US20210373019A1

US20210373019A1 - SYSTEMS AND PROCESSES TO SCREEN FOR SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 (SARS-CoV-2) OF 2019 (COVID-19)

Info

Publication number: US20210373019A1
Application number: US17/336,443
Authority: US
Inventors: Melicia R. Gainey; Dalia Natour
Original assignee: Battelle Memorial Institute Inc
Current assignee: Battelle Memorial Institute Inc
Priority date: 2020-06-02
Filing date: 2021-06-02
Publication date: 2021-12-02
Also published as: US20240192212A1; CA3181239A1; US20210373020A1; CA3181246A1; EP4158348A1; EP4158346A1; JP2023528876A; AU2021283288A1; KR20230018522A; JP2023529148A; KR20230018521A; US11921114B2; WO2021247631A1; AU2021283210A1; WO2021247712A1

Abstract

Systems and processes to screen for SARS-CoV-2, which includes a process that uses an alternative antibody for ELISA. The alternative antibody is an alternative to a mouse antibody, thereby expanding the ability to test for COVID-19 using non-human antibodies.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Ser. No. 63/041,551, filed Jun. 19, 2020, having the title SYSTEMS AND PROCESSES TO SCREEN FOR SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 (SARS-CoV-2) OF 2019 (COVID-19), and claims the benefit of U.S. Provisional Patent Application Ser. No. 63/033,276, filed Jun. 2, 2020, having the title SYSTEMS AND PROCESSES TO SCREEN FOR SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 (SARS-CoV-2) OF 2019 (COVID-19), the disclosures of which are hereby incorporated by reference.

BACKGROUND

Field of the Disclosure

The present disclosure relates generally to Severe Acute Respiratory Syndrome (SARS) Coronavirus 2 (Cov-2) and, more particularly, to systems and processes to screen for SARS-CoV-2.

Description of Related Art

Screening for a virus, such as the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) of 2019 (COVID-19), can be done using Enzyme Linked Immunosorbent Assay (ELISA). ELISA involves at least one antibody with specificity for a particular antigen. Consequently, during a pandemic (such as the COVID-19 pandemic) there can be a shortage of supplies needed for ELISA.

