Antigen detection

Abstract

The present disclosure provides immunoassays with exceptional sensitivity, for the detection of target antigens in samples. In one teaching the capture and detection steps of these assays use recombinant antibodies. The disclosed immunoassays may find particular application in the detection of HBV antigens.

Description

[0001] ANTIGEN DETECTION

[0002] FIELD

[0003] The present disclosure provides an improved method of detecting the presence of Hepatitis B virus, or an antigen therefrom, in a sample.

[0004] BACKGROUND

[0005] Hepatitis B virus (HBV) is one of the world's most widespread infectious agents that can cause lifelong, chronic infection, cirrhosis of the liver, liver cancer, liver failure, and death (Liang et al., Hepatology, 49(5): S13- 21 (2009)). Early screening and detection of HBV infection is critical in disease management and the control of the spread of HBV (Franco et al., World J. Heptaol., 4: 74-80 (2012)). Methods of detection of HBV infection typically involve serological detection of HBV antigen(s), such as ELISA-based techniques or other immunoassays that detect HBV surface antigen (HBsAg), hepatitis core antigen (HBcAg) and / or hepatitis B surface antibody (Mao et al., Anal. Chim. Acta, 909: 101-108 (2016)). Alternatively, real-time PCR or various sequencing techniques may be adopted to detect the presence of HBV DNA, such as to aid in the management of patients with chronic HBV infection undergoing anti-viral therapy, to measure HBV DNA levels at baseline, or during treatment to aid assessment of response to treatment (Terrault et al., Hepatology, 63(1): 261-282 (2015)).

[0006] While there are a large number of commercially available antibodies raised against Hepatitis B antigen(s), there remains a need in the art for improved methods that enable accurate detection of HBV infection and provide the required level of sensitivity, and / or methods that can detect HBV infection rapidly and cost effectively.

[0007] It is amongst the objectives of the present disclosure to provide an improved method for the detection of hepatitis B virus in a sample comprising the steps provided herein.

[0008] SUMMARY

[0009] The present disclosure is based on the finding that immunoassays comprising target capture and target detection steps, which use recombinant antibodies in both said capture and detection steps, represent an improvement over prior art immunoassays which, at best, may only use recombinant antibodies in one of the capture or detection steps.

[0010] It should be noted that the terms “comprise”, “comprising” and / or “comprises” is / are used to denote that aspects and embodiments of this invention “comprise” a particular feature or features. It should be understood that this / these terms may also encompass aspects and / or embodiments which “consist essentially of” or “consist of’ the relevant feature or features. The various aspects and embodiments of this disclosure are applicable to immunoassays for the detection of target antigens in a sample. As such, the term ‘target’ may include a ‘target antigen’ or ‘target antigens’.

[0011] In view of the above, one aspect of the disclosure provides an immunoassay, for the detection of a target in a sample, wherein said immunoassay comprises target capture and target detection steps and both the target capture and target detection steps comprise recombinantly generated antibodies (or ‘recombinant antibodies’).

[0012] A second aspect of the disclosure provides recombinant antibodies for use in the target capture and target detection steps of an immunoassay for the detection of a target in a sample.

[0013] As used herein, the term ‘immunoassay’ refers to an assay method which is used to determine whether or not a sample contains a particular target. One of skill will appreciate that there a vast number of immunoassays and the disclosure presented here is applicable to any assay which exploits an antibody based capture and detection system. By way of example only, the term ‘immunoassay’ may embrace an enzyme immunoassay (EIA: for example an ELISA), a radioimmunoassay (RIA), a fluoroimmunoassay (FIA); a Western blot (WB); a chemiluminescent immunoassay (CIA) and counting immunoassay (CIA); immunohistochemistry-type assays (IHC); immunocytochemistry-type assays (ICC). Within the context of this disclosure, the term immunoassay may not embrace a radioimmunoassay (RIA).

[0014] As stated and in one teaching, the target may be an antigen. In a further teaching, the target may comprise an antigen known to be expressed by a pathogen, for example a fungal, bacterial or viral antigen.

[0015] An immunoassay of this disclosure may comprise a target capture step in which a sample is contacted with a target capture agent under conditions which permit binding between any target present in the sample and the target capture agent.

[0016] As stated, the target capture agent may comprise a recombinant antibody. The recombinant antibody for use as a target capture agent may bind to the target (e.g. the target antigen). Binding between any target present in the sample and the target capture agent will yield a target / target capture agent complex.

[0017] The target capture agent may be immobilised to a substrate.

[0018] An immunoassay of this disclosure may further comprise a detection step in which target / target capture agent complexes (formed during the capture step of the immune assay) are contacted with a detection agent. As stated, the detection agent may comprise a recombinant antibody. The recombinant antibody for use as a detection agent may bind to the target (e.g. target antigen). The recombinant antibody for use as a detection agent may bind to the target at a site or epitope, which is different from that bound by the capture agent.

