METHOD OF DETECTING PATHOGENS AND/OR ANTIGENS IN SAMPLES

Abstract

The present disclosure provides new and improved methods for the detection of pathogens, for example viral pathogens, in samples. The methods, tests and assays permit the identification of an infection, for example a Coronavirus infection, in a sample, but can also be used as a means to identify the infectious status or “infectivity level” of a subject. Disclosed is a method for detecting an antigen in a sample, said method comprising: contacting the sample with an antigen binding agent, wherein the antigen binding agent is not an antibody.

Claims

1. A method for detecting Coronavirus or a Coronavirus antigen in a sample, said method comprising: contacting the sample with a Coronavirus/Coronavirus antigen binding agent under conditions which permit binding between the binding agent and any Coronavirus or Coronavirus antigen present in the sample and detecting complexes comprising binding agent/Coronavirus/Coronavirus antigen, wherein the Coronavirus/Coronavirus antigen binding agent is not an antibody and detection of complexes comprising binding agent and Coronavirus/Coronavirus antigen, indicates that the sample contains Coronavirus and/or Coronavirus antigen and/or that the sample may have been provided by or obtained from a subject that has or has been infected with Coronavirus.

2. The method of claim 1, wherein the Coronavirus is SARS-CoV-2, SARS-CoV and/or MERS-CoV.

3. The method of claim 1, wherein the Coronavirus antigen is the spike (S)-protein, the S1 domain of the S-protein, the S2 domain of the S-protein and/or receptor binding domain (RBD) of the S-protein.

4. The method of claim 1, wherein the binding agent comprises ACE2 or a Coronavirus/Coronavirus antigen binding fragment thereof.

5. The method of claim 1, wherein the binding agent comprises a protein having a sequence of SEQ ID NO: 1, SEQ ID NO: 2 or a Coronavirus/Coronavirus antigen binding fragment thereof.

6. The method of claim 1 wherein the binding agent comprises DPP4 or a Coronavirus/Coronavirus antigen binding fragment thereof.

7. The method of claim 1, wherein the binding agent comprises a protein having a sequence of SEQ ID NO 3, SEQ ID NO: 4 or a Coronavirus/Coronavirus antigen binding fragment thereof.

8. The method of claim 1, wherein the binding agent is conjugated or bound to a nanoparticle.

9. The method of claim 8, wherein the nanoparticle is coloured.

10. The method of claim 1, wherein the sample comprises a biological fluid, saliva, blood and/or a fraction thereof.

11. The method of claim 1, wherein the sample is combined with a buffer prior to contacting the sample with a Coronavirus/Coronavirus antigen binding agent.

12. The method of claim 11, wherein the buffer comprises a detergent to disrupt any virus present in the sample.

13. The method of claim 1, wherein the method further comprises the use of a second Coronavirus/Coronavirus antigen binding agent.

14. The method of claim 13, wherein any step of adding second Coronavirus/Coronavirus antigen binding agent yields complexes which comprise the second binding agent and/or any Coronavirus/Coronavirus antigen.

15. The method of claim 14, wherein the second Coronavirus/Coronavirus antigen-binding agent is contacted with the sample.

16. The method of claim 15, wherein any step comprising the second Coronavirus/Coronavirus antigen binding agent is executed before, concurrently with or after any step comprising a or the first, Coronavirus/Coronavirus antigen binding agent.

17. The method of claim 16, wherein the step of adding a second Coronavirus/Coronavirus antigen binding agent is executed before the step of adding the a or the first Coronavirus/Coronavirus binding agent.

18. The method claim 13, wherein before the detecting step, the method comprises contacting the sample and/or any binding agent/Coronavirus complexes and/or first binding agent/Coronavirus antigen complexes, with the second Coronavirus/Coronavirus antigen binding agent.

19. The method of claim 13, wherein the second binding agent comprises a molecule selected from the group consisting of: (i) ACE2 or a Coronavirus/Coronavirus antigen binding fragment thereof; (ii) DPP4 or a Coronavirus/Coronavirus antigen binding fragment thereof; (iii) SEQ ID NO: 1, SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4 or a or a Coronavirus/Coronavirus antigen binding fragment thereof; (iv) an antibody which binds the spike (S)-protein, the S1 domain of the S-protein, the S2 domain of the S-protein and/or the receptor binding domain (RBD) of the S-protein. (v) an antibody which binds the same target as the first binding agent; and (vi) an antibody which binds the S2 domain of the S-protein

20. The method of claim 13, wherein the second binding agent is biotinylated.

21. The method of claim 13, wherein the second binding agent comprises an antibody which binds the S2 domain of the Coronavirus S-protein.

