can be lyophilized, in which case the kit can further comprise reagents suitable for the reconstitution of the lyophilized components.
  • kits for holding or storing a sample (e.g., a container or cartridge for a urine, whole blood, plasma, or serum sample).
  • a sample e.g., a container or cartridge for a urine, whole blood, plasma, or serum sample.
  • the kit optionally also can contain reaction vessels, mixing vessels, and other components that facilitate the preparation of reagents or the test sample.
  • the kit can also include one or more instruments for assisting with obtaining a test sample, such as a syringe, pipette, forceps, measured spoon, or the like.
  • the kit can contain a solid phase, such as a magnetic particle, bead, test tube, microtiter plate, cuvette, membrane, scaffolding molecule, film, filter paper, disc, or chip.
  • a solid phase such as a magnetic particle, bead, test tube, microtiter plate, cuvette, membrane, scaffolding molecule, film, filter paper, disc, or chip.
  • the kit (or components thereof) and methods for detecting the presence of SARS- CoV-2 in a test sample as described herein can be adapted for use in a variety of automated and semi-automated systems or platforms (including those wherein the solid phase comprises a microparticle), as described, e.g., U.S. Patent No. 5,063,081, U.S. Patent Application Publication Nos. 2003/0170881, 2004/0018577, 2005/0054078, and 2006/0160164 and as commercially marketed e.g., by Abbott Laboratories (Abbott Park, IL) as Abbott Point of Care (i-STAT® or i- STAT Alinity, Abbott Laboratories) as well as those described in U.S. Patent Nos. 5,089,424 and 5,006,309, and as commercially marketed, e.g., by Abbott Laboratories (Abbott Park, IL) as ARCHITECT® or the series of Abbott Alinity devices.
  • Abbott Laboratories Abbott Point of Care
  • kits, and kit components can be employed in other formats, for example, on electrochemical or other hand-held or point-of-care assay systems.
  • the present disclosure is, for example, applicable to the commercial Abbott Point of Care (i-STAT®, Abbott Laboratories) electrochemical immunoassay system that performs sandwich immunoassays.
  • This example demonstrates the addition of tergitol to a transport culture medium to inactivate SARS-CoV-2.
  • Experiments were conducted to determine if tergitol inactivates SARS-CoV-2 (especially high titer stocks) by assessing infectivity on Vero cells and to determine the optimal tergitol exposure time for virus inactivation.
  • Vero cells ATCC CCL-81 were plated overnight.
  • a 96-well white-walled plate with clear bottom was labeled for luminescence detection. 200 ⁇ l of cells were dispensed at 1.5 x 10 5 cells/mL concentration into each well using a trough and multichannel pipette.
  • virus infection and inactivation assessment were performed.
  • SARS-CoV-2 virus stocks maintained by Abbott Laboratories were diluted in 2 mL screw cap vials using media lacking fetal bovine serum (‘no fetal bovine serum (FBS)’ media) as diluent.
  • FBS fetal bovine serum
  • a separate SARS-CoV-2 lysate purchased from BEI Resources Isolate USA-WA1/2020; Catalog No. NR- 52281; Lot: 70036318, referred to in tables below as “BEI”) was diluted in the same manner.
  • VTM+2% (v/v) tergitol 100 ⁇ l of VTM+2% (v/v) tergitol was dispensed to a 96-well dilution plate in all wells except columns 1 and 7 for non-inactivated controls. 100 ⁇ l of diluted virus was transferred to a 96-well dilution/inactivation plate, beginning with a 2-hour timepoint working backwards to 10 minutes. Virus was incubated at room temperature until infection.
  • Inoculum was removed from one column at a time by aspiration, starting with the lowest concentration of virus.
  • the non-filter tip on the Pasteur pipette was changed after each sample.
  • 20 mL PBS was poured into the trough and 150 pl PBS was dispensed into each well with a multichannel pipette, gently down the right side. PBS was then aspirated.
  • About 20 mL +10% FBS media was poured into the trough and 200 pl of complete media were dispensed to each well with a multichannel pipette, gently down the left side. There was no need to change tips.
  • plates were placed back into an incubator at 37 °C with 5% CO 2 for 3-4 days.
