Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56983—Viruses
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
  • G01N2333/08—RNA viruses
  • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus

Definitions

• the present invention relates to a method of detecting the presence of an antibody in a sample, wherein said antibody has affinity for a coronavirus antigen, for example, a SARS-CoV antigen, a SARS-CoV2 antigen or a MERS- CoV antigen.
• a coronavirus antigen for example, a SARS-CoV antigen, a SARS-CoV2 antigen or a MERS- CoV antigen.
• the present invention also relates to a substrate having immobilised on its surface said coronavirus antigen and methods for producing said substrate.
• SARS-CoV-2 coronavirus pandemic and the incipient mortality and post-infection wellbeing impact has highlighted the fundamental importance of medical healthcare.
• a burgeoning and mobile global population has presented new challenges compared to previous pandemics, and world-wide diagnostic testing and vaccination programmes have emerged as the principle means being used to combat the virus.
• Coronaviruses are a group of related RNA viruses that cause respiratory tract infections in their host, the severity of which can range from mild to lethal. Examples of viruses relating to this family, in addition to SARS-CoV-2, include SARS-CoV and MERS-CoV, both of which have been responsible for viral outbreaks in 2002 and 2012, respectively. Coronaviruses are positive sense single-stranded RNA virus.
• the virion has an average diameter of approximately 80 -120 nm and incorporates four structural proteins, the spike, envelope (“E” protein), membrane (“M” protein) and nucleocapsid (“N” protein) proteins.
• the immune system produces antibodies to various component parts of the coronavirus virion in an individual who has either been infected by the virus or who has been vaccinated for virus protection.
• diagnostic tests relating to detection of coronaviruses, such as SARS-CoV-2, including real-time polymerase chain reaction (RT-PCR), antibody or serology tests and antigen tests, there is a continued need for improved diagnostic assays that can accurately determine if an individual who is/has been infected with a coronavirus has antibodies that confer protective immunity upon the individual.
• Nab neutralising antibodies
• Bab binding antibodies
• Nab are defined as antibodies that prevent entry of the virion into the host cell whereas Bab bind to the coronavirus but do not prevent host-cell infection by the virion.
• Nab can exert their neutralising effect and prevent virion docking onto the host cell receptor, for example, ACE-2, through binding to the coronavirus and directly blocking attachment or by an ‘indirect’ mechanism by altering coronavirus conformation and disabling potential attachment.
• ACE-2 a host cell receptor
• coronavirus diagnostic assays that are amenable to high through-put processing, efficient, reliable and provide a strong indicator as to the level of protective immunity an individual may possess.
• the present invention is based on the surprising discovery that the antibody detection assay herein described provides a highly sensitive and efficacious assay. Furthermore, said assay does not require a pre-incubation step, as is the case with other known assays, and therefore provides for a rapid and energy efficient test format.
• the present invention provides for a method of detecting the presence of an antibody in a sample obtained from a subject, wherein said antibody has affinity for a coronavirus antigen, or portion thereof, and wherein said method comprises bringing the sample obtained from the subject into contact with a coronavirus antigen, or portion thereof, which is immobilised on a substrate support, in the presence of a detectably-labelled molecule that can compete with any antibody present for binding to the coronavirus antigen, or portion thereof, and detecting the binding of antibody to the coronavirus antigen, or portion thereof, by measuring the amount of binding of said molecule compared to a control, wherein the coronavirus antigen, or portion thereof, is immobilised to the substrate surface by means of a linking agent.
• the present invention provides for a substrate having immobilised on its surface a coronavirus antigen, or portion thereof, as defined herein.
• the present invention provides for a method for producing a substrate having a coronavirus antigen immobilised thereon, comprising attaching the coronavirus antigen, or portion thereof, to the substrate surface via a sulphone or sulphonate-based cross-linker.
• Figure 1 shows a schematic of an exemplary assay format in which the receptor binding domain (RBD) is spotted onto a discrete test region (DTR) on the substrate surface.
• the RBD may be covalently attached to the substrate DTR by way of a substrate linking agent and optionally a cross-linker.
• the substrate surface can be optionally covered with an ink layer on the non-DTR area, to reduce non-specific binding of reagents to the substrate.
• the upper part of the RBD comprises the receptor binding motif (RBM).
• the neutralising antibody (Nab) prevents ACE-2 protein from binding to the RBD, whereas the binding antibody (Bab) does not.
• the Nab anti-ACE-2 effect can operate through either binding directly to the RBM (so-called ‘direct’ effect) or to a non-RBM portion of the RBD (so-called ‘indirect’ effect).
• Figure 2 shows the cross-linker 4-[bis(4-methylphenylsulphonylmethyl)-1- oxoethyl] benzoic acid, which can incorporate a silane moiety prior to addition to the substrate (see Figure 7, substrate linking agent B) or can be bonded to a substrate-linking agent (such as a silane moiety) already present on the substrate surface.
• Figure 3 shows the synthesis of 4-[bis(4-methylphenylsulphonylmethyl)-1- oxoethyl] benzoic acid and subsequent addition of a silyl moiety (APTES) to form a substrate linking agent.
• Figure 4 shows calibration curves for competitive neutralisation assay using RBD on substrate and ACE-2-HRP conjugate/anti-RBD antibodies in assay buffer.
• Figure 5 shows generic assay device components for an immunoassay incorporating a sulphone or sulphonate-based cross-linker.
• Figure 6 shows a device that can be used in a SARS-CoV-2 neutralisation antibody assay. Includes the S1 spike subunit with poly-His tag, a sulphone-silyl cross-linker/substrate linking agent and optional ink layer (w/wo means with or without).
• Figure 7 shows examples of suitable sulphone substrate linking agents.
• the terms “patient” and “subject” are used interchangeably herein and refer to any animal (e.g. mammal), including, but not limited to, humans, non-human primates, canines, felines, rodents and the like, which is to be where the sample is obtained from.
• the subject or patient is a human.
• a sample includes biological samples obtained from a patient or subject, which may comprise blood, plasma, serum, urine, saliva, mucous, wax, tears, hair, sweat or sputum.
• the sample is a blood or serum sample.
• detecting refers to qualitatively analysing for the presence or absence of a substance, for example, a signal, usually above a set threshold value to account for background signal noise, indicating presence or absence of the substance.
• determining refers to quantitatively analysing for the amount of substance present.
• detectably-labelled molecule refers to a molecule that has a label covalently attached to said molecule to enable its detection.
• labels may include, but are not limited to, radionuclides, fluorophores, dyes, quantum dots, polymers or enzymes, including, for example, horse-radish peroxidase (HRP) and alkaline phosphatase.
