A lateral flow test strip to detect a threshold titre of neutralizing antibody to SARS-COV-2 in a biological sample from an individual

Abstract

A point of care lateral flow test strip is configured to semi-quantitatively detect whether or not a subject has a sufficient level (titre) of neutralising antibodies directed at a spike region of SARS CoV-2 virus (anti-spike protein neutralising antibody) in a biological sample obtained from the subject. The lateral flow test strip comprises three test zones that are configured to generate three visually detectable lines when greater that 500 binding antibody units per millilitre (BAU / ml) of anti- spike protein neutralising antibody is present in the biologial sample obtained from the subject. The appearance of three lines on the test strip indicates that the subject has a sufficient titer of neutralising antibody in their blood to confer protection from developing severe COVID-19 after exposure to the SARS-CoV-2 virus. The test provides a fast and easy to use point of care assay for subject to determine whether they are protected against SARS-COV-2 disease or whether they may require a booster vaccination.

Description

[0001] TITLE

[0002] A lateral flow test strip to detect a threshold titre of neutralizing antibody to SARS-

[0003] CoV-2 in a biological sample from an individual

[0004] Field of the Invention

[0005] The present invention relates to a lateral flow test strip to detect a threshold of antibody to SARS-CoV-2 in a biological sample from an individual.

[0006] Background to the Invention

[0007] The COVID-19 pandemic, also known as the coronavirus pandemic, relates to coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The novel virus was first identified in an outbreak in the Chinese city of Wuhan in December 2019. The resulting pandemic triggered severe social and economic disruption around the world, including the largest global recession since the Great Depression. Widespread supply shortages, including food shortages, were caused by supply chain disruptions. COVID-19 vaccines have been approved and widely distributed internationally since December 2020 and are widely credited for their role in reducing the severity of disease and death caused by COVID-19. However, the vaccines only provide protection against COVID-19 for a limited period of time necessitating that most people considered at risk of severe disease are administered booster vaccinations at regular intervals (from 4-6 months in most cases). As the protection conferred by prior SARS-CoV-2 infections and repeated vaccinations varies from person to person and varies over time, it is difficult to know if an individual has had an adequate response to vaccination and at what interval it is ideal to administer a booster vaccination. This is further complicated by the different levels of protection conferred to individuals administered different commercially available vaccines. The FDA currently recommends against using antibody testing to guide decisions around vaccination timing, use of prophylactic monoclonal antibody therapy and other therapies. This is primarily due to a lack of research evaluating the use of available tests for this purpose.

[0008] It is an objective of the invention to overcome at least one of the above-referenced problems by developing a test that can identify if someone has insufficient antibodies against SARS-CoV-2.

[0009] Summary of the Invention

[0010] The invention is based on the Applicants identification of a threshold of SARS-CoV- 2 neutralising antibody that can accurately predict a clinically significant host viral neutralising capacity against wild type SARS-COV-2 that was retained against two immune escape variants (Nature Communications | (2023)14:7015). The invention therefore broadly provides a point of care lateral flow assay (LFA) that can detect whether or not a subject has insufficient levels (titre) of neutralising antibodies directed at the spike region of SARS COV-2. The test is a semi-quantitative LFA that, in one embodiment, semi-quantitatively detects neutralising antibodies to the SARS-COV-2 spike protein in the blood, or in serum or plasma derived from whole blood of a subject. The test employs three test zones that are configured to generate three visually detectable lines when 500 or more binding antibody units per millilitre (BAU / rnl), which are equivalent to World health Organisation (WHO) international units (III), of anti-spike protein neutralising antibody is present in the blood sample. If three test lines do not appear, it is indicative that the subject has less than 500 BAU / rnl of neutralising antibody against SARS COV-2, which is considered to offer insufficient protection against COVID-19 disease, therefore indicating that the subject requires a booster vaccination for SARS-COV-2. Likewise, if three test lines do appear, it is indicative that the subject has 500 or more BAU / ml of neutralising antibody against SARS COV-2, which is considered to offer sufficient protection against COVID-19 disease. The result of the test assists clinical decision making to determine if an individual needs a booster vaccination or how effective prior vaccination or vaccinations have been for an individual.

