CORONAVIRUS IgG/IgM MULTIPLEXED DUAL PATH IMMUNOASSAY DEVICE

Abstract

Test cells with first and second sorbent materials defining a first flow path for a solution, a second flow path distinct from the first flow path for a sample, and a test site with immobilized antigens or antibodies or other ligand-binding molecules located at the junction of the sorbent materials for identifying one or more ligands. In one embodiment, a single highly sensitive immunoassay device is provided that detects the presence in a body fluid sample of two or more COVID-19 (Coronavirus disease 2019) antibodies including immunoglobulin M (IgM) and/or immunoglobulin G (IgG) antibodies to nucleocapsid protein (NP) and spike protein receptor binding domain (RBD), and optionally spike protein S1 subunit (S1) COVID-19 virus antigens. The immunoassay device is sensitive in detecting early infection using IgM antibody detection and continuing infection using IgG antibody detection. Additionally, successful inoculation is distinguished from infection after inoculation by comparing NP and RBD results.

Claims

1. A test device for determining the presence of at least two different antibodies of a particular virus in a liquid sample, comprising: a first sorbent strip having a first location for receiving a solution and defining a first migration path; a first marker conjugate adapted to move along said first migration path and bind to said at least two different antibodies; a second sorbent strip distinct from said first sorbent strip and having a second location for receiving the liquid sample and defining a second migration path; a first test zone having first and second test sites located on or in one at least one of said first sorbent strip and said second sorbent strip, said first test site having an immobilized first ligand binding mechanism for a first of said at least two different antibodies of the particular virus and said second test site having an immobilized second ligand binding mechanism for a second of said at least two different antibodies of said particular virus and said first and second sorbent strips touching each other at said first test zone, wherein said second location is removed from said first and second test sites such that sample applied to said second location requires time to migrate to said first and second test sites and does not immediately wet said first and second test sites.

2. The test device according to claim 1, wherein said virus is a COVID-19 (Severe acute respiratory syndrome coronavirus-2) virus.

3. The test device according to claim 2, wherein the test device determines the presence of at least three different antibodies of a virus and includes a third test site located adjacent the second test site, said third test site having an immobilized third ligand binding mechanism for a third of said at least three different antibodies.

4. The test device according to claim 2, wherein said two different antibodies include antibodies against a nucleocapsid protein (NP) and against a spike protein receptor binding domain (RBD) of COVID-19 IgG antibodies.

5. The test device according to claim 2, wherein said two different antibodies include antibodies against nucleocapsid protein (NP) and against spike protein receptor binding domain (RBD) of COVID-19 IgM antibodies.

6. The test device according to claim 3, wherein said three different antibodies include an antibody against nucleocapsid protein (NP), and against spike protein S1 subunit (S1), and against spike protein receptor binding domain (RBD) of COVID-19 virus IgG antibodies.

7. The test device according to claim 3, wherein said three different antibodies include antibodies against a nucleocapsid protein (NP), against a spike protein S1 subunit (S1), and against a spike protein receptor binding domain (RBD) of COVID-19 virus IgM antibodies.

8. The test device according to claim 2, further comprising a third sorbent strip which receives the solution and defines a third migration path; wherein the third sorbent strip contains a second marker conjugate different than said first marker conjugate second sorbent strip touches the third sorbent strip at a second test zone having a plurality of additional test sites for a plurality of additional different antibodies to the same virus.

9. The test device according to claim 8, wherein the first test zone includes antigens to a nucleocapsid protein (NP) and a spike protein receptor binding domain (RBD) of COVID-19 IgG antibodies and the second test zone includes antigens to a nucleocapsid protein (NP) and a spike protein receptor binding domain (RBD) of COVID-19 IgM antibodies.

10. The test device according to claim 8, wherein the first test zone includes antigens to a nucleocapsid protein (NP), a spike protein S1 subunit (S1), and a spike protein receptor binding domain (RBD) of COVID-19 virus IgG antibodies, and the second test zone includes antigens to a nucleocapsid protein (NP), a spike protein S1 subunit (S1), and a spike protein receptor binding domain (RBD) of COVID-19 virus IgM antibodies.

11. The test device according to claim 8, wherein the second sorbent strip includes depletion molecules or conjugates located between said second location and said first test zone for broadly depleting IgG antibodies and/or between said second location and said second test zone, for broadly depleting IgM antibodies.

12. The test device according to claim 9, wherein the second sorbent strip includes depletion molecules or conjugates located between said second location and said first test zone for broadly depleting IgG antibodies and/or between said second location and said second test zone, for broadly depleting IgM antibodies.

13. The test device according to claim 8, wherein the second sorbent strip includes depletion molecules or conjugates located between said second location and either one or both of said first test zone and said second test zone comprising antigens or conjugates that attach to Human Coronavirus HKU1, 229E, NL63, and OC43 antigens.

14. The test device according to claim 9, wherein the second sorbent strip includes depletion molecules or conjugates located between said second location and either one or both of said first test zone and said second test zone comprising antigens or conjugates that attach to Human Coronavirus HKU1, 229E, NL63, and OC43 antigens.

15. The test device according to claim 10, wherein the second sorbent strip includes depletion molecules or conjugates located between said second location and either one or both of said first test zone and said second test zone comprising antigens or conjugates that attach to Human Coronavirus HKU1, 229E, NL63, and OC43 antigens.

