Rapid Viral Assay

Abstract

The present invention provides a method for rapid, highly specific and sensitive detection and quantification of a virus by observing viral substrate binding to its host receptor protein. The invention also provides a method for rapid, highly specific and sensitive detection and quantification of a virus in an individual suspected of being infected with a virus. The invention further provides a test kit for rapid, highly specific and sensitive point-of-care detection of a virus in an individual. The viruses and their host receptor proteins that can rapidly be detected include SARS-CoV-2 and its host receptor protein ACE2. The surprisingly rapid, specific, sensitive method and kit of the invention provide a point-of care test capable of diagnosing individuals suffering from COVID-19 by observation of a color change in the assay, which color change occurs in about five minutes, and which test can be completed by a user in about 60 minutes.

Claims

1. A method for rapid, highly specific and sensitive, detection and quantification of a virus in an individual suspected of being infected with a virus by observing binding with a host receptor protein of a viral substrate of the virus contained in a specimen taken from the individual, comprising the steps of: coating a plurality of microtiter wells with a host receptor protein contained in a coating buffer; incubating the plurality of microtiter wells overnight; washing the microtiter wells; adding a blocking solution to the plurality of microtiter wells; washing the plurality of microtiter wells three times; adding the viral substrate to the plurality of microtiter wells; incubating the plurality of microtiter wells for 20 minutes; washing the plurality of microtiter wells three times; adding an antibody directed against the viral substrate to the plurality of microtiter wells; incubating the plurality of microtiter wells for 20 minutes; adding a horseradish peroxidase (HRP)-conjugated antibody directed against the antibody to the plurality of microtiter wells; incubating the plurality of microtiter wells for 20 minutes; washing the plurality of microtiter wells three times; adding a TMB solution to the plurality of microtiter wells; adding a Stop solution to the plurality of microtiter wells; and detecting the viral substrate in the microtiter wells by observing those microtiter wells that undergo a color change or quantifying the concentration of the viral substrate by reading optical density at 450 nm, wherein the method following the overnight incubation is completed by a user in about one hour.

2. The rapid method of claim 1, wherein after adding the blocking solution to the microtiter wells, the microtiter plate can be stored, after which it can be shipped to a user at another site, as the assay start time begins when the viral substrate is added to the microtiter wells.

3. The rapid method of claim 1, wherein the virus and its viral substrate, and the host receptor protein to which the viral substrate binds is selected from SARS-CoV-2:Spike protein and angiotensin-converting enzyme 2 (ACE2), SARS-CoV-2:Spike protein and other host protein candidates; Betacoronaviruses (lineage A):Hemagglutinin (HA) esterase and sialic acid receptors; Influenza:HA protein and sialic acid receptors and HA2; Murine hepatitis virus (MHV):Spike protein and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1); and Middle East respiratory syndrome (MERS):Spike protein and dipeptidyl peptidase 4 (DPP4/CD26).

4. The rapid method of claim 3, wherein the virus and its viral substrate have undergone one or more mutations so that the binding between the virus and its viral substrate and the host receptor protein is positively affected resulting in a stronger avidity of the virus and its viral substrate with its host receptor protein which causes a faster color change and a higher reading of the concentration of the viral substrate than what is observed with the virus and its viral substrate that have not undergone one or more mutations, said stronger avidity indicating that the mutated virus and its viral substrate may be more transmissible and/or more virulent than the virus and its viral substrate that have not undergone one or more mutations.

5. The rapid method of claim 3, wherein the virus and its viral substrate have undergone one or more mutations so that the binding between the virus and its viral substrate and the host receptor protein is negatively affected resulting in a weaker avidity of the virus and its viral substrate with its host receptor protein which causes a slower color change and a lower reading of the concentration of the viral substrate than what is observed with the virus and its viral substrate that have not undergone one or more mutations, said weaker avidity indicating that the mutated virus and its viral substrate may be less transmissible and/or less virulent than the virus and its viral substrate that have not undergone one or more mutations.

6. The rapid method of claim 3, wherein the virus and its viral substrate have undergone one or more mutations so that the binding between the virus and its viral substrate and the host receptor protein is unaffected resulting in substantially the same avidity of the virus and its viral substrate with its host receptor protein which causes substantially the same color change and substantially the same reading of the concentration of the viral substrate than what is observed with the virus and its viral substrate that have not undergone one or more mutations, said substantially the same avidity indicating that the mutated virus and its viral substrate may have substantially the same transmissibility and/or the same virulence as the virus and its viral substrate that have not undergone one or more mutations.

