Novel Coronavirus Testing

Abstract

The invention proposes a testing device and method for detecting infection with or immunity to SARS-Cov-2 of a subject, the method comprising the following steps: (i) contacting a urine sample from the subject with an application area of a testing device comprising a urine sorption material defining a sequence of said application area, a conjugate area and a testing area, the areas being in direct or indirect capillary flow communication with each other, (ii) allowing the urine sample to flow by capillarity from said application area to said conjugate area, said conjugate area comprising a testing conjugate movably held therein, wherein said testing conjugate comprises or consists of a polypeptide having an amino acid sequence that shares sequence identity of at least 90%, preferably at least 95%, with a SARS-CoV-2 protein or fragment thereof, coupled to a first colored marker, (iii) allowing the urine sample to continue to flow by capillarity to the testing area, said testing area comprising a testing sub-area with immobilized anti-human IgA antibodies, and (iv) determining infection with or immunity SARS-CoV-2 of the subject by visually inspecting the testing sub-area of the testing area for a color build-up, wherein the presence of a color build-up is indicative of said infection with or immunity to SARS-CoV-2.

Claims

1. A method for detecting infection with or immunity to SARS-Cov-2 of a subject, the method comprising the following steps: (i) contacting a urine sample from the subject with an application area of a testing device comprising a urine sorption material defining a sequence of said application area, a conjugate area and a testing area, the areas being in direct or indirect capillary flow communication with each other, (ii) allowing the urine sample to flow by capillarity from said application area to said conjugate area, said conjugate area comprising a testing conjugate movably held therein, wherein said testing conjugate comprises or consists of a polypeptide having an amino acid sequence that shares sequence identity of at least 90%, preferably at least 95%, with a SARS-CoV-2 protein or fragment thereof, coupled to a first colored marker, (iii) allowing the urine sample to continue to flow by capillarity to the testing area, said testing area comprising a testing sub-area with immobilized anti-human IgA antibodies, and (iv) determining infection with or immunity to SARS-CoV-2 of the subject by visually inspecting the testing sub-area of the testing area for a color build-up, wherein the presence of a color build-up is indicative of said infection with or immunity to SARS-CoV-2.

2. The method as claimed in claim 1, wherein the SARS-CoV-2 protein or fragment thereof has an amino acid sequence comprising that of SARS-CoV-2 surface glycoprotein (Coronavirus S protein), of SARS-CoV-2 surface glycoprotein S1 (Coronavirus S1 protein), of SARS-CoV-2 surface glycoprotein S2 (Coronavirus S2 protein), of SARS-CoV-2 RBD protein, of SARS-CoV-2 Nucleocapsid phosphoprotein N (Coronavirus N protein), or of fragments of at least 150 consecutive amino acids thereof.

3. The method as claimed in claim 1 wherein the polypeptide has an amino acid sequence that shares sequence identity of at least 90%, preferably at least 95%, with SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or any fragment of at least 150 consecutive amino acids thereof.

4. The method as claimed in claim 1 wherein said first colored marker is selected among nanometer-sized gold particles and latex particles.

5. The method as claimed in claim 1 wherein said conjugate area further comprises a control conjugate movably held therein, wherein said control conjugate comprises a control polypeptide coupled to a second colored marker and said testing area comprising a control sub-area with immobilized anti-(control polypeptide) antibodies, and wherein the method further comprises, before or after step (iv), the step of (iv′) determining a correct sample flow within the testing device by visually inspecting the control sub-area of the testing area for a color build-up, wherein the presence of a color build-up is indicative of said correct sample flow within the testing device.

6. The method as claimed in claim 5, wherein the control polypeptide is selected among immunoglobulins, preferably IgA, IgG, more preferably non-human IgG, such as rabbit IgG or mouse IgG.

7. The method as claimed in claim 5 wherein said second colored marker is selected among nanometer-sized or colloid gold particles or latex particles.