  • VTM+tergitol Three concentrations of VTM+tergitol were made (2%, 1% 0.2%, and 0.02%) and 100 pl was dispensed to four rows (rows 1, 4, 7, 9). When diluted 1:1 with virus, final inactivation concentrations were 1%, 0.5% 0.1%, and 0.01%. 1.2 mL of each buffer dilution was needed (140 pl +1.26 mL of no tergitol VTM). To be able to dilute the tergitol prior to plating, 180 pl of media (+10% FBS) was dispensed to rows 2, 3, 6, 7. Row 5 had 160 ⁇ l of media.
  • Virus (10 8 /100 ⁇ l) was added at the appropriate time to each of the four concentrations, starting with a 60-minute inactivation time period. For no virus, media was added.
  • CPE Cytopathic effects
  • Clause 1 A method of inactivating any SARS-CoV-2 in a biological sample prior to testing the sample for the presence of a S ARS-CoV-2 antigen or antibody, the method comprising maintaining the sample in a medium comprising at least about 0.1% (v/v) of at least one non-ionic surfactant under conditions appropriate for and for an amount of time sufficient for the at least one non-ionic surfactant to inactivate the SARS-CoV-2 in the sample as determined by an inability of the SARS-CoV-2 to replicate in culture, without substantially interfering with testing the sample for the presence of a SARS-CoV-2 antigen or antibody.
  • Clause 3 The method of clause 1 or clause 2, wherein the non-ionic surfactant is tergitol.
  • Clause 4 The method of any one of clauses 1-3, wherein the at least one non-ionic surfactant is tergitol and polyethylene glycol sorbitan monolaurate.
  • Clause 5 The method of any one of clauses 1-4, wherein the medium comprises from about 0.1% to about 1.0% (v/v) non-ionic surfactant.
  • Clause 6 The method of clause 5, wherein the medium comprises from about 0.1% to about 0.75% (v/v) non-ionic surfactant.
  • Clause 7 The method of clause 6, wherein the medium comprises from about 0.1% to about 0.5% (v/v) non-ionic surfactant.
  • Clause 10 The method of any one of clauses 1 -9, wherein the at least one non-ionic surfactant is: (a) present in or added to a viral transport medium (VTM) or universal transport medium (UTM); (b) present in or added to a pretreatment solution; (c) present in or added to an assay-specific reagent; or (d) any combination of (a) to (c).
  • VTM viral transport medium
  • UDM universal transport medium
  • Clause 11 The method of any one of clauses 1-10, wherein the sample comprises saliva, an oropharyngeal specimen, a nasopharyngeal specimen, an anal swab specimen, or a nasal mucus specimen.
  • Clause 12 The method of any one of clauses 1-11, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using an immunoassay or a clinical chemistry assay.
  • Clause 13 The method of any one of clauses 1-12, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using single molecule detection, a lateral flow assay, or a point-of-care assay.
  • Clause 14 The method of any one of clauses 1-13, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is adapted for use in an automated system or a semi-automated system.
  • Clause 15 In an improvement of a method of assaying a biological sample for the presence of a SARS-CoV-2 antigen or antibody, the improvement comprising incubating the biological sample in a medium containing at least 0.1% (v/v) of at least one non-ionic surfactant prior to assaying the biological sample for the presence of a SARS-CoV-2 antigen or antibody, wherein the at least one non-ionic surfactant inactivates the SARS-CoV-2 present in the biological sample as determined by an inability of the SARS-CoV-2 to replicate in culture.
  • Clause 16 In the improvement of clause 15, wherein the non-ionic surfactant is a secondary alcohol ethoxylate.
  • Clause 17 In the improvement of clause 15 or clause 16, wherein the non-ionic surfactant is tergitol.
  • Clause 18 In the improvement of any of clauses 15-17, wherein the at least one non- ionic surfactant is tergitol and polyethylene glycol sorbitan monolaurate.
  • Clause 20 In the improvement of clause 19, wherein the medium comprises from about 0.1% to about 0.75% (v/v) non-ionic surfactant.
  • Clause 23 In the improvement of clause 22, wherein the medium comprises about 0.1% (v/v) tergitol and about 0.1% (v/v) polyethylene glycol sorbitan monolaurate.
  • Clause 24 In the improvement of any one of clauses 15-23, wherein the at least one non-ionic surfactant is: (a) present in or added to a VTM or UTM; (b) present in or added to a pretreatment solution; (c) present in or added to an assay-specific reagent; or (d) any combination of (a) to (c).