• HRP horse-radish peroxidase
• alkaline phosphatase preferably, the label is HRP
• antibody refers to an immunoglobulin which specifically recognises an epitope on a target as determined by the binding characteristics of the immunoglobulin variable domains of the heavy and light chains (V H S and V L S), more specifically the complementarity-determining regions (CDRs).
• the term “antibody” refers to an immunoglobulin which specifically recognises an epitope on a coronavirus antigen, for example, the spike protein of SARS-CoV, SARS-CoV2 or MERS- CoV, the nucleocapsid protein (“N” protein) of SARS-CoV, SARS-CoV2 or MERS-CoV, the HE protein of SARS-CoV, SARS-CoV2 or MERS-CoV, the Plpro protein of SARS-CoV, SARS-CoV2 or MERS-CoV, the 3CLPro protein of SARS-CoV, SARS-COV2 or MERS-CoV, the E protein of SARS-CoV, SARS- CoV2 or MERS-CoV, or the M protein of SARS-CoV, SARS-CoV2 or MERS- CoV.
• the antibody may recognise an epitope on the N protein, E protein or M protein of SARS-CoV, SARS-CoV2 or MERS- CoV2
• the term “has affinity for”, in the context of antibody-coronavirus antigen interactions, refers to an interaction wherein the antibody and epitope of the coronavirus antigen associate more frequently or rapidly, or with greater duration or affinity, or with any combination of the above, than when either antibody or coronavirus antigen is substituted for an alternative substance, for example an unrelated protein, such as a non-coronavirus related protein, or a coronavirus protein other than the coronavirus antigen in question.
• reference to binding means specific recognition.
• specific binding, or lack thereof may be determined by comparative analysis with a control comprising the use of an antibody which is known in the art to specifically recognise said target and/or a control comprising the absence of, or minimal, specific recognition of said target (for example wherein the control comprises the use of a non-specific antibody).
• Said comparative analysis may be either qualitative or quantitative. It is understood, however, that an antibody or binding moiety which demonstrates exclusive specific recognition of a given target is said to have higher specificity for said target when compared with an antibody which, for example, specifically recognises both the target and a homologous protein.
• coronavirus antigen refers to any part, or portion thereof, of the coronavirus virion that is capable of producing an immune response in the host/subject i.e. the production of antibodies in response to said antigen.
• coronavirus antigen may refer to the spike protein of SARS-CoV, SARS-CoV2 or MERS-CoV, the nucleocapsid protein (“N” protein) of SARS-CoV, SARS-CoV2 or MERS-CoV, the E protein of SARS-CoV, SARS-Co /2 or MERS-CoV, the M protein of SARS-CoV, SARS-CoV2 or MERS- CoV, the HE protein of SARS-CoV, SARS-CoV2 or MERS-CoV, the Plpro protein of SARS-CoV, SARS-CoV2 or MERS-CoV, or the 3CLPro protein of SARS-CoV, SARS-CoV2 or MERS-CoV
• coronavirus spike protein and “S1 protein” are used interchangeably and refers to a glycoprotein found on the surface of all coronaviruses.
• the spike protein is known to be important for the coronavirus gaining entry to the host cell via attaching to the complementary host cell receptor, for example, the host cell receptor for the spike protein of SARS-C0V2 is the angiotensin converting enzyme (ACE-2) receptor, and subsequent fusion with the host cell membrane.
• the spike protein may have the amino acid sequence according to Uniprot accession number P0DTC2, of which the RBD section spans amino acids 319-541 and the RBM section spans amino acids 437-508.
• the spike protein may be from any coronavirus, including variants of the spike protein containing one or more sequence variations or mutations.
• the coronavirus spike protein may be in monomeric form or, it may be a homotrimer to represent its native conformation on the virion.
• the coronavirus spike protein is an RBD or RBM.
• nucleocapsid protein and “N protein” are used interchangeably and refers to a highly conserved immunogenic phosphoprotein found in all coronaviruses that binds to viral RNA and leads to the formation of the helical nucleopcapsid.
• the nucleocapsid protein is composed of two separate domains, an N-terminal domain (NTD) and a C-terminal domain (CTD). It is thought that the abundance and high hydrophilicity of the N protein are key contributors in provoking an immune response in a subject following coronavirus infection.
• M protein or “membrane protein” are used interchangeably and refers to an integral membrane protein that is known to play a crucial role in coronavirus assembly.
• M proteins have three transmembrane domains and are glycosylated in all coronaviruses, either by N-linked or by O- linked oligosaccharides. These oligosaccharides have been shown to be important for folding, structure, stability, intracellular sorting of proteins and to play a role in the generation of immune responses in a subject following coronavirus infection.
• ⁇ protein or “envelope protein” are used interchangeably and refers to a small, hydrophobic membrane protein that contains at least one alpha-helical transmembrane domain.
• the E protein is known to play an important role in the assembly of virions, as well as in the host stress response.
• control refers to a value or values previously determined or determined simultaneously during the assay to which the assay results can be assessed and quantified against.
• the control will typically provide a measure of the extent of binding between the coronavirus spike protein and the detectably-labelled molecule in the absence of antibody. This can be used to provide a measure of the amount of binding that occurs in the assay between the coronavirus spike protein and antibody.
• the signal determined in the assay will be reduced due to the competitive binding of the antibody.
• the extent of the reduction compared to the control value(s) will indicate whether antibody is present in the sample and optionally the concentration of the antibody present.
• the control can be established as a calibration, alternatively, a calibration curve can be generated using neutralizing antibody preparations at multiple concentrations (see Figure 4).
• the assay signal output generated from a sample can be applied to the calibration curve to enable quantification of the Nab activity of said sample.
• epitope refers to the portion of a target which is specifically recognised by a given antibody.
• substrate linking agent As used herein, the terms “substrate linking agent”, “linker”, “linking agent” and “linking group” are used interchangeably and refers to a chemical moiety that connects the coronavirus spike protein to the substrate surface.
• cross-linker refers to any suitable chemical entity capable of binding a substrate linking agent to a coronavirus antigen, or portion thereof, for example, the RBD or RBM of the spike protein, the N protein, the M protein or the E protein.
• competitive and “competitive assay” are used interchangeably, and in the context of the present invention refers to the detectably labelled molecule and antibodies present in a sample competing for binding to the chip on which is immobilised a coronavirus spike protein.
• Such a competitive assay format may be a simultaneous competitive assay format (for example, the sample and detectably-labelled molecule are added at the same time) or a sequential competitive assay (for example, the detectably-labelled molecule is added to the substrate subsequently to the sample). Accordingly, the skilled person will readily understand that the assay herein disclosed is considered a “competitive assay” regardless of the order or timing of the addition of the competing moieties.
• the present invention provides for an assay device by which antibodies having affinity for a coronavirus antigen, or portion thereof, can be efficiently and accurately determined.