16. The test device according to claim 11, wherein the second sorbent strip includes depletion molecules or conjugates located between said second location and either one or both of said first test zone and said second test zone comprising antigens or conjugates that attach to Human Coronavirus HKU1, 229E, NL63, and OC43 antigens.

17. The test device according to claim 2, wherein the test device determines the presence of at least four different antibodies of a virus and includes a third test site located adjacent the second test site and a fourth test site located adjacent the third test site, said third test site having an immobilized third ligand binding mechanism for a third of said at least three different antibodies, and the fourth test site having an immobilized fourth ligand binding mechanism for a fourth of said at least four different antibodies, wherein said four different antibodies include antibodies against a nucleocapsid protein (NP), against a spike protein S1 subunit (S1), against a spike protein S2 subunit (S2) and against a spike protein receptor binding domain (RBD) of COVID-19 virus IgM or COVID-19 virus IgG antibodies.

18. A method using a test device according to claim 4, comprising: obtaining a body fluid or sample from a subject inoculated with a vaccine against COVID-19; applying the body fluid or sample to the second location of the test device; applying a solution to the first location of the test device; detecting the presence or lack of presence of positive signals at at least the first and second site; based on the detecting, determining whether or not the subject is presently infected with the COVID-19 virus.

19. A method according to claim 18, wherein said first site includes antigens to a nucleocapsid protein (NP) and said second site includes antigens to a spike protein receptor binding domain (RBD) of COVID-19 IgG antibodies or to spike protein S1, wherein the lack of a positive signal for the first site and the presence of a positive signal for the second site indicates effective vaccination, and wherein the presence of a positive signal for the first site and the presence of a positive signal for the second site indicates infection.

20. A method of monitoring post-vaccinated patients regularly over a period of months, comprising: obtaining nasal swabs from one or both nostrils of a subject at least twice over the period of months to obtain samples; applying the samples to test devices having a test line having antigens to a spike protein receptor binding domain (RBD) of COVID-19 IgG antibodies or antigens to the S1 protein of COVID-19 IgG antibodies; reading the test devices with a test reader; providing a booster shot to the subject if test reader indicates that the presence of IgG RBD or IgG S1 antibodies has dropped below a predetermined threshold value or by a predetermined numeric factor from a highest reader value recorded for the subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 is a top schematic view of a first embodiment.

[0032] FIG. 1A is a cross-sectional view taken along line 1A-1A of FIG. 1.

[0033] FIG. 1B is a cross-sectional view taken along line 1B-1B of FIG. 1.

[0034] FIG. 2 is a diagram of a COVID-19 IgM/IgG multiplexed immunoassay device test cell.

[0035] FIG. 2A is a view of a test cassette incorporating the test cell of FIG. 2.

[0036] FIG. 3 is a diagram of a confirmatory test kit system utilizing the test device of FIG. 2A.

[0037] FIG. 4A is an instruction manual describing a method of using the test kit system of FIG. 3.

[0038] FIG. 4B is a manual showing a method of reading results from the immunoassay device of FIG. 3.

[0039] FIG. 5 is a chart of possible results provided by the reader of FIG. 3.

[0040] FIG. 6 is a chart of actual test results from samples run through the test kit system of FIG. 3.

[0041] FIG. 7 is a graph of IgM RBD and IgG RBD antibody readings using the test kit system of FIG. 3 versus days since vaccination for a subject vaccinated with a Moderna SARS-Cov-2 Vaccine.

[0042] FIG. 8 is a test cell of FIG. 2A marked with reading values for six antibodies of a COVID-19 as determined by a reader of FIG. 3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Turning now to FIGS. 1, 1A and 1B, an immunoassay device test cell 10 is provided and includes: a T-shaped housing 20 having a top wall 21 defining first and second holes 24, 26, and a window 28; and first and second sorbent or bibulous materials 30, 32 defining perpendicular horizontal flow paths in the housing. The first sorbent material 30 includes at least two and preferably three or four zones and may be made from a plurality of materials. A first zone 31 (sometimes called a filter zone) is located at the first hole 24 and extends to a second zone 33 (sometimes called a test zone) which is located at the junction of the “T”. The first zone 31 preferably includes a filter 31a, a pad 31b on or in which a conjugate 39 having desired antigens or antibodies with attached colored markers is deposited and immobilized, and a first portion of a thin membrane or sorbent or bibulous material 30 typically made from nitrocellulose with a plastic backing (not shown). The first zone 31 is adapted to receive a buffer solution, to cause the buffer solution to contact the conjugate, thereby mobilizing the conjugate, and to wick the conjugate-carrying buffer solution to the second zone 33. The second (test) zone 33 includes a second portion of the thin membrane 30 which is preferably printed with a test lines 50a, 50b having immobilized antigens on the membrane as is well known in the art. The test lines 50a, 50b may be seen through the window 28 of clear plastic provided in the housing. An optional third zone 35 (sometimes called a control zone) which includes a third portion of the thin membrane 30 may also be printed with a control line 60 typically containing antibodies to the conjugate antigens (or in some cases antibodies which will bind to conjugate antibodies, or even antigens which will bind to conjugate antibodies) as is well known in the art. Where the third zone 35 is provided, window 28 extends above the control line 60. If desired, an optional fourth zone 37 (sometimes called a reservoir zone) may be provided as a wicking reservoir as is also well known in the art. The fourth zone 37 includes a relatively thicker absorbent paper 31d. Preferably overlying all the zones is a thin, preferably transparent plastic film or card 38a having an adhesive which keeps the sorbent materials in place. The card 38a may be cut with an opening at hole 24 so that it does not block liquid access to the hole 24.