8. The method as claimed in claim 5 wherein the absence of color build-up in the control sub-area in step (iv′) is indicative that the test result of step (iv) should be disregarded and the method repeated with a fresh testing device.

9. The method as claimed in claim 1, wherein the urine sorption material of the testing device is supported on a backing pad, preferably made of glass fiber, polyester film or a non-woven.

10. A testing device for detecting infection with or immunity to SARS-Cov-2 of a subject, the testing device comprising a urine sorption material defining a sequence of an application area, a conjugate area and a testing area, the areas being in direct or indirect capillary flow communication with each other, wherein said application area is configured for receiving a urine sample of a subject, wherein said conjugate area comprises a testing conjugate movably held therein, wherein said testing conjugate comprises or consists of a polypeptide having an amino acid sequence that shares sequence identity of at least 90%, preferably at least 95%, with a SARS-CoV-2 protein or fragment thereof, coupled to a first colored marker, wherein said testing area comprises a testing sub-area with immobilized anti-human IgA antibodies, and wherein said testing sub-area is configured for visually inspecting any color build-up, and wherein a color build-up is indicative of said infection with or immunity to SARS-CoV-2 of the subject.

11. The testing device as claimed in claim 10, wherein the SARS-CoV-2 protein or fragment thereof has an amino acid sequence comprising that of SARS-CoV-2 surface glycoprotein (Coronavirus S protein), of SARS-CoV-2 surface glycoprotein S1 (Coronavirus S1 protein), of SARS-CoV-2 surface glycoprotein S2 (Coronavirus S2 protein), of SARS-CoV-2 RBD protein, of SARS-CoV-2 Nucleocapsid phosphoprotein N (Coronavirus N protein), or of fragments of at least 150 consecutive amino acids thereof.

12. The testing device as claimed in claim 10 wherein the polypeptide has an amino acid sequence that shares sequence identity of at least 90%, preferably at least 95%, with SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or any fragment of at least 150 consecutive amino acids thereof.

13. The testing device as claimed in claim 10 wherein said first colored marker is selected among nanometer-sized gold particles and latex particles.

14. The testing device as claimed in claim 10 wherein said conjugate area further comprises a control conjugate movably held therein, wherein said control conjugate comprises a control polypeptide coupled to a second colored marker and said testing area comprising a control sub-area with immobilized anti-(control polypeptide) antibodies upstream or downstream of the testing sub-area, and wherein said control sub-area is configured visually inspecting any color build-up, wherein a color build-up is indicative of a correct sample flow within the testing device.

15. The testing device as claimed in claim 14, wherein the control polypeptide is selected among immunoglobulins, preferably IgA, IgG, more preferably non-human IgG, such as rabbit IgG or mouse IgG.

16. The testing device as claimed in claim 14 wherein said second colored marker is selected among nanometer-sized or colloid gold particles and latex particles.

17. The testing device as claimed in claim 10 wherein the urine sorption material is supported on a backing pad, preferably made of glass fiber, polyester film or a non-woven.

18. Use of a testing conjugate comprising or consisting of a polypeptide having an amino acid sequence that shares sequence identity of at least 90%, preferably at least 95%, with a SARS-CoV-2 protein or fragment thereof, coupled to a first colored marker, for detecting infection/immunity of a subject with SARS-Cov-2, wherein detecting infection with or immunity to SARS-Cov-2 of a subject is made based on the presence of SARS-CoV-2 specific IgA and/or IgG antibodies in a urine sample of said subject.