7. The rapid method of claim 3, wherein the suspected virus is SARS-CoV-2 and the host receptor protein is ACE2.

8. The rapid method of claim 7, wherein the infection causes COVID-19 disease.

9. The rapid method of claim 1, wherein the specimen is selected from a nasopharyngeal swab, a nares swab, saliva, urine, tears, cerebrospinal fluid, amniotic fluid, serum, plasma, whole blood, bronchopulmonary lavage, vaginal sampling and a rectal/stool sampling obtained from the individual.

10. The rapid method of claim 1, wherein the antibody is a rabbit polyclonal antibody and the HRP-conjugated antibody is an HRP-conjugated anti-rabbit polyclonal goat antibody.

11. The rapid method of claim 7, wherein the binding of SARS-CoV-2 to ACE2-coated microtiter wells is studied in the presence of antibodies contained in convalescent sera or plasma obtained from individuals who have recovered from COVID-19 or from purified monoclonal antibodies.

12. The rapid method of claim 7, wherein the binding of SARS-CoV-2 to ACE2-coated microtiter wells is studied in the presence of drug candidates which may compete for binding and negatively influence the interaction between the viral substrate and its receptor.

13. A test kit for rapid, highly specific and sensitive, point-of-care detection of a virus in an individual suspected of being infected with a virus, comprising: a plurality of microtiter wells in a microtiter plate, said microtiter wells coated with a host receptor protein specific for a suspected virus deposited on surfaces of the plurality of microtiter wells; an antibody directed against the suspected virus; a wash liquid for washing the plurality of microtiter wells and for preparing a mixture consisting of the wash liquid, an HRP-conjugated antibody directed against the antibody, and a specimen obtained from an individual suspected of being infected with the virus, said mixture made into one or more serial dilutions that are deposited atop the coating in the plurality of microtiter wells; a TMB solution; and a STOP solution, wherein the detection of the virus in the specimen is achieved by observing those microtiter wells that undergo a color change, said color change occurring in about five minutes and said detection accomplished by a user in about thirty minutes.

14. The test kit of claim 13, wherein the virus and its viral substrate, and its host receptor protein to which the viral substrate binds is selected from SARS-CoV-2:Spike protein and angiotensin-converting enzyme 2 (ACE2); SARS-CoV-2:Spike protein and other host protein candidates; Betacoronaviruses (lineage A):Hemagglutinin (HA) esterase and sialic acid receptors; Influenza:HA protein and sialic acid receptors and HA2; Murine hepatitis virus (MHV):Spike protein and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1); and Middle East respiratory syndrome (MERS):Spike protein and dipeptidyl peptidase 4 (DPP4/CD26).

15. The test kit of claim 13, wherein the virus and its viral substrate have undergone one or more mutations so that the binding between the virus and its viral substrate and the host receptor protein is positively affected resulting in a stronger avidity of the virus and its viral substrate with its host receptor protein which causes a faster color change and a higher reading of the concentration of the viral substrate than what is observed with the virus and its viral substrate that have not undergone one or more mutations, said stronger avidity indicating that the mutated virus and its viral substrate may be more transmissible and/or more virulent than the virus and its viral substrate that have not undergone one or more mutations.

16. The test kit of claim 13, wherein the virus and its viral substrate have undergone one or more mutations so that the binding between the virus and its viral substrate and the host receptor protein is negatively affected resulting in a weaker avidity of the virus and its viral substrate with its host receptor protein which causes a slower color change and a lower reading of the concentration of the viral substrate than what is observed with the virus and its viral substrate that have not undergone one or more mutations, said weaker avidity indicating that the mutated virus and its viral substrate may be less transmissible and/or less virulent than the virus and its viral substrate that have not undergone one or more mutations.

17. The test kit of claim 13, wherein the virus and its viral substrate have undergone one or more mutations so that the binding between the virus and its viral substrate and the host receptor protein is unaffected resulting in substantially the same avidity of the virus and its viral substrate with its host receptor protein which causes substantially the same color change and substantially the same reading of the concentration of the viral substrate than what is observed with the virus and its viral substrate that have not undergone one or more mutations, said substantially same avidity indicating that the mutated virus and its viral substrate may have substantially the same transmissibility and/or same virulence as the virus and its viral substrate that have not undergone one or more mutations.

18. The test kit of claim 14, wherein the suspected virus is SARS-CoV-2 and the host receptor protein is ACE2.

19. The test kit of claim 13, wherein the specimen is selected from a nasopharyngeal swab, a nares swab, saliva, urine, tears, cerebrospinal fluid, amniotic fluid, serum, plasma, whole blood, bronchopulmonary lavage, vaginal sampling and a rectal/stool sampling obtained from the individual.