[0044] The second sorbent material 32 may also be made from a plurality of materials and preferably includes two zones 61, 63. The first zone 61 (sometimes called a filter zone) includes a filter or pad 62 and a first portion of a thin membrane or sorbent or bibulous material 32 typically made from nitrocellulose with a backing (not shown). The first zone 61 is located at the second hole 26 and extends to the second zone 63. The second zone 63 includes a second portion of the thin membrane 32 which is in contact with the second zone 33 of the first sorbent material 30. As is seen in FIGS. 1A and 1B, the first sorbent material 30 overlies the second sorbent material 32 such that the membranes are in contact with each other (as opposed to the backings contacting the membranes or each other), and such that the test lines 50a, 50b are effectively located between the membranes. Thus, test lines 50a, 50b could be printed on the second zone 63 of the second sorbent material 32 instead of, or in addition to the second zone 33 of the first sorbent material 30. If desired, a thin plastic film or card 38b having an adhesive which keeps the second sorbent material in place may be utilized.

[0045] Where standard-type nitrocellulose strips with a backing are utilized as the first and second membranes, it is desirable for the membranes to have different pore sizes. For example, and as discussed in more detail hereinafter, if membrane 31 (for the conjugate migration) has a 3μ pore size, and membrane 32 (for the sample migration) has a 15μ pore size, sample applied to membrane 32 will tend to migrate and stay in the sample membrane 32 and will tend not to migrate into the conjugate membrane 31.

[0046] The immunoassay of FIG. 1 is preferably utilized as follows. First, a sample (not shown—and constituting blood, nasal mucus, urine, saliva, feces, or any other bodily fluid or excretion that could contain antibodies) possibly containing antibodies (or antigens) is provided to the second opening or hole 26 and allowed to migrate through the second sorbent material 32 to its second zone 63 which is contact with the second zone 33 of the first sorbent material 30. Optionally, after providing the sample to hole 26, a preferably measured amount of liquid such as a buffer solution may be added to hole 26 to help in the migration of the sample. Regardless, the sample reaches the test lines 50a, 50b which are printed atop the second zone 33 of the first sorbent material or infused therein. After a desired amount of time, by which time the antibodies in the sample (if present) will have had an opportunity to bind to the antigens immobilized at the test lines 50a, 50b, a preferably measured amount of liquid such as a buffer solution (not shown) is added to the first opening 24. After another period of time, sufficient to permit the conjugate to migrate to the test lines 50a, 50b (and control site 60 if provided), the test lines 50a, 50b (and control site 60 if provided) are inspected via window 28 in order to determine whether the sample is “positive” or not. Typically, a “positive” test indicating the presence of the antibody in the sample is obtained when one or both of the test lines 50a, 50b and the control site 60 show lines of color. A “negative” test indicating the lack of the presence of the antibody (or antigen) in the sample is obtained when only the control site 60 shows a line of color.

[0047] The methods of use may be expedited by providing the housing with numbering and/or lettering to indicate that hole 26 is for receiving the sample (and optionally some buffer) and is to be used first, and that hole 24 is for receiving the buffer solution and is to be used second.

[0048] Those skilled in the art will appreciate that the immunoassay 10 functions as follows. Because the test lines 50a, 50b are provided with antigens immobilized on a membrane, if the test sample contains antibodies to the antigens, the antibodies will bind themselves to the antigens at the test line. Thereafter, when the conjugate 39 containing an antigen for the antibody coupled to a colored marker is caused to migrate to the test line, if the test sample contains the antibodies which are now held at one or both of the test lines 50a, 50b, the antigen of the conjugate will bind itself to the antibodies and the colored marker will cause a colored line to appear at one or both of the test sites 50a, 50b. If the test sample does not contain antibodies, the conjugate will not have the antibodies to bind to at the test lines, and no colored line(s) will appear at the test site(s). On the other hand, because the control line 60 is provided with antibodies, the antigens of the conjugate will always bind to the antibodies in the control line 60, thereby causing a colored line to appear at the control site 60 if the conjugate reaches the control site 60. Thus, if sufficient buffer solution is provided to the test cell, a colored line should always appear at the control site 60, thereby providing a control for the test.

[0049] In one embodiment, the test lines 50a, 50b are antigens that will respectively specifically capture two different antibodies of a single virus. For example, the antigens may specifically capture two different proteins of COVID-19 virus antibodies such as the NP and RBD proteins, or the NP and S1 proteins, or the RBD and S1 proteins, etc. In embodiments, and as described in more detail hereinafter, the antigens may specifically capture two different proteins of COVID-19 virus IgG antibodies or two different proteins of COVID-19 virus IgM antibodies. That the different proteins of a single virus may be captured and detected with high sensitivity and specificity is a surprising result, and the information that may be gleaned from those results is also surprising. For example, as described in more detail hereinafter, a test showing a lack of antibodies to the NP protein but the presence of antibodies to the RBD and/or S1 proteins can show that the subject has received a vaccination, whereas a test showing the presence of antibodies to the NP protein may show an ongoing COVID-19 viral infection.