  • Clause 25 In the improvement of any one of clauses 15-24, wherein the sample comprises saliva, an oropharyngeal specimen, a nasopharyngeal specimen, an anal swab specimen, or a nasal mucus specimen.
  • Clause 26 In the improvement of any one of clauses 15-25, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using an immunoassay or a clinical chemistry assay.
  • Clause 27 In the improvement of any of clauses 15-26, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using single molecule detection, a lateral flow assay, or a point-of-care assay.
  • Clause 28 In the improvement of any of clauses 15-27, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is adapted for use in an automated system or a semi-automated system.
  • a method of inactivating any SARS-CoV-2 in a biological sample prior to testing the sample for the presence of a SARS-CoV-2 antigen or antibody comprising maintaining the sample in a medium comprising at least about 0.1% (v/v) of at least one non-ionic surfactant under conditions appropriate for and for an amount of time sufficient for the at least one non-ionic surfactant to inactivate the SARS-CoV-2 in the sample as determined by an inability of the SARS-CoV-2 to replicate in culture, without substantially interfering with testing the sample for the presence of a SARS-CoV-2 antigen or antibody, wherein the non-ionic surfactant is a secondary alcohol ethoxylate.
  • Clause 30 The method of clause 29, wherein the secondary alcohol ethoxylate is tergitol.
  • Clause 31 The method of clause 29 or clause 30, wherein the at least one non-ionic surfactant is: (a) present in or added to a viral transport medium (VTM) or universal transport medium (UTM); (b) present in or added to a pretreatment solution; (c) present in or added to an assay-specific reagent; or (d) any combination of (a) to (c).
  • VTM viral transport medium
  • UDM universal transport medium
  • Clause 32 The method of any one of clauses 29-31, wherein the sample comprises saliva, an oropharyngeal specimen, a nasopharyngeal specimen, an anal swab specimen, or a nasal mucus specimen.
  • Clause 33 The method of any one of clauses 29-32, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using an immunoassay or a clinical chemistry assay.
  • Clause 34 The method of any one of clauses 29-33, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using single molecule detection, a lateral flow assay, or a point-of-care assay.
  • Clause 35 The method of any one of clauses 29-34, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is adapted for use in an automated system or a semi-automated system.
  • a method of inactivating any SARS-CoV-2 in a biological sample prior to testing the sample for the presence of a SARS-CoV-2 antigen or antibody comprising maintaining the sample in a medium comprising at least about 0.1% (v/v) of at least one non-ionic surfactant under conditions appropriate for and for an amount of time sufficient for the at least one non-ionic surfactant to inactivate the SARS-CoV-2 in the sample as determined by an inability of the SARS-CoV-2 to replicate in culture, without substantially interfering with testing the sample for the presence of a SARS-CoV-2 antigen or antibody, wherein the non-ionic surfactant is tergitol.
  • Clause 37 The method of clause 36, wherein the at least one non-ionic surfactant is: (a) present in or added to a viral transport medium (VTM) or universal transport medium (UTM); (b) present in or added to a pretreatment solution; (c) present in or added to an assay- specific reagent; or (d) any combination of (a) to (c).
  • VTM viral transport medium
  • UDM universal transport medium
  • Clause 38 The method of clause 36 or clause 37, wherein the sample comprises saliva, an oropharyngeal specimen, a nasopharyngeal specimen, an anal swab specimen, or a nasal mucus specimen.
  • Clause 39 The method of any one of clauses 36-38, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using an immunoassay or a clinical chemistry assay.
  • Clause 40 The method of any one of clauses 36-39, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using single molecule detection, a lateral flow assay, or a point-of-care assay.
  • Clause 41 The method of any one of clauses 36-40, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is adapted for use in an automated system or a semi-automated system.
  • a method of inactivating any SARS-CoV-2 in a biological sample prior to testing the sample for the presence of a SARS-CoV-2 antigen or antibody comprising maintaining the sample in a medium comprising at least about 0.1% (v/v) of at least one non-ionic surfactant under conditions appropriate for and for an amount of time sufficient for the at least one non-ionic surfactant to inactivate the SARS-CoV-2 in the sample as determined by an inability of the SARS-CoV-2 to replicate in culture, without substantially interfering with testing the sample for the presence of a SARS-CoV-2 antigen or antibody, wherein the non-ionic surfactant is tergitol and polyethylene glycol sorbitan monolaurate.