22. A method of for detecting Coronavirus or a Coronavirus antigen in a sample, said method comprising: contacting the sample with a first Coronavirus/Coronavirus antigen binding agent under conditions which permit binding between the first binding agent and any Coronavirus or Coronavirus antigen present in the sample to form a first binding agent/Coronavirus or a first binding agent/Coronavirus antigen, complex, wherein the first Coronavirus/Coronavirus antigen binding agent is not an antibody; contacting the sample and/or any complex, with a second Coronavirus/Coronavirus antigen binding agent under conditions which permit binding between any complexes and the second binding agent; and detecting first binding agent/Coronavirus/second agent complexes and/or first binding agent/Coronavirus antigen/second agent complexes, wherein detection of a first binding agent/Coronavirus/second agent complex and/or first binding agent/Coronavirus antigen/second agent complex indicates that the sample contains Coronavirus and/or Coronavirus antigen and/or that the sample may have been provided by or obtained from a subject that has or has been infected with Coronavirus or has had, a Coronavirus infection and/or a disease or condition associated therewith.

23. A method for detecting SARS-CoV-2 or a SARS-CoV-2 antigen in a sample, said method comprising: contacting the sample with ACE2 or a SARS-CoV-2/SARS-CoV-2 antigen binding fragment thereof, under conditions which permit binding between the ACE2/ACE2 fragment and any SARS-CoV-2 or SARS-CoV-2 antigen present in the sample and detecting ACE2/ACE2 fragment::SARS-CoV-2 complexes and/or ACE2/ACE2 fragment::SARS-CoV-2 antigen complexes.

24. The method of claim 23, wherein the method further comprises the use of an antibody binding the S2 domain of the S-protein.

25. The method of claim 23, wherein the detection of ACE2/ACE2 fragment::SARS-CoV-2 complexes and/or ACE2/ACE2 fragment::SARS-CoV-2 antigen complexes, indicates that the sample may have been provided by or obtained from a subject that has or has been infected with SARS-CoV-2.

26. A method for detecting MERS-CoV or a MERS-CoV antigen in a sample, said method comprising: contacting the sample with DPP4 or a MERS-CoV/MERS-CoV antigen binding fragment thereof, under conditions which permit binding between the DPP4/DPP4 fragment and any MERS-CoV or MERS-CoV antigen present in the sample and detecting DPP4/DPP4 fragment::MERS-CoV complexes and/or DPP4/DPP4 fragment::MERS-CoV antigen complexes.

27. The method of claim 26, wherein the detection of DPP4/DPP4 fragment::MERS-CoV complexes and/or DPP4/DPP4 fragment::MERS-CoV antigen complexes, indicates that the sample may have been provided by or obtained from a subject that has or has been infected with MERS-CoV.

28. A lateral flow assay for the detection of Coronavirus, a Coronavirus antigen, SARS-CoV-2, SARS-CoV, MERS-CoV, the S-protein of SARS-CoV-2, the S-protein of SARS-CoV or the S-protein of MERS-CoV in a sample, the lateral flow assay comprising: a first Coronavirus binding agent, which is not an antibody; and a second Coronavirus binding agent.

29. The lateral flow assay of claim 28, wherein the first binding agent comprises ACE2 or a Coronavirus, a Coronavirus antigen, SARS-CoV-2, SARS-CoV, MERS-CoV, SARS-CoV-2 S-protein, S-protein of SARS-CoV S-protein or MERS-CoV S-protein, binding fragment thereof.

30. The lateral flow assay of claim 28, wherein the first binding agent is conjugated to a nanoparticle.

31. The lateral flow assay of claim 28, wherein the nanoparticle is a coloured nanoparticle.

32. The lateral flow assay claim 28, wherein the second binding agent comprises an antibody which binds to a different domain of the Coronavirus spike protein to the first binding agent.

33. The lateral flow assay of claim 28, wherein the second binding agent comprises an antibody which binds the S2 domain of the Coronavirus spike protein.

34. The lateral flow assay of claim 28, wherein the lateral flow assay comprises an immobilised capture agent.

35. The lateral flow assay of claim 28, wherein the immobilised capture agent is immobilised at a test line of the lateral flow assay and comprises polystreptavidin (PSA).

36. The lateral flow assay of claim 28, wherein the second binding agent is biotinylated.

37. A lateral flow assay for the detection of Coronavirus or the S-protein of SARS-CoV-2, in a sample, the lateral flow assay comprising: a Coronavirus binding agent comprising a nanoparticle with ACE2, or an S-protein or S1 domain binding fragment thereof, bound thereto; and a Coronavirus binding agent comprising a biotinylated antibody which binds the S2 domain of the Coronavirus S-protein.

38. The lateral flow assay of claim 37, wherein the assay further comprises a test line comprising PSA.

39. The lateral flow assay of claim 37, wherein one Coronavirus binding agent is spaced apart from the other Coronavirus binding agent of the assay.

40. The lateral flow assay of claim 37, wherein the assay is configured to receive a sample and contact that sample with a biotinylated antibody which binds the S2 domain of the Coronavirus S-protein to yield antibody/Coronavirus or S-protein complexes.

41. The lateral flow assay of claim 40, wherein any antibody/Coronavirus or S-protein complexes assay are contacted with the nanoparticle to yield complexes comprising the antibody, nanoparticle and any Coronavirus or Coronavirus or S-protein.