[0050] FIG. 2 is a diagram of one embodiment of a COVID-19 IgM/IgG multiplexed immunoassay device test cell. The test cell 2110, as described hereinafter provides the surprising results that antibodies to multiple proteins of a single virus are detected with high sensitivity and specificity from a single blood droplet sample. More particularly, test cell 2010 includes a first sorbent strip 2030a with marker conjugate 2039a, a test zone 2050a with test lines 2050a-1, 2050a-2, 2050a-3, and control line 2060a, a second sorbent strip 2042 for receiving a sample, with the second sorbent strip optionally containing one or more separate depletion molecule zones, e.g., with depletion molecules or conjugates 2041a, 2041b on either side of a sample receiving location 2042a, a third sorbent strip 2030b with marker conjugate 2039b, a test zone 2050b with test lines 2050b-1, 2050b-2 and 2050b-3, and control line 2060b, and an optional fourth sorbent strip 2030c which is coupled to the first and third sorbent strips or integral therewith. In the embodiment of FIG. 2, test lines 2050a-1, 2050b-2 and 2050b-3 contain immobilized antigens that respectively will specifically capture the nucleocapsid protein (NP), spike protein S1 subunit (S1), and spike protein receptor binding domain (RBD) of COVID-19 virus IgM antibodies. By way of example only, the antigens may include recombinant SARS-CoV-2 (2019-nCoV) spike protein (RBD, His Tag) consisting of 234 amino acids with a predicted molecular mass of 26 kDa, a recombinant SARS-CoV-2 (2019-nCoV) nucleocapsid protein (His Tag) consisting of 430 amino acids with a predicted molecular mass of 47 kDa, and a recombinant SARS-CoV-2 (2019-nCoV) spike protein (S1 subunit, His Tag) consisting of 681 amino acids and a predicted molecular mass of 76 kDa. Similarly, test lines 2050b-1, 2050b-2 and 2050b-3 contain immobilized antigens that respectively will specifically capture the nucleocapsid protein (NP), spike protein S1 subunit (S1), and spike protein receptor binding domain (RBD) of COVID-19 virus IgG antibodies. In the embodiment of FIG. 2, marker conjugate 2039a may be a monoclonal anti-IgM (against mu-chain) gold conjugate and conjugate 2039b may be Protein-A Gold conjugate or a similar conjugate that will bind to the FC region of IgG antibodies.

[0051] In accord with one aspect, depletion molecules 2041a and 2041b are optional. When utilized, depletion molecules 2041a are conjugates for broadly depleting IgG antibodies (such as Goat anti-human IgG FC mixed with stabilizing solution containing sucrose, detergent, preservative). Similarly, when utilized, depletion molecules 2041b are conjugates for broadly depleting IgM antibodies. If desired, depletion molecules 2041a which deplete IgG antibodies could be used while depletion molecules 2041b for depleting IgM antibodies are not used so that the IgG test lines actually pick up both IgG and IgM antibodies, whereas the IgM test lines pick up IgM antibodies only. The conjugates may be sprayed on strip 2042 or may be immobilized thereon. In this manner, only IgM antibodies in the sample will travel from the sample receiving site 2042a on second sorbent strip 2042 to test zone 2050a (i.e., the IgG antibodies will be either trapped or will have been rendered inactive by mating with conjugate), while only IgG antibodies in the sample will travel from the sample receiving site 2042a to test zone 2050b. Whether antibodies such as Anti-human IgG or the conjugates are sprayed or immobilized, it may be useful to use white latex particles as part of the depletion conjugate.

[0052] In another embodiment, in addition or as an alternative to depletion zone 2041a containing conjugates for broadly depleting IgG antibodies, depletion zone 2041a may contain (additional) conjugates for depleting antibodies of viruses that are similar to (i.e., cross-reactive with) but different than the COVID-19 antibodies. By way of example, depletion zone 2041a may additionally contain antigens or conjugates that attach to Human Coronavirus HKU1, 229E, NL63, and OC43 antigens. Similarly, in addition or as an alternative to depletion zone 2041b containing conjugates for broadly depleting IgM antibodies, zone 2041b may contain (additional) antigens or conjugates for depleting e.g., Human Coronavirus HKU1, 229E, NL63, and OC43 antibodies. In this manner, the specificity of the test 2010 is increased.