• Such an assay allows for an effective method of diagnosing subjects or patients suffering from a coronavirus related disease, for example, COVID-19, severe acute respiratory syndrome or Middle East respiratory syndrome, as well as an additional means of identifying subjects who may now have active immunity from said coronavirus.
• the present invention provides for a method of detecting the presence of an antibody in a sample obtained from a subject, wherein said antibody has affinity for a coronavirus antigen, or portion thereof, and wherein said method comprises bringing the sample obtained from the subject into contact with a coronavirus antigen, or portion thereof, which is immobilised on a substrate support, in the presence of a detectably-labelled molecule that can compete with any antibody present for binding to the coronavirus antigen, or portion thereof, and detecting the binding of antibody to the coronavirus antigen, or portion thereof, by measuring the amount of binding of said molecule compared to a control, wherein the coronavirus antigen, or portion thereof, is immobilised to the substrate surface by means of a linking agent.
• the coronavirus antigen, or portion thereof may be any part of a coronavirus that is capable of generating antibodies in a subject.
• the coronavirus antigen, or portion thereof may be a coronavirus spike protein, a coronavirus N protein, a coronavirus E protein, a coronavirus M protein and/or combinations thereof.
• the antigen, or portion thereof is the coronavirus spike protein.
• the method herein disclosed uses a device in which the coronavirus antigen, or portion thereof, is immobilised on the surface and is brought into contact with an ex vivo sample and a detectably-labelled molecule that has affinity for the coronavirus antigen, or portion thereof. It is therefore understood that, in one format, such an assay is a competitive binding assay, with the potential antibodies in the sample competing with the detectably-labelled molecule for binding to the coronavirus antigen, or portion thereof. Accordingly, a reduction in the signal produced by the detectably-labelled molecule signifies that there is a higher level of antibodies having affinity for a coronavirus antigen in the sample, and vice versa.
• the format of the assay herein described provides for an improved assay where the sample and detectably-labelled molecule do not require a pre-incubation step, thus allowing for a more efficient assay to be provided.
• the sample and detectably-labelled molecule can be mixed immediately prior to addition to the device/substrate surface, or added at the same time.
• the assay herein disclosed may be a simultaneous competitive assay format.
• the sample may be added first to the substrate, with the detectably-labelled molecule added to the substrate in a sequential step.
• the coronavirus spike protein, or portion thereof, immobilised on the substrate support may be brought into contact with the sample prior to the addition of the detectably-labelled molecule to the substrate support.
• the assay disclosed herein may be a sequential competitive binding assay. This format provides a large reduction in the expected binding when the detectably-labelled molecule is added if said sample contains antibodies having affinity for the coronavirus antigen, or portion thereof.
• the method herein disclosed provides an assay whereby the coronavirus antigen, or portion thereof, is immobilised to the substrate surface by means of a linking agent and optionally a cross-linker.
• Such a format allows for the projection of the coronavirus antigen, or portion thereof, away from the substrate surface, exposing the binding portion of the coronavirus antigen, or portion thereof, as well as producing a greater engagement of the coronavirus antigen, or portion thereof with the sample compared to a passive adsorption, resulting in a more sensitive assay. Additionally, this particular format minimises the influence of the substrate surface, leading to improved assay kinetics. Further details of appropriate linking agents and cross-linkers are provided below.
• the method herein disclosed may be suitable for the detection of both neutralising and binding antibodies, however, it is particularly envisaged that the method may be used to detect an antibody which is a neutralising antibody.
• the presence of Nab are said to demonstrate the individual having protective immunity against said coronavirus, and therefore have obvious benefits in being able to accurately and efficiently determine both their presence and amount in which they are present.
• the present invention also provides for a method of detecting whether an individual has protective immunity against a coronavirus, for example, SARS-CoV, SARS-CoV2, and MERS-CoV. Nab exert their neutralising effect by preventing the coronavirus from docking to its corresponding host cell receptor.
• Nab can prevent the coronavirus docking to the host cell by both direct and indirect mechanisms.
• the direct mechanism may involve the Nab binding to the RBM of the coronavirus spike protein and therefore preventing any interaction between the coronavirus spike protein and the corresponding host cell receptor.
• indirect mechanisms may involve the Nab binding elsewhere on the coronavirus, thus resulting in a conformational change of the coronavirus proteins important for coronavirus-host cell interaction, and preventing binding to the corresponding host cell receptor in this manner.
• the method herein may be used to detect antibodies having affinity for any coronavirus antigen, or portion thereof.
• the coronavirus antigen, or portion thereof may be a SARS-CoV antigen, SARS-CoV2 antigen or MERS- CoV antigen.
• the coronavirus antigen, or portion thereof may be a SARS-CoV2 spike protein, N protein, M protein and/or E protein.
• it is the SARS-C0V2 spike protein.
• the method herein may be used to detect neutralising antibodies having affinity for any coronavirus antigen, or portion thereof, for example, SARS-CoV antigen, SARS-CoV2 antigen or MERS-CoV antigen.
• the method herein disclosed may be used to detect neutralising antibodies having an affinity for SARS-C0V2.
• the method herein disclosed is exemplified in relation to SARS-CoV, SARS-C0V2 and MERS-CoV, it is envisaged that said method will also be applicable to any coronavirus, including any as of yet unknown future novel coronaviruses.
• the detectably-labelled molecule may be dependent on the coronavirus in question, for example, if the coronavirus is SARS-CoV, SARS-CoV2 or MERS-CoV. Accordingly, if the method is for the detection of antibodies, for example, neutralising antibodies, having affinity for the SARS-CoV spike protein or the SARS-Co /2 spike protein, the detectably-labelled molecule can be ACE-2. However, if the coronavirus spike protein is the MERS-CoV spike protein, then the detectably labelled molecule can be DPP4.
• coronavirus antigen, or portion thereof is not a coronavirus spike protein
• other suitable molecules can be used to compete with the antibodies in the patient sample, wherein said suitable molecules have affinity for the coronavirus antigen, or portion thereof, for example, wherein said suitable molecules have affinity for an epitope present on the N protein, M protein and/or E protein of SARS-CoV, SARS-CoN/2 or MERS-CoV.
• a detectably-labelled antibody (or suitable fragment or derivative thereof) that has affinity for the coronavirus antigen, or portion thereof could be used in the competition assay format as disclosed herein. This concept applies to any other coronavirus that may be used in the context of the method herein disclosed, including any future unknown coronavirus to which the target receptor is as of yet also unknown.
• the antibodies to be detected in a sample obtained from a subject may have an affinity for any epitope on the coronavirus antigen in question, for example, the SARS-CoV spike protein, N protein, M protein, or E protein, the SARS-CoV2 spike protein, N protein, M protein or E protein, or the MERS-CoV spike protein, N protein, M protein or E protein.
• the antibody may have an affinity for the RBD of the coronavirus spike protein in question.