SUMMARY

The present disclosure provides systems and processes to screen for SARS-CoV-2. Briefly described, one embodiment comprises a process that uses an alternative antibody for Enzyme Linked Immunosorbent Assay (ELISA). Specifically, the alternative antibody is an alternative to a human antibody, thereby expanding the ability to test for COVID-19 using non-human antibodies.
One embodiment of a process comprises coating a plate with a cell culture or other suitable growth medium that is suitable as hosts for growing viruses. For some embodiments, the plate is coated with one or more layers (preferably, a monolayer) of VERO E6 cells. The coated plate is then inoculated with a predetermined amount of inoculum. In some embodiments, the inoculum is a serum from a subject, preferably a diluted serum. For other embodiments, the inoculum is a serum-included inoculum that comprises a diluted serum and a predetermined amount of a virus, such as, for example, Severe Acute Respiratory Syndrome C.oronavirus-2 (SARS-CoV-2). Preferably, the predetermined amount of SARS-CoV-2 is a predetermined number of fifty-percent-tissue-culture-infective-dose-assays-per-millileter (TCID₅₀/mL) SARS-CoV-2. In another embodiment, the inoculum is a diluted virus (e.g., SARS-CoV-2).
In embodiments where the inoculum includes a serum (i.e., a microneutralization assay), the serum is diluted (as indicated above) and is incubated with a constant concentration of SARS-CoV-2 (known as “neutralization”) for an initial incubation period at a controlled incubation temperature. The incubation temperature is, preferably, approximately thirty-seven degrees Celsius, give or take approximately two degrees Celsius (˜37±2° C.). The initial incubation period in one embodiment is approximately one hour (˜1 hr).
In embodiments without serum or after the initial incubation period in embodiments with serum, the growth medium is removed from the VERO E6 cells, in its place, the inoculum replaces the growth medium. For one embodiment, the fresh inoculum medium includes the virus sample that is being tested (e.g., SARS-CoV-2). Preferably, the sample is diluted to 2000 (or 2e3) TCID₅₀/mL. Thereafter, the sample can be further diluted to 1:100. If a more diluted sample is desired, then the 1:100 dilution can be further serially titrated down two-fold to 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, and so on. It should also be appreciated that the dilution need not be two-fold but, instead, can be other multiples, such as 1:500, 1:600, 1:700, 1:900, 1:1000, 1:1250, 1:1500, 1:2000, 1:4000; 1:8000, 1:10000, 1:12000, 1:16000, 1:20000, and so on.
The inoculum is incubated at a controlled incubation temperature (e.g., ˜37±2° C.) until an expiration of a total incubation period (e.g., ˜16 hr, ˜24 hr, ˜40 hr, ˜46 hr, ˜48 hr, etc.). For some embodiments, the incubation environment comprises a carbon-dioxide (CO₂) content of approximately five percent, give or take two percent (˜5±2%) CO₂.
After the total incubation period, the inoculum medium is removed and the plate is washed or rinsed using a solution, such as, for example, a Hanks Buffered Salt Solution (HBSS), which is thereafter removed. The plate can be washed multiple times (e.g., three (3) times, five (5) times, etc.). The plate is then fixed using a fixative (e.g., acetone, methanol, formalin, etc. or combinations thereof) (e.g., eighty percent (80%) cold acetone, which is commonly available for purchase). At this point, the fixed plate comprises a fixed amount of the virus under study.
A primary antibody is added to the fixed plate. Upon adding the primary antibody (preferably diluted), the plate is again incubated at an incubation temperature (e.g., ˜37±2° C.) for ˜60±5 minutes. During this particular incubation period, the added primary antibody binds to the virus and forms a virus-antibody complex. For some embodiments, the primary antibody is one selected from the group consisting of: (a) Mouse Species Coronavirus (COVID-19, MERS, and SARS-CoV NP) Antibody, Catalog Number HM1056 (“E3”), from EastCoast Bio, Inc., PO Box 489, North Berwick, Me. 03906, USA (“EastCoast Bio”); (b) Mouse Species Coronavirus (COVID-19, MERS, and SARS-CoV NP) Antibody, Catalog Number HM1057 (“E1”), from EastCoast Bio; E3 in combination with E1 (“E3/E1”); (d) Mouse Species Anti-SARS-Cov-2 NP mAb, clone 4B21, Catalog Number CABT-CS027 (“C4”), from Creative Diagnostics, 45-1 Ramsey Road, Shirley, N.Y. 11967, USA (“Creative Diagnostics”); (e) Mouse Species Anti-SARS-Cov-2 NP mAb, clone 4B21, Catalog Number CABT-CS026 (“C5”), also from Creative Diagnostics; and (f) C4 in combination with C5 (“C4/C5”). It should be appreciated that the primary antibody can be any of the individual antibodies that are shown in FIG. 2A or 2B, or any combination of the antibodies in FIGS. 2A and 2B. In an alternative embodiment, the primary antibody is a horseradish peroxide (HRP)-conjugated primary antibody. Depending on the embodiment, the primary antibody or HRP-conjugated primary antibody is prepared in ELISA blocking buffer and diluted to 1:100, 1:200, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:1250, 1:1500, 1:1600, 1:2000, 1:3000, 1:3200, 1:4000, 1:5000, 1:6400, 1:7000, 1:7500, 1:8000, 1:9000, 1:10000, 1:11000, 1:12000, 1:12500, 1:12800, 1:13000, 1:15000, 1:16000, 1:17000, 1:17500, 1:19000, 1:20000, 1:21000, 1:23000, 1:25000, 1:27000, 1:29000, 1:31000, 1:33000, or any other desired dilution. The plate is then washed again with a wash buffer or rinsate (e.g., HBSS or other suitable rinsate).
For some embodiments, a secondary antibody is added to the plate and incubated at ˜37±2° C. for another incubation period (e.g., ˜60±5 min). Similar to the primary antibody or the HRP-conjugated primary antibody, the secondary antibody is prepared in ELISA blocking buffer and diluted to a desired dilution (e.g., 1:100, 1:200, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:1250, 1:1500, 1:1600, 1:2000, 1:3000, 1:3200, 1:4000, 1:5000, 1:6400, 1:7000, 1:7500, 1:8000, 1:9000, 1:10000, 1:11000, 1:12000, 1:12500, 1:12800, 1:13000, 1:15000, 1:16000, 1:17000, 1:17500, 1:19000, 1:20000, 1:21000, 1:23000, 1:25000, 1:27000, 1:29000, 1:31000, 1:33000, etc.). Again, the secondary antibody can be any of the antibodies shown in FIG. 2A, or any combination of those antibodies. After the incubation period for the secondary antibody, the plate is washed (e.g., 3 times, 5 times, etc.).
It should be appreciated that, for some embodiments, the secondary antibody is optional.
After washing the plate, a solution having a chromogenic substrate is applied. The chromogenic substrate binds to the horseradish peroxidase, thereby facilitating detection of the virus-antibody complex. For some embodiments the solution is ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) solution, while in other embodiments the solution is TMB (3,3′,5,5′-Tetramethylbenzidine) solution. Both ABTS substrate and TMB substrate are known in the art and, therefore, further details of ABTS and TMB are omitted herein. For purposes of this disclosure, ABTS substrate, ABTS solution, solution with ABTS, and solution with ABTS substrate are all used interchangeably. Similarly, TMB substrate, TMB solution, solution with TMB, and solution with TMB substrate are also used interchangeably. After application of the chemogenic substrate, the plate is incubated for ˜30±5 min (preferably, at ˜37±2° C. in ˜5±2% CO₂), to allow sufficient reaction between the chemogenic substrate and the virus-antibody complex. A stop solution is applied to stop the reaction.
The plate is then read. More specifically, an optical density (OD) of the plate is read. For a plate that used the ABTS substrate, the OD of the plate is read at 405 nanometers (405 nm) with a 490 nm reference filter. For a plate that used the TMB substrate, the OD of the plate is read at 450 nm with a 650 nm reference filter.
From the OD, the process determines whether or not the OD is greater than a predefined threshold, which provides an indication of whether or not the test is positive for SARS-CoV-2 infection. Specifically, for some embodiments, an average value of a cell culture control (AVG CC) is calculated and an OD reading that is above two standard deviations (2SD) is considered positive for SARS-CoV-2 (meaning, AVD CC+2SD). A fifty percent (50%) neutralizing plate threshold for a microneutralization (MN) assay is determined with an average OD value of a cell culture control (AVG CC) and an average OD of a virus control (AVG VC), such that any value below the 50% neutralization cutoff is considered positive for neutralization of SARS-CoV-2 (meaning, below (AVG VC+AVG CC)/2).
Other systems, devices, methods, features, and advantages will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Many aspects of the disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.

FIG. 1 is a chart showing one embodiment of various reagents, along with their respective suppliers, catalog numbers, and lot numbers.

FIG. 2A is a chart showing one embodiment of VERO E6 cells, SARS-CoV-2 viruses, SARS-CoV-2 naïve human serum, and antibodies, along with their respective suppliers, concentrations, catalog numbers, and lot numbers.

FIG. 2B is a chart showing one embodiment of various antibody panels tested, along with their respective abbreviations, species, suppliers, and catalog numbers.

FIG. 3 is a chart showing one embodiment of software resources used to evaluate experimental data.

FIG. 4A is a chart showing results for one embodiment with a checkerboard titration of primary and secondary antibodies with ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) substrate on Quidel Vero E6 cells.

FIG. 4B is a chart showing average values of the results from FIG. 4A.

FIG. 4C is a chart showing signal-to-noise ratio (SNR) for the values in FIGS. 4A and 4B.

FIG. 4D is a chart showing results for another embodiment with a checkerboard titration of primary and secondary antibodies with ABTS substrate on Quidel Vero E6 cells.

FIG. 4E is a chart showing average values of the results from FIG. 4D.

FIG. 4F is a chart showing SNR for the values in FIGS. 4D and 4E.

FIG. 5A is a chart showing results for one embodiment with a checkerboard titration of primary and secondary antibodies with TMB (3,3′,5,5′-Tetramethylbenzidine) substrate on Quidel Vero E6 cells.

FIG. 5B is a chart showing results for another embodiment with a checkerboard titration of primary and secondary antibodies with TMB substrate on Quidel Vero E6 cells.

FIG. 6 is a chart showing results for one embodiment of a microneutralization (MN) assay using a 1:700 dilution of a primary antibody and a 1:15000 dilution of a secondary antibody with ABTS substrate.

FIG. 7A is a chart showing results for one embodiment with a refined checkerboard titration of primary and secondary antibodies with TMB substrate on Quidel Vero E6 cells.