[0011] In one embodiment, the LFA has three test zones; the first one detects the presence of any neutralising antibody and the second and third test zones detect whether an individual has sufficient (>500 BAU / rnl) neutralising antibodies present. The use of three test lines results in high sensitivity and specificity of 92.9% (95% confidence interval (Cl) 90.0% - 95.9%) and 94.7% (95% Cl 90.7% - 98.8%) respectively to correctly determine if an individual’s blood sample has >500 BAU / rnl of binding antibody against SARS-CoV-2 when tested in a group of people with known history of COVID-19. The use of three test lines also results in high sensitivity and specificity of 86.7% and 89.7% respectively to correctly determine if an individual’s blood sample has >500 BAU / rnl of binding antibody against SARS- CoV-2 when tested in another group of people with a mixed history of infection and vaccination.

[0012] In a first aspect, there is provided a lateral flow test strip to detect the presence or absence of a threshold titre of a neutralizing antibody to SARS-CoV-2 in a biological sample from an individual by semi-quantitative detection of a target neutralising antibody to a SARS-CoV-2 spike antigen. The test strip, in one embodiment, comprises: a sample application region; a conjugation region located downstream of the sample application region comprising a conjugate comprising (a) a capture ligand (e.g. antigen) with antigenic specificity to the target neutralizing antibody conjugated to (b) a detectable label; a first test region located downstream of the conjugation region comprising a first test zone comprising a first binding ligand immobilized at the first test zone region, wherein the first binding ligand has antigenic specificity to the capture antigen / target neutralizing antibody conjugate; a second test region located downstream of the conjugation region comprising a second test zone comprising a second binding ligand immobilized at the second test zone region, wherein the second binding ligand has antigenic specificity to the capture antigen / target neutralizing antibody conjugate; optionally, a third test region located downstream of the conjugation region comprising a third test zone comprising a third binding ligand immobilized at the third test zone region, wherein the third binding ligand has antigenic specificity to the capture antigen / target neutralizing antibody conjugate; and a control region located downstream to the two test zones comprising a ligand capable of specifically binding to a control protein in the sample to generate a visually detectable control line.

[0013] The first test region is typically configured to generate a visually detectable line when the biological sample comprises a detectable titre of target neutralizing antibodies to the SARS COV-2 spike protein (e.g. at least 20, 30, 40 or 50 BAU / rnl of the target neutralising antibody). The second and third test regions are each typically configured to generate a visually detectable line when the biological sample comprises a threshold (e.g. protective) titre of target neutralising antibody (e.g. at least 500 BAU / rnl of the target neutralising antibody). Thus appearance of the first line and no appearance of the second and optional third lines indicates that the sample contains neutralizing antibodies to the SARS COV-2 spike protein that are less than 500 BAU / rnl and therefore the individual may not have an adequate level of protective antibodies to SARS-CoV-2 and may require e.g. an additional booster vaccination, and the appearance of all three lines indicates that the sample contains a sufficient titre of neutralizing antibodies to the SARS COV-2 spike protein and, for example, suggests that the individual does not need a booster vaccine.

[0014] In any embodiment, the test strip comprises the third test region

[0015] In any embodiment, the capture antigen is Protein A. Other capture antigens that may be employed include SARS COV-2 antigens such as the spike protein, or domains thereof.

[0016] In any embodiment, the test strip comprises a buffer application region located downstream from the sample application region.

[0017] In any embodiment, the first test zone is configured to generate a visually detectable line when the biological sample comprises a level of target neutralising antibody of at least 20 BAU / rnl of the target neutralizing antibody.

[0018] In any embodiment, the ligand in the control zone comprises an immobilized Protein A.

[0019] In any embodiment, the control protein is a plasma protein.

[0020] In any embodiment, the first, second and third binding ligands are the same or different. In any embodiment, the first, second and third binding ligands are SARS- COV-2 spike protein (or a domain thereof such as the RBD domain)..

[0021] In any embodiment, the capture antigen is a recombinant protein.

[0022] In any embodiment, the lateral flow test strip comprises nitrocellulose or cellulose.

[0023] In any embodiment, the second and third test regions are disposed between the first test region and the control region. In another aspect, there is provided a point-of-care lateral flow assay device comprising a lateral flow test strip according to the invention.

[0024] In another aspect, there is provided a diagnostic kit comprising: a point-of-care lateral flow assay device according to the invention; a buffer for incubating the biological sample or for direct application to the lateral flow test; and optionally, a sample collection device for the biological sample.