• the antibody may have an affinity for the RBM of the coronavirus spike protein in question.
• the antibodies to be detected in a sample obtained from a subject may have an affinity for any epitope on a coronavirus antigen, for example, the antibodies to be detected may have an affinity for one or more of the coronavirus N protein, M protein and/or E protein.
• the different epitopes to which the antibodies bind may affect the mechanism by which the antibody exerts its effect. For example, for Nab, this may directly prevent the coronavirus from binding to the host cell receptor, for example, ACE-2 or DPP4.
• a conformational change may be induced which affects the binding capability of the coronavirus, thus preventing the binding to the host cell receptor in this way, for example, a partial blocking effect may occur, in which binding of the Nab reduces the efficiency of the virion approach, for example, to ACE-2 or DPP4.
• the presence of a linking agent and optionally a cross linker on the substrate surface allows for the coronavirus antigen, or portion thereof to be projected away from the substrate surface, exposing the binding portion of the coronavirus antigen, or portion thereof and producing a greater engagement of the coronavirus antigen, or portion thereof, with the sample compared to if no linking agent was present, i.e. passive adsorption.
• the present invention provides for a method in which the coronavirus antigen, or portion thereof, may be covalently attached to the substrate. This feature inevitably results in a more sensitive assay.
• the substrate linking agent may be any suitable chemical moiety that can support/bond to the coronavirus antigen, or portion thereof, or bond to a cross linker.
• the linking agent is an epoxy silane derivative, an epoxy oligomer or an epoxy polymer. Examples include, but are not limited to, (EtO) 3 Si-(CH2)n-NH 2 (see EP0874242 for silane derivative examples) and EPON-SU8 (also referred to as Epoly-8).
• the linking agent is EPON- SU8 (CAS No. 28906-96-9, commercially available from Hexion Inc).
• the coronavirus antigen, or portion thereof engages with the substrate (i.e. is supported by the substrate) by way of covalent bonds provided by a linking agent covalently attached to the substrate surface (the substrate linking agent) prior to coronavirus antigen addition.
• the coronavirus antigen, or portion thereof, for example, a SARS-CoV, a SARS- CoV2 or a MERS-CoV antigen may bond to the substrate linking agent and optionally a cross-linker through inherent functional groups such as carboxy, amino or sulphur present in the amino acids that constitute the protein.
• the assay effectiveness may be improved via the incorporation of a chemical activating group in the coronavirus antigen, or portion thereof.
• the attachment of the coronavirus antigen, or portion thereof, to a substrate linking agent and optionally a cross-linker requires that the binding characteristics of the coronavirus antigen, or portion thereof, are not affected, and that the specificity and affinity of the coronavirus antigen, or portion thereof, following the bonding process remain fit for purpose.
• Possible deleterious effects upon the coronavirus antigen, or portion thereof, upon chemical activation and or bonding to the substrate include coronavirus antigen tertiary structure disruption leading to reduced bonding and/or specificity to the target antibody.
• the method herein disclosed may use a coronavirus antigen, or portion thereof, immobilised to the substrate surface that further comprises a protein tag.
• the method herein disclosed may use a coronavirus antigen, or portion thereof, immobilised to the substrate surface that further comprises a poly-His tag or a coronavirus antigen, or portion thereof, immobilised to the substrate surface that has been modified to have a poly-His tag.
• the SARS-CoV spike protein, N protein, M protein or E protein may further comprise a poly-His tag or be modified to have a poly-His tag
• the SARS-CoV2 spike protein, N protein, M protein or E protein may further comprise a poly-His tag or be modified to have a poly-His tag
• the MERS-CoV spike protein, N protein, M protein or E protein may further comprise a poly-His tag or be modified to have a poly-His tag. Accordingly, such a modification results in a recombinant coronavirus antigen, or portion thereof, that helps to reduce structural disruption of the coronavirus antigen, or portion thereof, when bound to the linking agent/cross-linker.
• poly-His tag preferably a poly-His tag
• recombinant DNA methods comprising inserting the DNA encoding a protein into a suitable vector encoding a His-tag (Loughran and Walls, (2011), Purification of poly-histidine-tagged proteins, Methods Mol Biol, 681 :311 -35).
• the method herein disclosed may comprise a recombinantly produced coronavirus antigen, or portion thereof, incorporating a poly-His tag attached to a substrate linking agent/cross-linker.
• the poly-His tag can be varied in the number of histidine units but it is preferable to incorporate between 2 and 10 histidines, more preferably 6, 7 or 8 histidines; a 6 unit histidine tag is most preferred.
• the method herein disclosed also discloses an assay in which the coronavirus antigen, or portion thereof, may be immobilised to the substrate surface by means of a linking agent and a cross-linker.
• the method herein disclosed may use any cross-linker that is capable of binding the substrate linking agent to the coronavirus antigen, or portion thereof, for example, a coronavirus spike protein, N protein, M protein or E protein.
• the cross-linker is a bifunctional cross-linker.
• a bifunctional cross linker we refer to a molecule that has two reactive groups, each reactive group capable of reacting with a moiety of the substrate linking agent or moiety of the coronavirus antigen, or portion thereof, such that the substrate linking agent and coronavirus antigen, or portion thereof, are bound together by the cross-linker.
• the two reactive groups may be the same or different.
• Suitable bifunctional cross-linkers are well known in the art (for example, suitable bifunctional cross linkers are disclosed in Bioconjugate Techniques, G.T. Hermanson, Third Edition 2013).
• the cross-linker may be bonded to the surface linking agent prior to their attachment to the substrate surface; alternatively, the cross-linker may be bonded to a substrate linking agent, the substrate linking agent having already been attached (covalently bonded) to the substrate surface, or the cross-linker agent may be attached to the coronavirus antigen, or portion thereof, prior to their attachment to the substrate linking agent.
• “Sulphone-based cross-linker” refers to a cross-linker comprising at least one sulphone moiety
• “sulphonate-based” refers to a cross-linker comprising at least one sulphonate moiety.
• Suitable examples of sulphone-based cross-linkers include, but are not limited to: a,b-unsaturated ketone sulphones and precursors to a,b-unsaturated ketone sulphones, such as the bis-sulphone of Figure 3. Accordingly, in a preferred embodiment, a,b- unsaturated ketone sulphones and precursors to a,b-unsaturated ketone sulphones are used.
• sulphonate-based cross-linkers include, but are not limited to: a,b- unsaturated ketone sulphonates and precursors to a,b-unsaturated ketone sulphonates.
• Such cross-linkers incorporate strong leaving groups which promote favourable reaction kinetics and bonding to the poly-His tag if present in the coronavirus spike protein. It is noted that, for this reason, sulphonate-based cross-linkers may also be used as the substrate linking agent. Without being bound by theory, it is believed that the use of sulphonate-based cross-linkers in this manner achieve this effect due to their strong leaving group properties.