42. A method of for detecting Coronavirus or a Coronavirus antigen in a sample, said method comprising: contacting the sample with a first Coronavirus/Coronavirus antigen binding agent under conditions which permit binding between the first binding agent and any Coronavirus or Coronavirus antigen present in the sample to form a first binding agent/Coronavirus or a first binding agent/Coronavirus antigen, complex; contacting the sample and/or any complex, with a second Coronavirus/Coronavirus antigen binding agent under conditions which permit binding between any complexes and the second binding agent, wherein the second Coronavirus/Coronavirus antigen binding agent is not an antibody; and detecting first binding agent/Coronavirus/second agent complexes and/or first binding agent/Coronavirus antigen/second agent complexes, wherein detection of a first binding agent/Coronavirus/second agent complex and/or first binding agent/Coronavirus antigen/second agent complex indicates that the sample contains Coronavirus and/or Coronavirus antigen and/or that the sample may have been provided by or obtained from a subject that has or has been infected with Coronavirus or has had, a Coronavirus infection and/or a disease or condition associated therewith.

43. The method of claim 42, wherein the first binding agent is selected from the group consisting of: (i) ACE2 or a Coronavirus/Coronavirus antigen binding fragment thereof; (ii) DPP4 or a Coronavirus/Coronavirus antigen binding fragment thereof; (iii) SEQ ID NO: 1, SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4 or a or a Coronavirus/Coronavirus antigen binding fragment thereof; (iv) an antibody which binds the spike (S)-protein, the S1 domain of the S-protein, the S2 domain of the S-protein and/or the receptor binding domain (RBD) of the S-protein. (v) an antibody which binds the same target as the first binding agent; and (vi) an antibody which binds the S2 domain of the S-protein.

44. The method of claim 42, wherein the second binding agent is selected from the group consisting of: (i) ACE2 or a Coronavirus/Coronavirus antigen binding fragment thereof; and (ii) a protein having a sequence of SEQ ID NO: 1, SEQ ID NO: 2 or a Coronavirus/Coronavirus antigen binding fragment thereof.

Description

DRAWINGS

[0211] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The present invention will now be described by reference to the following figures which show:

[0212] FIG. 1: A) positive line indicating the presence of biotinylated S1 antibody, B) Biotinylated Rabbit IgG was used for control.

[0213] FIG. 2: Visual representation of 100 uL of BSA blocked solutions of S1 polyclonal antibody or S1 antibody conjugated with GNP 40 nm OD=3.3 A) Unsuccessful conjugation as an indication of gold NP crashing out after addition of polyclonal S1 antibody B) Successful conjugation of monoclonal S1 antibody as gold remains read after addition of the antibody.

[0214] FIG. 3: Titration of S-RBD into 10 uL an 20 uL of ACE2-GNP OD=3.6 using halfrate 0.5 mg/mL PSA nitrocellulose membrane. S-RBD (0.25 mg/mL) was diluted 1:10000. 20-2.5 uL (test 1-5) of 1:10000 S-RBD was mixed with 2 uL of 1:50 dilution of (1 mg/mL) biotinylated S1 mab. 10 or 20 uL was added to the sample and the mixture was incubated for 15 min before running the strip. Sample was run through the strip and washed with 10 uL of drying buffer.

[0215] FIG. 4. Schematic diagram demonstrating the sensitivity of the lateral flow assay. A) demonstration of the sandwich formation after 2-15 min incubation of a solution prepared for wet assay followed by a subsequent run and PSA plotted assay strip, B) demonstrate the assay in absence S-RBD, C) indicates a successful asymmetric sandwich assay in presence of S-RBD protein, D) Test strip format containing 1 line of 0.5-2 mg PSA in 1% Sucrose, 1% PBS in filtered water.

[0216] FIG. 5: Schematic illustration of A) Wet assay of Viral protein, B) Lateral flow assay format demonstrating viral antigen-NP and S-RBD specific antibody sandwiched around the virus, C) Lateral flow assay with viral antigen-NP and antibody sandwiched around the viral protein.

[0217] FIG. 6: Comparison of A) Wet assay with B) dry assay.

[0218] FIG. 7: Comparison of asymmetric assay with S1 protein present versus S-RBD protein.

[0219] FIG. 8: A) Buffer RBD (50:50), 15 pg/ml OD20 gold colloid. 5 minutes incubation w/10 minute run, n=3, error=SD: B) Spiked saliva with RBD (50:50), OD20 gold 15 μg/ml using 8 μl deposition, 1% T20 PBS-B running buffer, 20 minute run time.

[0220] FIG. 9: Comparison of spiked saliva sample application: 15 μg/ml OD20 gold 4 μl dried.

[0221] FIG. 10: Comparison of gold volumes, 10 and 20 minute read times, RBD saliva spike 250 ng/ml (50:50)

[0222] FIG. 11: spiked saliva with RBD (1;1), OD20 gold 15 μg/ml using 8 μl deposition, 1% T20 PBS-B running buffer, 20 minute run time.