[0053] The immunoassay of FIG. 2 may be utilized as follows. First, a sample (not shown) possibly containing IgG and/or IgM antibodies for COVID-19 is optionally diluted (e.g., with buffer) and provided to the second sorbent strip 2042 at 2042a between the depletion zones 2041a, 2041b. The sample does not immediately wet the test sites but is allowed to take time to migrate from its location of application to the depletion zones 2042a, 2042b, and then to the test sites 2050a, 2050b. If the sample is not first diluted, optionally, after providing the sample to the second sorbent strip, a measured amount of liquid such as a buffer solution may be added to the second sorbent strip to help in the migration of the sample. Regardless, if the sample includes antigens or antibodies that react with the antigens in one or both of the depletion zones, those antibodies are captured at the depletion zones and are depleted from the sample before reaching the respective test lines. To the extent that the antigens of the depletion zones are not immobilized, or loosen from the sorbent strip and travel down to the test sites, many of the reactive sites on the antibodies are occupied with depletion zone recombinant antigens so that they will not bind to the antigen at the test line. Conversely, to the extent that antibodies of interest are present in the sample, they will generally not be depleted significantly by the specific antigens in the depletion zone, but will travel down to the test line and bind to the antigens immobilized at the test lines. A sufficient time after application of the sample to the second sorbent strip of the immunoassay, a liquid such as a buffer solution is added to the first and third sorbent strips 2030a, 2030b (e.g., via the fourth sorbent strip 2030c). The solution is added to a location which permits it to cause the conjugates 2039a, 2039b on the first and third sorbent strips to migrate to the respective test sites (and control sites 2060a, 2060b, if provided), and to bind with the antibodies of the sample (if present) that are captured at the respective test sites. The test sites and control sites are then inspected in order to determine whether the sample is “positive” or not. Typically, a “positive” test indicating the presence of the tested antibody protein in the sample is obtained when both the test site and the control site show spots (lines) of color. A “negative” test indicating the lack of the presence of the antibody protein in the sample is obtained when only the control site shows a line of color. As with the previously described embodiments, the use of the immunoassay apparatus may be expedited by providing a housing for the sorbent strips, with the housing having holes and numbering and/or lettering to indicate that one hole in the housing is for receiving the sample (and optionally some buffer) and is to be used first, and that another hole (or holes) is for receiving the buffer solution that moves the marker conjugate and is to be used second.

[0054] It is noted that the combination of indications at the IgM test site and at the IgG test site provide a large amount of information, and that the information relating to the different specific proteins at the IgM and/or IgG test sites provides even more information. For example, since IgM antibodies are the first antibodies that the human body produced after infection, a positive or relative high signal at the IgM test site relative to the IgG test site may indicate an acute infection. A positive signal at the IgM test site in conjunction with a similar positive signal at the IgG test site may indicate an ongoing infection. A negative signal at the IgM test site and a positive signal at the IgG test site may indicate an infection that is no longer active, and possible immunity. Further, assuming that vaccinations against RBD and/or Spike protein (S1) may become available in the future for COVID-19, it is probable that a negative signal at the NP test line but a positive signal at the S1 and/or RBD test lines will indicate an effective vaccination, whereas a positive with NP antigen shows a natural infection rather than a signal caused by vaccine immunity. In fact, as discussed hereinafter, this scenario has proven to be correct.

[0055] FIG. 2A shows a test cassette 2011 that incorporates the test cell 2010 of FIG. 2. As seen, test cassette 2011 is provided with a housing having a first opening (marked 1) over the one or more sorbent strips which receive the sample (and buffer), a second opening (marked 2) over the one or more sorbent strips which receive the buffer, and third and fourth openings or windows (marked IgM and IgG) over the IgM and IgG test lines (and control lines) from which results may be seen/read.

[0056] FIG. 3 is a diagram of a confirmatory test kit system utilizing the test device of FIG. 2. In particular, test kit system is shown having test device cassette 2011 containing the test device 2010, a safety lancet 2080, a sample vial 2082, a transfer pipette 2083, a sample loop 2084, a vial of buffer solution 2085, a bandage 2086, an RFID card 2087 and a test reader 2088. The reader may be a reader such as the DPP Microreader available from Chembio Diagnostics Gmbh which may provide a relative quantitative reading of test results.

[0057] FIG. 4A is an instruction manual describing a method of using the test kit system of FIG. 3. At 2090a, five drops of buffer solution from buffer vial 2085 are supplied to the sample vial 2082. At 2090b, the safety lancet 2080 is used to perform a fingerstick prick per standard procedures, and the subject (patient) may be given the bandage 2086 for application on the finger. The blood sample is collected with the sample loop 2084. Standard venous phlebotomy procedures are used to collect 10 microliters of blood. At 2090c, the loop containing the blood is immersed into the sample vial with the buffer. The contents are mixed using the loop. At 2090d, the transfer pipette is filled to 100 microliters and delivered to a hole (well) in the housing of the test device cassette 2011 located over the sample site 2042a (labeled “1 Sample+Buffer). At 2090e, the user waits for approximately five minutes to see whether the colored control lines 2060a, 2060b (e.g., colored with soluble dye) lose their color (visually disappear). If so, at 2090f, approximately nine drops of buffer from buffer vial 2085 are added to a buffer well in the housing of the test device cassette 2011 located over strip 2030c (labeled “2 Buffer”). At 2090g, the user waits for approximately ten to fifteen minutes to see whether any of the test lines 2050a-1, 2050a-2, 2050a-3, 2050b-1, 2050b-2, 2050b-3 are visible. At 2090g, the control lines 2060a and 2060b may also be inspected to see whether they are now visible. If they are visible, the test results should be valid. The test results may be viewed by the user or supplied to the test reader 2088 as described with respect to FIG. 5A.

[0058] FIG. 4B is a manual showing one method of reading results from the immunoassay device of FIG. 3. FIG. 4B shows that the test reader 2088 may be cleaned at 2091a, and at 2091b, an RFID card accompanying the test device cassette 2011 may be inserted into the reader. At 2091c, the test reader is activated and the measurement/reading sequence starts at 2091d. At 2091e, the measurement is complete when the test results for each of the six test lines is shown with a relative number.