[0019] The detection agent may be conjugated to a detectable moiety, for example an optically detectable moiety. A detection system for use in any of the methods or assays described herein may comprise, for example, biotin-based systems, HRP-based systems; streptavidinbased systems; silver and / or gold staining-based systems and / or alkaline phosphatase-based systems. In one example, a section system for use in a method or assay of this disclosure may comprise a streptavidin Poly-HRP conjugate (a biotin-binding protein conjugated with polymers of horseradish peroxidase).

[0020] In view of the above, a third aspect of the disclosure provides a method of detecting a target, for example a target antigen in a sample, said method comprising: contacting a sample with a first recombinant antibody under conditions suitable to permit binding between the first recombinant antibody and any target in the sample; and then contacting any first recombinant antibody / target complexes (formed by binding events between the first antibody and any target present in the sample) with a second antibody under conditions suitable to permit binding between said second antibody and any first recombinant antibody / target complexes; and then detecting second antibody bound to first recombinant antibody / target complexes; wherein detection of second antibody bound to a first recombinant antibody / target complexes indicates that the sample contains the target.

[0021] In one teaching of the method of detecting a target (e.g. a target antigen) in a sample, the first recombinant antibody may be immobilised to a substrate.

[0022] In a further teaching, the above method (or indeed any of the immunoassays described herein) may comprise one or more washing step(s). For example, after the step of contacting a sample with a first recombinant antibody, the assay may be subject to a wash designed to wash away any unbound first antibody. The method may comprise a second wash after the step of contacting any first recombinant antibody / target complexes with a second recombinant antibody, the additional wash being designed to remove unbound second recombinant antibody.

[0023] The use of recombinant antibodies in both the capture and detection steps of an immunoassay as described herein, is advantageous as: (i) immunoassays which exploit (in any capture and detection step) recombinant antibodies exhibit improved sensitivity;

[0024] (ii) recombinant antibody technology allows customisation of the desired specifications for both the capture and detection antibodies; in this way the very best target binders can be selected. This delivers an immunoassay with (relative to prior art assays which do not use recombinant antibodies in both capture and detection steps) improved sensitivity and specificity; and

[0025] (iii) recombinant antibodies can be easily manipulated and / or converted to adopt any desired predetermined format, fragment type or class;

[0026] In addition, antibodies generated using recombinant technology can be generated quickly and cost effectively.

[0027] One of skill will appreciate that immunoassays for the detection of antigens in samples may be used in the diagnosis of certain diseases or conditions. As stated, the antigen may comprise a pathogen antigen (e.g. a bacterial, viral or fungal antigen) and the presence of that antigen in a sample may be associated with a specific disease, infection or condition caused or contributed to by the pathogen expressing that antigen. Therefore, the finding that a sample comprises a particular antigen may indicate that the sample has been provided by or obtained from, a subject infected with a pathogen expressing that antigen or suffering / convalescing from a disease or condition caused or contributed to by a pathogen expressing that antigen.

[0028] A recombinant antibody for use as a capture or detection agent may be generated using established recombinant methods. By way of example, recombinant methods for the generation of useful antibodies may include the use of molecular techniques to generate recombinant antibody libraries, antibody display technology, antibody selection protocols, affinity maturation techniques. Further detail on the recombinant techniques used to generate antibodies, may be found in Hoogenboom, (2005); Nature Biotechnology,

[0029] By way of example suitable antibody display technologies may include those referred to as phage (antibody) display, ribosome (antibody) display and / or microbial (antibody) cell display.

[0030] Techniques for recombinant antibody selection may include exposing the antibodies of the display library to antigen. Such techniques may include, for example biopanning to select for clones (e.g. phage clones) which exhibit the desired binding characteristics.

[0031] Selection procedures may comprise repeated rounds of selection in order to derive antibodies exhibiting the very best binding properties. Such selection techniques may be used to select antibodies for use in capture and detection steps of the immunoassays described herein. Affinity maturation is a process by which properties such as antibody potency and antibody affinity may be improved, developed and / or manipulated. The processes may comprise the generation of libraries with focused diversity at a small number of residues most likely to be involved in target / antigen interaction. The CDRs are built using, for example, amplification techniques (e.g. PCR) and libraries screened to identify variants with the desired characteristics. Other approaches include the use of ‘selection pressures’ in order to select antibodies which bind best (to their targets) under specific conditions. These approaches can result in antibodies exhibiting substantial gains in affinity / potency etc.

[0032] Antibodies for use in an immunoassay of this disclosure may comprise antibodies obtainable using recombinant techniques, including those described herein. By way of further example, antibodies for use as capture and detection agents in an immunoassay of this invention may be obtained as antibody (capture and detection) pairs exhibiting, for example, specific and predetermined target binding and / or potency and / or affinity properties. In this way, an immune assay of this disclosure (versus prior art methods which do not use capture and detection antibodies which are recombinant and generated using the techniques described herein) may exhibit an improved level of sensitivity.