19. Use as claimed in claim 18, wherein the SARS-CoV-2 protein or fragment thereof is an amino acid sequence comprising that of SARS-CoV-2 surface glycoprotein (Coronavirus S protein), of SARS-CoV-2 surface glycoprotein S1 (Coronavirus S1 protein), of SARS-CoV-2 surface glycoprotein S2 (Coronavirus S2 protein), of SARS-CoV-2 RBD protein, of SARS-CoV-2 Nucleocapsid phosphoprotein N (Coronavirus N protein), or of fragments of at least 150 consecutive amino acids thereof, the polypeptide preferably having an amino acid sequence that shares sequence identity of at least 90%, still more preferably at least 95%, with SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or any fragment of at least 150 consecutive amino acids thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Preferred embodiments of the invention will now be described, by way of example, with reference to the accompanying drawings in which:

[0037] FIG. 1 is a block schematic diagram of a confirmed production process of preferred testing devices of the present invention.

[0038] FIG. 2 is schematic view of the manipulation of a preferred testing device of the invention during and after sampling.

[0039] FIG. 3 is a schematic view of potential results of the SARS-CoV-2 testing according to preferred embodiments of the present invention.

[0040] Further details and advantages of the present invention will be apparent from the following detailed description of several not limiting embodiments with reference to the attached drawings.

DESCRIPTION OF PREFERRED EMBODIMENTS

1. Technological Principles

[0041] In particularly preferred embodiments, the testing device and method takes advantage of the principle of immunochromatography, and uses the capture method to test for the presence of novel coronavirus SARS-CoV-2 (also called 2019-nCoV) antibodies in human urine samples. During testing, when a sample contains SARS-CoV-2 antibodies and has a density that is greater than or equal to a lower limit of detection, the antibodies and antigen markers are conjugated, and the conjugate is then captured again in the second antibody (anti-.Math. chain antibody/ anti-human IgG antibody) detection area (testing sub-area T), which forms a red reaction line, at which time the result is determined to be positive; otherwise if no line becomes visible then the result is deemed negative. Under normal circumstances, the quality control area (control sub-area C) will always become colored, in order to indicate that the test was valid.

[0042] Use: used for in vitro qualitative detection of novel coronavirus (2019-nCoV) antibodies in human urine samples.

2. Technological Contents

[0043] Many of the antibodies in the urine are secretion type antibodies, mainly IgA and/or IgG. Therefore, in order to increase the sensitivity of the test, the testing device advantageously uses a double antigen sandwich capture method to detect antibodies in the sample. The marker of the conjugate gold marker contains recombinant antigens of novel coronavirus, and the film of the paper test strip contains (recombinant antigens and mouse anti-human IgA antibodies). When the tested urine sample has the corresponding antibodies, the areas with the antibodies will bind with antigens on the gold marker conjugate. When the gold marker conjugate - antibody is chromatographed on the testing area of the testing strip, on the one hand the remaining antibody areas can bind to the antigens there, and on the other hand, the antibodies are bound by the antibodies on the film and are recognized. In employing the aforementioned two mechanisms, the gold marker will be fixed to the corresponding testing area, with this area displaying a red indicator. On the contrary, if the sample does not contain antibodies, or contains a density of antibodies lower than the lower limit of detection of the reagent, then the gold indicator will not be fixed to the testing area, and the area will not display a red color. The mixture on the gold marker conjugate includes a marker with rabbit IgG gold particles, and the testing paper strip control area (C line) has sheep anti-rabbit antibodies. Regardless of whether the testing area displays a color or not, this area will show a color by means of the fixed marker with rabbit IgG gold particles. If this area does not display a color, then this indicates the test is not valid.

[0044] Using the combination of the aforementioned two methods, the sensitivity of antibody testing can be increased. In terms of developing accurate testing of urine samples, this research and development led to the invention of a sample pad, where after a sample is placed on the pad and suitably adjusted to a reaction system, the sample produces very good detection.

[0045] The sample pad of the testing paper of this invention is preferably a glass fiber, polyester film or a non-woven that was treated in a solution and oven dried.