[0059] FIG. 5 is a chart of possible results provided by the reader of FIG. 3. As seen in FIG. 5, test lines 2050a-1 and 2050b-1 may show a “reactive” (positive) or “non-reactive” (negative) reading result for the COVID-19 nucleocapsid IgM and IgG proteins; test lines 2050a-2 and 2050b-2 may show a “reactive” or “non-reactive” reading result for the IgM and IgG spike protein S1 subunit; and test lines 2050a-3 and 2050b-3 may show a “reactive” or “non-reactive” reading result for the IgM and IgG spike protein receptor binding domain. The particle values displayed may be relative, and a cut-off may be selected for purposes of declaring “reactive” or “non-reactive”. Thus, as shown in FIG. 36B, the cut-off value of twenty-five is selected as the minimum value for “reactive”, whereas any value under twenty-five (e.g., 0 to twenty-four) is declared “unreactive”.

[0060] Using the test kit of FIG. 3, including the reader, seventy-four samples that were previously subject to polymerase chain reaction (PCR) testing for COVID-19 were tested using test cells 2010 as previously described and read by reader 2088. Tables 1A and 1B below provides the readings for each of six test lines (IgM NP, S1, RBD, and IgG NP, S1, RBD). From left to right, the columns show the sample ID #, the PCR COVID-19 data results, the IgM NP, S1, and RBD results from the test cells 2010 (DPP Covid-19), the IgG NP, S1, and RBD results from the test cell 2010 (DPP Covid-19), the IgM result interpretation, the IgG interpretation, and the Total Antibody interpretation. For purposes of Tables 1A and 1B, a reader value of twenty-five or more was considered a “positive” test result, whereas a reader value of less than twenty-five was considered a “negative” test result. In the results interpretation, and IgG or IgM test was considered “positive” if any of the NP, S1 or RBD values was at twenty-five or greater, and a total antibody result was considered positive if either the IgG or IgM test result was positive. The total antibody result was considered negative only when both the IgG and IgM test results were negative. As shown in FIG. 6, which summarizes the results of Tables 1A and 1B, of the seventy-four samples obtained, fifty-six tested “positive” through PCR testing, while eighteen tested negative. The test results from the test cells 2010 adhered exactly to the PCR testing results showing that the test cells had a 100% sensitivity. It is noted that the IgG positive and negative results complied exactly with the PCR testing results, whereas the IgM results were slightly different. This result does not mean that the IgM testing was not as sensitive, as the PCR tests look for virus but does not detect antibodies. Rather, the test results using the test cells described herein provides additional and potentially valuable information. Similarly, the differences among the NP, S1, and RBD values does not necessarily reveal a lack of sensitivity. Rather, it is additional and potentially valuable information that is not provided by the PCR testing. It will be also be appreciated that the “total antibody” result provides a better sensitivity result than the result from any single protein test line. Also, in one aspect higher TgM readings relative to IgG readings may be interpreted as early infection or infection onset, whereas higher IgG readings relative to IgM readings may be interpreted as continuing or past infection as opposed to early infection or onset.

TABLE-US-00001 Table 1A Summary of result for COVID-19 positive and negative samples DPP COVID-19 IgM/IgG Immunostat Confirmatory System PCR NP-Rec S1-Rec RBD-Rec NP-Rec S1-Rec RBD-Rec Results positive Ag Ag Ag Ag Ag Ag Interpretation Sample COVID-19 IgM Pos ≥2.5 IgG Pos ≥2.5 Total ID Data DPP Micro Reader II Value DPP Micro Reader II Value IgM IgG Antibody 1 Positive 111 300 289 203 178 158 POS POS POS 2 Positive 77 113 116 246 225 187 POS POS POS 3 Positive 41 176 215 213 278 236 POS POS POS 4 Positive 149 101 213 297 112 148 POS POS POS 5 Positive 55 49 84 272 29 78 POS POS POS 6 Positive 37 108 178 203 241 235 POS POS POS 7 Positive 72 91 69 180 307 286 POS POS POS 8 Positive 70 151 184 117 267 251 POS POS POS 9 Positive 50 147 152 238 301 255 POS POS POS 10 Positive 42 241 173 171 239 239 POS POS POS 11 Positive 200 204 305 310 314 218 POS POS POS 12 Positive 95 207 233 240 208 201 POS POS POS 13 Positive 52 34 119 338 78 116 POS POS POS 14 Positive 43 59 32 234 161 164 POS POS POS 15 Positive 33 73 159 203 95 169 POS POS POS 16 Positive 75 6 12 277 4 6 POS POS POS 17 Positive 71 192 204 258 244 210 POS POS POS 18 Positive 18 38 51 219 140 246 POS POS POS 19 Positive 159 86 75 104 238 249 POS POS POS 20 Positive 59 188 233 139 61 79 POS POS POS 21 Positive 65 251 262 228 297 269 POS POS POS 22 Positive 41 78 123 245 226 207 POS POS POS 23 Positive 71 3 7 289 3 2 POS POS POS 24 Positive 13 13 31 7 3 26 POS POS POS 25 Positive 107 99 54 236 264 213 POS POS POS R 1 Positive 1 3 3 4 9 43 NEG POS POS R2 Positive 40 140 176 238 14 67 POS POS POS R 3 Positive 7 1 71 38 2 171 POS POS POS R4 Positive 2 1 2 2 3 71 NEG POS POS R 5 Positive 24 13 25 194 9 98 POS POS POS R6 Positive 0 1 63 5 2 163 POS POS POS R7 Positive 2 2 6 28 27 45 NEG POS POS R8 Positive 3 6 36 65 64 270 POS POS POS 189690 positive 21 22 88 16 38 51 POS POS POS 21230 Positive 13 5 17 140 10 12 NEG POS POS