FIG. 7B is a chart showing SNR results for the values of FIG. 7A.

FIG. 8 is a chart showing results for one embodiment of a MN assay using a 1:12500 dilution of a primary antibody and both a 1:4000 and a 1:8000 dilution of a secondary antibody with TMB substrate.

FIG. 9 is a chart showing results for one embodiment of MN assay using a conjugated primary antibody with ABTS substrate.

FIG. 10 is a chart showing results for one embodiment of fifty-percent-tissue-culture-infective-dose-assays-per-millileter (TCID₅₀/mL, also designated as median-tissue-culture-infectious dose) SARS-CoV-2 using a conjugated primary with ABTS substrate.

FIG. 11A is a chart showing results for one embodiment with a checkerboard titration of conjugated primary antibody with ABTS substrate.

FIG. 11B is a chart showing average values of the results from FIG. 11A.

FIG. 11C is a chart showing SNR for the values in FIGS. 11A and 11B.

FIG. 12A is a chart showing results for one embodiment with a checkerboard titration of conjugated primary antibody with TMB substrate.

FIG. 12B is a chart showing average values of the results from FIG. 12A.

FIG. 12C is a chart showing SNR for the values in FIGS. 12A and 12B.

FIG. 13 is a chart showing results for one embodiment of MN assay using a 1:3000 dilution of a conjugated primary antibody with TMB substrate.

FIG. 14A is a chart showing results for one embodiment with a refined checkerboard titration of conjugated primary antibodies with TMB substrate.

FIG. 14B is a chart showing average values of the results from FIG. 14A.

FIG. 14C is a chart showing SNR for the values in FIGS. 14A and 14B.

FIG. 14D is a chart showing results for another embodiment with a refined checkerboard titration of conjugated primary antibodies with TMB substrate.

FIG. 14E is a chart showing average values of the results from FIG. 14D.

FIG. 14F is a chart showing SNR for the values in FIGS. 14D and 14E.

FIG. 15 is a chart showing results for one embodiment of MN assay using 1:11000 and 1:17000 dilution of a conjugated primary antibody with TMB substrate.

FIG. 16 is a chart showing results for one embodiment of MN assay using 1:10000 dilution of a conjugated primary antibody with TMB substrate.

FIG. 17 is a chart showing results for another embodiment of MN assay using 1:10000 dilution of a conjugated primary antibody with TMB substrate, but with a starting confluence that is greater than approximately ninety percent (>90%).

FIG. 18 is a chart showing results for one embodiment in which additional washing cycles were used.

FIG. 19 is a chart showing results for another embodiment in which additional washing cycles were used.

FIG. 20 is a chart showing results that compare non-human primate (NHP) lung tissue without SARS-CoV-2 (naïve NHP lung tissue) and NHP lung tissue with SARS-CoV-2 (spiked NHP lung tissue).

FIG. 21 is a chart showing results that compare naïve NHP lung tissue and spiked NHP lung tissue, but with the removal of inoculum one (1) hour post absorption.

FIG. 22 is a chart showing a comparison of storage results for fixed plates at various times and various temperatures.

FIG. 23 is a chart showing a comparison of storage results for diluted serum at various times and various temperatures.

FIG. 24 is a chart showing results of a certified titer of MN for one embodiment.

FIG. 25A is a chart showing results of a certified titer of MN for another embodiment.

FIG. 25B is a chart showing results of cytopathic effect (CPE) for the results of FIG. 25A.

FIG. 26 is a chart showing results of cell culture controls of one embodiment of SARS-CoV-2.

FIG. 27A is a chart showing results of performance characteristics in one embodiment with different Vero E6 cells.

FIG. 27B is a chart showing results of performance characteristics in another embodiment with different Vero E6 cells.

FIG. 27C is a chart showing results of performance characteristics in yet another embodiment with different Vero E6 cells.