[0223] FIG. 12: cartoon showing the arrangement at the test line of an example lateral flow assay. In this figure, nitrocellulose strip 16 contains a test line having immobilised thereof a quantity PSA (14) immobilised thereon. In previous steps, a nanoparticle (NP) having immobilised thereof a quantity of ACE2 (2: or a Coronavirus/S-protein binding fragment thereof) is contacted with a sample thought to contain either Coronavirus and/or the Coronavirus S-protein under conditions which permit binding between any Coronavirus and/or the Coronavirus S-protein present in the sample and the ACE2 bound to the nanoparticle. In this figure, the antigen (the Coronavirus S-protein) is represented by components 4 (the RBD domain), 6 (the S1 domain) and 8 (the S2 domain). The result of this step of the assay (if the sample contains the target antigen (the S-protein)) is the formation of a complex between the S-protein (specifically the S1 domain thereof) and the ACE-2 component bound to the nanoparticle. The assay system further comprises an antibody (10) with specificity for the S2 domain of the S-protein (8). That antibody (10) is biotinylated (12). The assay permits the formation of a further complex with antibody 10. This complex is then captured at the test line by binding between the biotin moiety (12) of the antibody (10) and the PSA (14) at the test line (18). Because a test line contains multiple PSA (14) moieties, multiple nanoparticles become immobilised and can be detected. The method of detection may vary depending on the type of nanoparticle, but it may be optically detectable—the nanoparticle being a coloured bead or the like.

[0224] FIG. 13: Cartoon showing an exemplar assay format. In this case, the Coronavirus spike protein is shown as comprising three domains, the RBD domain (42), the S1 domain (44) and the S2-domain (46). In this assay, a sample (20) comprising spike protein is applied to a device which comprises a nitrocellulose strip (21). The strip (21) comprises a sample pad (22) for receiving the sample, a conjugation pad (24) comprising a first spike protein binding agent (26) and a second conjugation pad (28) comprising a molecule which itself comprises a nanoparticle (32) with ACE2 (30) (or a spike binding fragment thereof) immobilised thereto.

[0225] The sample (20) moves through the nitrocellulose strip (21) by capillary action/wicking. The sample maybe ‘pulled’ or drawn through by an absorption pad located at a distal point of the strip (21: not shown). The sample 20 and any spike protein therein (42, 44, 46) arrives at conjugation pad (24) which comprises an antibody (26) with specificity for the S2 domain (46) of the spike protein. In this example, the antibody is biotin labelled. The result of this interaction is the formation of a spike protein (42, 44, 46)::antibody complex (26). This complex will then move through to conjugation pad 2 (28) which comprises the Ace2 (30) bound nanoparticle (32). At conjugation pad 2 (28) there will form a further complex comprising not only the spike protein (42, 44, 46) and antibody (26), but also the Ace2 (30) bound nanoparticle (32). This larger complex will then continue to move through the strip (210 towards the text line (34) and control line (36). Test line (34) may have immobilised thereon s quantity of PSA—this will bind the biotin moiety of the antibody (26). The control line (36) may comprise an Ace2 binding moiety (38). In this regard, a test line forms because of complex binding to the PSA of the test line (via biotin on the anti-S2 antibody (26)) free ACE2/nanoparticle conjugates (30/32) bind to moiety (38) at the control line to form a red line.

DETAILED DESCRIPTION

Example 1

[0226] Biotinylation of antibodies specific to S-RBD is prepared by using Lightning-Link @ Rapid Biotin Conjugation Kit (Type B) following advised protocol: [0227] 1. Addition of 1 uL of LL Rapid Modifier reagent for each 10 uL of S1 antibody to be labeled and mixed. [0228] 2. Add the mixture of the mixture of S1 antibody and LL Rapid Modifier reagent was added to the lyophilised LL Rapid mix and redispersed with a pipette. [0229] 3. The sample was left for 15 min at room temperature (21 C). [0230] 4. After incubation 1 uL of Rapid Quencher reagent was added for every 10 uL of antibody used. The sample was left for 5 min and subsequently stored at 4 C until use. [0231] 5. The biotinylation was checked using a biotinylation test kit supplied by abcam.

[0232] See also FIG. 1.

[0233] Gold Antibody Conjugate Buffer Optimisation

[0234] In initial studies the passive conjugation of NSP3, polyclonal S1 antibody, ACE-2, S1 antibody and CD147 on gold nanoparticles was optimised. This aims to select the conjugate with the highest specificity and sensitivity. These studies were carried out by mixing 1.5 uL—2.5 uL of antibody/antigen with conjugation buffers Tris, Borate, MES, TAPS and BES at pH ranges 5-10. Then 200 uL of gold nanoparticles were added to the mixtures. The samples were left on a shaker for 10 min at 21 C and analysed by eye and the optimal aggregation ratios were obtained by taking the ratio of 550 and 600 nm absorption intensity obtained by UV-vis. See FIG. 2.

