Hepatitis b core-related assay for inclusive diagnosis of occult hepatitis b

Abstract

Provided herein is a method for detecting Hepatitis B Virus (HBV) infection, particularly Occult hepatitis B infection (OBI) based on detection of hepatitis B core-related antigen (HBcrAg) epitopes. Also provided is a diagnostic device for detecting HBV infection, using the method provided.

Description

[0001] HEPATITIS B CORE-RELATED ASSAY FOR INCLUSIVE DIAGNOSIS OF OCCULT HEPATITIS B

[0002] BACKGROUND OF THE INVENTION

[0003] The present invention relates generally to a method for detecting Hepatitis B Virus (HBV) infection. Particularly, the invention relates to a method for detecting HBV infection in a sample that does not have the hepatitis B surface antigen (HBsAg) by detecting the hepatitis B core-related antigen (HBcrAg).

[0004] HBV infection remains a significant global health burden, with the World Health Organisation (WHO) estimating that, globally, 296 million individuals are living with chronic HBV, with 820 000 deaths reported annually due to liver disease. HBV accounts for approximately 30% of liver cirrhosis cases and 53% liver cancers. Africa is reported as the second largest population with HBV chronic carriers and thus holds the world’s highest reported cases of hepatocellular carcinoma (HCC). In South Africa, approximately 1.9 million individuals are chronically infected with HBV, and about 600 000 individuals die from HBV and associated complications such as liver diseases and liver cancer. HBV is reported as the 7th leading cause of death globally exceeding HIV, malaria and TB cases. Furthermore, occult HBV infection (OBI) has been reported as a confounding factor of HCC development. HBV infection is considered a notifiable 'routine' disease, as it continues to be a global health concern, especially with cases of OBI that pose a risk of transmission.

[0005] Accurate diagnosis of HBV infection is essential for effective management and prevention of transmission. While the standard approach hepatitis B surface antigen (HBsAg) screening test is effective for identifying HBV infection in most cases, occult HBV infection (OBI) can go undetected owing to the absence of HBsAg. OBI is thus described as the presence of HBV DNA in the absence of hepatitis B surface antigen (HBsAg). It is characterized by low levels of closed circular deoxyribonucleic acid (cccDNA) in the liver even though it is replication-competent. True OBI patients present with HBV DNA viremia of less than 200 lU / ml. HBV DNA above 200 lU / ml in the absence of HBsAg is defined as false OBI. OBI poses a diagnostic challenge as HBsAg antigen used in screening assays may be negative where there is presence of hepatitis B virus deoxyribonucleic acid (HBV DNA) in the liver of an infected patient. The current available solutions in the market are faced with limitations in sensitivity as they cannot reliably diagnose low levels of HBV infection.

[0006] The true burden of HBV is unknown due to issues of under-reporting and inaccurate records in Africa. Another reason is that HBV in adults can be self-limiting with different prognosis compared to infants. In adults it can present with little to no clinical manifestations. Defining the epidemiology of OBI is difficult because it relies on the sensitivity of the serological and HBV DNA assays. Most prevalence studies on OBI are conducted on HIV positive patients, hepatocellular carcinoma (HOC) patients, health care workers, and blood donors and not on the general population.

[0007] The global prevalence rate of OBI is estimated at 0-89% based on different populations. In Africa multiple studies have reported on the prevalence of OBI, with a study in Kenya reporting 18.7% among high-risk populations, Gambia reporting 18.3% in a general population and Burkina Faso 4-32.8% in blood donors. A systematic review on the overall prevalence of OBI in Africa reported 14.8% in different populations. Studies in South Africa have reported a high prevalence of OBI in HIV-positive population with a study reporting 88.4% OBI cases in its population. In 2006 a study from South Africa reported 33.3% occult HBV in HBsAg negative HIV positive patients, while in 2016 a further study on health care workers reported 6.7% occult HBV. At the Department of Virology NHLS / DGM, an average 3188 HBV tests are requested monthly with an average of 252 negative tests for HBsAg. In March 2021 to May 2022, 444 HBsAg negative routinely screened samples were collected for HBV DNA testing. Sixty-nine (69) of the samples tested positive for HBV DNA. Thus 16% of the patients were misdiagnosed as not having HBV infection.

[0008] HBV belongs to the Hepadnaviridae family and is a small 3.2 kb double-shelled viral particle with a spherical structure of 30 to 42 nm in diameter. The viral genome has four overlapping open reading frames (ORF); surface (S), core (C), polymerase (P), and X- region coding for different proteins. HBV is a highly diverse virus with significant genetic variability. This variability is due to the high mutational rate of the virus during replication as well as its ability to recombine with other HBV strains. There are ten genotypes of HBV (A- J), which are distinguished by their geographic distribution, genetic variability, and clinical features. The genotypes are classified into sub-genotypes i.e. A1-A7, B1-B9, C1-C16, D1- D8 and F1-F4 which differ by 4%. Genotype A, subtype A1 presents with low viral loads and low levels of Hepatitis B e-Antigen (HBeAg). Infection with genotype A and B pose high risk of development of liver cancer and HCC. The highly reported genotype in Southern Africa according to published data is genotype A. In South Africa, multiple genotypes (A, B, C, D and E) of HBV have been reported, but the predominant genotypes vary by region. Sub type A1 is reported in 70% of the black population infected with HBV in South Africa.