FIG. 28 is a chart showing a comparison of in situ ELISA titer to CPE titer.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Screening for a virus, such as the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) of 2019 (COVID-19), can be done using Enzyme Linked Immunosorbent Assay (ELISA). ELISA involves at least one antibody with specificity for a particular antigen. When there is no global emergency, such as a pandemic, there is sufficient supply of materials to perform ELISA virus-screening processes. However, as one can imagine, during a pandemic (such as the COVID-19 pandemic) demand for the materials becomes far greater than the supply for ELISA screening processes. The antibody-antigen specificity further exacerbates the supply-and-demand problem because only a limited number of suitable materials can be used during ELISA screening. Furthermore, the problems associated with over-demand is amplified when the cause of the pandemic is a novel virus (such as in COVID-19).
To mitigate this problem, the present disclosure provides alternative antibodies for ELISA, thereby alleviating the supply-and-demand problems that can arise (and have indeed arisen during the COVID-19 pandemic). For some embodiments, one alternative antibody is Mouse Species Coronavirus (COVID-19, MERS, and SARS-CoV NP) Antibody, Catalog Number HM1056 (“E3-Antibody” or “E3”), from EastCoast Bio, Inc., PO Box 489, North Berwick, Me. 03906, USA (“EastCoast Bio”). For other embodiments, another alternative antibody is a combination of the E3-Antibody and Mouse Species Coronavirus (COVID-19, MERS, and SARS-CoV NP) Antibody, Catalog Number HM1057 (“E1-Antibody” or “E1”), from EastCoast Bio (the combination of the E1-Antibody and the E3-Antibody is designated as “E1/E3-Antibody” or simply “E1/E3”). For other embodiments, yet another alternative antibody is a combination of Mouse Species Anti-SARS-CoV-2 NP mAb, clone 4B21, Catalog Number CABT-CS027 (“C4-Antibody” or “C4”), from Creative Diagnostics, 45-1 Ramsey Road, Shirley, N.Y. 11967, USA (“Creative Diagnostics”), and Mouse Species Anti-SARS-CoV-2 NP mAb, clone 7G21, Catalog Number CABT-CS026 (“C5-Antibody” or “C5”), also from Creative Diagnostics (the combination of the C4-Antibody and the C5-Antibody is designated as “C4/C5-Antibody” or simply “C4/C5”). By providing at least three (3) additional alternative antibodies (namely, E3, E1/E3, and C4/C5), this disclosure expands considerably the supply of materials that can be used for ELISA-based COVID-19 testing.
Having provided a broad technical solution to a technical problem, reference is now made in detail to the description of the embodiments as illustrated in the drawings. Although several embodiments are described in connection with these drawings, there is no intent to limit the disclosure to the embodiment or embodiments disclosed herein. On the contrary, the intent is to cover all alternatives, modifications, and equivalents.
FIGS. 1, 2A, and 2B show one embodiment of experimental materials, along with their respective suppliers, concentrations (if applicable), catalog numbers, and lot numbers. FIG. 3 shows one embodiment of software resources that were used to evaluate the experimental data.
Particularly, FIG. 2B shows six (6) different antibody panels tested with the description, abbreviation, species, supplier, and catalog number shown for each antibody panel tested. The antibody panels include:
(1) Mouse Species Coronavirus (COVID-19, MERS, and SARS-CoV NP) Antibody, Catalog Number HM1056 (“E3”), from EastCoast Bio;
(2) Mouse Species Coronavirus (COVID-19, MERS, and SARS-CoV NP) Antibody, Catalog Number HM1057 (“E1”), from EastCoast Bio, in combination with Mouse Species Coronavirus (COVID-19, MERS, and SARS-CoV NP) Antibody, Catalog Number HM1058 (“E2”), also from EastCoast Bio (the combination of E1 and E2 is designated as “E1/E2”);
(3) E1 in combination with E3 (designated as “E1/E3”);
(4) E2 in combination with E3 (designated as “E2/E3”);
(5) Mouse Species Anti-SARS-Cov-2 NP mAb, clone 4B21, Catalog Number CABT-CS027 (“C4”), from Creative Diagnostics, in combination with Mouse Species Anti-SARS-Cov-2 NP mAb, clone 4B21, Catalog Number CABT-CS026 (“C5”), also from Creative Diagnostics (the combination of C4 and C5 is designated as “C4/C5”); and
(6) E1 in combination with C5 (designated as “E1/C5”).
To determine which antibodies or combinations of antibodies can be used to detect SARS-CoV-2 using in situ ELISA, a plate was coated with a monolayer of Vero E6 cells. For some embodiments, the Vero E6 cells were obtained from BEI Resources (“BEI”), which was established by the National Institute of Allergy and Infectious Diseases (“NIAID”) and managed under contract by American Type Culture Collection (ATCC). For other embodiments the Vero E6 cells were obtained from Quidel Corporation of 9975 Summers Ridge Road, San Diego, Calif. 92121, USA (“Quidel”). The different sources of Vero E6 cells are shown in FIG. 2A. Regardless of the source, the Vero E6 cells served as host cells for growing the virus under study.
It should be noted that three (3) different Vero E6 cell lines from BEI and seven (7) different Quidel Vero E6 cell lots were tested, with the Quidel Vero E6 cells showing comparable results. However, plates having the Quidel Vero E6 resulted in data that was difficult to interpret with a starting confluence of seventy percent (70%) and, therefore, a starting confluence of at least ninety percent (>90%) for the Vero E6 cells is preferable for in situ ELISA, regardless of which Vero E6 source is used. Results for less-than-90% starting confluence and results for >90% starting confluence are shown in FIGS. 16 and 17, respectively. The three (3) BEI Vero E6 cell lines and seven (7) Quidel Vero E6 cell lines that were tested are shown in FIGS. 27A, 27B, and 27C, along with their respective lot numbers, passage numbers, expiration dates, starting confluences (in percent), plate IDs, relevant means, standard deviations, SNR, neutralizing plate thresholds, etc.
Continuing, the Vero E6-coated plates were inoculated with the virus under study. For some embodiments, the Vero E6-coated plates were inoculated with 2,000 (2e3) fifty-percent-tissue-culture-infective-dose-assays-per-millileter (TCID₅₀/mL, also designated as median-tissue-culture-infectious dose) SARS-CoV-2 or more. For other embodiments, the Vero E6 cells were inoculated with a mixture of SARS-CoV-2 and human or non-human primate (NHP) serum.
For some embodiments, two (2) 96-wells microtiter Vero E6 plates were prepared in which each plate was fixed with a fixative of eighty percent (80%) acetone and incubated at room temperature. One of the 96-wells configurations was incubated for fourteen (14) hours, while the other of the 96-wells configurations was incubated for twenty-four (24) or forty-eight (48) hours prior to fixation with acetone. Incubation temperature was maintained at approximately 37±2° C. in a humidified incubator with an environment having approximately 5±2% CO₂. The volume per well was approximately 150 microliters (˜150 μL).
After incubation, the plates were fixed by adding an 80% cold acetone. The fixative was then removed and the plates were allowed to air dry in a Class II biological safety cabinets (BSC II) for a maximum time of approximately two (2) hours and twenty (20) minutes (˜140 min total).
Each plate was washed with a wash buffer. Specifically, for some embodiments, each plate was washed at least three (3) times with ˜300 μL/well of wash buffer for each wash. For other embodiments, additional wash cycles (up to five (5) total wash steps) were tested. Results for tests with additional wash cycles are shown in FIGS. 18 and 19. These results showed that there was no clear advantage or disadvantage over three (3) wash cycles.
After the wash, a primary antibody was added. Specifically, ˜297 μL of blocking buffer and ˜3 μL of antibody (for a dilution of 1:100) was then serially titrated to obtain different dilutions. The different dilutions for the primary antibody (namely, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900) are shown in FIGS. 4A through 4F for ABTS substrate (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)). A preferred embodiment used a 1:700 dilution of the primary antibody for ABTS substrate. Furthermore, FIGS. 4A through 4F show secondary antibody dilutions for ABTS substrate, which are discussed in greater detail below.