  • Clause 43 The method of clause 43, wherein the at least one non-ionic surfactant is: (a) present in or added to a viral transport medium (VTM) or universal transport medium (UTM); (b) present in or added to a pretreatment solution; (c) present in or added to an assay- specific reagent; or (d) any combination of (a) to (c).
  • VTM viral transport medium
  • UDM universal transport medium
  • Clause 44 The method of clause 42 or clause 43, wherein the sample comprises saliva, an oropharyngeal specimen, a nasopharyngeal specimen, an anal swab specimen, or a nasal mucus specimen.
  • Clause 45 The method of any one of clauses 42-44, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using an immunoassay or a clinical chemistry assay.
  • Clause 46 The method of any one of clauses 42-45, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using single molecule detection, a lateral flow assay, or a point-of-care assay.
  • Clause 47 The method of any one of clauses 42-46, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is adapted for use in an automated system or a semi-automated system.
  • a method of inactivating any SARS-CoV-2 in a biological sample prior to testing the sample for the presence of a SARS-CoV-2 antigen or antibody comprising maintaining the sample in a medium comprising from about 0.1% (v/v) to about 1.0% (v/v) of at least one non-ionic surfactant under conditions appropriate for and for an amount of time sufficient for the at least one non-ionic surfactant to inactivate the SARS-CoV-2 in the sample as determined by an inability of the SARS-CoV-2 to replicate in culture, without substantially interfering with testing the sample for the presence of a SARS-CoV-2 antigen or antibody, wherein the non-ionic surfactant is a secondary alcohol ethoxylate.
  • Clause 49 The method of clause 48, wherein the secondary alcohol ethoxylate is tergitol.
  • Clause 50 The method of clause 48 or clause 49, wherein the medium comprises from about 0.1% to about 0.75% (v/v) non-ionic surfactant.
  • Clause 51 The method of clause 50, wherein the medium comprises from about 0.1% to about 0.5% (v/v) non-ionic surfactant.
  • Clause 52 The method of clause 51, wherein the medium comprises about 0.2% (v/v) of the at least one non-ionic surfactant.
  • Clause 53 The method of any one of clauses 48-52, wherein the at least one non-ionic surfactant is: (a) present in or added to a viral transport medium (VTM) or universal transport medium (UTM); (b) present in or added to a pretreatment solution; (c) present in or added to an assay-specific reagent; or (d) any combination of (a) to (c).
  • VTM viral transport medium
  • UDM universal transport medium
  • Clause 54 The method of any one of clauses 48-53, wherein the sample comprises saliva, an oropharyngeal specimen, a nasopharyngeal specimen, an anal swab specimen, or a nasal mucus specimen.
  • Clause 55 The method of any one of clauses 48-54, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using an immunoassay or a clinical chemistry assay.
  • Clause 56 The method of any one of clauses 48-55, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using single molecule detection, a lateral flow assay, or a point-of-care assay.
  • Clause 57 The method of any one of clauses 48-56, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is adapted for use in an automated system or a semi-automated system.
  • Clause 58 A method of inactivating any SARS-CoV-2 in a biological sample prior to testing the sample for the presence of a SARS-CoV-2 antigen or antibody, the method comprising maintaining the sample in a medium comprising from about 0.1% (v/v) to about 1.0% (v/v) of at least one non-ionic surfactant under conditions appropriate for and for an amount of time sufficient for the at least one non-ionic surfactant to inactivate the SARS-CoV-2 in the sample as determined by an inability of the SARS-CoV-2 to replicate in culture, without substantially interfering with testing the sample for the presence of a SARS-CoV-2 antigen or antibody, wherein the non-ionic surfactant is tergitol.
  • Clause 59 The method of clause 58, wherein the medium comprises from about 0.1% to about 0.75% (v/v) of tergitol.
  • Clause 60 The method of clause 59, wherein the medium comprises from about 0.1% to about 0.5% (v/v) of tergitol.
  • Clause 61 The method of clause 60, wherein the medium comprises about 0.2% (v/v) of tergitol.
  • Clause 62 The method of any one of clauses 58-61, wherein the at least one non-ionic surfactant is: (a) present in or added to a viral transport medium (VTM) or universal transport medium (UTM); (b) present in or added to a pretreatment solution; (c) present in or added to an assay-specific reagent; or (d) any combination of (a) to (c).