TABLE-US-00005 TABLE 1 Gold aggregate ratios obtained for ACE-2 (1.5 uL added) and S1pAB (1.5 uL, 2.0 uL and 2.5 uL added). Borate Borate Borate Borate 9.3 9.0 8.5 MES6.7 MES5.3 TES7.1 BES7.8 TES7.5 TAPS8.5 BES6.5 9.24 TES7.1 ACE-2 3.8630849 3.974217 4.01119 4.05075 4.1286  3.95833 3.91091 3.90235 4.0814 4.08818 4.2164329 4.04699 1.5 uL S1pAB 3.2467018 2.777533 2.92532 2.71992 2.27637 2.45236 2.8516  2.74611 3.03073 2.55753 3.1649485 2.88229 1.5 uL 3.5624123 3.250627 3.30869 2.71919 1.57018 2.4276  3.00229 2.68333 3.34241 2.20983 3.4888889 2.71013 2.0 uL 3.4193989 3.45   3.1788  2.81142 2.02534 2.34347 3.07317 2.64666 3.18017 2.16944 3.5169492 2.74587 2.5 uL Gold 4.4733475 4.433476 4.35759 4.46316 4.40421 4.21937 4.27912 4.23633 4.31174 4.30498 4.2409639 4.22088   0 uL NP The numbers in bold font represent successful aggregation with a 550 nm/600 nm UV vis ratio   above 3.5, the numbers in italic font represent 550/600 nm ratios 3-3.49 and the numbers in underlined font are unsuccessful conjugation. MES, TES, TASPS and Borate represents the conjugation buffers added as 5 uL and the gold NP used is 40 nm with OD = 5.

TABLE-US-00006 TABLE 2 Gold aggregate ratios obtained for NSP3 (0-2.0 uL added) and CD147 (0 uL-2.0 uL added). Borate Borate Borate 9.3 9.0 8.5 MES6.7 MES5.5 TES7.1 BES7.8 TES7.5 TAPS8.5 BES6.5 TES7.1 NSP3 4.342975 4.38758  4.378723 4.446623 4.398693 4.390558 4.277551 4.324324 4.305263 4.26506  4.288066   0 uL 4.208835 4.167323 4.201597 4.13852  4.136719 4.223735 4.050373 4.099065 4.15458  4.125   3.970534 0.5 uL 4.286885 4.009328 4.129482 4.083969 4.128352 4.285132 4.322034 3.979817 4.256674 4.07457  4.27789  1.5 uL 3.99812  4.033962 4.046422 4.092453 4.100569 4.293634 4.330526 4.313402 4.280665 3.59271  4.403846 2.0 uL CD147 3.88764  4.322034 4.324324 4.409766 4.41649  4.374732 4.261224 3.227205 3.360601 3.851779 3.265139   0 uL 1.895075 2.602378 2.087659 1.893548 1.88609 2.191638 1.7892  2.04662  1.913734 1.703264 1.689616 0.5 uL 4.4375  4.118   4.442731 4.345041 4.481319 4.129845 4.383475 4.331959 4.420601 4.352321 4.33543  1.5 uL 1.060606 1.057143 1.057143 1.057143 1.057143 1.057143 1.055556 1.051282 1.046512 1.083333 1.088235 2.0 uL The numbers in bold font represent successful aggregation with a 550 nm/600 nm UV vis ration above 3.5, the numbers in italic font represent 550/600 nm ratios 3-3.49 and the underlined numbers are unsuccessful conjugation. MES, TES, TASPS and Borate represents the conjugation buffers added as 5 uL and the gold NP used is 40 nm with OD = 5.

TABLE-US-00007 TABLE 3 Gold aggregate ratios obtained for Spike S1 AB (0-2.5 uL added), NSP3 antibody (2.5 uL added) and CD147 (2.5 uL added). Borate Borate Borate 9.3 9.0 8.5 MES6.7 MES5.5 TES7.1 BES7.8 TES7.5 TAPS8.5 BES6.5 BES6.5 Spike S1 AB 4.462687 4.444444 4.32766  4.4267  4.39957 4.44541 4.49666 4.47682 4.42217 4.46154 4.42117   0 uL 3.858736 3.414089 3.570191 2.01941 3.40193 3.39187 3.43954 3.20821 3.58944 3.85316 3.08309 0.5 uL 4.298174 4.223969 4.246377 4.30769 4.32985 4.34232 4.39195 4.28016 4.34728 4.13917 4.25355 1.5 uL 4.353814 4.346639 4.347639 4.42949 4.41365 4.3264  4.43226 4.38205 4.23434 4.27216 4.14147 2.0 uL NSP3 4.473913 4.412527 4.399142 4.4586  4.39019 4.41432 4.31818 4.21138 4.202  4.23926 4.27071 2.5 uL CD147 1.918098 1.66581  1.541568 1.66884 1.62973 1.58495 1.63464 1.60638 1.74668 1.53791 1.58945 2.5 uL Spike S1 AB 1.561381 3.842004 1.519303 1.70496 1.66279 1.75232 N/A N/A N/A N/A N/A 2.5 uL The numbers in bold font represent successful aggregation with a 550 nm/600 nm UV vis ration above 3.5, the numbers in italic font represent 550/600 nm ratios 3-3.49 and the underline numbers are unsuccessful conjugation. MES, TES, TASPS and Borate represents the conjugation buffers added as 5 uL and the gold NP used is 40 nm with OD = 5.