• the cross-linker when the cross-linker is a sulphone-based cross-linker, preferably an a,b- unsaturated ketone sulphone or precursor thereof, the cross-linker may have a structure according to Formula wherein X is -CH 2 -;
• R 2 is selected from -Ce- ⁇ aryl-Z, -Ci-isalkyl-Z, and -C2-2oalkenyl-Z, wherein Z is selected from COR 3 , -NH 2 and -OH, and R 3 is selected from H, OH, NH 2 , -Ci- ealkyl-OH and (EtO) 3 Si-(CH 2 ) n -NH- in which n is 1-6; and R is H or is optionally substituted with one or more Ci-ealkyl, N0 2 , F, Cl or Br;.
• Ri is CH 3 -Ph
• Ph is substituted with a Ci-ealkyl group, preferably methyl.
• R 2 is selected from -Ph-Z, -CioH s -Z, -Ci-ealkyl-Z and -C 2 -ealkenyl-Z, more preferably from -Ph-Z and -CioH s -Z, and more preferably -Ph-Z.
• Z is selected from COR 3 .
• the cross-linker is a sulphone-based cross-linker, preferably an a,b- unsaturated ketone sulphone or precursor thereof, the cross-linker may have a structure according to Formula (2): wherein X is -CH 2 -;
• Ri is CH 3 or CH 3 -Ph, wherein the Ph is optionally substituted with one or more Ci-ealkyl;
• R 3 is selected from H, OH, NH 2 , -Ci-ealkyl-OH and (EtO) 3 Si-(CH 2 ) n -NH- in which n is 1-6.
• Ri is CH 3 -Ph, and Ph is substituted with a Ci-ealkyl group, preferably methyl.
• R 3 is OH, H or (EtO) 3 Si-(CH 2 )n-NH- in which n is 1-6.
• the cross-linker when the cross-linker is a sulphone-based crosslinker, preferably an a,b- unsaturated ketone sulphone or precursor thereof, the cross-linker may have a structure according to Formula (3) or Formula (4): wherein R 3 is selected from H, OH, NH 2 , -Ci-ealkyl-OH and (EtO) 3 Si-(CH 2 ) n -NH- in which n is 1-6, preferably OH, H or (EtO) 3 Si-(CH 2 ) n -NH- in which n is 1-6.
• the cross-linker is a sulphone-based cross-linker. More preferably, the cross-linker is a sulphone-based crosslinker of Formulas (1), (2), (3) or (4) as detailed above.
• Ph refers to phenyl
• CioH s refers to a naphthalene or napthyl group.
• Ci-is alkyl demotes a straight or branched saturated alkyl group having from 1 to 18 carbon atoms; For parts of the range Ci-is alkyl, all sub-groups thereof are contemplated, such as Ci-e alkyl, C 5-15 alkyl, C 5-10 alkyl, and C 1-6 alkyl.
• Examples of said C 1-4 alkyl groups include methyl, ethyl, n- propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and tert-butyl.
• the alkyl groups may be optionally substituted with one or more functional groups, including C 1-18 alkyl groups, "Ce- 12 aryl", and "C 1-18 alkoxy", halogen, and "C 3-18 cycloalkyl".
• C 2 -isalkenyl denotes a "Ci-is alkyl” group containing some degree of unsaturation (partial unsaturation) i.e. containing one or more alkene/alkenyl moiety(s).
• Ce- 12 aryl denotes a monocyclic or polycyclic conjugated unsaturated ring system having from 6 to 12 carbon atoms.
• Ce- 12 aryl all sub-groups thereof are contemplated, such as Ce-io aryl, C 10-12 aryl, and Ce-s aryl.
• An aryl group includes condensed ring groups such as monocyclic ring groups, or bicyclic ring groups. Examples of Ce- 12 aryl groups include phenyl, biphenyl, indenyl, naphthyl or azulenyl.
• Condensed rings such as indan and tetrahydro naphthalene are also included in the Ce- 12 aryl group.
• the aryl groups may be optionally substituted with other functional groups.
• the aryl groups may be optionally substituted with one or more functional groups, including C 1-18 alkyl groups, halogen, and "C 1-18 alkoxy".
• the aryl groups may be substituted with these substituents at a single position on their unsaturated ring system, or may be substituted with these substituents at multiple positions on their unsaturated ring system.
• the term “fully saturated” refers to rings where there are no multiple bonds between ring atoms.
• the cross-linker may be subject to chemical activation prior to attachment to the substrate linking agent.
• chemical activation is meant a process whereby the cross-linker is altered such that it has increased propensity for subsequent reaction, i.e. increased propensity for bonding with the substrate linking group and/or coronavirus spike protein.
• Suitable methods of chemical activation will be well known by a skilled person, for example, the EDC method or a maleimido-group incorporation.
• Z of formula (1) is COR 3 and R 3 is OH
• R 3 of formula (2) is OH
• the EDC method or a maleimido-group incorporation may be used to activate the cross-linker.
• the present method also provides for the incorporation of a substrate linking agent in the sulphone- or sulphonate-based cross-linker prior to the addition to the substrate. This has the advantage of omitting the substrate chemical-activation step, which in turn benefits efficiency of device production.
• Figures 3 and 5 present examples of silyl-sulphone compounds.
• the method herein provides for an immunoassay in which the antibodies in a sample and a detectably-labelled molecule compete with one another to bind to the immobilised coronavirus antigen, or portion thereof, bonded to the substrate surface via the linking agent and optionally the cross-linker described above (see Figure 1). Accordingly, the detectably-labelled molecule provides a detectable and measurable signal which is reduced in the presence of antibodies, thus allowing the antibodies present in said sample to be detected and/or quantified.
• the measurable signal may be electromagnetic radiation based on, for example, phosphorescence, fluorescence, chemiluminescence (e.g. HRP/luminol/peroxide system).
• fluorescence or chemiluminescence is used.
• a detecting agent suitable for use with the present invention is the streptavidin- biotin-enzyme complex
• avidin may also be used in place of streptavidin, resulting in a complex with the molecule to be detected, for example, an ACE-2 - biotin-streptavidin-enzyme complex, a DPP4-biotin-streptavidin-enzyme complex or an antibody-(or suitable fragment or derivative thereof)-biotin-streptavidin- enzyme complex.
• the method herein disclosed may include a detectably-labelled molecule labelled with a streptavidin-biotin-enzyme complex or an avidin-biotin-enzyme complex.
• the enzyme of said complex may be HRP, which when exposed to luminol/peroxide system produces a detectable signal.