20. The test kit of claim 18, wherein the infection causes COVID-19 disease.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] A fuller understanding of the invention can be gained from the following description when read in conjunction with the accompanying drawings, in which:

[0020] FIG. 1 is a bar graph showing detection of two different concentrations of a SARS-CoV-2 Spike protein using arbitrary dilutions of a polyclonal rabbit anti-Spike antibody and an HRP-conjugated goat anti-rabbit antibody, which illustrates the surprising specificity of the invention; and

[0021] FIG. 2 is a bar graph showing the surprising sensitivity when the concentration of a SARS-CoV-2 Spike protein is held constant, while changing the concentration of ACE2.

DETAILED DESCRIPTION OF THE INVENTION

[0022] As used herein, the terms “COVID-19,” “SARS-CoV-2,” and “novel coronavirus” are meant to be interchangeable.

[0023] As used herein, the terms “host cell receptor,” “host receptor protein,” “viral host receptor protein,” “cellular host receptor protein” and “ligand” are meant to be interchangeable.

[0024] As used herein, the words “infection” and “disease” are meant to be interchangeable.

[0025] As used herein, a “user” is defined as an individual that wishes to determine whether he/she, or some other individual, is infected with a virus, such as the SARS-CoV-2 virus. Thus, a user includes, without limitation, front-line workers such as EMT technicians, police officers, firemen, health care workers, doctors, nurses, or any other individual wishing to determine viral status for themselves or others.

[0026] The present invention provides a rapid, highly specific and sensitive assay that demonstrates the interaction between a virus and its cellular host receptor protein. This interaction may be shown for many different types of viruses, including, without limitation, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus (HIV), respiratory syncytial virus (RSV) and human papilloma virus (HPV).

[0027] In an embodiment of the present invention, viruses and their viral substrates, and the host receptor proteins to which the viral substrates bind (virus:viral substrate and host receptor protein), which may be rapidly assayed by the methods and the kit of the present invention include, without limitation, SARS-CoV-2:Spike protein and ACE2; SARS-CoV-2:Spike protein and other host protein candidates; Betacoronaviruses (lineage A):Hemagglutinin esterase and sialic acid receptors; Influenza:Hemagglutinin protein and sialic acid receptors and HA2; Murine hepatitis virus (MHV):Spike protein and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1); and Middle East respiratory syndrome (MERS):Spike protein and dipeptidyl peptidase 4 (DPP4/CD26).

[0028] In this embodiment, the rapid viral assay comprises the following steps. First, a host receptor protein contained in a coating buffer, which is chosen based on previous studies, or selected from a panel of protein candidates, is used to coat microtiter wells at various concentrations. In addition to proteins, other molecules may be used, including, without limitation, aptamers, affimers, polysaccharides, DNA/RNA, and the like. Next, a viral substrate is added at various concentrations. The viral substrate can include, without limitation, a purified viral protein, a purified, recombinantly-generated viral protein, or intact virus-live-attenuated, heat-killed viable virus, or a virus-like particle. Purified, recombinantly-generated viral proteins and virus/virus-like particles can include, without limitation, a Spike protein, a nucleocapsid protein, an envelope protein, an RNA-dependent RNA polymerase (RdRP)-protein for SARS-CoV2 or a hemagglutinin esterase for influenza. After washing, any remaining viral substrate that still is bound to the coating antigen then is detected with a primary antibody directed against the viral substrate and an HRP-conjugated secondary antibody directed against the primary antibody.

[0029] With the use of this assay, the quality of the binding can be studied by changing the biological matrix, salt concentration and/or adding detergents, so that hydrophilic/hydrophobic interactions may be assessed. Furthermore, additional molecules may be added immediately before, during and after the viral substrate is added in order to determine the nature of the binding. By focusing on the interaction between the virus and its receptor protein, i.e., ligand, various chemicals/therapeutic candidates, such as anti-viral monoclonal/polyclonal antibodies, antibody purified from convalescent sera, hydroxychloroquine, chloroquine, or remdesivir, may be added into the assay, and any effect from the chemical/therapeutic candidate on either the cellular receptor protein/ligand or virus may be ascertained.

[0030] In another embodiment of the present invention, there is provided a rapid assay which can determine if an intact virus has been isolated. This may provide insight as to whether an individual has an infection, and if the infectious agent still is intact. This provides essential information to clinicians, as intact virions are a prerequisite for transmission of viral disease. PCR or RT-PCR testing is unable to make this distinction between viable and non-viable viruses, and serology studies, i.e., determination of the presence of IgM/IgG antibodies in serum, also are unable to make this distinction.