[0033] In view of the above, the disclosure provides a method for the selection of capture and detection antibodies for use in an immunoassay for the detection of a target, said method comprising, preparing recombinant capture antibodies and recombinant detection antibodies and selecting a capture / detection recombinant antibody pair which, in the immunoassay, exhibit the desired level of target detection sensitivity and / or selectivity.

[0034] By way of example, this disclosure may relate to immunoassays for the detection of hepatitis B antigens in samples. It should be noted that the various definitions provided with respect to an immunoassay for the detection of hepatitis antigens, might also apply to the general disclosure of immunoassays which use recombinant antibodies for both the capture and detection steps (for example, sample source and subject type).

[0035] In view of the above, an immunoassay for the detection of a hepatitis B antigen in a sample may comprise the use of antigen capture and detection steps both of which comprise recombinant antibodies - in other words a recombinant antibody for use as a capture agent (an agent for capturing and / or immobilising a target from a sample) and a recombinant antibody for use in the detection of said target.

[0036] For example, an immunoassay for the detection of a hepatitis B antigen in a sample may comprise a capture step in which a recombinant antibody is used as the capture agent (to capture any hepatitis B antigen in the sample) and a detection step in which any captured target is labelled for detection using a recombinant antibody. As stated, the recombinant antibody for use as the capture agent may be immobilised to a substrate. The recombinant antibody for use as the detection agent may be conjugated to a detectable moiety.

[0037] The present disclosure is further based on the unexpected finding that a specific combination of capture and detection (recombinant) antibodies against a hepatitis B antigen exhibits exceptional sensitivity. Without wishing to be bound by theory, it is suggested that the improved methods described herein are the result of the combined high binding capacity of the antibodies.

[0038] In one aspect, there is provided a method of detecting a hepatitis B virus (HBV) antigen (for example a HBV target antigen) in a sample, said method comprising contacting the sample with first (capture) and second (detection) antibodies, wherein both the first and second antibodies bind the HBV antigen.

[0039] In one teaching, the first and second antibodies are recombinant antibodies or fragments thereof.

[0040] In one teaching, the step of contacting comprises contacting the first and second antibodies with the sample under conditions which permit binding between the first and second antibodies and any of the HBV target antigen present in the sample.

[0041] The contacting step(s) may yield an antigen-antibody complex, wherein the antigen-antibody complex comprises the first and second antibodies bound to the HBV antigen.

[0042] The method may further comprise a detecting step. The detecting step may be used to detect the presence of antigen-antibody complexes as described herein. The detection of an antigenantibody complex indicates that the sample comprises the HBV antigen.

[0043] In view of the above, one embodiment of the described method comprises contacting the sample with a first and a second antibody under conditions which permit binding between the first and second antibody and any HBV antigen present in the sample, and detecting the resulting antigen-antibody complexes, wherein detection of antigen antibody complexes indicates that the sample comprised the HBV antigen.

[0044] In one example, an antibody, for example the first antibody, may be immobilised on or to a substrate.

[0045] The substrate may comprise glass, polystyrene or any other material suitable for the immobilisation of an antibody. The substrate may be suitable for use in imaging, microscopic observation and / or optical procedures / measurements and / or in cell culture. In some examples, the substrate may comprise a coating, such as to promote the binding and / or immobilisation of the antibody (for example the first antibody) to the substrate and / or to inhibit the immobilisation or binding of other antibodies, proteins (peptides), molecules and / or reagents. These other antibodies, proteins (peptides), molecules and / or reagents may be present in the sample.

[0046] In one teaching, once an antibody (for example the first antibody) has been immobilised to a surface of the substrate, the substrate may be blocked, in order to mask further binding sites on the substrate. Blocking may also help reduce non-specific binding events which may contribute to false positive results. A ‘blocking’ procedure may use, for example, gelatin, BSA, skimmed milk or some other blocking agent.

[0047] Accordingly, in one embodiment this disclosure provides a method of detecting a hepatitis B virus (HBV) antigen in a sample, said method comprising contacting the sample with first and second antibodies, wherein both the first and second antibodies bind the HBV antigen and the first antibody is immobilised to a substrate.

[0048] One of skill will appreciate that by immobilising, for example, the first antibody to a substrate, any antigen-antibody complexes formed as the method is used (i.e. by binding events between the first and second antibodies and any target HBV antigen present in the sample) will also become immobilised at the substrate.

[0049] As stated, the step of detecting, may comprise detecting antigen-antibody complexes, wherein successful detection of antigen-antibody complexes indicates that the sample contains the target HBV antigen (and may have been obtained from or provided by a subject suffering from an HBV infection). The detecting step need not be limited to a particular method of detection and antigen-antibody complexes of the type described herein may be detected using any suitable technique known in the art. In one embodiment, the first and / or second antibody (preferably the second antibody) may comprise a detectable moiety. For example, the first and / or second antibody (preferably the second antibody) may be bound or conjugated to a detectable moiety.