Corresponding dilutions for the primary antibody (and secondary antibody, discussed below) are shown in FIGS. 5A and 5B for TMB substrate (3,3′,5,5′-Tetramethylbenzidine)). Refined checkerboard titrations of primary (and secondary) antibodies are shown in FIGS. 7A and 7B for TMB substrate on a Quidel Vero E6 cells. Specifically, FIGS. 7A and 7B designated SARS-CoV-2 inoculated human serum (also designated as spiked serum) as VS, while naïve serum (without SARS-CoV-2) is designated as V. As shown in FIGS. 7A and 7B, the primary dilutions for the refined checkerboard titrations were 1:1000, 1:2500, 1:5000, 1:7500, 1:10000, 1:12500, 1:17500, and 1:20000. A preferred dilution of 1:12500 was used for the primary antibody for TMB substrate. Also, as discussed below, the secondary antibody (for TMB substrate) was diluted to 1:2000, 1:4000, 1:8000, 1:12000, 1:16000, and 1:20000.
After the primary antibody was added, the plates were incubated at ˜37±2° C. for ˜60±5 min.
Returning to the in situ ELISA procedure, to the extent that there was a secondary antibody, each plate was washed at least three (3) times with ˜300 μL of wash buffer per well for each wash. The different dilutions of the secondary antibody for ABTS (as noted above) are shown in FIGS. 4A through 4F, with a corresponding microneutralization (MN) assay for a preferred dilution pair shown in FIG. 6. The different dilutions of the secondary antibody for TMB (as noted above) are shown in FIGS. 5A through 5B and 7A through 7B, with a corresponding MN assay for a preferred dilution pair shown in FIG. 8.
After washing the plates three (3) times, the secondary antibody (diluted as noted above) was added and incubated at ˜37±2° C. for ˜60±5 min. For the secondary antibody, the volume of blocking buffer for each plate was ˜11 mL, with the volume of anti-mouse immunoglobulin G conjugate being ˜11 μL. A preferred embodiment used the 1:1500 dilution of the secondary antibody and ABTS substrate, and 1:4000 or 1:8000 dilution of the secondary antibody and TMB substrate.
In yet other embodiments, horseradish peroxide (HRP)-conjugated primary antibody was tested for both ABTS substrate and TMB substrate. The results for HRP-conjugated antibody for ABTS substrate are shown in FIGS. 10 and 11A through 11C. For comparison, some results for MN assays using HRP-conjugated primary antibodies for ABTS substrate are shown FIG. 9. The results for HRP-conjugated primary antibody for TMB substrate are shown in FIGS. 12A through 12C and 14A through 14F. Again, for comparison, some results for MN assays for HRP-conjugated primary antibody for TMB substrate are shown in FIGS. 13 and 15.
Returning to the in situ ELISA procedure, each plate was then washed at least three (3) times with ˜300 μL per well of wash buffer for each wash. Thereafter, either ABTS substrate or TMB substrate was then added and the plate was incubated at ˜37±2° C. For ABTS substrate, the incubation period was ˜30±5 min, while the incubation period was ˜10±2 min for TMB substrate. After the relevant incubation period, a stop solution was prepared per the manufacturer's instructions and applied to the plates.
For ABTS substrate, the optical density (OD) of each plate was then read at 405 nanometers (nm) with a 490 nm reference filter. For TMB substrate, the OD of each plate was read at 450 nm with a 650 nm reference. Results of the measured OD for all of the tested dilution combinations (e.g., different primary antibody dilutions, different secondary antibody dilutions, and different HRP-conjugated antibody dilutions) for different substrates (e.g., ABTS substrate, TMB substrate) and comparative MN assays, are shown in FIGS. 4A through 15.
From the results of FIGS. 4A through 15, one can see that the OD values at 14-hours were below 0.7. In other words, both the infected wells and the uninfected wells for all of the titrations exhibited a low OD at 14-hours. At the 14-hour mark, although a few infected wells exhibited an OD that was greater than the OD of its corresponding uninfected well, all of the infected OD exhibited a less-than-two-fold OD when compared to its corresponding uninfected well. Stated differently, viral infection of COVID-19 could not be readily determined at 14-hours of incubation.
Additionally, TMB substrate was found to be more sensitive than ABTS substrate and, thus, TMB substrate was used in a preferred embodiment. Also, the comparative results shown in FIGS. 9, 13, and 15 demonstrate that the HRP-conjugated antibodies did not perform as well as the individual primary antibodies and, thus, the individual primary antibodies were preferable over the HRP-conjugated antibodies.
Further tests were also performed to determine whether or not the in situ ELISA could determine viral titer in NHP lung tissue. These tests compared naïve NHP lung tissue homogenate and spiked lung tissue homogenate (spiked with 1e5 TCID₅₀/mL SARS-CoV-2). Both the naïve homogenate and the spiked homogenate were incubated for approximately 44 hours and, thereafter, fixed in accordance with the procedures set forth above. As shown in the results of FIG. 20, there was insufficient variability between the naíve tissue and the spiked tissue, thereby indicating non-specific binding of the primary antibody to the fixed cells. Consequently, the tests were repeated with an additional step of removing the inoculum that contained the tissue homogenate after one (1) hour of absorption. Results from the repeated tests are provided in FIG. 21, which shows that the removal of the inoculum containing the tissue homogenate reduces non-specific binding. Consequently, when in situ ELISA is used for tissue viral load analysis, background non-specific binding should be subtracted by using naïve tissue homogenate as a negative control for the assay.
Next, storage conditions for the fixed plates were also tested by fixing the plates with cold 80% acetone and storing the fixed plates at temperatures of: (a) room temperature (RT); (b) between approximately two degrees Celsius (2° C.) to approximately eight degrees Celsius (8° C.); (c) below approximately negative twenty degrees Celsius (−20° C.); and (d) below approximately negative seventy degrees Celsius (−70° C.). The fixed plates were stored for seven (7) days and thirteen (13) days with the fixative in the plate before conducing the in situ ELISA. A primary dilution of 1:400 and a secondary dilution of 1:1000 for ABTS substrate was used to test the storage conditions for the fixed plates. Although all storage conditions provided acceptable results, the highest noise was measured for room temperature, which indicated that a preferred storage condition for up to two (2) weeks was for temperatures that were below approximately −20° C. FIG. 22 shows results of the various storage conditions of the fixed plates.
One can also determine from the data that, unlike results at 14-hours after incubation, several of the results at 24-hours after incubation exhibited both: (a) a larger-than-0.7 OD; and (b) a greater-than-two-fold increase in OD in the infected wells when compared to the OD of the corresponding uninfected wells. Signal-to-noise ratio (SNR) was calculated as a straightforward division of the OD of the infected well (which is designated as the signal) by the OD of the uninfected well (which is considered as noise). Thus, SNR=OD_infected/OD_uninfected.
As shown from the data, high binding ratios (and consequently high SNR) was observed from the C4/C5 combination, the E1/E3 combination, and the E3-Antibody. These results show that there are now alternative antibody panels that can be used to detect SARS-CoV-2 in a Vero E6 monolayer after 24-hours of infection. These alternative antibody panels provide additional options for manufacturers and, consequently, relieve some of the supply-and-demand problems associated with reagents that are needed for COVID-19 screening.