[0235] Protocol for Passive Conjugation of Antigens and Antibodies to Nanoparticles (Such as Gold Nanoparticles).

[0236] Passive conjugation of antigens and antibodies such as Angiotensin-Converting Enzyme 2 (ACE2), any versions of S-RBD and S1 antibodies in the volume range 0.5-5 uL to conjugation buffers (Tris, Borate, MES, TAPS and BES) in the pH ranges 2-10 where used in the quantity of 5 uL. After mixing the protein with the conjugation buffer we add OD1-OD10 gold nanoparticles in size ranges 10-150 nm (100-500 uL). The mixture was left on a shaker for 45 min at room temperature (21 C). 1 uL of (300 mg/mL) of Probumin was added to the mixture and vortexed. Then left for 30 min at room temperature (21 C). Then 900 uL drying buffer (pH range >6, 1 mM Borate, 2-5% sucrose, 1-3% BSA, 0.1-2% Tween 20 or Tween 80, 0.1% sodium azide in filtered deionised water) was added to make up 1 mL in an eppendorf tube and the sample was centrifuged for 10-15 min at 4000 g. Then 950 uL of the supernatant was removed and 950 uL of drying buffer was added. The sample was centrifuged for 10-15 min at 4000 g. Then 950 uL of sample was removed and 50 uL of drying buffer was added. The sample was vortexed and stored at 4 C until use.

[0237] Plotting of PSA on nitrocellulose strips was performed by preparing a solution of 4.7 uL of 4.36 mg/ml of PSA, 4.1 uL of 10% sucrose, 3.6 uL of PBS buffer and 29 uL filtered and deionised water. The mixture was used to plot a line on 30 cm nitrocellulose membrane CN140 with a plotting rate of 0.1 mm/s. The membrane was dried at 40 C and attached to a card together with an absorbent pad. The membrane was cut in 3 mm to yield 100 strips. See FIG. 3.

Example 2—Parts of a Useful Assay May Include

[0238] 1. Passive conjugation of SARS-CoV2 specific antigens such as, ACE-2, to nanoparticles such as gold nanoparticles.

[0239] 2. Biotinylation of S-RBD antibodies.

[0240] 3. Plotting of 0.5-2 mg/mL of PSA, S-RBD antibodies or antigens, biotinylated S-RBD antibodies or antigens to nitrocellulose strips.

[0241] 4. Spraying a solution of ACE-2 or SARS-CoV2 specific antigens with or without TMPRSS2 protein or other serine proteases.

[0242] 5. Procedure for the formation of asymmetric sandwich. Mix together 20-2.5 uL of saliva, serum or throat swab containing S-RBD, S1+S2 and S1 protein solution in 0-20 uL drying buffer with 2 uL of conjugated S-RBD specific antibodies and 5-20 uL of ACE-2 conjugated to gold nanoparticles 40 nm.

[0243] 6. Add to the nitrocellulose strips and if the saliva, serum or throat swab contained viral proteins then a line appears on the strip.

[0244] See FIGS. 4, 5 and 6 for additional details.

[0245] Sample/Antigen Activation with TMPRSS2

[0246] Experiments show that the open (activated) form (S-RBD) binds stronger than S1 to ACE2 in lateral flow assay. See FIG. 7. Without wishing to be bound by theory, when TMPRSS2 is present, it cleaves a residue on S1 that leads to a conformational change that results in the S-RBD binding site to get exposed and bind tight to ACE2. TMPSSR2 could be replaced by a whole range of different proteases that will give the same results.

[0247] Optional Features [0248] 1. The amount and type of gold nanoparticle could be modified such that the intensity of the band generated in the presence of SARS-COV-2 proteins increased more progressively based on the viral protein concentration. This would allow the LFA to be used to measure how well another compound binds to ACE2 in competition with viral proteins. This could be used to test potential drug candidates for treatment of SARS-COV-2 rapidly and cheaply compared to other lab based techniques (mostly ELISA). Note current formulation designed to give as much signal as possible with as little viral protein as possible. [0249] 2. This LFA setup described is likely to be applicable to other viruses (other than SARS-COV-2). As viruses all need to bind to some protein to gain entry into cells such a protein might substitute for ACE2 and along with an antibody specific to alternative virus the same type of sandwich LFA could be constructed.