TABLE-US-00002 TABLE 1B Summary of result for COVID-19 positive and negative samples DPP COVID-19 IgM/IgG Immunostat Confirmatory System PCR NP-Rec S1-Rec RBD-Rec NP-Rec S1-Rec RBD-Rec Results positive Ag Ag Ag Ag Ag Ag Interpretation Sample COVID-19 IgM Pos ≥2.5 IgG Pos ≥2.5 Total ID Data DPP Micro Reader II Value DPP Micro Reader II Value IgM IgG Antibody 21231 Positive 62 120 148 79 55 67 POS POS POS 21248 Positive 21 23 99 115 96 136 POS POS POS 21259 Positive 62 129 171 321 132 128 POS POS POS 21271 Positive 41 89 159 277 77 91 POS POS POS 21277 Positive 50 19 54 24 46 11 POS POS POS 21285 Positive 17 9 22 158 86 75 NEG POS POS 21287 Positive 100 39 144 254 16 29 POS POS POS 2000738100 Positive 41 91 110 215 69 67 POS POS POS 2000737900 Positive 29 4 6 184 93 50 IND POS POS 2000738000 Positive 30 8 22 248 141 83 IND POS POS 2000738200 Positive 12 5 4 26 10 5 NEG POS POS 2000738400 Positive 23 24 25 196 96 96 POS POS POS 2000738300 Positive 75 29 42 299 133 73 POS POS POS 2000739300 Positive 40 6 8 203 109 50 POS POS POS 2000739200 Positive 9 3 6 31 14 8 NEG POS POS 2000739600 Positive 42 8 28 222 8 6 POS POS POS 2000738500 Positive 25 4 5 151 22 11 POS POS IND 2000739500 Positive 35 90 78 238 88 72 POS POS POS 2000739400 Positive 30 7 17 233 138 92 POS POS POS 2000740800 Positive 53 23 31 308 167 118 POS POS POS 2000826100 Positive 88 142 202 151 16 31 POS POS POS 205-13 Negative 21 5 3 23 2 4 NEG NEG NEG 05-0394 Negative 3 1 3 3 16 1 NEG NEG NEG 04-0142 Negative 23 5 2 7 3 3 NEG NEG NEG 04-0113 Negative 9 2 2 4 7 3 NEG NEG NEG 01-0106 Negative 11 4 4 4 4 2 NEG NEG NEG 20779 Negative 8 9 3 6 2 3 NEG NEG NEG 20959 Negative 6 2 4 5 3 2 NEG NEG NEG 21115 Negative 7 3 2 11 2 3 NEG NEG NEG 21184 Negative 5 2 3 9 2 2 NEG NEG NEG 21187 Negative 16 1 2 9 1 2 NEG NEG NEG R288286 Negative 3 3 1 4 4 1 NEG NEG NEG R288292 Negative 8 4 1 2 2 1 NEG NEG NEG R288305 Negative 9 2 1 3 1 6 NEG NEG NEG R288307 Negative 7 4 1 3 2 2 NEG NEG NEG R288309 Negative 5 2 1 9 1 8 NEG NEG NEG R288316 Negative 9 4 1 5 2 7 NEG NEG NEG R288322 Negative 6 4 3 4 2 9 NEG NEG NEG R294175 Negative 8 4 2 10 1 8 NEG NEG NEG

[0061] It will be appreciated that in various embodiments, instead of utilizing three separate test lines for testing three different proteins of a single virus such as COVID-19, only two separate test lines may be used for testing different proteins of the virus. By way of example, the test lines may be arranged to test for just the NP and RDB proteins of COVID-19. Similarly, instead of using two or three separate lines for testing two or three different proteins of a single virus, four or more lines for testing for or more different proteins of the same virus may be used. Thus, by way of example only, four lines for testing the NP, S1, S2 and RBD proteins of the COVID-19 virus antibodies may be utilized.

[0062] According to additional embodiments, rather than testing for the presence of both IgG and IgM antibodies to multiple proteins of a single vims, a test may just test for the IgG or IgM antibodies to the multiple proteins of the single virus. Alternatively, the test may test for multiple of IgG, IgM, IgA antibodies to the multiple proteins of the single virus in any combination; e.g., IgG and IgM, IgG and IgA, IgM and IgA, IgG, IgM and IgA.