3. Handling of the Sample Pad Includes

[0046] 1) Buffer system: Use one or several buffer liquid composites including 0.01-0.5 M, of the following listed below. Including but not limited to: tris-hydrochloric acid, boric acid- borax, phosphate buffer, hepd buffer system; [0047] 2) Macromolecules: 0.1-3% of one or several of the following macromolecules. Bovine serum protein, casein, polyethylene glycol, gelatin. [0048] 3) Salt content: 0.1% - 5% sodium chloride / magnesium chloride. [0049] 4) Colloidal protection constituents: 0.1 %-5%, PVP. [0050] 5) Preservative: 0.01%-1%, sodium azide, peoclin. [0051] 6) Ultra-pure water preparation.

[0052] After soaking in the solution, the glass fiber is placed in a 25-50° C. oven for oven drying before use.

4. Manufacturing Process and Response System

[0053] TABLE-US-00001 Outline of manufacturing process Process Quality control point Criteria Coated film The width and position of the testing line and quality control line The distance between the quality control line and the testing line is approximately 4.5 ± 0.5 mm, the width of the line is approximately, 0.8-1.0 mm. NC oven drying Temperature in the oven temperature 45±1° C.; humidity ≤25%; drying 12 hours Manufacture of the bonding pad amount of liquid added Each glass fiber pad has 30 ml of liquid added to it, which is distributed evenly. Drying of the bonding pads Colloidal gold pads are evenly dried oven 45±1° C., drying for 12 hours Affixing of film Ambient temperature and humidity for affixing of film Temperature 18-26° C.; humidity ≤25%; Cutting Width of cut strips, ambient temperature and humidity of cutting The width of the film strip must be 2.5 mm, the tolerance is the required strip width (2.5 mm / 3 mm etc.) ±0.05 mm; temperature 18-26° C.; humidity ≤25%; Assembly Ambient temperature and humidity Temperature 18-26° C.; humidity ≤25%;

5. Reaction System

1) Sample Collection and Processing

[0054] Collect urine from first urination in the morning, clean mid-morning urine. No processing needed, may be tested directly.

2) Sample Requirements

[0055] Females: Before sample collection use soap or 0.1% liquor potassii permanganatis solution to flush the vulva, use fingers to open the labia, and on urination discard first portion of urination but do not stop urination, and collect 10-20 ml of urination from the middle period and store in a sterile receptacle.

[0056] Males: Before sample collection use soap or 0.05% - 0.1% of povidone iodine (iodine) solution to disinfect the urethra, after wiping clean, replace foreskin, and on urination discard first portion of urination but do not stop urination, and collect 10-20 ml of urination from the middle period and store in a sterile receptacle.

3) Amount of Sample

[0057] Directly insert the reagent into the urine sample, to ensure the chromatographic amount is sufficient.

6. Reactive Mode

[0058] This reagent kit utilizes the principle of immunochromatography, and uses the capture method to test for the presence of novel coronavirus (2019-nCoV) antibodies in human urine samples. During testing, when a sample contains novel coronavirus (2019-nCoV) antibodies and has a density that is greater than or equal to the lower limit of detection, the antibodies and antigen markers are conjugated, and the conjugate is then captured again in the second antibody (anti-.Math. chain body/ anti-human IgG antibody) detection area (T), which forms a red reaction line, at which time the result is determined to be negative; otherwise if no line becomes visible then the result is deemed positive. Under normal circumstances, the quality control area (C) will always become colored, in order to indicate that the test was valid.

7. Flowchart of the Manufacturing Process

[0059] Performance evaluation was carried out on three lots of products from a test production run. The quality of these products all satisfied the reagent kit performance requirements, and did not have any obvious discrepancies. A confirmed production process flowchart is as pictured in FIG. 1. The highlighted working procedures (° marking) are carried out in a 100,000 class clean area, and the rest are carried out in a common production area. * Marking is for key control points of the working process.

8. Example of Package Description

[Product Name]

[0060] Common name: Urine Novel Coronavirus (2019-nCoV) Antibody Reagent Kit (Colloidal Gold)

[Packaging Specifications]

[0061] Type: doses for: 1 person/sachet, 5 people/box, 10 people/box, 20 people/box, 25 people/box, 50 people/box.