  • VTM viral transport medium
  • UDM universal transport medium
  • Clause 63 The method of any one of clauses 58-62, wherein the sample comprises saliva, an oropharyngeal specimen, a nasopharyngeal specimen, an anal swab specimen, or a nasal mucus specimen.
  • Clause 64 The method of any one of clauses 58-63, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using an immunoassay or a clinical chemistry assay.
  • Clause 65 The method of any one of clauses 58-64, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using single molecule detection, a lateral flow assay, or a point-of-care assay.
  • Clause 66 The method of any one of clauses 58-65, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is adapted for use in an automated system or a semi-automated system.
  • a method of inactivating any SARS-CoV-2 in a biological sample prior to testing the sample for the presence of a SARS-CoV-2 antigen or antibody comprising maintaining the sample in a medium comprising at least one non-ionic surfactant under conditions appropriate for and for an amount of time sufficient for the at least one non- ionic surfactant to inactivate the SARS-CoV-2 in the sample as determined by an inability of the SARS-CoV-2 to replicate in culture, without substantially interfering with testing the sample for the presence of a SARS-CoV-2 antigen or antibody, wherein the non-ionic surfactant is tergitol and polyethylene glycol sorbitan monolaurate and the medium comprises about 0.1% (v/v) tergitol and about 0.1% (v/v) polyethylene glycol sorbitan monolaurate.
  • Clause 68 The method of clause 67, wherein the at least one non-ionic surfactant is: (a) present in or added to a viral transport medium (VTM) or universal transport medium (UTM); (b) present in or added to a pretreatment solution; (c) present in or added to an assay- specific reagent; or (d) any combination of (a) to (c).
  • VTM viral transport medium
  • UDM universal transport medium
  • Clause 69 The method of clause 67 or clause 68, wherein the sample comprises saliva, an oropharyngeal specimen, a nasopharyngeal specimen, an anal swab specimen, or a nasal mucus specimen.
  • Clause 70 The method of any one of clauses 67-69, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using an immunoassay or a clinical chemistry assay.
  • Clause 71 The method of any one of clauses 67-70, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using single molecule detection, a lateral flow assay, or a point-of-care assay.
  • Clause 72 The method of any one of clauses 67-71, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is adapted for use in an automated system or a semi-automated system. [0179] Clause 73.
  • a method of assaying a biological sample for the presence of a SARS-CoV-2 antigen or antibody comprising incubating the biological sample in a medium containing at least 0.1% (v/v) of at least one non-ionic surfactant prior to assaying the biological sample for the presence of a SARS-CoV-2 antigen or antibody, wherein the at least one non-ionic surfactant inactivates the SARS-CoV-2 present in the biological sample as determined by an inability of the SARS-CoV-2 to replicate in culture, wherein the non-ionic surfactant is a secondary alcohol ethoxylate.
  • Clause 74 The improvement of clause 73, wherein the secondary alcohol ethoxylate is tergitol.
  • Clause 75 The improvement of clause 73 or clause 74, wherein the at least one non- ionic surfactant is: (a) present in or added to a viral transport medium (VTM) or universal transport medium (UTM); (b) present in or added to a pretreatment solution; (c) present in or added to an assay-specific reagent; or (d) any combination of (a) to (c).
  • VTM viral transport medium
  • UDM universal transport medium
  • Clause 76 The improvement of any one of clauses 73-75, wherein the sample comprises saliva, an oropharyngeal specimen, a nasopharyngeal specimen, an anal swab specimen, or a nasal mucus specimen.
  • Clause 77 The improvement of any one of clauses 73-76, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using an immunoassay or a clinical chemistry assay.
  • Clause 78 The improvement of any one of clauses 73-77, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using single molecule detection, a lateral flow assay, or a point-of-care assay.
  • Clause 79 The improvement of any one of clauses 73-78, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is adapted for use in an automated system or a semi-automated system.
  • Clause 80 In an improvement of a method of assaying a biological sample for the presence of a SARS-CoV-2 antigen or antibody, the improvement comprising incubating the biological sample in a medium containing at least 0.1% (v/v) of at least one non-ionic surfactant prior to assaying the biological sample for the presence of a SARS-CoV-2 antigen or antibody, wherein the at least one non-ionic surfactant inactivates the SARS-CoV-2 present in the biological sample as determined by an inability of the SARS-CoV-2 to replicate in culture, wherein the non-ionic surfactant is tergitol.