[0031] In this embodiment, the rapid viral assay comprises the following steps. First, microtiter wells are coated with a viral host receptor protein candidate, such as, without limitation, ACE2, which is the host receptor protein for SARS-CoV-2. After blocking, a virus is added. After washing with PBS/PBS-Tween, an antibody directed against either the envelope (E) protein, membrane (M) protein, or Spike (S) protein of the virus is added, an antibody then is added, after which an HRP-conjugated antibody directed against the antibody is added, and any bound, intact virions are identified.

[0032] While this assay is able to detect intact virions, it is possible that these virions may not be infectious, i.e., they are empty virions or remnants of viral particles. Thus, in a further embodiment of the present invention, there is provided a rapid assay which can serve as a test to determine if an intact, infectious virus has been isolated.

[0033] In this embodiment, the rapid viral assay comprises the following steps. First, microtiter wells are coated with a viral host receptor protein candidate. After blocking, a virus is added. After washing with a viral lysis buffer, an antibody directed against a nucleocapsid protein, or an RNA-dependent RNA Polymerase (RdRP) protein complex, is added. Next, the liquid from these wells is transferred to wells which are coated with antibody against the same protein (i.e. anti-nucleocapsid/RdRP complex antibody). This pulls down and captures non-structural viral proteins. Importantly, it is necessary to use a capture antibody (i.e., a primary antibody) of a specific species, for example, a rabbit, and a detection antibody (i.e., secondary antibody) from a different species, for example, a goat, as an additional HRP-labeled antibody directed specifically against the detection antibody, may be needed.

[0034] The present invention therefore provides a unique approach of focusing on the interaction between a virus and its host-cell receptor. By “pulling down” the virus and capturing it onto receptor-coated wells, it allows for an entirely new set of questions to be answered. In using this approach, the target antigen used for detection can be changed, so that an entirely different component of the virus may be detected. For example, if an intact virion binds to ACE2-coated wells through the Spike protein, then distant envelope proteins within the viral membrane may be detected. In addition, bound virus may be lysed, allowing the release and detection of genomic material/nucleocapsid/RdRP.

[0035] Utilization of this novel approach may allow for the more accurate determination of infectivity. One of the main drawbacks of PCR and RT-PCR is that viral genomic material may be isolated days to weeks after infection has resolved, which is less likely when antigen-binding assays are utilized. This is an incredibly important distinction to make when employers are requiring their employees to have negative test results before returning to work. The present invention thus provides the ability for individuals suspected of being infected with the virus to provide a sample, and if this sample contains intact virus with functional Spike protein (judged to be functional by its ability to bind to its host-cell receptor), then that individual most likely still is contagious.

[0036] The present invention further provides the ability to determine whether a virus and its viral substrate have undergone one or more mutations. In such a case the binding between the virus and its viral substrate and the host receptor protein may be positively affected, negatively affected, or unaffected, which may indicate altered transmissibility and/or virulence.

[0037] If the binding between the virus and its viral substrate is positively affected, this results in a stronger avidity of the virus and its viral substrate with its host receptor protein, which causes a faster color change and a higher reading of the concentration of the viral substrate than what is observed with the virus and its viral substrate that have not undergone one or more mutations. A stronger avidity indicates that the mutated virus and its viral substrate may be more transmissible and/or more virulent than the virus and its viral substrate that have not undergone one or more mutations.

[0038] If the binding between the virus and its viral substrate is negatively affected, this results in a weaker avidity of the virus and its viral substrate with its host receptor protein, which causes a slower color change and a lower reading of the concentration of the viral substrate than what is observed with the virus and its viral substrate that have not undergone one or more mutations. A weaker avidity indicates that the mutated virus and its viral substrate may be less transmissible and/or less virulent than the virus and its viral substrate that have not undergone one or more mutations.

[0039] If the binding between the virus and its viral substrate is unaffected, this results in substantially the same avidity of the virus and its viral substrate with its host receptor protein, which causes substantially the same color change and substantially the same reading of the concentration of the viral substrate than what is observed with the virus and its viral substrate that have not undergone one or more mutations. Substantially the same avidity indicates that the mutated virus and its viral substrate may have substantially the same transmissibility and/or the same virulence as the virus and its viral substrate that have not undergone one or more mutations.

[0040] By observing the interaction between the Spike protein and the host cell receptor in accordance with the methods of the present invention, the influence that certain therapeutic candidates have on the SARS-CoV-2 virus can be investigated. If, for instance, remdesivir negatively influences the binding of the viral Spike protein to its host cell receptor, then this may prove to be a beneficial relationship to exploit in treating those infected/exposed to the virus.