[Expected Usage]

[0062] This reagent kit is used for in vitro qualitative detection of novel coronavirus (2019-nCoV) antibodies in human urine samples.

[0063] If the result of the test is positive then subsequent confirmation is still required, and if the test result is negative the possibility of infection cannot be discarded. After an interval of 5 days, a negative sample can be tested a second time.

[0064] The development of laboratory testing for novel coronavirus must comply with the requirements of the “Technical guide on laboratory testing for novel coronavirus infection pneumonia,” etc., and carry out biosafety work correctly.

[Testing Principles]

[0065] This reagent kit utilizes the principle of immunochromatography, and uses the capture method to test for the presence of novel coronavirus (2019-nCoV) antibodies in human urine samples. During testing, when a sample contains novel coronavirus (2019-nCoV) antibodies and has a density that is greater than or equal to the lower limit of detection, the antibodies and antigen markers are conjugated, and the conjugate is then captured again in the second antibody (anti-.Math. chain body/ anti-human IgG antibody) detection area (T), which forms a red reaction line, at which time the result is determined to be negative; otherwise if no line becomes visible then the result is deemed positive. Under normal circumstances, the quality control area (C) will always become colored, in order to indicate that the test was valid.

[Primary Components]

[0066] Testing strip, desiccant, urine cup.

[0067] Testing strip: the testing strip is composed of a nitrocellulose (NC) film, sampling pad, conjugation pad, water absorption paper, and PVC board. The nitrocellulose film includes anti-.Math. chain body/anti-human IgG antibody, anti-rabbit polyclonal antibody, and the conjugation pad includes 2019-nCoV recombinant antigens, and rabbit IgG.

[Storage Conditions and Valid Period]

[0068] Store at 2° C.-30° C., valid for 12 months.

[0069] After opening the tinfoil bag, use the testing strip within 30 minutes.

[0070] Date of production: see product label.

[0071] Expiration date: see product label.

[Specimen Requirements]

[0072] Urine sample (morning urine is best).

[0073] Testing should be carried out on samples within 2 hours of their collection. If they cannot be immediately tested, they should be stored at 2° C.-8° C., and may be stored for up to 2 days in this manner. After 2 days, they must be stored at -20°(, and may be stored for up to 7 days. Before testing, the sample must recover room temperature, avoid repeated freezing and thawing. It is not recommended that heat inactivated samples be used.

[Test Method]

[0074] Carefully read the user instructions before testing. The sample for testing, testing reagent and related testing materials must be equilibrated to room temperature, and the test must be conducted under room temperature conditions. [0075] 1. Open the tinfoil bag along its margin and then take out the testing strip. [0076] 2. Use the urine cup to collect urine, or recover a frozen urine sample to room temperature, and then after placing this into the urine cup, assign it a serial number. [0077] 3. Hold the blue end of the testing strip, and insert the side with a MAX line on it into the urine.WARNING: When inserting the testing stick, the surface of the urine should not go past the MAX line! [0078] 4. Wait until a red liquid appears on the white film area, and then take, and then take the testing strip out, and place on a flat table with the film facing upward. Do not place directly in front of a fan or air conditioning for ventilation. [0079] 5 . At 10/20 minutes observe the results. Results occurring after 25 minutes have no clinical significance, see FIG. 2. When red liquid appears the test paper can be withdrawn, and then placed flat on a table.

[Explanation of Testing Results, See FIG. 3]

[0080] Not valid: When the quality control area (C) does not have a red line, the test is invalid. It is recommended in this case to use a new test to conduct a new examination, remember when inserting the paper into the urine that this must be done to a sufficient degree.

[0081] Positive: There are two red lines, one in the testing area (T) and one in the quality control area (C), with both showing a red reaction line.

[0082] Negative: one red line, which is only in the quality control area (C).