[0025] In another aspect, there is provided a method to detect the presence or absence of a threshold titre of a neutralizing antibody to SARS-CoV-2 in a biological sample from an individual, wherein the method employs a lateral flow test strip or a diagnostic kit of the invention, the method comprising the steps of: applying the biological sample to the sample application region of the lateral flow test strip; adding buffer to the lateral flow test strip; and monitoring the test zones and control zones for the appearance of one or more lines (typically no more than 15 minutes after application of the sample and buffer to the lateral flow test device), wherein the appearance of a line in the control zone, the first test zone, the second test zone and the third test zone indicates that the sample contains a threshold titre of neutralizing antibody to SARS-CoV-2; or wherein the appearance of a line in the control zone and the first test zone but not in the second test zone and third test zone indicates that the sample does not contain a threshold titre of neutralizing antibody to SARS-CoV-2; or wherein the appearance of a line in the control zone but not in the first test zone, the second test zone and third test zone indicates that the sample does not contain a threshold titre of neutralizing antibody to SARS-CoV-2.

[0026] In any embodiment, the method comprises monitoring the test zones and control zones for the appearance of one or more lines for no more than 15 minutes after application of the sample and buffer to the lateral flow test device. In any embodiment, the absence of a line in the control zone indicates an invalid test result.

[0027] In any embodiment, the buffer is added to a buffer application region of the test strip.

[0028] In any embodiment, the buffer is added to the sample before a sample / buffer mixture is added to the sample application region.

[0029] In any embodiment, the biological sample is selected from blood, serum, plasma and urine.

[0030] In any embodiment, the biological sample is blood, wherein the method comprises applying 10 to 20 pl, or about 15 pl, of blood to the sample application zone.

[0031] In any embodiment, the biological sample is serum or plasma, wherein the method comprises applying 5 to 10 pl, or about 7.5 pl, of serum or plasma to the sample application zone.

[0032] In any embodiment, the capture antigen is protein A and each of the binding ligands is a SARS-COV-2 antigen (typically SARS-COV-2 spike protein, or a domain thereof).

[0033] Other aspects and preferred embodiments of the invention are defined and described in the other claims set out below.

[0034] Brief Description of the Figures

[0035] FIG.1 illustrates one embodiment of the lateral flow test strip of the invention. Fig. 2A illustrates the appearance of a test strip tested with a sample from an individual that has a sufficient titre of neutralizing antibodies to the SARS COV-2 spike protein, in which both the control line (11 ) the first test line (5) and both the second test line (6) and the third test line (7) are visually detectable. This test indicates that the sample from the individual has more than 500 BAU / rnl of neutralising antibody to the SARS-COV-2 spike protein.

[0036] Fig. 2B illustrates the appearance of a test strip tested with a sample from an individual who has an insufficient and undetectable titre of neutralizing antibodies to the SARS COV-2 spike protein, in which only the control line (11 ) is visually detectable. This test indicates that the sample from the individual contains less than 20 BAU / rnl of neutralising antibody to the SARS-CoV-2 spike protein.

[0037] Fig. 2C illustrates the appearance of a test strip tested with a sample from an individual who has a detectable but insufficient titre of neutralizing antibodies to the SARS COV-2 spike protein, in which both the control line (11 ) and first test line (5) are visually detectable but both the second test line (T2) and the third test line (T3) are not visually detectable. This test indicates that the sample from the individual contains neutralising antibodies against SARS-CoV-2 spike protein, but the titre of the antibodies is less than 500 BAU / rnl.

[0038] Fig. 2D indicates that the test result is invalid as the control line (C) has not appeared.

[0039] Detailed Description of the Invention

[0040] All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full. Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

[0041] Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0042] As used herein, the term "comprise," or variations thereof such as "comprises" or "comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method / process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method / process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open- ended and does not exclude additional, unrecited integers or method / process steps.

[0043] As used herein, the term “lateral flow test strip” primarily refers to the sandwich lateral flow assays of the type shown in Figure 1 (prior art) that generally comprise a strip of capillary flow materials having a longitudinal dimension and a capillary flow path along the longitudinal dimension. The strip generally comprises discrete overlapping sections including a sample pad, a buffer pad, (or a combined sample / buffer pad), a conjugate release pad, a detection membrane (e.g. cellulose or a cellulose derivative such as nitrocellulose), and an adsorbent pad. The sample pad is generally formed from a wicking material, the details of which will be known to those skilled in the art, although it may also be provided by a proximal part of the detection membrane. The conjugate release pad generally comprises a conjugate dispersed within a dissolvable matrix (often salt or sugar) that releases the conjugate upon dissolution by the sample fluid. The conjugate comprises an analyte-specific ligand (generally an analyte-specific antigen - referred to herein as a “capture antigen”) conjugated to a detectable label (such as gold nanoparticles). The conjugate release pad may also be provided by a proximal part of the detection membrane. Further description of the components of the sections of the lateral flow assay are not provided herein but will be known to a person skilled in the art and are described in the literature referenced below and in EP0349215. The capture antigen may comprise Protein A, a SARS-CoV-2 Spike antigen (protein fragment), SARS-CoV-2 receptor binding domain (RBD), spike S1 subunit and spike S2 subunit (Sino Biological Inc., Germany) which is generally conjugated to nanoparticle gold.