• a calibrator or standard which can be used for effecting assay calibration, is well known in the art and enables a threshold concentration or the exact or calibrator equivalent amount of analyte(s) to be determined. The determination of an exact or calibrator equivalent amount of analyte(s) usually requires the construction of a calibration curve (also known as a standard curve). The number of calibrator points vary but is preferably from 5 to 9. Alternatively, the calibrator value can be a single pre-determined threshold value.
• the surface of the substrate for use in the method of the present invention may be selectively covered with a coating composition.
• the coating composition may be any coating that would be expected to enhance or maintain the desirable properties of the surface to which the coronavirus antigen, or portion thereof, is immobilised, i.e. the DTR.
• the term “selectively covered”, as used in the context of the present invention, refers to areas of the substrate where the coronavirus antigen, or portion thereof, is immobilised.
• the coating composition may be an ink formulation.
• Ink formulations have been added to the surfaces of substrates, such as chips and biochips used in medical diagnostics, to minimise the attachment of chemical and biological components present in patient samples which can affect assay performance. It is understood that the choice of ink can be dependent upon the assay configuration of the substrate and the assay objectives and is usually characterised as being on the hydrophilic-hydrophobic continuum using the water contact angle. It has been found that the assay devices and methods of the current invention can further benefit from an increase in performance if the substrate surface supports an ink layer. This leads to yet a further increase in the sensitivity of the assay.
• the ink formulation may comprise a pigment, a resin, an ink solvent, an ink additive and/or a structuring agent.
• pigments may be selected from the group of inorganic artificial pigment, inorganic natural pigment, organic artificial pigment, organic natural pigment, black pigment, blue pigment, brown pigment, green pigment, orange pigment, red pigment, violet pigment, white pigment or yellow pigment.
• resins include, but are not limited to, acrylics, alkyds, epoxides, hydrocarbons, phenolics or fluoropolymers such as a polytetrafluoroethylene (PTFE).
• Suitable ink solvents include, but are not limited to, cyclohexanone, butoxyethanol and aromatic distillates.
• Suitable ink additives include, but are not limited to, carbon black (black pigment), mineral oil (wetting agent), petroleum distillate, dibutyl phthalate (plasticizer), salts of cobalt, manganese or zirconium (drying agent), aluminium and titanium chelator (chelating agent), antioxidants, surfactants and defoamers.
• Suitable structuring agents include, but are not limited to CERAFLOUR® 965.
• the ink formulation may comprise one or more resins, pigments, ink solvents/additives and/or structuring agents selected from the lists above, and combinations thereof.
• the ink formulation may be a composition according to those disclosed in EP3377900 A1.
• the ink formulation may comprise an epoxy or acrylic resin, a pigment and a structuring agent.
• the ink composition may comprise an acrylic resin, a pigment and a structuring agent.
• One pigment may be present or multiple pigments may be used.
• Epoxy and acrylic resin are used to increase ink viscosity, rheological properties and adhesion to the substrate.
• the pigment imparts a dark colour, preferably a black colour, and hence imparts optical opacity to the ink.
• the structuring agent provides hydrophilic/hydrophobic properties to the surface of the substrate and also help adhesion to the substrate.
• the pigment may be present in an amount of 1 to 15% w/w of the ink formulation; the epoxy resin may be present in an amount of 10 to 60 % w/w, the acrylic resin may be present in an amount of 1 to 20% w/w, and the structuring agent may be present in an amount of 10 to 60% w/w.
• the pigment preferably black pigment, is present in an amount of 1 to 8% w/w of the ink formulation; the epoxy resin is present in an amount of 15 to 50 % w/w of the ink formulation, the acrylic resin is present in an amount of 2 to 15% w/w of the ink formulation, and the structuring agent is present in an amount of 15-50% w/w of the ink formulation.
• the pigment preferably black pigment, is present in an amount of 5% w/w of the masking composition; the epoxy resin is present in an amount of 30 % w/w of the ink formulation, the acrylic resin is present in an amount of 10% w/w of the ink formulation, and the structuring agent is present in an amount of 20% w/w of the ink formulation.
• carbon black pigment is used in the ink formulation, preferably Elftex 285.
• the acrylic resin is B-67.
• the structuring agent is a PTFE wax, such as CERAFLOUR® 965.
• the ink formulation comprises an epoxy resin, preferably Epikote 1004.
• the pigment is Elftex 285, the acrylic resin is B-67, the epoxy resin is Epikote 1004 and the structuring agent is CERAFLOUR® 965.
• the ink formulation may further comprise one or more agents selected from the list of solvents, such as ethanol, propanol, xylene, diglycol, butyl ether; dispersing agents; pigment wetting agents; levelling agents; pigment wetting agents and/or crosslinking agents.
• the ink formulation has a contact angle of 20- 175°, more preferably 20-170° more preferably 90-120°, even more preferably about 1 10°.
• the measurement is taken using the following protocol: The contact angle is measured using a KSV CAM200 contact angle meter equipped with automated dispenser controlled using stepper motor, LED source and CCD camera.
• the contact angle meter is connected to a software tool for dispense controller, image grabbing and image analysis. A droplet of deionised water of 3.5 pi is dispensed on the substrate at a predefined location and the image is captured using a CCD camera. Image analysis is performed using software to estimate the contact angle of the water droplet.
• the thickness of the ink formulation applied to the substrate is 1-100 pm thick, preferably, 2-50 pm thick. This creates a discrete reaction zone that is a well having a depth of 1 -100 pm, preferably 2-50 pm, respectively. Most preferably the thickness of coating is 3 to 20 pm thick and the resulting depth of the well is 3 to 20 pm in depth.
• the ink is applied to the substrate such that there are exposed areas of the substrate that define the reaction sites.
• the areas coated with the ink are not intended to be the reaction sites and provide a typically hydrophobic surface that prevents non-specific binding from occurring and helps retain solutions at the reaction site to provide a clear image of the reaction site.
• the sample for use in the method of the present invention may be any biological sample taken from the individual in which antibodies may be detected.
• the biological samples require no pre-processing and can be used neat in the assay herein disclosed.
• the sample may be a serum sample, plasma sample, whole blood sample, urine sample, mucous sample, saliva sample, CSF sample, sputum sample, ear wax sample, hair sample, sweat sample, tear sample, meconium, skin, solid tumour extracts, peripheral blood mononuclear cells, bone marrow mononuclear cells, cerebrospinal fluid, cystic fluid or any suitable cell lysate.
• the sample is a serum or plasma sample; alternatively, it is a whole blood sample or a saliva sample.
• the sample may be obtained from the subject or patient by methods routinely used in the art, for example, via venous blood collection, swab testing or tissue biopsy.
• the determination and/or detection of antibodies, for example, neutralising antibodies may be done on one or more samples obtained from the subject.
• the substrate for use in the present invention may be of a planar conformation, such as a glass slide, microtitre plate or a chip/biochip.