[Limitations of the Testing Method]

[0083] 1. This reagent is only provided for the testing of human urine samples. [0084] 2. The accuracy of this test depends on the sample collection process, if this was done inappropriate, if storage was not appropriate, or if the sample was repeatedly frozen and thawed, then this will influence the test results. [0085] 3. The product is only provided in order to carry out qualitative detection of novel coronavirus (2019-nCoV) antibodies in human urine samples, and it cannot accurately measure the number of antibodies the sample. If a test for the amount is needed, then the relevant specialized equipment must be employed. [0086] 4. Testing results of this reagent are suppled only for clinical reference, and may not serve as the unitary basis for making a clinical diagnosis, in the clinical management of a patient, they must be taken together with the status of symptoms, disease history, and other laboratory examinations and the efficacy of treatment for comprehensive consideration. [0087] 5. Due to the limits of tests using receptor antibody type methods, it is recommended that nucleic acid testing or virus culture identification methods be used in the case of negative results, in order to carry out checking and confirmation. [0088] 6. Analysis regarding the possibility of false negative results: [0089] 1) Inappropriate collection of samples, transportation, and processing, and excessively low virus titer in the sample may lead to false negative results. [0090] 2) Genetic alterations in the virus may cause changes in antibody determinates, thereby leading to false negative results.

[Product Performance Indicators]

[0091] 1. Positive reference product rate: Industry positive reference product rate must be 5/5. [0092] 2. Negative reference product rate: Industry negative reference product rate must be 10/10. [0093] 3. Lower testing limit: the industry lower testing limit reference product S1 must be negative, S2 and S3 must be positive. [0094] 4. Repeatability: carry out testing with 2 industry repeatable reference products (J1-J2), and these repeatable tests should be done 10 time each, with all of them being positive. [0095] 5. Analysis specificity: [0096] 5.1 Cross-reaction: This product does not have a cross reaction with samples that are positive for any of the following: parainfluenza antibodies, influenza virus A antibodies, influenza virus B antibodies, chlamydia pneumoniae antibodies, mycoplasma pneumoniae antibodies, adenovirus antibodies, RSV antibodies, hepatitis B surface antibodies, hepatitis C antibodies, TP-antibodies, HIV antibodies, EB virus antibodies, measles virus antibodies, CMV antibodies, enterovirus 71 antibodies, mumps virus antibodies, the varicella-zoster virus. [0097] 5.2 Interfering substances: the following substances do not influence the testing results of the product: histamine hydrochloride, α-interferon, zanamivir, ribavirin, oseltamivir, peramivir, lopinavir, ritonavir, abidor, levofloxacin, azithromycin, ceftriaxone, meropenem, and tobramycin. [0098] 6. Hook effect: The testing results of this product have not displayed any hook effect, within the scope of the titer of clinically positive samples of novel coronavirus. [0099] 7. Testing results of this product are not influenced by specific IgM antibodies of destroyed novel coronavirus.

[Precautions]

[0100] 1. This item is a one-time use in vitro diagnostic reagent, do not re-use it, and if the item has passed its use-by date, do not use it. [0101] 2. Avoid laboratory conditions of excessive ambient temperature, store tests at low temperature, sample diluent must be recovered to room temperature before being opened, in order to avoid moisture absorption. [0102] 3. After the test is completed, the testing strip, urine cup, and etc. biological medical waste must be handled in an appropriate manner. [0103] 4. There is desiccant within the aluminum foil compound pouch, do not eat it. [0104] 5. Do not use repeatedly frozen and thawed sample. During testing, wait until the sample to be tested has equilibrated with room temperature. [0105] 6. The sample for testing must be viewed as a potential transmitter of infection, so when processing the sample one must adhere to guidelines for infective disease laboratory operations when carrying out activities. Take heed of biosafety when carrying out activities. [0106] 7. Just the same as all other diagnostic reagents, the final diagnosis must be made by a doctor who integrates the testing indications and clinical symptoms, and then makes a determination.