[0009] A standardized approach to laboratory- based detection of hepatitis B infection (HBV) is hepatitis B surface antigen (HBsAg) screening. The gold standard for HBsAg screening is the Enzyme-linked immunoabsorbent assay (ELISA) and Chemiluminiscence immunoassay (CLIA). In the case of negative screening, the patient is considered negative, and HBV infection is ruled out. The problem lies with the diagnosis of OBI, whereby HBsAg used in screening assay tests negative, but there is presence of HBV DNA in the liver of an infected patient. Therefore, this poses a risk of HBV transmission and increases the burden of disease. Mutations in the S-region antigen gene of the hepatitis B virion can be problematic for laboratories using commercial diagnostic HBsAg assays that are unable to recognize these mutational changes because the assay constituents target the antigenic epitopes in the ‘a’ determinant. Mutations in the s-region are one of the factors resulting in OBI with the most commonly reported OBI mutation to date is G145R. This mutation occurs in the major hydrophilic region (MHR), which is the antigenic determinant of an HBV strain and results in conformational changes. G145R including T118K, K141 E, D144G, C147R and C149R mutation are associated with vaccine escape in OBI and thus decrease virion production. Mutations such as sC124R, sC124Y, sK141 E and sD144A have a severe impact on the sensitivity on seven HBsAg assays.

[0010] According to the current definition of OBI, the most direct method of diagnosis appears to be the screening of liver tissue for replication-competent HBV DNA. The most reliable test to diagnose OBI is HBV DNA amplification. However, liver tissue may not always be available, and the test is unstandardized, unapproved by the food and drug administration (FDA), and lacks any internal or external validity. Thus, there is a need for enhanced and sensitive detection of HBV.

[0011] The present invention thus relates to the development of an improved serologybased diagnostic assay utilizing the hepatitis B core-related antigen (HBcrAg) as a marker for enhanced detection of HBV, where HBsAg detection is negative, with a focus on OBI.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention relates generally to a method for detecting Hepatitis B Virus (HBV) infection and also relates to a diagnostic device for detecting HBV. The invention further relates to a method for detecting HBV infection in a sample that does not have the hepatitis B surface antigen (HBsAg) by detecting the hepatitis B core-related antigen (HBcrAg).

[0014] According to a first aspect of the present invention there is provided for a method of diagnosing Hepatitis B Virus (HBV) infection, preferably occult hepatitis B (OBI), in a subject, the method comprising: a) providing a sample from the subject; b) contacting the sample with a selective agent which specifically binds to one or more hepatitis B corerelated antigen (HBcrAg) epitopes; and c) detecting binding of the selective agent with the one or more HBcrAg epitopes, wherein binding of the selective agent with the one or more HBcrAg epitopes indicates HBV infection in the subject.

[0015] In a first embodiment of the method of diagnosing HBV in a subject, the one or more HBcrAg epitopes may be B cell epitopes. In one embodiment, the one or more HBcrAg epitopes may be selected from HBV genotype A. Preferably, the one or more HBcrAg epitopes has an amino acid sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, and SEQ ID NO:3.

[0016] According to a second embodiment of the method of diagnosing HBV in a subject, the subject has previously tested negative for HBV using an HBsAg assay.

[0017] In a third embodiment of the method of diagnosing HBV in a subject, the sample may be selected from the group consisting of blood, serum, plasma, and / or liver tissue extract, preferably the sample is a serum sample or a plasma sample.

[0018] According to a fourth embodiment of the method of diagnosing HBV in a subject, the selective agent may be an antibody and the antibody binds to the HBcrAg epitope to form an HBcrAg-antibody complex. For example, the selection agent may be an antibody, and the antibody may be a capture antibody and / or a detection antibody. In one embodiment, the detection antibody may be conjugated covalently or non-covalently to a detection label. It will be appreciated that numerous detection labels are known to those of skill in the art. For example, in some embodiments, the detection label may be selected from the group consisting of colourimetric labels, fluorescent labels, chemiluminescent labels, biotin, phosphor-based labels, thermal-based labels, enzymatic labels, gold nanoparticles, silver nanoparticles and magnetic beads.

[0019] According to a further embodiment of the method of diagnosing HBV in a subject, the antibody may be a polyclonal antibody. The antibody may further be a full-length antibody or fragment thereof that binds to the HBcrAg epitope.

[0020] In yet another embodiment of the method of diagnosing HBV in a subject, the method is preferably a point-of-care method of diagnosing HBV, such as one in which the binding of the antibody to the HBcrAg epitope may be detected within 20 minutes.

[0021] According to a second aspect of the present invention there is provided for a diagnostic device for detecting Hepatitis B Virus (HBV) infection, preferably occult hepatitis B (OBI), in a sample of a subject, comprising: a) a selective agent which specifically binds to one or more HBcrAg epitope; and b) an indicator for detecting binding of the selective agent with the one or more HBcrAg epitopes, wherein binding of the selective agent with the one or more HBcrAg epitopes indicates HBV infection in the subject.

[0022] In a first embodiment of the diagnostic device of the invention, the one or more HBcrAg epitopes may be B cell epitopes. In one embodiment, the one or more HBcrAg epitopes may be selected from HBV genotype A. Preferably, the one or more HBcrAg epitopes has an amino acid sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, and SEQ ID NO:3.