  • Clause 81 The improvement of clause 80, wherein the at least one non-ionic surfactant is: (a) present in or added to a viral transport medium (VTM) or universal transport medium (UTM); (b) present in or added to a pretreatment solution; (c) present in or added to an assay-specific reagent; or (d) any combination of (a) to (c).
  • VTM viral transport medium
  • UDM universal transport medium
  • Clause 82 The improvement of clause 80 or clause 81, wherein the sample comprises saliva, an oropharyngeal specimen, a nasopharyngeal specimen, an anal swab specimen, or a nasal mucus specimen.
  • Clause 83 The improvement of any of clauses 80-82, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using an immunoassay or a clinical chemistry assay.
  • Clause 84 The improvement of any of clauses 80-83, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using single molecule detection, a lateral flow assay, or a point-of-care assay.
  • Clause 85 The improvement of any of clauses 80-84, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is adapted for use in an automated system or a semi-automated system.
  • Clause 86 In an improvement of a method of assaying a biological sample for the presence of a SARS-CoV-2 antigen or antibody, the improvement comprising incubating the biological sample in a medium containing at least 0.1 % (v/v) of at least one non-ionic surfactant prior to assaying the biological sample for the presence of a SARS-CoV-2 antigen or antibody, wherein the at least one non-ionic surfactant inactivates the SARS-CoV-2 present in the biological sample as determined by an inability of the SARS-CoV-2 to replicate in culture, wherein the non-ionic surfactant is tergitol and polyethylene glycol sorbitan monolaurate [0193] Clause 87.
  • Clause 86 wherein the at least one non-ionic surfactant is: (a) present in or added to a viral transport medium (VTM) or universal transport medium (UTM); (b) present in or added to a pretreatment solution; (c) present in or added to an assay-specific reagent; or (d) any combination of (a) to (c).
  • VTM viral transport medium
  • UDM universal transport medium
  • Clause 88 The improvement of clause 86 or clause 87, wherein the sample comprises saliva, an oropharyngeal specimen, a nasopharyngeal specimen, an anal swab specimen, or a nasal mucus specimen.
  • Clause 89 The improvement of any of clauses 86-88, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using an immunoassay or a clinical chemistry assay.
  • Clause 90 The improvement of any of clauses 86-89, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using single molecule detection, a lateral flow assay, or a point-of-care assay.
  • Clause 91 The improvement of any of clauses 86-90, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is adapted for use in an automated system or a semi-automated system.
  • Clause 92 In an improvement of a method of assaying a biological sample for the presence of a SARS-CoV-2 antigen or antibody, the improvement comprising incubating the biological sample in a medium containing from about 0.1% (v/v) to about 1.0% (v/v) of at least one non-ionic surfactant prior to assaying the biological sample for the presence of a SARS- CoV-2 antigen or antibody, wherein the at least one non-ionic surfactant inactivates the SARS- CoV-2 present in the biological sample as determined by an inability of the SARS-CoV-2 to replicate in culture, wherein the non-ionic surfactant is a secondary alcohol ethoxylate.
  • Clause 93 The improvement of clause 92, wherein the secondary alcohol ethoxylate is tergitol.
  • Clause 95 The improvement of clause 94, wherein the medium comprises from about 0.1% to about 0.5% (v/v) non-ionic surfactant.
  • Clause 97 The improvement of any one of clauses 91-96, wherein the at least one non-ionic surfactant is: (a) present in or added to a viral transport medium (VTM) or universal transport medium (UTM); (b) present in or added to a pretreatment solution; (c) present in or added to an assay-specific reagent; or (d) any combination of (a) to (c).
  • VTM viral transport medium
  • UDM universal transport medium
  • Clause 98 The improvement of any one of clauses 91-97, wherein the sample comprises saliva, an oropharyngeal specimen, a nasopharyngeal specimen, an anal swab specimen, or a nasal mucus specimen.
  • Clause 99 The improvement of any one of clauses 91-98, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using an immunoassay or a clinical chemistry assay.
  • Clause 100 The improvement of any one of clauses 91-99, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using single molecule detection, a lateral flow assay, or a point-of-care assay.
  • Clause 101 The improvement of any one of clauses 91-100, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is adapted for use in an automated system or a semi-automated system.