[0050] The detectable moiety may produce, either directly or indirectly, a detectable signal, such as an optically detectable signal. The detectable moiety may be suitable for fluorescence detection, absorbance detection, enzyme-mediated detection, chromogenic and / or chemiluminescent detection. By way of example, a detectable moiety may be radio-opaque or a radioisotope, such as 3H, 14C, 32P, 35S, 1231, 1251, 1311; biotin; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, p-galactosidase or horseradish peroxidase; an imaging agent; or a metal ion. In view of the above, one teaching of the methods described herein exploits an immobilised (first) antibody and another (second) antibody (which is not immobilised) conjugated to a detectable moiety.

[0051] The antibodies used in the methods of this disclosure may be used in any order. For example the antibodies may be added to the sample at the same time. Alternatively, the sample may be contacted with one of the antibodies before it is contacted with the other antibody. In one teaching, the second antibody may be provided together with the sample and / or the second antibody may be provided subsequent to contacting the first antibody with the sample. Accordingly, a method of this disclosure may comprise multiple contacting steps, for example a contacting step in which the sample is contacted with the first (optionally immobilised) antibody and the sample / first antibody mix incubated under conditions which permit binding between the first antibody and any target HBV antigen. After a suitable period of incubation, the sample / first antibody mix may be subject to a wash step. Where the first antibody has been immobilised to a substrate, washing will not remove the immobilised first antibody; indeed washing at this point will remove the sample leaving the immobilised first antibody which (depending on the antigen content of the sample) is either bound or unbound to the target antigen. In other words, washing will leave a mix of immobilised first antibody and / or immobilised first antibody / target antigen complexes. The number of immobilised first antibody / target antigen complexes will depend on the target HBV antigen content of the sample.

[0052] The method may further comprise a subsequent contacting step in which the sample is contacted with the washed and immobilised first antibody and / or any immobilised first antibody / target antigen complexes. This step may be conducted under conditions which permit binding between the second antibody and any target antigen present in an (immobilised) first antibody / target antigen complex. After a suitable period of time, the system (or assay) may be washed. Washing at this stage will remove unbound second antigen, leaving only the second antibody bound to target antigen bound to the first (and immobilised) antibody. The method may then be subjected to the described detection step in order to detect the presence of first antibody / target antigen / second antibody complexes.

[0053] Accordingly, a method of this disclosure may comprise one or more wash steps. For example, after the, or each of the, contacting step(s), the assay may be washed to remove antibody that has not bound to its target. The one or more wash steps will reduce the occurrence of false positive results.

[0054] The present invention is based on the identification of a combination of anti-hepatitis B antigen antibodies which can be used to enhance the detection of HBV antigen in a sample. Importantly, the methods described herein can detect a hepatitis B antigen in a sample obtained from early infection patients at the required regulatory limit. The improved methods and / or assays of this disclosure may be used to facilitate the diagnosis of hepatitis B in a subject - wherein the detection of a HBV target antigen in a sample using a method described herein) may indicate that the subject providing the sample or from whom the sample was obtained, may be suffering or convalescing from an HBV infection.

[0055] A sample to be subject to a method of this disclosure may be provided or obtained from any suitable subject, including, for example healthy subjects with no apparent signs or symptoms of a hepatitis B viral infection. The sample may be provided or obtained from a subject who has, or is suspected of having, a hepatitis B virus infection.

[0056] The term “sample” may embrace any sample type thought capable of harbouring the HBV antigen. By way of example, the sample may comprise a bodily fluid, such as serum, blood (including whole blood or a fragment or fraction thereof, for example serum or plasma), saliva or urine from a subject, for example. Alternatively, or in addition, the sample may comprise a cell (or cells) or a tissue, for example a tissue biopsy. In one teaching, the sample may comprise a liver biopsy.

[0057] The term "subject" may embrace any human or animal subject, including (as stated) healthy subjects or subjects thought to be predisposed, susceptible to an HBV infection, subjects known to have had or with an HBV infection and / or subjects convalescing from an HBV infection.

[0058] According to the method of the present disclosure, a sample (of whatever type) is contacted with a first and a second antibody. The first and second antibodies provided herein specifically bind a HBV antigen. The first and second antibody may bind the same antigen or different HBV antigens in a sample. The first and the second antibody may bind the same epitope of a particular HBV antigen or different epitopes on the same HBV antigen.

[0059] As used herein, the term "antigen" refers to a substance that can elicit an immune response and promotes the generation of antibodies in a subject. An “epitope”, also known as an “antigenic determinant”, as used herein refers to the portion of an antigen that elicits an immune response and is the portion of the antigen to which an antibody specifically binds. For use in a method of this disclosure, an antigen, or any antigenic fragment thereof, may comprise an epitope or epitopes recognised and specifically bound by the antibodies of this disclosure.