[0023] According to a second embodiment of the diagnostic device, the subject has previously tested negative for HBV using an HBsAg assay. In a third embodiment of the diagnostic device, the diagnostic device may comprise a zone for loading the sample.

[0024] According to a fourth embodiment of the diagnostic device, the sample may be selected from the group consisting of blood, serum, plasma, and / or liver tissue extract, preferably the sample is a serum sample or a plasma sample.

[0025] In a further embodiment of the diagnostic device, the selective agent may be an antibody and the antibody binds to the HBcrAg epitope to form an HBcrAg-antibody complex. For example, the selection agent may be an antibody, and the antibody may be a capture antibody and / or a detection antibody. In one embodiment, the detection antibody may be conjugated covalently or non-covalently to a detection label. It will be appreciated that numerous detection labels are known to those of skill in the art. For example, in some embodiments, the detection label may be selected from the group consisting of colourimetric labels, fluorescent labels, chemiluminescent labels, biotin, phosphor-based labels, thermal-based labels, enzymatic labels, gold nanoparticles, silver nanoparticles and magnetic beads.

[0026] According to another embodiment of the diagnostic device, the antibody may be a polyclonal antibody. The antibody may further be a full-length antibody or fragment thereof that binds to the HBcrAg epitope.

[0027] In yet another embodiment of the diagnostic device, the device is preferably a point- of-care device for diagnosing HBV, such as a device in which the binding of the antibody to the HBcrAg epitope may be detected within 20 minutes.

[0028] BRIEF DESCRIPTION OF THE FIGURES

[0029] Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures:

[0030] Figure 1 : A schematic diagram of the components of the HBcrAg.

[0031] Figure 2: An exemplary schematic representation of a prototype of the device and assay of the present invention. The sample and buffer are added into the sample window (S), followed by migration of the sample towards polyclonal antibodies specific for HBcrAg, resulting in formation of an antigen-antibody complex (HBcrAg-2yev). The reaction test-line (T) comprises a labelled capture antibody. Also provided is a control line (C) to validate whether the assay works, and monitor that proper migration occurs.

[0032] Figure 3: An illustration of gel electrophoresis of HBV amplicons with 615 bp from the HBcAg region. MW- molecular weight marker; PC- positive control; NC- negative control; Bp- base pairs.

[0033] Figure 4: HBcrAg multiple sequence alignment and consensus genotype A sequence. The HBcrAg sequences are provided as follows:, ZDGM0602 (SEQ ID NO:4), ZDGM9750 (SEQ ID N0:5), ZDGM0138 (SEQ ID N0:6), RD 3793 (SEQ ID N0:7), MA 6465 (SEQ ID N0:8), MA 6725 (SEQ ID N0:9), MA 6438 (SEQ ID NQ:10), MA 9879 (SEQ ID N0:11) MA 5525 (SEQ ID N0:12), MA 8235 (SEQ ID N0:13), and consensus genotype A sequence (SEQ ID NO:14).

[0034] Figure 5: Predicted epitope region from the consensus sequence of genotype A (SEQ ID NO:14). E- predicted epitopes.

[0035] Figure 6: Secondary structure of consensus genotype A HBcrAg region. Highlighted in white are the epitope regions (HB5-18, HB32-47, HB74-92) to be targeted by the assay.

[0036] Figure 7: Predicted solvent accessibility of genotype A consensus sequence (SEQ ID NO:14).

[0037] Figure 8: Predicted solvent accessibility of genotype A consensus sequence (SEQ ID NO: 14). For relative surface accessibility the black is the exposed regions and the grey are buried region . The secondary structure is shown by the Helix , the strand coils — . The grey line shows the disorder of the residues with the thickness of the line equals probability of disorder residue.

[0038] Figure 9: 3D molecular structure in two different orientations of the antigenantibody docking.

[0039] DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.

[0041] The invention as described should not be limited to the specific embodiments disclosed and modifications and other embodiments are intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0042] As used throughout this specification and in the claims which follow, the singular forms “a”, “an” and “the” include the plural form, unless the context clearly indicates otherwise.

[0043] The terminology and phraseology used herein is for the purpose of description and should not be regarded as limiting. The use of the terms “comprising”, “containing”, “having” and “including” and variations thereof used herein, are meant to encompass the items listed thereafter and equivalents thereof as well as additional items. It is, however, contemplated as a specific embodiment of the present disclosure that the term “comprising” encompasses the possibility of no further members being present, i.e., for the purpose of such an embodiment “comprising” is to be understood as having the meaning of “consisting of”.

[0044] In its narrowest sense, the present invention relates to the development of a serology-based diagnostic assay utilizing the hepatitis B core-related antigen (HBcrAg) as a novel epitope for enhanced detection of HBV, with a focus on OBI. The inventors have investigated HBV DNA and mutations in HBsAg negative specimens and have shown that HBcrAg shows promise as a sensitive indicator of HBV replication and transcription, correlating with HBV DNA levels in liver tissue. HBcrAg is more sensitive and specific than other HBV antigens, such as HBsAg and anti-HBc, and can detect HBV infection even in patients with low viral loads. Further, HBcrAg is more sensitive and specific for chronic patient monitoring and is effective in detecting HBV replication in patients with low viral loads (2.3 log U / rnL to 6.4 log U / rnL). HBcrAg therefore compensates for limitations of liver biopsy, which carries risks of sampling error and lacks standardization. Thus, the assay and device of the present invention aim to enhance the process of HBV detection and management, offering high sensitivity, specificity, reliability, accuracy, and scalability.