In yet other embodiments, specific steps in the in situ ELISA process are modified for use in both TCID₅₀assays and MN assays. Insofar as TCID₅₀-related processes are discussed above (in which the inoculum does not include serum), the following description focuses on processes that are applicable to MN assays (in which the inoculum includes both serum and virus).
By way of example, one embodiment of the process applicable to MN assays (designated herein as MN-assay-process for convenience) comprises coating a plate with a monolayer of Vero E6 cells and inoculating the coated plate with a predetermined amount of an inoculum. For some embodiments applicable to MN assays, the inoculum comprises both diluted serum (preferably from a human patient or other suitable subject (e.g., animal)) and a fixed amount of virus (in this case, for Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2)). The actual experimental inoculum included de-identified patient serum samples obtained from a hospital at Ohio State University (OSU), which were numerically labeled (to exclude patient-identifying information (e.g., 1817, 1818, 1827, 1899, 1942, or some other numerical designation that is de-coupled from patient information). Specifically, the de-identified patient samples were from patients who had tested positive for COVID-19 by an acceptable polymerase-chain reaction (PCR).
For microneutralization assays, the diluted serum with virus mixtures were incubated for an initial incubating period of approximately one (˜1) hour, at which point the serum-included inoculum was transferred to the VERO E6 cell plate. Thereafter, the plate was incubated further for a total incubating period of approximately forty-eight (˜48) hours (meaning, the initial incubation period and the additional incubation period totaled ˜48 hours). In the actual experiment, both the initial incubation and the additional incubation occurred at a temperature of ˜37±2° C. and a carbon-dioxide (CO₂) content of ˜5±2% CO₂.
After the total incubation period, the inoculation medium is removed and the plate is washed with Hanks Buffered Salt Solution (HBSS) at approximately one-hundred-and-fifty microliters per well (˜150 μL/well). The HBSS wash is removed and the plate is fixed with approximately eighty percent (˜80%) acetone. The process continues with the steps of adding an antibody to the fixed plate. Although any of the antibodies recited above can be used, the data below shows results for E3 in combination (and substantially equal proportions) with E1 and, more particularly, to the E3/E1 combination diluted to 1:400.
The process continues with applying ABTS substrate or TMB substrate, applying a stop solution to the washed plate, and thereafter reading the optical density of the plate. If the optical density is greater than a predefined threshold (here, approximately 0.7), then the process provides an indication that the sample is positive for COVID-19.
One point to note is that, to test sufficiency of storage conditions for the diluted serum, the diluted serum was refrigerated and stored for three (3) weeks at temperatures of: (a) between approximately two degrees Celsius (2° C.) to approximately eight degrees Celsius (8° C.); (b) below approximately negative twenty degrees Celsius (−20° C.); and (c) below approximately negative seventy degrees Celsius (−70° C.). FIG. 23 shows results from various storage conditions for the diluted serum. As shown in FIG. 23, all storage conditions provided comparable results, thereby indicating that storage conditions were acceptable for up to three (3) weeks for all of the tested conditions.
Further tests were conducted to compare the MN assay results to those obtained using cytopathic effect (CPE) readout. This test plan was conducted using Vero E6 cells and SARS-CoV-2 with different operators preparing different dilution series of each control on multiple separate days. The serum/virus mixtures were inoculated and incubated for ˜48 hours prior to rinsing the monolayer and fixing the plate for Day-1. In an effort to maximize the SNR and reduce any non-specific binding of the primary antibody in wells containing serum for Day-2 and Day-3, the inoculum was removed after one hour of incubation and replaced with fresh inoculation media. The plates were then incubated for ˜48 hours on Day 2 and ˜72 hours on Day 3 prior to rinsing the monolayer and fixing the plate. Primary dilution of 1:400 and secondary dilution of 1:1000 for ABTS substrate was used for comparing the MN assay with CPE readout.
The resulting endpoints and MN₅₀titers for each different dilution series are shown in FIGS. 24, 25A, 25B, and 26. As shown, the CPE readouts were inconclusive, but neutralization was detectable using in situ ELISA, thereby inferring that in situ ELISA is more sensitive than the CPE readout. Removal of the inoculum after the first hour of incubation on the VERO E6 cells did not provide an obvious advantage and may have been detrimental to the SNR as Day-3 plates were treated in this manner and were not reportable.
FIG. 28 shows a general comparison of OD results between in situ ELISA titers as compared to CPE titers under various conditions.
Based on the processes described with reference to FIGS. 4A through 28 and the results shown in FIGS. 4A through 28, in situ ELISA detects SARS-CoV-2 in a more objective manner than previous subjective CPE readouts for both TCID₅₀and MN assays. This is because the in situ ELISA appeared to have a greater sensitivity than CPE readouts. It should be noted, however, that a starting confluence of at least 90% is recommended for reliable SNR. The various embodiments also demonstrated that alternative SARS-CoV-2 antibodies can be used to detect presence of the virus as early as 24-hours post-infection and, further, the nucleoprotein is detectable reliably at 36-hours to 48-hours post-infection. Additionally, the disclosed test results suggest that SNR is better when antibody clones are paired in a 1:1 ratio than when primary antibodies are tested individually. For BEI Resources Vero E6 cells using ABTS substrates, dilution ratios of 1:400 for the primary antibody and 1:1000 for the secondary antibody are preferred. For Quidel Vero E6 cells using TMB substrates, dilution ratios of 1:12500 for the primary antibody and 1:8000 for the secondary antibody are preferred. Moreover, three (3) wash cycles were shown to be sufficient, as no additional advantage was shown with five (5) wash cycles. Also, the analysis of spiked NHP lung tissue compared with naïve NHP lung tissue suggested that the naïve NHP lung tissue be used as a negative control and, thus, be subtracted from the spiked NHP lung tissue. Lastly, storage temperatures of between −20° C. and −70° C. were sufficient to preserve the fixed plates for 7 to 13 days and the diluted serum for up to three (3) weeks.
As shown in the various disclosed embodiments, the present disclosure provides alternative antibodies for ELISA, thereby alleviating the supply-and-demand problems that can arise (and have indeed arisen during the COVID-19 pandemic). Furthermore, the disclosed embodiments provide systems and processes for detecting SARS-CoV-2 using in situ ELISA, which is more sensitive and more objective than CPE readouts. These embodiments improve upon current testing methods.
To reiterate some of the different embodiments, systems and processes to screen for SARS-CoV-2 are disclosed. Briefly described, one embodiment comprises a process that uses an alternative antibody for Enzyme Linked Immunosorbent Assay (ELISA). Specifically, the alternative antibody is an alternative to a human antibody, thereby expanding the ability to test for COVID-19 using non-human antibodies.
One embodiment of a process comprises coating a plate with a cell culture or other suitable growth medium that is suitable as hosts for growing viruses. For some embodiments, the plate is coated with one or more layers (preferably, a monolayer) of Vero E6 cells. The coated plate is then inoculated with a predetermined amount of inoculum. In some embodiments, the inoculum is a serum from a subject, preferably a diluted serum. For other embodiments, the inoculum is a serum-included inoculum that comprises a diluted serum and a predetermined amount of a virus, such as, for example, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Preferably, the predetermined amount of SARS-CoV-2 is a predetermined number of fifty-percent-tissue-culture-infective-dose-assays-per-millileter (TCID₅₀/mL) SARS-CoV-2.