[0060] As used herein, the term "antibody" (including the “first” and “second” antibodies) refers not only to recombinant (first and second) antibodies, but to any (recombinant) immunoglobulin or intact molecule as well as to fragments thereof that bind to the target HBV antigen and / or a specific epitope thereof. Such antibodies include, but are not limited to polyclonal, monoclonal, chimeric, humanised, single chain, Fab, Fab’, F(ab)’ fragments and / or F(v) portions of the whole antibody.

[0061] Additionally, the term "antibody fragment" as used herein refers to an incomplete or isolated portion of the full sequence of the antibody which retains the antigen binding function of the parent antibody. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Fragments of antibodies disclosed herein are encompassed by the invention so long as they retain the desired affinity of the full-length antibody. In particular, an antibody fragment may be shorter by at least one amino acid compared to the full-length antibody.

[0062] As used herein, the terms "specific binding" or "specifically binding" refer to the interaction between an antibody (for example the first and second antibodies described herein) and a target HBV antigen.

[0063] In the present disclosure, the term “HBV antigen” may embrace the hepatitis B surface antigen (HBsAg).

[0064] Accordingly, the first and second antibodies for use in a method or assay of this disclosure may bind (or have an affinity for) Hepatitis B surface antigen (HBsAg). In other words, each of the first and second antibodies may bind an epitope present on HBsAg.

[0065] In one example, the first and second antibodies of the present disclosure are typically monoclonal antibodies.

[0066] In one example, an antibody for use in a method of this disclosure (for example the first or capture antibody) may specifically bind an antigen comprising, consisting essentially of or consisting of the following sequence (SEQ ID NO: 1), or a fragment or variant thereof (which fragment or variant also binds the antibody and / or comprises the relevant epitopes):

[0067] MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHSP TSCPPICPGYRWMCLRRFI IFLFILLLCLI FLLVLLDYQGMLPVCPLIPGSTTTSTGPCKTC TTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGL SPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPI FFCLWVYI

[0068] (SEQ ID NO: 1)

[0069] As explained earlier in this disclosure, SEQ ID NO: 1 may comprise at least one, for example two epitope(s) recognised and / or bound by an antibody of this disclosure. As such, an antigen which is detectable using a method of this disclosure may comprise any one or more of the following:

[0070] (i) SEQ ID NO: 1 ; and / or

[0071] (ii) an antibody-binding fragment of SEQ ID NO: 1 ; and / or

[0072] (iii) a first antibody-binding fragment of SEQ ID NO: 1 ; and / or

[0073] (iv) a second antibody-binding fragment of SEQ ID NO: 1 ; and / or

[0074] (v) an epitope defined by SEQ ID NO: 1 ; and / or

[0075] (vi) a first antibody-binding epitope defined by SEQ ID NO: 1 ; and / or

[0076] (vii) a second antibody-binding fragment of SEQ ID NO: 1.

[0077] In one teaching, the HBsAg antigen to be detected by a method of this disclosure, may comprise a sequence which exhibits a level of sequence identity or homology with SEQ ID NO: 1. For example an HBsAg antigen to be detected by a method of this disclosure may comprise a sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identify / homology with the sequence of SEQ ID NO: 1.

[0078] In one teaching, an HBsAg antigen to be detected by a method of this disclosure may comprise a sequence comprising any suitable portion or fragment of the full 226 amino acids of SEQ ID NO: 1 . A detectable portion or fragment of SEQ ID NO: 1 , should always bind or be bound by the antibodies described herein and may comprise ‘n’ amino acids from the full SEQ ID NO: 1 sequence. The term ‘n’ may represent any number of amino acids from about 5 (consecutive) amino acids of SEQ ID NO: 1 to about 225 (consecutive) amino acids of SEQ ID NO: 1. A detectable portion or fragment of SEQ ID NO: 1 may comprise 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 221 , 222, 223 or 224 (consecutive) amino acids from SEQ ID NO: 1. A detectable portion or fragment may comprise a first antibody-binding epitope and / or a second-antibody binding epitope.

[0079] The term a "variant" as used herein refers to a detectable antigen / amino acid sequence that is altered by one or more amino acids. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). Additionally or alternatively, a detectable variant may have "nonconservative" changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. The skilled person would recognise various methods by which an amino acid sequence may be altered (e.g. substitution, insertion, deletion) without abolishing the biological activity (in this case an ability to bind the antibodies described herein), such as by using bioinformatics software available in the art (e.g. PROVEAN, DNASTAR). For example, a detectable variant of an antigen, for example an HBsAg antigen described herein may have 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to a reference protein or peptide sequence (e.g. SEQ ID NO: 1). In all cases a variant and detectable form of SEQ ID NO: 1 or any detectable fragment or portion therefore, should bind or be bound by any of the antibodies described herein..