[0045] An epitope, also known as an antigenic determinant, as used herein is a portion of an antigen that is recognized by the immune system in the appropriate context, specifically by antibodies, B cells, or T cells. Epitopes may include B cell epitopes (e.g., predicted B cell reactive epitopes) and T cell epitopes (e.g., predicted T cell reactive epitopes). B-cell epitopes (e.g., predicted B cell reactive epitopes) are peptide sequences which are required for recognition by specific antibody producing B-cells. B cell epitopes (e.g., predicted B cell reactive epitopes) refer to a specific region of the antigen that is recognized by an antibody. T-cell epitopes (e.g., predicted T cell reactive epitopes) are peptide sequences which, in association with proteins on APC, are required for recognition by specific T-cells. T cell epitopes (e.g., predicted T cell reactive epitopes) are processed intracellularly and presented on the surface of APCs, where they are bound to MHC molecules including MHC class II and MHC class I molecules. The portion of an antibody that binds to the epitope is called a paratope. An epitope may be a conformational epitope or a linear epitope, based on the structure and interaction with the paratope. A linear, or continuous, epitope is defined by the primary amino acid sequence of a particular region of a protein. The sequences that interact with the antibody are situated next to each other sequentially on the protein, and the epitope can usually be mimicked by a single peptide. Conformational epitopes are epitopes that are defined by the conformational structure of the native protein. These epitopes may be continuous or discontinuous (i.e., may be components of the epitope can be situated on disparate parts of the protein, which are brought close to each other in the folded native protein structure). The present invention has identified B cell epitopes within the targeted sequences using BepiPred-2.0 and IEDB, thus, yielding several promising candidates, which are the targeted antigens by the antibodies of the present invention. These predicted epitopes are critical for understanding the immunogenic properties of the antigen and for the development of the present invention. The identified epitopes represent regions of the antigen that can be recognised by immune B cells. The locations, lengths, and BepiPred scores of the predicted epitopes suggest that they are highly immunogenic. The epitope regions: HB5-18, HB32-47, and HB74-92 have been shown to be good epitope regions that elicit a high immune response. The antigen has been selected on the genotypes circulating in South Africa thus making it possible to achieve a more accurate and sensitive detection. Such a specificity increases the accuracy since there are few chances of false positive results. The predicted epitopes were reported to be 92.86%-84.21% conserved in different sequences, which shows that the epitope is widely shared across different sequences, suggesting it being a good target.

[0046] Incorporating these epitopes into diagnostic assays will improve the specificity and sensitivity of tests designed to detect HBcrAg. The predicted epitope regions within HBcrAg present promising targets for further immunological studies. Their high BepiPred scores, surface accessibility, and conservancy make them attractive candidates for antibody development. Furthermore, working towards the development of this new assay where novel antigens and antibodies were used is a remarkable accomplishment in the diagnosis of HBV infection. The strong binding affinities of antigen-antibody interactions reported in the present invention and the increased accuracy of the results will allow this assay to be used for reliable and early diagnosis. In the success of this work, antibodies are key components, providing greater sensitivity and less cross-reactivity. In this light, the assay of the present invention promises to be a transformative and advanced technology, which will have a significant intra-banner scope for improvement in diagnostics and patient management.

[0047] The term ’’antibody” includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for instance, bispecific antibodies and polyreactive antibodies), and antibody fragments. In a preferred embodiment of the invention, the antibody is a polyclonal antibody. Accordingly, the term “antibody” as used in this specification includes, but is not limited to, any specific binding member, immunoglobulin class and / or isotype (for instance: lgG1 , lgG2, lgG3, lgG4, IgM, IgA, IgD, IgE and IgM) or an antibody fragment thereof. Also included in the definition of “antibody” are chimeric antibodies, humanized antibodies, recombinant antibodies, human antibodies generated from a transgenic non-human animal and antibodies selected from libraries using enrichment technologies available to those skilled in the art. In an embodiment of the invention the antibodies of the invention may be used in a diagnostic composition. A diagnostic composition is a composition containing a compound or antibody, e.g., a labelled compound or antibody, that is used to detect the presence in a sample, such as a biological sample, of an antibody that binds to the compound or an immunogen, antigen or epitope that binds to the antibody, for instance, an HBcrAg antigen or epitope.

[0048] The inventors have shown that the assay of the present invention, based on HBcrAg epitopes, has a significant enhancement in target specificity due to the nature of the antigenantibody interaction, which minimizes the possibility of detecting false-positive and falsenegative results. This is especially needed for early detection of hepatitis B virus infection, where diagnostic accuracy is essential. Furthermore, the reporting of the test results in the present invention will be enhanced as the protocols used are efficient to allow rapid response times. This efficiency could ultimately be beneficial to clinical practice, as it would reduce turnaround times and streamline the diagnosis and treatment decision process. In addition to that, the use of modern techniques in antibody construction increases the resilience of the assay system, making the results consistent and reproducible regardless of batch or type of the sample.