[0250] The methods as described herein could be further modified as follows:

[0251] 1. Both the second binding agent (for example the specific antibody) as well as first binding agent (e.g. angiotensin-Converting Enzyme 2 (ACE2)) could be replaced with other types/versions of antibodies. All that would be required is that there were two separate binding sites where the new/alternative antibodies could bind. Specific binding domains (e.g. short peptides/aptamers/etc.) could be used instead of whole antibodies/proteins. This could improve overall device stability and potentially binding affinity.

[0252] 2. Gold nanoparticles (an option for tagging or labelling the first binding agent) could be changed to a wide range of other materials. Other metals could potentially be used. Also, other fluorescent or coloured compounds are potential substitutes. Any nanoparticles could be used.

[0253] 3. The test could be arranged in a variety of formats and conjugations. For example, the methods of this invention could be adapted to take place in ELISA format, ELIspot format, as Dot blots, radioimmunoassays and the like. Additional information regarding immunoassays which may be adapted or used to permit the detection of Coronavirus and/or Coronavirus antigens in samples may be found in The Handbook of Immunoassay technologies, (Vashist and Luong (2018), published by Academic press: see in particular chapter 1: Immunoassays: an overview: the contents of which is incorporated herein by reference.

Example 3

Summary

[0254] The titration of RBD in an assay with a gold conjugated ACE2 component as a signalling moiety and a human monoclonal anti-SARS-CoV-2 Spike antibody as the capture moiety on a nitrocellulose format has been completed.

[0255] The wet assay format has been optimised into a dry assay format. Moreover, sensitivity has been optimised by titrating RBD, attenuated SARS-CoV-2 or SARS-CoV-2 viruses spiked into negative saliva samples on the dry assay format to evaluate the assay performance.

[0256] The assay is evaluated using clinical samples to confirm those results obtained from the abovementioned mentioned titrations.

[0257] Assay Components: [0258] 1 mg/ml of human monoclonal anti SARS-CoV-2 Spike antibody. [0259] CN140 nitrocellulose membrane prepared as a 60 mm height and 5 mm broad strip [0260] Sink pad [0261] Conjugation pad (GF conjugate pad) [0262] Backing card [0263] Sample pad [0264] 40 nm OD20 gold colloid conjugated with 15 μg/ml ACE2 with 20 mM MES pH 5.3)

[0265] Plotting Procedure:

[0266] 1 mg/ml of human monoclonal anti SARS-CoV-2 Spike antibody is plotted on a CN140 membrane with a rate of 0.1 uL/mL to form a test line.

[0267] Spray Procedure:

[0268] 40 nm OD20 gold colloid conjugated with 15 μg/ml ACE2 with 20 mM MES pH 5.3 was prepared and sprayed onto the conjugation pad.

[0269] Strips were closed using 1-well Kanani housing tops and bottoms and passed through a cassette sealer.

[0270] The devices were placed in a labelled aluminium pouch with sufficient desiccant before sealing.

[0271] Procedure for Run of Dry 40 nm ACE-2 Gold Assay Using Healthy/Negative Saliva Spiked with RBD: [0272] The concentration of gold is at OD20 15 μg/ml ACE2 and 8 μl of the material was deposited centrally onto the conjugate pad. [0273] RBD spikes are diluted to the required concentration, or if testing with saliva, the initial concentration is doubled as RBD and saliva are combined 50:50. [0274] Sample/saliva is deposited onto the dried conjugate using 20 μl and an incubation time of 5 minutes and 20 min (see FIGS. 8A and 8B).

[0275] Procedure for Run of Dry ACE2 Gold Assay Spiked with Saliva Spiked with Attenuated Virus: [0276] Strips ran with 80 μl of 1×PBS, 1% Tween 20, 1% BSA. (other buffers have also been used in the optimisation process) [0277] Gold is at OD20 (15 μg/ml ACE2 from R&D); 8 μl deposited centrally onto the conjugate pad. [0278] Heat killed and irradiated virus stored at −80° C. [0279] Diluting irradiated virus requires 5×PBS 1% BSA 1% Pluronic 68. This buffer was also used as the running buffer.

[0280] Optimisation of the Sample and Gold Colloid Volume (for Saliva) [0281] Saliva was collected and filtered through a 0.45 μm filter [0282] Spiked with RBD to final concentration of 250 ng/ml (50:50) [0283] 15 μg/ml OD20 gold colloid dried (4-10 μl) on conjugate pad [0284] Compared 20, 40, 60, 80 μl sample volume, 5 minute incubations [0285] Assay ran with 80 μl running buffer

[0286] Assay Procedure: [0287] 20 μl is the current sample volume used for assay testing. [0288] Increasing the sample volume allows the assay to run, however this does not increase the signal. [0289] Larger conjugate pad possibly required to allow suitable incubation time. See FIG. 9.