• the term “biochip” refers to a chip whose use is biomedical and is made of a thin, wafer like substrate with a planar surface.
• the substrate may be a bio-chip due to its stability and adaptability.
• the biochip may be made of any suitable material, such as glass or any suitable polymer, preferably, the biochip is made of ceramic. Even more preferably, the biochip is made of aluminium oxide based ceramic and may be chemically activated.
• a ceramic substrate can be manufactured to provide a range of grain sizes.
• Typical grain sizes are 1 to 30 pm; ⁇ 10 pm is preferred as the reduced particle size imparts increased surface homogeneity which improves assay performance.
• the preferred ceramic material consists of about 94% alumina (AI 2 O 3 ) with a particle size in the range of 4-8 pm.
• the surface topography is usually withing the range of 0.6 to 0.8 pm after being ground. This can be improved through polishing to yield a surface with variation of 0.4-0.5 pm which can be further improved through by lapping and polishing to 0.05-0.1 pm.
• the present invention provides for a substrate having immobilised on its surface a coronavirus antigen, or portion thereof, as defined by any of the features herein described. Accordingly, the present invention provides for a substrate having immobilised on its surface a SARS-CoV antigen, a SARS CoV-2 antigen or a MERS-CoV spike antigen connected by a linking agent and optionally a cross-linker.
• the substrate may have immobilised on its surface a single type of coronavirus and/or coronavirus antigen, or portion thereof, for example, SARS-CoV spike protein, N protein, M protein, E protein, SARS-C0V2 spike protein, N protein, M protein, E protein or MERS-CoV spike protein, N protein, M protein or E protein.
• the substrate may have immobilised on its surface a combination of coronavirus and/or coronavirus anitgens, for example, SARS-CoV spike protein, N protein, M protein, E protein, SARS-Co /2 spike protein, N protein, M protein, E protein or MERS-CoV spike protein, N protein, M protein or E protein or a combination of variants of the coronavirus antigen.
• each detectably-labelled molecule having affinity for a specific coronavirus antigen, or portion thereof may have different labels, for example, different fluorophores, attached in order to differentiate the signal arising from different coronavirus antigens, or portions thereof.
• the substrate herein disclosed may be spatially arranged in such a manner that the same effect is achieved and the same detection labels may be used. The skilled person will recognise that being able to identify antibodies in a sample directed at multiple targets will help reduce the level of false negatives, thus resulting in a more sensitive, accurate and reliable assay.
• the present invention provides for a substrate having immobilised on its surface a SARS-CoV2 antigen, or portion thereof.
• the linking agent is EPON-SU8 and the cross-linker is a sulphone- or sulphonate-based cross-linker according to Formula (1), even more preferably, the cross-linker is a sulphone-based crosslinker according to Formula (2), (3) or (4).
• the substrate is a bio-chip, even more preferably, the substrate is a ceramic bio-chip.
• the substrate may be selectively covered with an ink formulation, as described above.
• a method for producing a substrate having a coronavirus antigen, or portion thereof, immobilised thereon, comprising attaching the coronavirus antigen, or portion thereof, to the substrate surface via a sulphone or sulphonate-based cross-linker comprising attaching the coronavirus antigen, or portion thereof, to the substrate surface via a sulphone or sulphonate-based cross-linker.
• the coronavirus antigen, or portion thereof may be a SARS-CoV antigen, or portion thereof, SARS-CoV2 antigen, or portion thereof, or a MERS-CoV antigen, or portion thereof.
• the coronavirus antigen, or portion thereof is a SARS-CoV2 antigen, or portion thereof.
• the present invention also discloses, a coronavirus antigen, or portion thereof recombinantly modified to include a poly-His tag.
• the coronavirus antigen, or portion thereof, recombinantly modified to include a poly-His tag may be a SARS-CoV antigen, or portion thereof, a SARS-CoV2 antigen, or portion thereof, or a MERS-CoV2 antigen, or portion thereof.
• the coronavirus antigen, or portion thereof, recombinantly modified to include a poly-His tag is a SARS-C0V2 antigen, or portion thereof.
• SEQ ID NO: 2 and SEQ ID NO: 6 were expressed in human cells using standard cloning of codon optimized SEQ ID NO: 4 and SEQ ID NO: 8 respectively into a constitutive high level mammalian expression vector with an artificial or native signal peptide sequence and C terminal His tag, resulting in the expression of SEQ ID NO: 2 and SEQ ID NO: 6 and after removal of the signal peptide sequence, SEQ ID NO: 3 and SEQ ID NO: 7.
• Transfected cells were cultured at 37°C and 8% CO2 in Expression Medium (Thermoscientific) containing glutamine for three to six days before harvesting.
• SEQ ID NO: 3 and SEQ ID NO: 7 comprised cell supernatant collection which was clarified by centrifugation for 30 minutes at 7,000rpm. Remaining particulate matter was removed by filtration through 1.2mM and 0.45mM low protein binding filter units respectively.
• SEQ ID NO: 3 and SEQ ID NO: 7 protein was purified from the clarified and filtered supernatant at 4°C by the Akta Avant system (Cytiva) using Nickel HisTrap Excel columns (Cytiva Lifesciences, USA) preequilibrated with 20mM Tris, 500mM Sodium chloride pH8.0.
• Contaminant non-specific binding protein was removed by a wash containing 20mM Tris, 500mM sodium chloride, 30mM imidazole and SEQ ID NO: 3 and SEQ ID NO: 7 were eluted by increasing the concentration of imidazole to 500mM. All fractions containing either SEQ ID NO: 3 or SEQ ID NO: 7 were pooled and concentrated by ultrafiltration (Vivaspin, Sartorius, Gottingen Germany) and buffer exchanged into PBS pH7.4 (Melford, UK).
• SEQ ID 3 and SEQ ID NO: 7 were further purified by fractionation on a HiLoad 26/600 Superdex 75 PG column (GE Healthcare, USA) pre-equilibrated in 2X PBS pH7.4 (Melford, UK). The column fractions were monitored for absorbance at A280nm. Selected fractions containing SEQ ID NO: 3 or SEQ ID NO: 7 were collected, pooled and concentrated by ultrafiltration (Vivaspin, Sartorius, Gottingen Germany). The final preparations of SEQ ID NO: 3 and SEQ ID NO: 7 proteins were evaluated by SDS gel electrophoresis. For spotting to the surface of the biochip, expressed protein SEQ ID NO: 3 was diluted in buffer.
• SARS-CoV-2 Nucleoprotein (Uniprot ID P0DTC9) was expressed in human cells using standard cloning methods into a constitutive high level mammalian expression vector with an artificial signal peptide sequence and C terminal His tag, resulting in the expression of SEQ ID NO: 9 after removal of the signal peptide.
• Transfected cells were cultured at 37oC and 8% C02 in Expression Medium (Thermoscientific) containing glutamine for three to six days before harvesting.