[0049] The prevalence of OBI in continents such as Africa and Asia is the highest. Furthermore, the prevalence of OBI differs from one population to the other. The highest prevalence of OBI is mostly reported in high-risk group in populations with liver disorder, malignancies and HIV. The present invention highlights the significant prevalence of true OBI and false OBI cases in surface antigen negative samples. The present study shows that the prevalence of true OBI is 16%, which is a significant proportion of the total number of HBV DNA positive samples. The use of highly sensitive assays such as in-house realtime PCR with a detection limit of 45 copies / mL was crucial in detecting OBI in the present study. These results are of concern, as they indicate potential risks of transmitting the virus due to misdiagnosis, and lack of treatment and monitoring of infected patients. Supporting the findings of the present study regarding the presence of HBV DNA in the absence of HBsAg, a review conducted by Raimondo et al. (2013), reported that the prevalence of OBI varies widely across different populations, ranging from less than 1% to more than 70%. The authors thereof also emphasized the importance of using sensitive diagnostic tests for OBI detection as standard tests, because HBsAg screening is not sufficient to detect OBI.

[0050] The main characteristic in OBI is the persistence of replication-competent cccDNA in the hepatocytes. HBV is non-cytopathic, and hepatocytes are known to have longevity, thus allowing cccDNA to persist for long periods. The low replicative state of cccDNA in OBI patients causes less HBV transcription and protein expression, which results in undetectable HBsAg levels. Several mechanisms have been suggested to contribute to OBI. Host factors, viral factors, and co-infection are some mechanisms that have been proposed for OBI. Host factors Include host immune and epigenetic factors. Viral factors, including mutations in the S region, are known to contribute to the occurrence of OBI. HBV S proteins have been found to contain a variety of mutations that affect immune response due to an unrecognizable T and B cells by the immune system, in vitro antigen detection, HBV infectivity, cell tropism, and virion morphogenesis. Mutational analysis in the present study found previously reported OBI-associated mutations; immune escape mutations (Y100C, T125M, M133I, P142L, D144A, P120T). Some of the other observed OBI associated mutations were K160R, K122R, R24K and F8L, previously reported to reduce virion production.

[0051] The present invention primarily relates to an assay and device for detecting hepatitis B core-related antigen (HBcrAg) in a sample, by contacting the sample with an antibody that binds HBcrAg in the sample and detecting binding of the antibody to HBcrAg to diagnose OBI.

[0052] HBcrAg assays rely on the detection of three structural and non-structural proteins: hepatitis B core antigen (HBcAg), hepatitis B e-antigen (HBeAg) and preC (p22cr) as shown in Figure 1. These proteins have 149 amino acid similar sequences, but different terminal regions. The protein HBcAg is present in the nucleus of infected hepatocytes and cannot be detected in the blood. The protein serological evaluations can be used as prognostic indicators during the acute stage of hepatitis B, and there are relationships between HBcAg levels and HBV DNA. HBeAg is a marker for active viral replication and correlates to high infectivity. The marker is primarily used to assess infectivity and response to antiviral therapy. The protein p22cr is a new marker and indicates acute infection and viral load. The performance of HBcrAg tests may differ based on the HBV genotypes being tested such as A, B, C, D, E, F, G, H and J. Each one can have differences, in how the disease progresses, responds to treatment and the amounts of these proteins present. The presence and levels of HBcAg, HBeAg, and p22cr in HBcrAg assays can be influenced by the specific genotype of HBV.

[0053] The first HBcrAg marker-based prototype was described in 2002 (Kimura et al., 2002). The HBcrAg was prescribed as a marker for monitoring of chronic hepatitis B patients and for management of patients under therapy. Research reported the marker to be more sensitive and specific for chronic patient monitoring. The marker was found to be effective in detecting HBV replication in patients with low viral loads (2.3 log U / rnL to 6.4 log U / rnL) and was found to compensate for limitations of liver biopsy, which carries risks of sampling error and lacks standardization.

[0054] Currently, an automated Lumipulse G1200 CLEIA analyser (Fujirebio, Tokyo, Japan) is available for research use only in Japan. However, recent data reported the limitations of HBcrAg used in this assay in comparison to HBV DNA detection, contrary to other studies that have shown that the two correspond (Caviglia et al., 2021 ; Inoue & Tanaka, 2019; Mak et al., 2018; Watanabe et al., 2021). The reported poor sensitivity and low positive predictive value (PPV) of the marker used in this assay, suggest this marker is unreliable in a South African population, as it misses a significant number of true positives. Thus, despite the high specificity, the use of available assays is limited by poor sensitivity, especially considering the existing assay was only verified for use in Japan with a focus on the population with predominant genotypes C and B (Kramvis et al., 2022). Further analysis has shown that discordance was due to unavailability of the target epitope region for antibody binding, attributed to the difference between the antigens used from the South African circulation genotype A HBcrAg. Structures of sequences of genotype D sequences used in the validation and verification of the existing assay showed availability of the antigenic region, while structures of sequences from the South African population genotypes A1 and A2 showed limited accessibility and unavailability of the antigenic regions. The targeted region of the existing assay is not 100% conserved, which in turn limits the binding affinity of the antibodies to the antigen epitopes.

[0055] Thus, the present invention primarily relates to an improved assay and device for detecting hepatitis B core-related antigen (HBcrAg), with an emphasis on HBcrAg epitopes predominant in Africa. The present invention seeks to address the shortfalls by utilizing novel antibodies to fit the population of HBsAg negative genotype A samples for inclusive diagnosis in Africa, to provide a point of care and end user-friendly diagnostic assay.