In embodiments where the inoculum includes a serum (i.e., a microneutralization assay), the serum is diluted (as indicated above) and is incubated with a constant concentration of SARS-CoV-2 (known as “neutralization”) for an initial incubation period at a controlled incubation temperature. The incubation temperature is, preferably, approximately thirty-seven degrees Celsius, give or take approximately two degrees Celsius (˜37±2° C.). The initial incubation period in one embodiment is approximately one hour (˜1 hr).
In embodiments without serum or after the initial incubation period in embodiments with serum, the growth medium is removed from the VERO E6 cells, in its place, the inoculum replaces the growth medium. For one embodiment, the fresh inoculum medium includes the virus sample that is being tested (e.g., SARS-CoV-2). Preferably, the sample is diluted to 2000 (or 2e3) TCID₅₀/mL. Thereafter, the sample can be further diluted to 1:100. If a more diluted sample is desired, then the 1:100 dilution can be further serially titrated down two-fold to 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, and so on. It should also be appreciated that the dilution need not be two-fold but, instead, can be other multiples, such as 1:500, 1:600, 1:700, 1:900, 1:1000, 1:1250, 1:1500, 1:2000, 1:4000; 1:8000, 1:10000, 1:12000, 1:16000, 1:20000, and so on.
The fresh inoculum medium is incubated at a controlled incubation temperature (e.g., ˜37±2° C.) until an expiration of a total incubation period (e.g., ˜16 hr, ˜24 hr, ˜40 hr, ˜46 hr, ˜48 hr, etc.). For some embodiments, the incubation environment comprises a carbon-dioxide (CO₂) content of approximately five percent, give or take two percent (˜5±2%) CO₂.
After the total incubation period, the inoculum medium is removed and the plate is washed or rinsed using a solution, such as, for example, a Hanks Buffered Salt Solution (HBSS), which is thereafter removed. The plate can be washed multiple times (e.g., three (3) times, five (5) times, etc.). The plate is then fixed using a fixative (e.g., acetone, methanol, formalin, etc. or combinations thereof) (e.g., eighty percent (80%) cold acetone, which is commonly available for purchase). At this point, the fixed plate comprises a fixed amount of the virus under study.
A primary antibody is added to the fixed plate. Upon adding the primary antibody (preferably diluted), the plate is again incubated at an incubation temperature (e.g., ˜37±2° C.) for ˜60±5 minutes. During this particular incubation period, the added primary antibody binds to the virus and forms a virus-antibody complex. For some embodiments, the primary antibody is one selected from the group consisting of: (a) Mouse Species Coronavirus (COVID-19, MERS, and SARS-CoV NP) Antibody, Catalog Number HM1056 (“E3”), from EastCoast Bio, Inc., PO Box 489, North Berwick, Me. 03906, USA (“EastCoast Bio”); (b) Mouse Species Coronavirus (COVID-19, MERS, and SARS-CoV NP) Antibody, Catalog Number HM1057 (“E1”), from EastCoast Bio; E3 in combination with E1 (“E3/E1”); (d) Mouse Species Anti-SARS-Cov-2 NP mAb, clone 4B21, Catalog Number CABT-CS027 (“C4”), from Creative Diagnostics, 45-1 Ramsey Road, Shirley, N.Y. 11967, USA (“Creative Diagnostics”); (e) Mouse Species Anti-SARS-Cov-2 NP mAb, clone 4B21, Catalog Number CABT-CS026 (“C5”), also from Creative Diagnostics; and (f) C4 in combination with C5 (“C4/C5”). It should be appreciated that the primary antibody can be any of the individual antibodies that are shown in FIG. 2A or 2B, or any combination of the antibodies in FIGS. 2A and 2B. In an alternative embodiment, the primary antibody is a horseradish peroxide (HRP)-conjugated primary antibody. Depending on the embodiment, the primary antibody or HRP-conjugated primary antibody is prepared in ELISA blocking buffer and diluted to 1:100, 1:200, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:1250, 1:1500, 1:1600, 1:2000, 1:3000, 1:3200, 1:4000, 1:5000, 1:6400, 1:7000, 1:7500, 1:8000, 1:9000, 1:10000, 1:11000, 1:12000, 1:12500, 1:12800, 1:13000, 1:15000, 1:16000, 1:17000, 1:17500, 1:19000, 1:20000, 1:21000, 1:23000, 1:25000, 1:27000, 1:29000, 1:31000, 1:33000, or any other desired dilution. The plate is then washed again with a wash buffer or rinsate (e.g., HBSS or other suitable rinsate).
For some embodiments, a secondary antibody is added to the plate and incubated at ˜37±2° C. for another incubation period (e.g., ˜60±5 min). Similar to the primary antibody or the HRP-conjugated primary antibody, the secondary antibody is prepared in ELISA blocking buffer and diluted to a desired dilution (e.g., 1:100, 1:200, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:1250, 1:1500, 1:1600, 1:2000, 1:3000, 1:3200, 1:4000, 1:5000, 1:6400, 1:7000, 1:7500, 1:8000, 1:9000, 1:10000, 1:11000, 1:12000, 1:12500, 1:12800, 1:13000, 1:15000, 1:16000, 1:17000, 1:17500, 1:19000, 1:20000, 1:21000, 1:23000, 1:25000, 1:27000, 1:29000, 1:31000, 1:33000, etc.). Again, the secondary antibody can be any of the antibodies shown in FIG. 2A or 2B, or any combination of those antibodies. After the incubation period for the secondary antibody, the plate is washed (e.g., 3 times, 5 times, etc.).
It should be appreciated that, for some embodiments, the secondary antibody is optional.
After washing the plate, a solution having a chemogenic substrate is applied. The chromogenic substrate binds to the horseradish peroxidase, thereby facilitating detection of the virus-antibody complex. For some embodiments the solution is ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) solution, while in other embodiments the solution is TMB (3,3′,5,5′-Tetramethylbenzidine) solution. Both ABTS substrate and TMB substrate are known in the art and, therefore, further details of ABTS and TMB are omitted herein. After application of the chemogenic substrate, the plate is incubated for ˜30±5 min (preferably, at ˜37±2° C.), to allow sufficient reaction between the chemogenic substrate and the virus-antibody complex. A stop solution is applied to stop the reaction.
The plate is then read. More specifically, an optical density (OD) of the plate is read. For a plate that used ABTS substrate, the OD of the plate is read at 405 nanometers (405 nm) with a 490 nm reference filter. For a plate that used TMB substrate, the OD of the plate is read at 450 nm with a 650 nm reference filter.
From the OD, the process determines whether or not the OD is greater than a predefined threshold, which provides an indication of whether or not the test is positive for SARS-CoV-2 infection. Specifically, for some embodiments, an average value of a cell culture control (AVG CC) is calculated and an OD reading that is above two standard deviations (2SD) is considered positive for SARS-CoV-2 (meaning, AVD CC+2SD). A fifty percent (50%) neutralizing plate threshold for a microneutralization (MN) assay is determined with an average OD value of a cell culture control (AVG CC) and an average OD of a virus control (AVG VC), such that any value below the 50% neutralization cutoff is considered positive for neutralization of SARS-CoV-2 (meaning, below (AVG VC+AVG CC)/2).
Any process descriptions or blocks in flow charts should be understood as being executable out of order from that shown or discussed, including substantially concurrently or in reverse order, depending on the functionality involved, as would be understood by those reasonably skilled in the art of the present disclosure.
Although exemplary embodiments have been shown and described, it will be clear to those of ordinary skill in the art that a number of changes, modifications, or alterations to the disclosure as described may be made. All such changes, modifications, and alterations should therefore be seen as within the scope of the disclosure.