[0044] As used herein, the term “analyte” primarily refers to a neutralizing antibody to a SARS COV-2 antigen, preferably the SARS COV-2 spike protein (s-protein) antigen or an antigenic domain thereof. The nucleic acid sequence and amino acid translation of the SARS COV-2 spike protein are provided below:

[0045] Nucleic acid sequence SEQUENCE ID NO:1 atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgacaaagttttca gatcctcagt tttacattca actcaggact tgttcttacc tttcttttcc aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgataaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata ataagaggct ggatttttgg tactacttta gattcgaaga cccagtccct acttattgtt aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg cacagaagtc cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct cctcggcggg cacgtagtgt agctagtcaa tccatcattg cctacactat gtcacttggt gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa ttttactatt agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt acacaattaa accgtgcttt aactggaata gctgttgaac aagacaaaaa cacccaagaa gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc cttggtgata ttgctgctag agacctcatt tgtgcacaaa agtttaacgg ccttactgtt ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa aaattgattg ccaaccaatt taatagtgct attggcaaaa ttcaagactc actttcttcc acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc ctttcacgtc ttgacaaagt tgaggctgaa gtgcaaattg ataggttgat cacaggcaga cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac agacaacaca tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt aaacattcaa aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc caagaacttg gaaagtatga gcagtatata aaatggccat ggtacatttg gctaggtttt atagctggct tgattgccat agtaatggtg acaattatgc tttgctgtat gaccagttgc tgtagttgtc tcaagggctg ttgttcttgt ggatcctgct gcaaatttga tgaagacgac tctgagccag tgctcaaagg agtcaaatta cattacacat aa

[0046] Amino acid translation SEQUECE ID NO: 2:

[0047] MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFL PFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDS KTQSLLIVNNATNWIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCT FEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSAL EPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYN ENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCP FGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCF TNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGG NYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNG VGYQPYRVWLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNK KFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQD

[0048] VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGI CASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILP VSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFA QVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDC LGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPF AM Q M AYR F N G I G VTQ N VLYE N Q KL I AN Q F N S Al G KI Q D S LS STAS ALG KLQ D VVN QNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQ QLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHV

[0049] TYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF VSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASW NIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLC CMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT The “biological sample” refers primarily to blood and blood derivatives (serum, plasma, etc) and urine, but may also include other biological fluids such as saliva, sweat, cerebrospinal fluid, semen, and lymph. In one embodiment, the liquid biological sample employed in the methods of the invention, is undiluted (especially when the sample is urine). This provides an advantage insofar as the user is not required to dilute the sample, and makes the test suitable as a home-use test. In one embodiment, the sample is diluted no more than 2, 3, 4, 5, 6, 7, 8, or 9-fold.

[0050] As used herein, the term “neutralising antibody” as applied to SARS COV-2 refers to antibodies produced by B-cells in mammals as part of the mammal’s humoral immune response to a pathogen. In an aspect of the invention, the neutralising antibody (analyte) is an antibody produced in response to either exposure to the SARS-COV-2 spike protein (antigen) or to SARS-CoV-2 vaccination that binds to the SARS-CoV-2 spike protein antigen.

[0051] As used herein, the term “threshold titre” as applied to a SARS COV-2 neutralising antibody refers to an amount of the neutralising antibody in the blood of the subject that confers protection to the subject from developing severe COVID-19 after exposure to the SARS-CoV-2 virus. The is also referred to herein as a “protective titre”. When the subject is human and the neutralising antibody is an anti-spike protein antibody, the threshold titre is typically about 500 BAU / rnl of neutralising antibody. It will be appreciated that the threshold level may vary between populations, for example the threshold level in a Hispanic population may be 450 BAU / rnl whereas a more appropriate threshold level in a Celtic population may be 500 BAU / rnl. In one embodiment, the threshold titre is 465 BAU / rnl. Thus, the test strip of the invention is configured to detect levels of neutralising antibody in a blood sample from the subject that are at least, or greater than, this threshold level. If levels equal to or greater than the threshold are detected, the subject is determined to have a sufficient titre of neutralising antibody to SARS COV-2 (e.g. the subject has a protective level of neutralising antibody and does not need a booster vaccination). Likewise, if levels lower than the threshold are detected, the subject is determined to have an insufficient titre of neutralising antibody to SARS COV-2 (e.g. the subject does not have a protective level of neutralising antibody and therefore requires need a booster vaccination).