[0056] The term “antigen” as used herein refers to all, or parts, of a peptide or protein, capable of eliciting an immune response against itself or portions thereof. This immune response may involve either antibody production, or the activation of specific immunologically competent cells, or both.

[0057] As used herein, “Hepatitis B core-related antigen” or “HBcrAg” refers to a protein antigen derived from the core of HBV, which is an important component of the viral core particle and which, according to the present invention is an antigen to be detected to predict the presence of active HBV infection and OBI.

[0058] The term “peptide” should be read to include “polypeptide” and “protein” and vice versa. As used herein, “peptide” refers to an amino acid sequence of a recombinant or nonrecombinant peptide having an amino acid sequence of i) a native peptide, ii) a biologically active fragment of a peptide, or iii) a biologically active variant of a peptide.

[0059] The point-of-care diagnostic device of the present invention, as shown in Figure 2, is intended for widespread clinical use. This point-of-care diagnostic assay utilizes a patient sample for the detection of HBcrAg in HBV infected patients. The S indicates the sample window were the sample and buffer will be added. Thereafter, the sample will migrate and a reaction with polyclonal antibodies with specificity and selectivity for HBcrAg will occur forming an antigen-antibody complex (HBcrAg-2yev). Further migration will occur towards the reaction test-line (T), resulting in a sandwich formation with the target analyte. To validate whether the assay works, and proper migration occurred, the control line appears as shown (C). For the visualization of the test result, a line will appear on the test-line window if the patient is positive and in the case of negative patients the test window will be blank.

[0060] As used herein, the term “sample” refers to a sample obtained from a biological source, or a “biological sample”. Typically, a biological sample is a saliva, sputum, blood, plasma, cerebrospinal fluid, serum, lymph, tissue or urine sample. In a preferred embodiment, the sample is a plasma or serum sample.

[0061] Typically, the detection comprises contacting the sample with a selective reagent. The “selective agent” may be selected from the group consisting of probes, primers, antibodies, aptamers or ligands, suitable for detecting the presence of an antigen or a nucleic acid encoding or expressing said antigen present in the sample. Preferably, the selective agent is an antibody. The contacting may be made under any condition suitable for obtaining a detectable complex, for example antibody-antigen complexation or nucleic acid hybridisation, formed between the reagent and the nucleic acids or proteins of the sample.

[0062] In an embodiment the antibody is a capture antibody and a further antibody, a detection antibody, is provided. The detection antibody may be conjugated either covalently or non-covalently to a detection label. Most preferably the detection label is selected from the group consisting or comprising of a colourimetric label, a fluorescent label, a chemiluminescent label, biotin, a phosphor-based label, a thermal-based label, an enzymatic label, a gold nanoparticle, a silver nanoparticle or a magnetic bead. Thus, the present invention specifically relates to diagnosing HBV by measuring HBcrAg by detecting an antigen-antibody complex.

[0063] Specifically, detecting binding of the antibody to HBcrAg may be performed using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. For example, such assays include, but are not limited to, lateral flow assays, Western blots, agglutination tests, enzyme- labelled and mediated immunoassays, such as ELISAs, biotin / avidin type assays, radioimmunoassays, immunoelectrophoresis, immunoprecipitation, etc. The reactions of the aforementioned assays generally include labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody. Such methods generally involve separation of unbound protein and transferring the protein to a solid support to which antigen-antibody complexes are bound. Solid supports suitable for use in these methods include substrates such as nitrocellulose (e.g. membrane or microtiter well), polyvinylchloride (e.g. sheets or microtiter wells), polystyrene latex (e.g. beads or microtiter plates), polyvinylidine fluoride, diazotized paper, nylon membranes, activated beads, magnetically responsive beads, etc. Preferably, a lateral flow test may be used.

[0064] The performance of HBcrAg tests may differ based on the HBV genotypes being tested such as A, B, C, D, E, F, G, H and J. Each one can have differences, in how the disease progresses, responds to treatment and the amounts of these proteins present. The presence and levels of HBcAg, HBeAg, and p22cr in HBcrAg assays can be influenced by the specific genotype of HBV.

[0065] Predominantly, genotype A HBcrAg is in circulation in South Africa, thus assays developed for other regions with different genotypes circulating will have poor sensitivity and low positive predictive value (PPV) and thus unreliable in a South African population. Thus, the present invention relates to the development of the HBcrAg detection assay with focus on a South African population by utilizing novel antibodies to fit the population of HBsAg negative genotype A samples for inclusive diagnosis. This entails the identification and isolation of the HBcrAg antigen for consensus HBV genotypes A, including characterization through various immunological and structural analysis. The HBcrAg is isolated from hepatitis B DNA-positive samples. The targeted antigen and antibodies of the present invention represent an improvement over those available in the market and display reduced immunogenicity making them ideal for therapeutic or diagnostic purposes in humans.