Claims

What is claimed is:

1. A process comprising:

coating a plate with a monolayer of Vero E6 cells;

inoculating the coated plate with a predetermined number of fifty-percent-tissue-culture-infective-dose-assays-per-millileter (TCID₅₀/mL) Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2);

incubating the inoculated plate;

fixing the Vero E6 cells plate with acetone;

adding an antibody to the fixed plate, the antibody being selected from the group consisting of:

Mouse Species Coronavirus (COVID-19, MERS, and SARS-CoV NP) Antibody, Catalog Number HM1056 (“E3”), from EastCoast Bio, Inc., PO Box 489, North Berwick, Me. 03906, USA (“EastCoast Bio”);

E3 in combination with Mouse Species Coronavirus (COVID-19, MERS, and SARS-CoV NP) Antibody, Catalog Number HM1057 (“E1”), from EastCoast Bio;

Mouse Species Anti-SARS-Cov-2 NP mAb, clone 4B21, Catalog Number CABT-CS027 (“C4”), from Creative Diagnostics, 45-1 Ramsey Road, Shirley, N.Y. 11967, USA (“Creative Diagnostics”), in combination with Mouse Species Anti-SARS-Cov-2 NP mAb, clone 4B21, Catalog Number CABT-CS026 (“C5”), also from Creative Diagnostics (“C4/C5”);

applying substrate;

applying a stop solution to the washed plate;

reading an optical density of the plate; and

determining whether the optical density is greater than a predefined threshold.

2. The process of claim 1, wherein incubating the inoculated plate comprises incubating for up to twenty-four (24) hours.

3. The process of claim 1, wherein incubating the inoculated plate comprises incubating for greater than 16 (sixteen) hours.

4. The process of claim 1 further comprising fixing the plate with eighty percent (80%) cold acetone before applying a primary antibody.

5. The process of claim 1, further comprising diluting the antibody to a 1:700 concentration from its initial concentration prior to adding the antibody.

6. The process of claim 1, wherein the predefined threshold is 0.7.

7. The process of claim 1, wherein the predetermined number of TCID₅₀/mL SARS-CoV-2 is 2,000 (or 2e3) TCID₅₀/mL SARS-CoV-2.

8. A process comprising:

coating a plate with a monolayer of Vero E6 cells;

inoculating the coated plate with a predetermined amount of a serum-included inoculum, the serum-included inoculum comprising:

serum from a subject, the serum being diluted; and

a predetermined amount of virus, the virus being Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2);

incubating the inoculated plate for an initial incubating period;

removing the serum-included inoculum after the initial incubation period;

transferring the serum-included inoculum to the plate with the monolayer of Vero E6 cells;

incubating further the inoculated plate until an expiration of a total incubating period;

removing the inoculation medium after the total incubation period;

washing the plate with Hanks Buffered Salt Solution (HBSS) after removing the inoculation medium;

fixing the Vero E6 cells plate with acetone;

adding an antibody to the fixed plate;

applying substrate;

applying a stop solution to the washed plate;

reading an optical density of the plate; and

determining whether the optical density is greater than a predefined threshold.

9. The process of claim 8, wherein the initial incubation period is approximately one (˜1) hour.

10. The process of claim 8, wherein the total incubation period is approximately forty-eight (˜48) hours.

11. The process of claim 8, further comprising diluting the antibody to a 1:700 concentration from its initial concentration prior to adding the antibody to the incubated plate.

12. The process of claim 8, wherein washing the plate with HBSS comprises washing the plate with HBSS at approximately one-hundred-and-fifty microliters per well (˜150 μL/well).

13. The process of claim 8, wherein fixing the Vero E6 cells plate with acetone comprises fixing the Vero E6 cells plate with eighty percent (80%) acetone.

14. The process of claim 8, wherein:

incubating the inoculated plate for the initial incubating period comprises incubating a at a temperature of ˜37±2° C. and a carbon-dioxide (CO₂) content of ˜5±2% CO₂; and

incubating further the inoculated plate until the expiration of the total incubating period comprises incubating at a temperature of ˜37±2° C. and a CO₂content of ˜5±2% CO₂.

15. The process of claim 8, wherein the predefined threshold is 0.7.

16. The process of claim 8, wherein the subject is a human patient.

17. The process of claim 8, wherein the subject is an animal.