[0052] As used herein, the term “conjugate” refers to an analyte-specific ligand conjugated to a detectable label. The analyte -specific ligand may be an antigen (anti-spike protein), or analyte-specific antigen or protein fragment, or a protein (e.g. protein A). The analyte-specific antigen is referred to herein as a “Capture Antigen). The antigen is generally a non-human antigen that is specific for the analyte in question. The purpose of the conjugate is to bind to any analyte in the sample, and travel along the flow path in the strip to the detection membrane where the conjugate binds to the binding ligand (e.g. antigen or antibody) that is immobilised in the test zone. The detection antibody or antibodies may be an anti-human IgG secondary antibody, e.g. goat (Thermofisher). The binding ligand may be a SARS COV-2 spike protein or domain thereof.

[0053] As used herein, the term “detectable label” refers to a label that can be detected (for example, some emit a signal that is detectable), for example an optical, fluorescent, luminescent, magnetic, or electrical signal. Examples of detectable labels useful for the present invention include gold nanoparticles, coloured latex beads, magnetic particles, carbon nanoparticles, selenium nanoparticles, silver nanoparticles, quantum dots, up converting phosphors, organic fluorophores, textile dyes, enzymes, liposomes and others.

[0054] As used herein, the term “visually detectable” as applied to a line in a test zone or control zone refers to a line that can be detected by the human eye.

[0055] As used herein, the term “test zone” refers to an area of the detection membrane containing a detection ligand for the analyte (e.g. binding ligand such as SARS- COV-2 spike protein or domain thereof). Generally, the detection ligand for the analyte is provided along a line (e.g. test line) that is generally perpendicular to the longitudinal axis of the test strip. Generally, the detection ligand for the analyte is an analyte-specific antibody (i.e. capture antibody) or analyte-specific antigen (i.e. capture ligand). When the analyte is a neutralising antibody to the SARS COV-2 spike protein, the binding ligand is generally an antibody with antigenic specificity to the human immunoglobulin G antibody or an antigen of the SARS COV-2 spike protein. When the analyte is a human antibody, the capture antibody is generally a non-human antibody, for example a mouse or goat antibody that is specific for human IgG. The detection membrane may contain one or more test zones, and each may contain different or the same concentrations of the binding ligand for the analyte.

[0056] As used herein, the term “control zone” refers to an area of the detection membrane, generally distal of the test region, containing a binding ligand for a control protein. Generally, the binding ligand is provided along a line (i.e. control line) that is generally perpendicular to the longitudinal axis of the test strip. Generally, the binding ligand for the capture (analyte-specific) antibody is an antibody, for example an antibody-binding protein (e.g. Protein A).

[0057] As used herein, the term “low level” as applied to neutralizing antibody in the biological sample refers to a level of less than 20 lll / ml neutralizing antibody in the sample. For example, the first test zone (T1 line) may be configured to detect a level of at least 20 BAU / rnl of neutralising antibody, which is indicative of the subject having detectable SARS-CoV-2 spike protein within the sample.

[0058] Exemplification

[0059] The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

[0060] FIG.1 illustrates one embodiment of the lateral flow test strip of the invention, indicated generally by the reference numeral 1 (hereafter referred to as “test strip”). The test strip 1 comprises a buffer and sample application pad 2, a conjugate pad 3, and a nitrocellulose strip 4 (Sartorius CN180DX, 25mm) having a control zone and three test zones, namely a first test zone configured to generate the first test line 5 when at least 20 BAU / rnl of neutralising antibody to the SARS-COV-2 spike protein is in the sample and a second and third test zones configured to generate two lines 6, 7 when at least 500 BAU / rnl of neutralising antibody to the SARS-COV- 2 spike protein is in the sample. The test strip 1 is 4.2 mm in width, comprises Ahlstrom 222 chromatography paper 8 and a 75 mm Lohmann backing card.

[0061] In more detail, the buffer and sample application pad 2 is a 15mm thick MDI FR1 blood separation membrane and the conjugate pad 3 is an 8980 conjugate pad from Ahlstrom. The conjugate 10 comprises 40nm colloidal gold nanoparticle conjugated to Protein A (8pg / ml OD5 Gold, 10 mM KPOM OD20, spray rate 8 pL / cm).