  • Clause 102 In an improvement of a method of assaying a biological sample for the presence of a SARS-CoV-2 antigen or antibody, the improvement comprising incubating the biological sample in a medium containing from about 0.1% (v/v) to about 1.0% (v/v) of at least one non-ionic surfactant prior to assaying the biological sample for the presence of a SARS- CoV-2 antigen or antibody, wherein the at least one non-ionic surfactant inactivates the SARS- CoV-2 present in the biological sample as determined by an inability of the SARS-CoV-2 to replicate in culture, wherein the non-ionic surfactant is tergitol.
  • Clause 103 The improvement of clause 102, wherein the medium comprises from about 0.1% to about 0.75% (v/v) of tergitol.
  • Clause 104 The improvement of clause 103, wherein the medium comprises from about 0.1% to about 0.5% (v/v) of tergitol.
  • Clause 106 In the improvement of any one of clauses 102- 105, wherein the at least one non-ionic surfactant is: (a) present in or added to a viral transport medium (VTM) or universal transport medium (UTM); (b) present in or added to a pretreatment solution; (c) present in or added to an assay-specific reagent; or (d) any combination of (a) to (c).
  • VTM viral transport medium
  • UDM universal transport medium
  • Clause 107 The improvement of any one of clauses 102-106, wherein the sample comprises saliva, an oropharyngeal specimen, a nasopharyngeal specimen, an anal swab specimen, or a nasal mucus specimen.
  • Clause 108 The improvement of any one of clauses 102-107, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using an immunoassay or a clinical chemistry assay.
  • Clause 109 The improvement of any one of clauses 102-108, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using single molecule detection, a lateral flow assay, or a point-of-care assay.
  • Clause 110 The improvement of any one of clauses 102-109, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is adapted for use in an automated system or a semi-automated system.
  • Clause 112. The improvement of clause 111, wherein the at least one non-ionic surfactant is: (a) present in or added to a viral transport medium (VTM) or universal transport medium (UTM); (b) present in or added to a pretreatment solution; (c) present in or added to an assay-specific reagent; or (d) any combination of (a) to (c).
  • VTM viral transport medium
  • UDM universal transport medium
  • Clause 113 The improvement of any one of clause 111 or clause 112, wherein the sample comprises saliva, an oropharyngeal specimen, a nasopharyngeal specimen, an anal swab specimen, or a nasal mucus specimen.
  • Clause 114 The improvement of any one of clauses 111-113, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using an immunoassay or a clinical chemistry assay.
  • Clause 115 The improvement of any one of clauses 111-114, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is performed using single molecule detection, a lateral flow assay, or a point-of-care assay.
  • Clause 116 The improvement of any one of the clauses of 111-115, wherein testing the sample for the presence of a SARS-CoV-2 antigen or antibody is adapted for use in an automated system or a semi-automated system.

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  • Tropical Medicine & Parasitology (AREA)
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Abstract

L'invention concerne des procédés d'inactivation de tous ß-coronavirus (par exemple, le SARS-CoV ou le SARS-CoV-2) dans un échantillon biologique avant le test de l'échantillon pour la présence d'un antigène ou d'un anticorps du ß-coronavirus (par exemple, le SARS-CoV ou le SARS-CoV-2), qui implique le maintien de l'échantillon dans un milieu comprenant au moins un tensioactif non ionique. Le ou les tensioactifs non ioniques activent le ß-coronavirus (par exemple le SARS-CoV ou le SARS-CoV-2) tel que déterminé par une incapacité du ß-coronavirus (par exemple, le SARS-CoV ou le SARS-CoV-2) à se répliquer en culture.
PCT/US2021/065581 2020-12-30 2021-12-29 Procédés, réactifs et kits améliorés pour l'inactivation à base de détergent de bêta-coronavirus avant et/ou pendant l'évaluation d'un échantillon biologique pour un antigène ou un anticorps du sars-cov-2 WO2022147178A1 (fr)

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US202063132155P 2020-12-30 2020-12-30
US63/132,155 2020-12-30
US202163180209P 2021-04-27 2021-04-27
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WO2024115344A1 (fr) * 2022-11-29 2024-06-06 Merck Patent Gmbh Procédé d'inactivation de virus enveloppés

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WO2024115344A1 (fr) * 2022-11-29 2024-06-06 Merck Patent Gmbh Procédé d'inactivation de virus enveloppés

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