[0290] Evaluation of Gold Volume Effects (Saliva) [0291] Repeated previous assay format for spiked saliva [0292] Dried varying volumes of gold: 4, 6, 8, 10 μl [0293] 20 μl spiked saliva with a 5-minute incubation, ran with 80 μl running buffer comparing [0294] 10 and 20 minute read times

[0295] The conclusion from this work is that 8 ul gold gives the optimal signal; 6 ul gold is also useful as the signal it generates is not too far below. See FIG. 10.

[0296] Assessment of Assay Sensitivity with Saliva [0297] Dried 8 μl of 15 μg/ml OD20 colloid [0298] Prepared saliva spiked with a starting concentration of 250 ng/ml RBD; doubling [0299] dilutions down. [0300] 5 minute incubation with 20 μl sample (saliva:buffer; 1:1), 80 μl running buffer run and [0301] read after 20 minutes.

TABLE-US-00008 ng/ml Cube units 250 168 125 97.4 62.5 58.2 31.3 21.5 15.6 15.5 7.8 8.3 3.9 6.6 2 5.5

[0302] See also, FIG. 11.

Example 3: Spiking of Fresh Virus into Saliva

[0303] Materials

[0304] SARS-CoV2 virus, drooled saliva, universal buffer, 12 SARS-CoV-2 Ag tests (as disclosed herein), eppendorf tubes, pipettes, tips and a timer.

[0305] Experimental Procedure

[0306] After addition of virus (used at 3×10.sup.4 PFU/mL) to the saliva, the sample was vortexed and inverted to ensure appropriate mixture to create a homogeneous sample. Dilutions were performed as follows to make up 5×10.sup.3, 2×10.sup.3 and 5×10.sup.2 and 0 pfu/mL from the provided 3×10.sup.4 PFU/mL viral stock solution as follows. [0307] 400 uL of universal buffer was added to 200 uL of the viral spiked samples and the sample was incubated for 60 s. [0308] 120 uL of the samples were added to a sample well and the device was left to run for 15 min. The device was read in the reading frame of 15-30 min and results were read by eye.

[0309] Titration of 3×10.sup.4 PFU/mL Virus

[0310] Dilutions were performed as follows to make up 5×10.sup.3, 2×10.sup.3 and 5×10.sup.2 and 0 PFU/mL from provided 3×10.sup.4 PFU/mL viral stock solution as follows:

[0311] Titration was completed as follows (Table 4):

TABLE-US-00009 TABLE 4 PFU/mL Sample/uL Saliva Cube units 1 0 0   200 uL 2.2 2 5 × 10.sup.3 83.33 uL (from 3 × 416.7 uL 13 10.sup.4 PFU/mL) 3 2 × 10.sup.3 33.33 (from 3 × 466.7 uL 11 10.sup.4 PFU/mL) 4 5 × 10.sup.2 8.33 uL (from 3 × 491.7 uL 3 10.sup.4 PFU/mL) 5 2 × 10.sup.2 3.33 uL (from 3 × 496.7 uL 1.7 10.sup.4)

[0312] Titration was performed in singles and a limit of detection of 5×10.sup.2 PFU/mL was visible.

[0313] Second titration on 20 healthy saliva samples were performed.

[0314] Cross Reactivity Studies

[0315] Materials

[0316] SARS-CoV-2 isolate, Culture materials, General consumables: filter tips, micropipette, vortex, PPE, reagent reservoirs, towels etc., Category 3 facilities and PPE, Human Coronavirus 229E, OC43, NL63, Influenza A and B, Rhinovirus and Adenovirus, 4 SARS-CoV-2 Rapid Antigen Tests devices (as described herein).

[0317] Protocol

[0318] This validation protocol will evaluate the specificity of the test by testing potentially cross-reactive microorganisms. The following microorganisms were tested in triplicate. The results were interpreted by two operators, each blinded to the result of the other. If a discrepant result was obtained, a third operator was called for a result. Invalid results are to be repeated once.

TABLE-US-00010 TABLE 5 The results of the Cross Reactivity assay Conc. Of Organisms likely in microorganisms circulating area PFU/ml Repeats Results Adenovirus 1.00 × 10.sup.5 3 Negative Influenza A 1.00 × 10.sup.5 3 Negative Influenza B 1.00 × 10.sup.5 3 Negative hCoV229 1.00 × 10.sup.5 3 Negative hCovOC43 1.00 × 10.sup.5 3 Negative hCoVNL63 1.00 × 10.sup.5 3 Negative Rhinovirus 1.00 × 10.sup.5 3 Negative

[0319] Antigen Tests devices (as disclosed herein), trimeric spike protein 1 mg/mL, Mcllvaine buffer with 0.3% DDAO pH 7.4.

[0320] 1 mg/mL of Trimeric spike was diluted in saliva to make a 5 ng/mL solution.

[0321] 100 uL of Mcllvaine buffer 0.3% DDAO pH 7.4 was added to 50 uL of the viral spiked samples and the sample was incubated for 60 s.

[0322] 120 uL of the samples were added to a sample well and the device was left to run for 15 min.

[0323] The device was read in the reading frame of 15-30 min and results were read by eye.