[0066] In another embodiment of the present invention, the HBcrAg antigen of the present invention may be measured indirectly by determining the quantity of mRNA expressing the antigen(s). Methods for measuring the quantity of mRNA are well known to those of skill in the art. For example, the nucleic acid in the sample is extracted using standard methods and the extracted mRNA is detected by hybridization (e.g. Northern blot analysis). Alternatively, the extracted mRNA may be subjected to reverse transcription and amplification, such as by polymerase chain reaction (qRT-PCR), using specific oligonucleotide primers that enable amplification of a region in the gene(s) of the antigen(s). Thereafter, the amplified sequences may be detected by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art. Other known methods of amplification may be used, including ligase chain reaction, transcription- mediated amplification, strand displacement amplification, and nucleic acid sequencebased amplification. Nucleic acids having at least 10 nucleotides that are complementary to the mRNA of interest may be used as hybridization probes or amplification primers and may be used in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators for detecting hybridization are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e.g. avidin / biotin). Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, whereas primers are typically shorter single-stranded nucleic acids, of between 10 to 25 nucleotides in length.

[0067] The methods of the invention may further comprise a step of comparing antigen levels with a control sample or with a reference sample.

[0068] As used herein, the term “control” may relate to a healthy subject, i.e. a subject who does not have HBV infection, or to a subject with OBI, or any other acceptable standard for differentiating between a positive and negative detection result.

[0069] As used herein the term “subject” includes mammals, preferably human or animal subjects, but most preferably the subjects are human subjects. The terms “subject” and “patient” are used interchangeably herein.

[0070] In the present disclosure HBV DNA was directly detected in samples negative for HBsAg using materials and methods for amplifying and detecting HBV in a sample. Herein, HBV DNA was determined by the detection of the HBcrAg gene. However, any means involving nucleic acid detection and / or amplification may be used to detect HBV DNA, such as but not limited to, polymerase chain reaction (PCR), reverse-transcriptase PCR (RT- PCR), real-time PCR (rt-PCR), transcription-mediated amplification (TMA), rolling circle amplification, nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), and ligase chain reaction (LCR). The HBV genome is an enveloped DNA virus that belongs to the Hepadnaviridae family, containing a small, partially doublestranded (DS), relaxed-circular DNA (rcDNA) genome that replicates by reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). The HBV genome is between 3182 and 3248 base pairs (bp) depending on the genotype. Following the detection of the HBcrAg gene, the detected amplicons were sequenced to generate a consensus sequence, depicting multiple sequence alignment of the HBcrAg used to generate a consensus genotype A sequence, for development or identification of anti- HBcrAg antibodies suitable for use in the assay.

[0071] In some embodiments, nucleic acid sequencing methods are utilized for determining the consensus sequence of HBcrAg epitopes, such as detection of HBV or in the case of one or more splice variants of HBV DNA, detection of HBV or hepatocellular carcinoma (HCC). In some embodiments, the sequencing is Sanger-based Sequencing, Second Generation (Next Generation or Next-Gen), Third Generation (Next-Next-Gen), or Fourth Generation (N3-Gen) sequencing technology including, but not limited to, pyrosequencing, sequencing-by-ligation, single molecule sequencing, sequence-by-synthesis (SBS), semiconductor sequencing, massive parallel clonal, massive parallel single molecule SBS, massive parallel single molecule real-time, massive parallel single molecule real-time nanopore technology, etc. In a preferred embodiment, the sequencing is Sanger-based sequencing.

[0072] In some embodiments, the reagents for detecting the antigen(s), such as the anti- HBcrAg antibodies, and / or measuring the expression level or quantity of the antigen(s) of the present invention may be provided as a device or in a kit, together with instructions for use

[0073] The following examples are offered by way of illustration and not by way of limitation.

[0074] EXAMPLE 1

[0075] Detection of the HBcrAg gene

[0076] Sample collection

[0077] This analysis included 20 HBV DNA positive samples from a previous study as detailed in Nkosi, N A (2025), which is incorporated in its entirety herein by reference. The study population consisted of stored serum samples from March 2021 to May 2022, which tested negative for HBsAg as part of the routine diagnosis of viral hepatitis infections at the Department of Virology, National Health Laboratory Services, at Dr George Mukhari (NHLS / DGM). The samples were stored at -20 °C.

[0078] Nucleic acid extraction from serum samples

[0079] Viral nucleic acid was extracted using a high pure viral nucleic acid extraction kit (Roche Diagnostics, Penzberg, Germany), following the manufacturer’s instructions.

[0080] Detection of the HBcrAg gene

[0081] A nested in-house PCR was performed to amplify the HBcrAg region. The amplification of the region, the first round of PCR 5 pl of RedMix (Meridian Bioscience, USA), 0.5 pl of primers C1 (5’CGGGATCCGAGGAGTTGGGGGAGGAGATT 3’ - SEQ ID NO: 15) and C2 (5’ GAGAATTCACCTTATGAGTCCAAGG 3’ - SEQ ID NO: 16) (Bioline, Luckenwalde, Germany), and 13.5 pl of nuclease free water (Thermoscientific, USA) were added. Subsequently, 20 pl of the prepared master mix was aliquoted into a 0.2ml PCR tube with 5 pl of extracted nucleic acid. A second master mix was made by combining 5 pl of RedMix with 0.5 pl of primers C3 (5’ CGGGATCCCTTTGTACTAGGAGGCTGTAG 3’ - SEQ ID NO: 17) and C4 (5’ GAGAATTCTACTAACATTGAGATTCCCG 3’ - SEQ ID NO: 18) (Bioline, Luckenwalde, Germany). Thereafter, 20 pl of the prepared master mix was aliquoted in a PCR tube followed by the addition of 5 pl nucleic acid extract. The master mix was then briefly centrifuged and loaded onto a thermocycler (Applied Biosystems™ ProFlex™ PCR System, Thermoscientific, USA) under the following conditions:

[0082] Table 1 : Thermocycling conditions

[0083] Hepatitis B core antigen HBcAg; minutes: seconds -mm:ss; degree Celsius - ° C.