[0062] The first test zone contains a line 5 of SARS-CoV-2 spike protein (RBG domain) immobilised across the nitrocellulose 18 mm from the top of the nitrocellulose (0.98 mg / ml protein, spray rate 0.8 pL / cm).

[0063] The second test zone contains a line 6 of SARS-CoV-2 spike protein (RBG domain) immobilised across the nitrocellulose 15 mm from the top of the nitrocellulose (0.6 mg / ml protein, spray rate 1.0 pL / cm).

[0064] The third test zone contains a line 7 of SARS-CoV-2 spike protein (RBG domain) immobilised across the nitrocellulose 12 mm from the top of the nitrocellulose (0.3 mg / ml protein, spray rate 0.8 pL / cm).

[0065] The control zone contains a line 11 of protein A immobilised across the nitrocellulose 9 mm from the top of the nitrocellulose (0.05 mg / ml Protein A in PBS, spray rate 0.8 pL / cm). In use, a sample of capillary blood is obtained is obtained from a subject. The sample may be processed to extract human plasma from the blood sample. In other embodiments, a urine sample may be employed, which may not need to be diluted in buffer. In the case of capillary blood, 15 pl of added to 100 pl of buffer. In the case of human plasma, 7.5 pl of plasma is added to 100 pl of buffer. The sample / buffer is then mixed and added to the sample / buffer application pad 2. After 15 minutes the test strip can be read.

[0066] Fig. 2A illustrates the appearance of the test strip 1 A tested with a blood sample from a previously vaccinated individual. The control line 11 and the three test lines 5, 6 and 7 are visually detectable. This test result indicates that the individual has more than 500 BAU / rnl of neutralising antibody to the SARS-COV-2 spike protein and does not therefore require a booster vaccination.

[0067] Fig. 2B illustrates the appearance of the test strip 1 B tested with a sample from an individual who has not been exposed previously to COVID disease and has not been vaccinated. Only the control line 11 is visually detectable. This test indicates that the sample from the individual contains less than 20 BAU / rnl of neutralising antibody to the SARS-CoV-2 spike protein, and that the therefore does not have a protective titre of neutralising antibodies to the SARS-CoV-2 spike protein and should be vaccinated

[0068] Fig. 2C illustrates the appearance of a test strip 1 C tested with a sample from a previously vaccinated individual. The control line 11 and test line 5 are visually detectable while the test lines 6 and 7 are not visually detectable. This test indicates that the sample from the individual contains neutralising antibodies against SARS-CoV-2 spike protein, but the titre of the antibodies is less than 500 BAU / rnl. The individual does not have a protective titre of neutralising antibodies to the SARS-CoV-2 spike protein and should be vaccinated

[0069] Fig. 2D indicates the appearance of a test strip 1 D in which the test result is invalid as the control line 11 has not appeared. Example 1

[0070] Sixty lateral flow tests according to Figure 1 were tested with plasma with the following conditions:

[0071] • 1^=10, RBD < 50 BAU / ml

[0072] • N=20, RBD < 500 BAU / ml

[0073] • N=15, 2000 BAU / ml < RBD > 500 BAU / ml

[0074] • N=15, RBD > 2000 BAU / ml

[0075] Read at 15 minutes with 2 scorers if 3 lines observed - 89.7% sensitivity, 86.7% specificity.

[0076] Read at 30 minutes with 2 scores if 3 lines observed - 95.8% sensitivity, 80.0% specificity.

[0077] Example 2

[0078] The invention was tested in 310 plasma samples derived from the whole blood of adult individuals who had a history of SARS-CoV-2 infection and / or recent vaccination and the results were compared to quantitative anti-SARS-CoV-2 antibodies targeting the receptor binding domain (RBD) region of the SARS-CoV-2 spike protein using published methodology. This quantitative antibody test can accurately quantify RBD spike antibody titres as low as 38 BAU / ml (lower limit of detection). The mean (standard deviation) of RBD spike antibody within the samples was 5170 BAU / ml, the maximum antibody titre was 52,935 BAU / ml and the lowest antibody titre was <38 BAU / ml. Of the 310 samples, 121 (39.0%) subjects had an RBD antibody titre below 500 BAU / ml.