[0080] In a preferred embodiment, the antibody, for example the first antibody for use (as a capture agent) in a method of this disclosure may comprise the following heavy chain CDR sequences:

[0081] CDRH1 : SYGVH (SEQ ID NO: 2)

[0082] CDRH2: VIWAGGITNYNSALMS (SEQ ID NO: 3)

[0083] CDRH3: GGGVYYGINYAMDY (SEQ ID NO: 4)

[0084] An antibody, for example the first antibody for use in a method of this disclosure may comprise or further comprise the following light chain CDR sequences:

[0085] CDRL1 : RASESVEYYGTSLMQ (SEQ ID NO: 5)

[0086] CDRL2: AASNVEP (SEQ ID NO: 6)

[0087] CDRL3: QQSRNVPWT (SEQ ID NO: 7)

[0088] A first antibody for use (as a capture agent) in a method of this disclosure may comprise the following heavy chain (HC) sequence:

[0089] LHQSGAGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWAGGITNYNSALMSRLSIRK DNFKSQVFLKMNSLQNDDTAMYYCARGGGVYYGINYAMDYWGQGTTVTV (SEQ ID NO: 8); and / or the following light chain (LC) sequence:

[0090] ELTQTPVSLAVSLGQRATISCRASESVEYYGTSLMQWYQQKPGQPPKLLIYAASNVEPGVPARFSGSG SGTDFSLNIQPVEEDDIAMYFCQQSRNVPWTFGGGTKLE (SEQ ID NO: 9).

[0091] Exemplary heavy and light chain sequences of a first (or capture) antibody for use in a method of this disclosure may comprise the sequences deposited in GenBank under accession numbers AF110502 (heavy chain sequence) and AF110503 (light chain sequence). Further exemplary heavy and light chain sequences of a first (or capture) antibody for use in a method of this disclosure are published in “Intracellular Expression of a Cloned Antibody Fragment Interferes with Hepatitis B Virus Surface Antigen Secretion": Jasper zu Putlitz, Arne Skerra, Jack R. Wands: Volume 255, Issue 3, 24 February 1999, Pages 785-791” - the contents of which are incorporated herein by reference.

[0092] A first (or capture) antibody for use in a method of this disclosure may comprise CDR, light and / or heavy chain sequences which show a degree of identity to the relevant sequence of any of SEQ ID NOS: 2-9. The term ‘degree of identity’ may embrace sequences which are at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the relevant sequence.

[0093] The antibody, for example the first antibody for use (as a capture agent) in a method of this disclosure may comprise the Anti-HBsAg monoclonal antibody known as “5C3” (catalogue no. Ab00769-2.0; Absolute antibody).

[0094] As stated, while a method of this disclosure may exploit the full or complete form of 5C3, the method may also use or exploit an antigen (i.e. HBsAg) binding fragment thereof.

[0095] In a preferred embodiment, the antibody, for example the second antibody, for use in (a detection step of) a method of this disclosure may comprise the following heavy chain CDR sequences:

[0096] CDRH1 : GFAFSDYSIN (SEQ ID NO: 10)

[0097] CDRH2: I I SYDGRITYYRDSVKG (SEQ ID NO: 11)

[0098] CDRH3: QYYDFWSGSSVGRNYDGMDV (SEQ ID NO: 12)

[0099] An antibody, for example the second antibody for use in a method of this disclosure may comprise or further comprise the following light chain CDR sequences:

[0100] CDRL1 : RSSQSLLHRSGNNYLD (SEQ ID NO: 13)

[0101] CDRL2: VGSNRAS (SEQ ID NO: 14)

[0102] CDRL3: MQALQTPRT (SEQ ID NO: 15)

[0103] A second antibody for use (as a detection agent) in a method of this disclosure may comprise the following heavy chain (HC) sequence:

[0104] QVQLVESGGGVVRPGRSLRLSCAASGFAFSDYSINWVRQAPGKGLEWVAI ISYDGRITYYRDSVKGRF TISRDDSKNTLYLQMNSLRTEDTAVYYCARQYYDFWSGSSVGRNYDGMDVWGLGTTVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 16); and / or the following light chain (LC) sequence:

[0105] DIVMTQSPLSLSVTPGEPASISCRSSQSLLHRSGNNYLDWYLQKPGHSPQLLIYVGSNRASGVPDRFS GSGSGTEYTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC (SEQ ID NO: 17).

[0106] A second (or detection) antibody for use in a method of this disclosure may comprise CDR, light and / or heavy chain sequences which show a degree of identity to the relevant sequence of any of SEQ ID NOS: 10-17. The term ‘degree of identity’ may embrace sequences which are at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the relevant sequence. The second (or detection) antibody, for use in a method of this disclosure may comprise the Anti-HBsAg monoclonal antibody known as Libivirumab (catalogue no. PX-TA1189; ProteoGenix). As stated, while a method of this disclosure may exploit the full or complete form of Libivirumab, the method may also use or exploit an antigen (i.e. HBsAg) binding fragment thereof.