[0041] Determining the nature of this relationship may also aid in determining which individuals who have recovered from infection have developed neutralizing antibodies. By purifying antibodies from these individuals, one can determine that certain individuals have produced an antibody response that more effectively targets the virus. Finally, this also may prove to be useful for vaccine manufacturers.

EXAMPLES

[0042] The present invention is more particularly described in the following non-limiting example, which is intended to be illustrative only, as numerous modifications and variations therein will be apparent to those skilled in the art.

Example 1

Rapid Binding Assay of ACE2 and SARS-CoV-2 Spike Protein to Detect COVID-19

[0043] Two experiments were conducted to observe the rapidity, specificity and sensitivity of the methods of the invention for detecting and quantifying viruses, as well as to set negative and positive controls for these methods.

[0044] In the first experiment, microtiter wells (Immulon, ThermoFisher, Waltham, Mass.) were coated with 100 μl of 1 μg/well of ACE2 (RayBiotech #230-30165-100) in bicarbonate buffer (Sigma, St. Louis) and incubated overnight at 4° C. The wells were washed and then blocked with 200 μ/well StartingBlock™ (ThermoScientific, Rockford, Ill.). Wells were washed three times with phosphate-buffered saline (PBS, Sigma Aldrich, Pa.) supplemented with 0.05% Tween-20 (PBS-Tw; Fisher Scientific, Pittsburg, Pa.) at room temperature. Next, serial dilutions of Spike protein (SARS-CoV-2 Spike protein recombinant S1 subunit purchased from RayBiotech, #230-01101) in PBS were added to the wells, starting at a concentration of 50 μg/well, and diluting out to 0.8 μg/well. Wells were incubated for 20 minutes at room temperature, and then washed three times with PBS-Tw. Next, rabbit polyclonal antibody (GeneTex #GTX135356) directed against SARS-CoV-2, diluted 1:100 in PBS was added, 100 μl/well, and incubated at room temperature for 20 minutes. Wells were again washed three times with PBS-Tw, and then HRP-conjugated anti-rabbit polyclonal goat antibody (Sigma Aldrich) diluted 1:2,000 in PBS was added, 100 μl/well, and incubated for 20 minutes at room temperature. Wells were again washed three times with PBS-Tw. TMB peroxidase substrate (Sigma Aldrich) then was added, 100 μ/well. Within 10 minutes, 100 μ/well Stop Solution (Thermo Scientific) was added, and ODs were measured at 450 nm on a BIO-RAD iMARK microplate reader.

[0045] As an alternative to reading the OD at 450 nm, a visual, qualitative detection of Spike protein was able to be made within 1-2 minutes after the addition of TMB, i.e., without the use of the microplate reader, by observing a color change in the microwells. Microwells that changed from light blue to dark blue in color indicated the presence of the Spike protein. This color change was fully observable in about five minutes after adding TMB.

[0046] FIG. 1. shows the detection of two different concentrations of Spike protein. In addition to testing the concentration of Spike protein, arbitrary dilutions of polyclonal anti-Spike antibody, and HRP-conjugated anti-rabbit antibody were also selected (1:100 and 1:2,000, respectively). A surprisingly high specificity for Spike protein of at least 98% was observed.

[0047] Once an optimal concentration of Spike protein was selected (1.5 μg/well), the second experiment was conducted. In this experiment, a series of dilutions of ACE2 protein was used to coat microtiter wells, and binding of Spike protein to ACE2 was assessed. FIG. 2 shows that when the concentration of Spike protein was held constant at 1.5 μg/well, more robust binding was detected as the concentration of ACE2 increased, and then it plateaued. A surprisingly high sensitivity of about 96% was shown in this experiment. i.e., about 80 ng of ACE2 was capable of binding about 800 ng Spike protein.

[0048] It is important to note that for the interaction between the Spike protein of SARS-CoV-2 and its host protein receptor ACE2 to be optimally studied, microtiter wells must first be coated with ACE2. Bound Spike protein is next detected. Performing the assay in reverse, i.e., coating wells with the Spike protein and then adding ACE2, places significant limitations on one's ability to study this interaction, and in doing so, does not allow one to determine if an individual is infected with the virus.

[0049] While specific embodiments have been described in detail, it will be appreciated by those skilled in the art that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure. Accordingly, the particular embodiments disclosed are meant to be illustrative only and not limiting as to the scope of the (device) and method described herein, which is to be given the full breadth of the appended claims and any and all equivalents thereof.