• SEQ ID NO: 9 Preparation of SEQ ID NO: 9 comprised cell supernatant collection which was clarified by centrifugation for 30 minutes at 7,000rpm. Remaining particulate matter was removed by filtration through 1.2mM and 0.45mM low protein binding filter units respectively.
• SEQ ID NO: 9 protein was purified from the clarified and filtered supernatant at 4°C by the Akta Avant system (Cytiva) using Nickel HisTrap Excel columns (Cytiva Lifesciences, USA) preequilibrated with 20mM Tris, 500mMSodium chloride pH8.0.
• Contaminant non-specific binding protein was removed by a wash containing 20mM Tris, 500mM sodium chloride, 30mM imidazole and SEQ ID NO: 9 was eluted by increasing the concentration of imidazole to 500mM. All fractions containing either SEQ ID NO:9 was pooled and concentrated by ultrafiltration (Vivaspin, Sartorius, Gottingen Germany) and buffer exchanged into PBS pH7.4 (Melford, UK). Expressed SEQ ID NO: 9 was further purified by fractionation on a Hil_oad26/600 Superdex 75 PG column (GE Healthcare, USA) pre-equilibrated in 2X PBSpH7.4 (Melford, UK).
• SEQ ID NO: 9 The column fractions were monitored for absorbance atA280nm. Selected fractions containing SEQ ID NO: 9 was collected, pooled and concentrated by ultrafiltration (Vivaspin, Sartorius, Gottingen Germany). The final preparations of SEQ ID NO: 9 proteins were evaluated by SDS gel electrophoresis. For spotting to the surface of the biochip, expressed protein SEQ ID NO: 9 was diluted in buffer.
• a ceramic substrate was washed with RBS 35 concentrate and water and plasma treated before addition of Epon SU8 (Hexion Incorporated, Ohio, US) or (OEt)3-Si-(CH 2 )3-NH 2 or a silyl-sulphone (B of Figure 7). Following stirring it was deposited on the ceramic substrate by spray coating and the chips cured for 1 hr at 140°C.
• Epon SU8 Hydrophilic Incorporated, Ohio, US
• OEt OEt3-Si-(CH 2 )3-NH 2 or a silyl-sulphone
• Substrate linking agent/Cross-linker preparation (see Figures 3 & 7) Substrate linking agent/Cross-linker A. To a cooled solution (0°C) of (3- aminopropyl) triethoxysilane (APTES) (22.131 g, 0.1 mol) and diisopropylethylamine (20.9mls, 0.12mol) in dichloromethane (300ml) under nitrogen was added dropwise a solution 2-chloroethylsulphonyl chloride (16.63g, 0.102mol) in dichloromethane (50mls). The mixture was than stirred at 0°C for two hours and then overnight at room temperature.
• APTES (3- aminopropyl) triethoxysilane
• 2-chloroethylsulphonyl chloride (16.63g, 0.102mol
• Substrate linking agent/Cross-linker B To a solution of bis-sulphone acid (2g, 4mmol) in dichloromethane was added N-hydroxysuccinimide (506mg, 1.1 eq) and dicyclohexylcarbodiimide (908mg, 1.1 eq) under nitrogen. The mixture was stirred at room temperature for 2h, the reaction was filtered to remove the urea by-product and the filtrate was evaporated to dryness in vacuo to give the crude product (2.74g) as a white solid.
• Substrate linking agent/Cross-linker C Monosul phone carboxylic acid (2.4027g, lOmmol) was dissolved in dichloromethane (40ml) containing N,N- dimethylformamide (1ml). To the suspension was added oxalyl chloride (5ml, 5.8eq) in dichloromethane (10ml) dropwise. On completion of addition the mixture was stirred at room temperature for 1h. The solvents were removed in vacuo to give the crude product (2.62g) as a solid. The crude acid chloride (2.62g, lOmmol) was dissolved in anhydrous tetrahydrofuran (50ml) under nitrogen.
• Biochips prepared in Example 2 using EPON-SU8 as the substrate linking agent, were incubated for 30 min at 37 °C in a thermoshaker at 370 rpm. The biochips were then washed with TBST wash buffer (BT020/000/UL, Randox) - 2 washes followed by 4 washes at 2 min intervals. 300mI of ACE-2-HRP conjugate was added to the appropriate discrete test region (DTR).
• DTR discrete test region
• the biochips were incubated again for 30min at 37°C in a thermoshaker at 370 rpm, followed by washing with TBST buffer (BT020/000/UL, Randox) - 2 washes followed by 4 washes at 2 min intervals.
• the biochips were developed with 250mI of a 1 :1 ratio of luminol: peroxide (EV841 , Randox) for 2 min in the dark and then imaged on the Randox Evidence Investigator.
• the S1 protein attached directly to a substrate linking agent or via the various substrate liking agents/cross-linking groups produced positive protein detection results without the need of a sample pre-incubation step. Improved detection sensitivity was achieved using EPON-SU8 or an ab-unsaturated keto-sulphone derivative; unsaturated sulphones without the ab configurations produced less sensitive assays than ab-unsaturated keto-sulphones.
• SARS-CoV-2 neutralising antibodies SARS-CoV-2 neutralising antibodies (Spike Receptor Binding Domain & Nucleocapsid Protein) were successfully detected in both sera and saliva samples from different patients using the commercially available assay EV4447 produced by Randox, which detects the two different sets of neutralising antibodies simultaneously.
• the Spike Receptor Binding Domain was detected with the assay detector ACE-2 and the nucleocapsid protein with an anti- nucleocapsid antibody (although it is understood that any other suitable binding ligands could also be used). Detection of spike RBD neutralising antibodies were particularly high.
• SEQ ID NO: 1 SARS-CoV-2 S1 Receptor Binding Domain
• SEQ ID NO: 2 SARS-CoV-2 S1 Receptor Binding Domain, C terminally his- tagged, as expressed in the cells
• SEQ ID NO: 3 SARS-CoV-2 S1 Receptor Binding Domain, C terminally his- tagged, after cleavage of the signal peptide
• SEQ ID NO: 4 SARS-CoV-2 S1 Receptor Binding Domain, nucleotide sequence encoding SEQ ID NO: 2
• SEQ ID NO: 5 Angiotensin converting enzyme-2
• SEQ ID NO: 6 Angiotensin converting enzyme-2, C terminally his-tagged, as expressed in the cells
• SEQ ID NO: 7 Angiotensin converting enzyme-2, C terminally his-tagged, after cleavage of the signal peptide
• RSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSHHHHHHHHHH SEQ ID NO: 8 Angiotensin converting enzyme-2, nucleotide sequence encoding SEQ ID NO: 6
• SEQ ID NO: 9 SARS-CoV-2 Nucleoprotein

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