[0107] It should also be noted that in a method of this disclosure, the abovementioned first and second antibodies may be interchanged - with the antibody referred to as the first antibody (e.g. 5C3) being used after the antibody referred to herein as the second antibody (e.g. Libivirumab).

[0108] The antibodies disclosed herein typically exhibit high affinity for a target epitope. More specifically, the antibodies disclosed herein typically exhibit high affinity for an epitope of an HBV antigen, for example, HBsAg. For example, the antibodies may exhibit a KD value in the range of 10'9to 10’13.

[0109] In view of the above, there is provided a method of detecting HBsAg in a sample, said method comprising contacting the sample with 5C3 and Libivirumab, wherein both 5C3 and Libivirumab bind HBsAg. The step of contacting may comprise firsts contacting the sample with 5C3 under conditions which permit binding between any HBsAg present in the sample and the 5C3 antibody to yield 5C3 / HBsAg complexes. The step of contacting may further comprise contacting any 5C3 / HBsAg complexes with Libivirumab under conditions which permit binding between the HBsAg component of any 53C / HBsAg complexes and Libivirumab to form 5C3 / HBsAg / Libivirumab complexes. The method may further comprise a step of detecting 5C3 / HBsAg / Libivirumab complexes, wherein the detection of a 5C3 / HBsAg / Libivirumab complex, indicates that the sample has been obtained from or provided by a subject suffering or convalescing from a HBV infection.

[0110] In addition to the steps of the method provided above, the method of the present disclosure may optionally further comprise one or more wash steps. In one example, the wash step may take place after contacting the first antibody but prior to contacting the second antibody with the sample. In addition, or alternatively, a wash step may take place after contacting the second antibody with the sample. By way of example, a method of this invention may comprise washing steps designed to, for example, remove unbound 53C and / or Libivirumab antibody. This helps reduce the occurrence of false positive results.

[0111] In one example, the method may optionally further comprise one or more control assays. For example a negative control assay may comprise contacting the first and second antibodies with a sample which is known not to contain the relevant target antigen. In such a scenario, no antigen antibody complexes will form and the detection (subject to appropriate wash steps) step will yield a negative result. A positive control may comprise the use of a sample known to contain or which has been spiked with the relevant target antigen. In this scenario, antigen antibody complexes will form and the detection step should yield a positive result.

[0112] It will be appreciated by those skilled in the art that in addition to the first and second antibodies of the present disclosure, the method may further comprise one or more additional antibodies to further enhance the sensitivity and / or specificity of the assay. By way of example only, the one or more additional antibodies may specifically bind another, different hepatitis B antigen, for example, the hepatitis B core antigen (HBcAg) and / or the hepatitis B e antigen (HBeAg).

[0113] It should be noted that in any of the disclosed methods, the method need not be limited to the examples provided herein and may be carried out in any suitable format, including in single receptacles, such as Eppendorf or other tubes, or in a multiple format such as multi-well plates, for example. Additionally, cartridge formats, such as lateral flow or microfluidic based cartridges may be envisaged. It will be appreciated by the skilled reader that depending on the method of detection used and / or sample provided (e.g. bodily fluid or tissue), the format of the method may need to be adapted using various methods known in the art.

[0114] In a further teaching, the present disclosure is also directed to kits for use in the methods as described herein. The kit may comprise, consist essentially of, or consist of: a first antibody and a second antibody, wherein the first and / or second antibody are selected from:

[0115] Libivirumab (catalogue no. PX-TA1189; ProteoGenix) and

[0116] Anti-HBsAg [5C3] (catalogue no. Ab00769-2.0; Absolute antibody), and optionally, the first antibody or the second antibody immobilised on a substrate, and further optionally, the first antibody and / or the second antibody labelled with a detection moiety.

[0117] The disclosure further provides use of Libivirumab (catalogue no. PX-TA1189; ProteoGenix) and / or 5C3 (catalogue no. Ab00769-2.0; Absolute antibody), in a method or assay for the detection of HBsAg in a sample.

[0118] DETAILED DESCRIPTION

[0119] The present invention will now be described in detail by reference to the following figures which show:

[0120] Figure 1 : Immunoassay (ELISA) for the detection of HBsAg comparing various combinations of capture and detection antibodies.

[0121] Figure 2: Immunoassay (ELISA) for the detection of HBsAg comparing various combination of capture and detection antibodies.

[0122] Figure 3: Immunoassay (MosaiQ) for the detection of HBsAg comparing various combination of capture and detection antibodies.

[0123] Figure 4: Immunoassay (MosaiQ) for the detection of HBsAg comparing various combination of capture and detection antibodies.