[0079] The lateral flow tests contained a buffer application region and a downstream sample application region. The test was placed on a flat surface and 7.5 microlitres of the subject’s plasma was added to the sample application region following which two drops (approximately 40 microlitres) of buffer was added to the buffer application region. The test was then left undisturbed for 15 minutes following which the test was visually inspected for presence or absence of the control and the three test lines.

[0080] The lateral flow tests were conducted by trained laboratory personnel who were blinded to the results of the quantitative antibody titres. Images of the lateral flow tests were recorded and grouped together into a single pdf document with standardised instructions for how to interpret the results. These instructions included three possible results:

[0081] 1 . Sufficient

[0082] Where the control line was visible along with all three test lines (T1 , T2 and T3)

[0083] 2. Insufficient

[0084] Where the control line was visible but all three test lines (T1 , T2 and T3) were not clearly visible.

[0085] 3. Invalid

[0086] Where the control line was not clearly visible.

[0087] The document containing the standardised instructions along with the images of the test results were provided to 11 healthcare workers, comprising 4 medical doctors, 4 nurses and 3 pharmacists, who acted as assessors. All of the assessors were only provided with the images of the lateral flow test results and were not provided with any details about the subjects apart from a code and were blinded to the results of the quantitative antibody levels linked to the sample. The assessors were asked to assign one of three possible results to each of the lateral flow tests and these were recorded on a standardised results log.

[0088] The results of the lateral flow test readings were entered onto an electronic database and compared to the respective quantitative RBD antibody results. For each assessor set of results, sensitivity and specificity of the lateral flow test result to correctly predict an RBD spike protein level of >500 BAU / ml, along with the rate of false positive results was estimated as follows:

[0089] Sensitivity = A / (A+C)

[0090] Specificity = D / (B+D)

[0091] False positive rate = B / (A+B)

[0092] Using the 11 individual sensitivity and specificity values from each set of assessor results, the average sensitivity and specificity was estimated, along with 95% confidence intervals (95% C.I.). The overall results showed that the lateral flow tests correctly predicted a RBD spike protein concentration of greater than >500 lU / ml with a sensitivity of 92.9% (95% C.l. 90.0-95.9%) and a specificity of 94.7% (95% C.l. 90.7-98.8%). The average false negative rate was 3.3% (95% C.l. 0.9- 5.7%).

[0093] In addition, a consensus result for each lateral flow test was estimated. A consensus result was assigned according to the result assigned by a majority of the 11 assessors. When analysed, 290 (94.1 %) tests were assigned the same result by 9 or more assessors. The consensus results correctly predicted an RBD spike protein concentration of >500 BAU / ml with a sensitivity of 95.2% and specificity of 98.3%, with a false negative rate of 1.1 %.

[0094] Equivalents

[0095] The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.

Claims

CLAIMS:1 . A point of care lateral flow test strip configured to semi-quantitatively detect whether or not a subject has a sufficient level (titre) of neutralising antibodies directed at a spike region of SARS CoV-2 virus (anti-spike protein neutralising antibody) in a biological sample obtained from the subject, wherein the lateral flow test strip comprises three test zones that are configured to generate three visually detectable lines when greater than 500 binding antibody units per millilitre (BAU / rnl) of anti-spike protein neutralising antibody is present in the biologial sample obtained from the subject.

2. A point of case lateral flow test strip according to Claim 1 , in which the biological sample is blood or a blood derivative such as serum or plasma.

3. A point of case lateral flow test strip according to Claim 1 or 2, in which the three test zones comprise a first test zone configured to detect the presence of any antispike protein neutralising antibody in the blood sample obtained from the subject and a second and third test zone each configured to detect the presence of at least 500 BAU / rnl of anti-spike protein neutralising antibody in the blood sample obtained from the subject.

4. A point of case lateral flow test strip according to any preceding Claim, which is a sandwich lateral flow assay comprising a strip of capillary flow materials having a longitudinal dimension and a capillary flow path along the longitudinal dimension.

5. A point of case lateral flow test strip according to any preceding Claim, comprising: a sample application region; a conjugation region located downstream of the sample application region comprising a conjugate comprising (a) a capture antigen with antigenicspecificity to the anti-spike protein neutralising antibody or human immunoglobulin conjugated to (b) a detectable label; a first of the three test zones located downstream of the conjugation region comprising a first binding ligand immobilized at the first test zone, wherein the first binding ligand has antigenic specificity to the anti-spike protein neutralising antibody; a second of the three test zones located downstream of the conjugation region comprising a second binding ligand immobilized at the second test zone, wherein the second binding ligand has antigenic specificity to the antispike protein neutralising antibody; a third of the three test zones located downstream of the conjugation region comprising a third binding ligand immobilized at the third test zone, wherein the third binding ligand has antigenic specificity to the anti-spike protein neutralising antibody; and a control region located downstream to the three test zones comprising a ligand capable of specifically binding to a control protein in the sample to generate a visually detectable control line.