[0063] As previously mentioned, various valuable results may be obtained from a test device such as previously described that is capable of simultaneously determining the presence of two or more antibodies to a single virus. In the case of the COVID-19 virus, and in according to aspects, blood samples are taken from a healthy subject over a period of approximately ten weeks, starting from a sample taken immediately before vaccination (inoculation) with the Moderna spike-based vaccine. The blood samples are applied to a test cassette 2011 such as shown in FIG. 2 according to the instructions of FIGS. 3 and 4A read by a reader 2088 according to the instructions of FIG. 4b. RBD, NP, and S1 IgG and IgM values are determined. Prior to vaccination, single digit values are obtained for all test results (IgG RBD, IgG NP, IgG S1, IgM RBD, IgM NP, and IgM S1). As seen in FIG. 7 which shows reader values for IgG and IgM RBD antibody values over a seventy-five day period, the readings over the first ten days were in the single digits (with values under twenty-five considered “negative”), and by day eighteen had increased to values of thirty-five and ninety-four for IgM and IgG RBD respectively. A second vaccination shot is administered at day twenty-eight, and antibody values for both IgM RBD and IgG RBD increase significantly through day thirty-eight for the IgM and day forty-five for the IgG. Thereafter, values decrease somewhat and generally plateau, but remain at a level of showing a positive test result. During this seventy-five day period, both the IgG NP and IgM NP results continue to read in the single digits, and the IgG S1 results remain track the RBD results, except that the numbers are slightly lower, and during the end of the period, the IgM S1 drops below the twenty-five value threshold, so that IgM S1 reads negative.

[0064] A test cassette such as shown in FIG. 2 to which a blood sample from the subject is applied at day seventy-eight is shown in FIG. 8. It shows values of two, twelve and twenty-eight for IgM NP, IgM S1 and IgM RBD respectively, and values of one, two hundred ten, and two hundred forty-two for IgG NP, IgG S1 and IgG RBD respectively.

[0065] Meanwhile, starting at seven weeks, oral fluid, nasal wall swab mucus from one or both nostrils, and nasopharyngeal mucus of a subject are obtained from the subject as well and likewise tested for IgG RBD and IgM RBD, resulting in IgG RBD levels similar to the blood sample results, but with IgM RBD values in single digits which is well below the blood sample results.

[0066] From the above, various valuable and surprising results may be obtained. By way of example, and as previously posited, a positive reading of IgG RBD and/or IgM RBD antibody results combined with a negative reading of IgG NP or IgM NP results is indicative of effective vaccination without infection, whereas a positive reading of NP along with positive reading of RBD indicates an ongoing COVID-19 infection. In this manner, and according to methods, it is possible to distinguish between whether a vaccinated individual is presently infected or not. If a vaccinated individual is infected, appropriate action such as treatment and/or quarantining may be implemented. Further, by way of example, in vaccinated individuals, the IgG RBD values are substantially higher than the IgM RBD values and it may be useful to test and consider only IgG values in vaccinated individuals to see whether the individual is effectively vaccinated, has a present infection, or neither. Further yet, by way of example, blood, nasal wall mucus (from nostrils), nasopharyngeal and saliva tests all appear to show similar results, and therefore, in various methods it may be possible to monitor post-vaccinated patients regularly over a long period of time (months and years) through nasal wall swabs (or saliva, blood samples, or nasopharyngeal) using the provided immunoassays and readers to see whether a booster shot is advised. In particular, in one embodiment, a booster shot is considered advisable if the IgG RBD and/or IgG S1 value drops below a threshold value (e.g., twenty-five for the reader) which would otherwise represent a positive immune response. In another embodiment, a booster shot is considered advisable if the IgG RBD and/or IgG S1 value drops by a predetermined factor (e.g., a factor of ten) from the highest value recorded for that individual.

[0067] There have been described and illustrated herein several embodiments of immunoassays and methods of their use. While particular embodiments have been described, it is not intended that the invention be limited thereto, as it is intended that the invention be as broad in scope as the art will allow and that the specification be read likewise. Thus, while the specification discusses ligand binding using antigen/antibody reactions, other ligand binding mechanisms such as aptamer binding, nucleic acid binding, enzymatic binding, etc. may also be used. Also, while specific test-line antigens, marker conjugates, and depletion antigens and conjugates have been described, other antigens, marker conjugates, and depletion antigens and conjugates could be utilized. Further, while the test cells are described as having two test lines for testing for two ligands of a single virus, three test lines for testing for three ligands of a single virus, and six test lines for testing six ligands of a single virus, it will be appreciated that different numbers of lines may be utilized for testing for different numbers of ligands. Further yet, while particular housing arrangements for sorbent strips are described, it will be appreciated that the housing could have different shapes, have different numbers of holes, and the sorbent strips may be laid out differently.

[0068] Those skilled in the art will also appreciate that the housing may be modified in additional ways to include separate windows for each test line. Also, while embodiments were described in conjunction with the use of a buffer solution which is added to the migration path of the conjugate and optionally to the migration path of the sample, it will be appreciated that that one or more buffers may be chosen as desired to be added to the migration paths depending upon the test or tests to be conducted. Thus, buffers such as phosphate buffers or TRIS (tris hydroxymethylaminomethane) buffers are often utilized. However, the disclosure is intended to encompass the use of any diluent including water. In addition, the diluent may, if needed, may be added to and mixed with the sample prior to adding the sample to the sorbent material or the sample may be deposited first and the diluent may be added thereafter. Likewise, any diluent capable of causing conjugate to migrate may be utilized, and may be premixed with the conjugate in a liquid conjugate system, or provided to the migration path for the conjugate in a dry conjugate system. It will therefore be appreciated by those skilled in the art that yet other modifications could be made without deviating from its spirit and scope of the claims.