[0124] Figure 5: Analysis demonstrating sensitivity of assays using difdernt combiantions of capture and detection agent. (A) capture: Absolute 5C3; Detection: proteogenix Libivirumab; Conjugate SDT Streptavidin poly-HRP 80; cycle time: 48s. (B) capture: Absolute 5C3; Detection: Meridian B65811 ; Conjugate SDT Streptavidin poly-HRP 80; cycle time: 48s.

[0125] Results

[0126] Table 1 (see also Figure 1)

[0127] Table 2 (see Figure 2) Table 3 Table 4 (see Figure 3)

[0128] Table 5 Table 6 (see Figure 4)

Claims

CLAIMS1. A method of detecting a hepatitis B virus (HBV) antigen in a sample, said method comprising contacting the sample with a first antibody comprising a sequence provided by any one of SEQ ID NOS 2-9; and a second antibody comprising a sequence provided by any one of SEQ ID NO: 10-17.

2. The method of claim 1 , wherein the first and a second antibodies, bind the HBV antigen.

3. The method of claim 1 or 2, wherein the first and second antibodies are respectively for the capture and detection of the HBV antigen.

4. The method of any preceding claim, wherein both the first and second antibodies are recombinant antibodies.

5. The method of any preceding claim, wherein the step of contacting comprises contacting the first and second antibodies with the sample under conditions which permit binding between the first and second antibodies and any HBV antigen present in the sample.

6. The method of any preceding claim, wherein binding between the first and second antibodies and an HBV antigen present in the sample yields an antibody-antigen complex, said complex comprising first and second antibodies bound to the HBV antigen.

7. The method of any one of claims 1-5, wherein binding between the first antibody and an HBV antigen present in the sample yields a first antibody-antigen complex, said complex comprising first antibody bound to the HBV antigen.

8. The method of claim 7, wherein the second antibody binds complexes comprising the first antibody bound to the HBV antigen.

9. The method of any preceding claim, wherein the first antibody is immobilised to a substrate.

10. The method of any one of any preceding claim, wherein the second antibody comprises a detectable moiety.11 . The method of any preceding claim, wherein the HBV antigen is the Hepatitis B surface antigen (HBsAg).

12. The method of any preceding claim, wherein the first antibody is the anti-HBsAg monoclonal antibody 5C3 (catalogue no. Ab00769-2.0; Absolute antibody) or an HBsAg- binding fragment thereof.

13. The method of any one of claims any preceding claim, wherein the second antibody is the anti-HBsAG monoclonal antibody Libivirumab (catalogue no. PX-TA1189; ProteoGenix) or an HBsAg-binding fragment thereof.

14. A method of detecting HBsAg in a sample, said method comprising: contacting a sample with monoclonal antibody 5C3 under conditions suitable to permit binding between monoclonal antibody 5C3 and any HBsAg in the sample, wherein a binding event between monoclonal antibody 5C3 and any HBsAg in the sample results in the formation of a 5C3 / HBsAg complex; contacting any 5C3 / HBsAg complexes with monoclonal antibody Libivirumab under conditions suitable to permit binding between said monoclonal antibody Libivirumab and any 5C3 / HBsAg complexes; and then detecting any Libivirumab bound to 5C3 / HBsAg complexes; wherein detection of Libivirumab bound to 5C3 / HBsAg complexes indicates that the sample contains the target.

15. An immunoassay, for the detection of a target in a sample, wherein said immunoassay comprises target capture and target detection steps, wherein both the target capture and target detection steps comprise recombinantly generated antibodies.

16. Recombinant antibodies for use in the target capture and target detection steps of an immunoassay for the detection of a target in a sample.

17. The immunoassay of claim 15 or use of claim 16, wherein the immunoassay is used to determine whether or not a sample contains a particular target or an antigen.

18. The immunoassay or use of claims 15-17, wherein the recombinant antibody for use in the target capture step is immobilised to a substrate and / or the recombinant antibody for use in the detection step is conjugated to a detectable moiety.

19. A method of detecting a target, for example a target antigen in a sample, said method comprising: contacting a sample with a first recombinant antibody under conditions suitable to permit binding between the first recombinant antibody and any target in the sample; and then contacting any first recombinant antibody / target complexes (formed by binding events between the first antibody and any target present in the sample) with a second antibody under conditions suitable to permit binding between said second antibody and any first recombinant antibody / target complexes; and then detecting second antibody bound to first recombinant antibody / target complexes; wherein detection of second antibody bound to a first recombinant antibody / target complexes indicates that the sample contains the target.

20. The method of claim 19, wherein the first recombinant antibody is immobilised to a substrate.

21. A method for the selection of capture and detection antibodies for use in an immunoassay for the detection of a target, said method comprising, preparing recombinant capture antibodies and recombinant detection antibodies and selecting a capture / detection recombinant antibody pair which, in the immunoassay, exhibit a desired or predetermined level of target detection sensitivity and / or selectivity.

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