6. A point of case lateral flow test strip according to Claim 5, in which the capture antigen is protein A.

7. A point of case lateral flow test strip according to Claim 5 or 6, in which the first, second and third binding ligand is SARS-CoV-2 spike protein.

8. A method to detect the presence of a protective titre of a neutralizing antibody to SARS-CoV-2 spike protein in a biological sample from an individual, wherein the method employs a lateral flow test strip of any of Claims 1 to 7, the method comprising the steps of:applying the biological sample to the sample application region of the lateral flow test strip; applying buffer to a buffer application region of the lateral flow test strip; and monitoring the test zones and control zones for the appearance of one or more lines no more than 15 minutes after application of the sample and buffer, wherein the appearance of a line in the control zone, the first test zone, the second test zone and the third test zone indicates that the sample contains a protective titre of neutralizing antibody to SARS-CoV-2 spike protein.

9. A method of assessing if a subject requires a booster vaccination for SARS-CoV- 2, wherein the method employs a lateral flow test strip of any of Claims 1 to 7, the method comprising the steps of: applying a biological sample obtained from the subject to the sample application region of the lateral flow test strip; applying buffer to the lateral flow test strip; and monitoring the test zones and control zone for the appearance of one or more lines no more than 15 minutes after application of the sample and buffer, wherein: the appearance of a line in the control zone, the first test zone, the second test zone and the third test zone indicates that the subject has sufficient antibodies present; orthe appearance of a line in the control zone and the absence of line in the second test zone and / or the third test zone indicates that the subject does need a booster vaccination.

10. A method according to Claim 8 or 9, wherein the absence of a line in the control zone indicates an invalid test result.11 . A method according to any of Claims 8 to 10, wherein the buffer is added to the sample and a sample / buffer mixture is added to the sample application region.

12. A method according to any of Claims 8 to 11 , wherein the test zones and control zones are monitored for the appearance of one or more lines for no more than 15 minutes after application of the sample and buffer to the lateral flow test device.

13. A method according to any of Claims 8 to 12, wherein the biological sample is blood, serum or plasma.

14. A lateral flow test strip to detect the presence or absence of a threshold titre of a neutralizing antibody to SARS-CoV-2 in a biological sample from an individual by semi-quantitative detection of a target neutralising antibody to a SARS-CoV-2 spike antigen, wherein the lateral flow test strip comprises: a sample application region; a conjugation region located downstream of the sample application region comprising a conjugate comprising (a) a capture antigen with antigenic specificity to the anti-spike protein neutralising antibody or human immunoglobulin conjugated to (b) a detectable label; a first of the three test zones located downstream of the conjugation region comprising a first binding ligand immobilized at the first test zone, whereinthe first binding ligand has antigenic specificity to the SARS COV-2 antispike protein neutralising antibody; a second of the three test zones located downstream of the conjugation region comprising a second binding ligand immobilized at the second test zone, wherein the second binding ligand has antigenic specificity to the SARS COV-2 anti-spike protein neutralising antibody; a third of the three test zones located downstream of the conjugation region comprising a third binding ligand immobilized at the third test zone, wherein the third binding ligand has antigenic specificity to the SARS COV-2 antispike protein neutralising antibody; and a control region located downstream to the three test zones comprising a ligand capable of specifically binding to a control protein in the sample to generate a visually detectable control line.

15. A lateral flow test strip according to Claim 14, in which the capture antigen is protein A.

16. A lateral flow test strip according to Claim 14 or 15, in which the first, second and third binding ligand is SARS-CoV-2 spike protein.

17. A lateral flow test strip according to Claim 14 or 15, in which the first, second and third binding ligand is SARS-CoV-2 spike protein RBD domain.

18. A lateral flow test strip according to Claim 14, in which the capture antigen is protein A and in which the first, second and third binding ligand is SARS-CoV-2 spike protein RBD domain.

19. Use of a lateral flow test strip according to any of Claims 14 to 18 to identify whether has a subject has protective titre of neutralizing antibody to SARS-CoV-2 spike protein.

20. Use of a lateral flow test strip according to any of Claims 14 to 18 to identify whether or not a subject requires a booster vaccination against SARS-COV-2.

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