[0084] Detection of amplicons

[0085] The PCR amplicons were visualised using gel electrophoresis using 2% agarose gel with an expected band size of 615 bp.

[0086] The isolated viral nucleic acid was run on gel to analyse the PCR products. Figure 3 shows gel electrophoresis results obtained from amplification of PCR products (polymerase chain reaction) products with amplicons of size 615 bp from the HBcAg region. MW- molecular weight marker; PC- positive control; NC- negative control; Bp- base pairs.

[0087] Amino acid sequencing

[0088] The sequencing of the PCR products was performed at Inqaba Biotechnical Industries (Pty) Ltd, Pretoria, South Africa. HBV DNA sequencing was performed on a Sanger-based sequencing platform with the ABI3500XL genetic analyzer (Applied Biosystems, 37 United States). Sequence analysis was done using different bioinformatics tools. Chromas Pro version 2.1 (Technelysium Pty Ltd) was used to manually edit the sequences of the HBcrAg positive samples. BioEdit was used to align the sequences, and a consensus sequence was created. Figure 4 shows the generated consensus sequence, depicting multiple sequence alignment of the HBcrAg used to generate a consensus genotype A sequence. The consensus sequence represents the common amino acids in the different sequences selected to create a sequence. A consensus protein sequence was performed for samples of genotype A as shown below (SEQ ID NO: 14).

[0089] >Genotype A consensus (SEQ ID NO:14)

[0090] MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALR QAILCWGELMTLATWVGNNLEDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFG RETVLEYLVSFGVWI RTPPAYRPPNAPI LSTLPET Epitope prediction

[0091] BepiPred-2.0 and IEDB, web-based tools that predict linear B cell epitopes, were used to predict epitope regions on the genotype A consensus sequence. Thereafter, to check whether the identified antigen has the capacity to specifically bind to antibodies sites, the VaxiJen (v2.0) software was used. This tool assesses the chemical characteristics of proteins to anticipate their effectiveness as antigens, independent of sequence alignment.

[0092] Figure 5 shows the predicted epitope region from the consensus sequence of genotype A. The predicted epitopes are indicated by the letter E and are the regions recognized by the immune system. These regions known to elicit immune response and are also recognised by the antibodies. Table 2 depicts epitopes predicted by IEDB based on the consensus sequence.

[0093] Table 2: Predicted peptides Epitope conservancy

[0094] The IEDB analysis resource is a web-based bioinformatics tool which provides information on the conservancy of immune epitopes based on the gathered database of experimentally identified immune epitopes. The IEDB was used to check for conservancy of the predicted epitopes above.

[0095] Table 3 shows the conservancy report of the three-epitope region to be targeted in the HBcrAg assay based on sequences from South African samples. The table shows the predicted epitope conservancy using IEDB. The first epitope reported to be 92%.86-100% conserved in all the database sequences used, with the second and third reporting reported as 84.62% and 84.21%-89.47% respectively.

[0096] Table 3: Epitope Conservancy Analysis Result

[0097] Prediction of antigen structure

[0098] The structure of the isolated antigen was then computed using the bioinformatics tool iTASSER server for antigen structure prediction based on function annotation. The predicted structure was then reviewed using PYMOL 2.0 for molecule visualization (The PyMOL Molecular Graphics System, Version 2.0 Schrodinger, LLC). Figure 6 shows the computed secondary structure for genotype A consensus sequence. Highlighted in white are the epitope regions (HB5-18, HB32-47, HB74-92) to be targeted by the assay. Solvent accessibility

[0099] To determine the protein folding and stability, a solvent accessibility prediction was performed using two methods: iTASSER and NetSurfP-3.0. The two methods reported the epitopes PYKEFGATVELLSF’ (HB5-18) - SEQ ID NO:1 , ‘DTASALYREALESP’ (HB32- 47) - SEQ ID NO:2, and ‘NNLEDPASRDLWNYVNTN’ (HB74-92) - SEQ ID NO:3) as residues that appear on the protein as shown in Figures 7 and 8. Referring to Figure 7, highlighting the accessibility scores of the sequence amino acids as shown, from which the desirable epitope regions for the HBcrAg assay were determined. These epitope regions are reported to be highly exposed as shown by the values. Figure 8 shows the genotype A consensus sequence structure and the accessibility of the residues on the surface of the core protein from which the regions with the epitopes of interest were determined.

[0100] EXAMPLE 2

[0101] Antibody selection

[0102] The assay uses polyclonal antibodies targeted at the predicted epitopes. First, a search for already available antibodies was performed on the protein data bank (PDB) and GenBank, and probable antibodies were identified and downloaded. Impurities such as salts were then removed from the sequence as probable inhibitors using PYMOL. To predict the structure of antibodies based on the identified epitopes Rosetta comms was used. Subsequently, Cluspro was used to verify the interaction of antigen and antibody, the antibody with the consensus antigen had a balanced lowest energy of -1744.6, showing to be the most stable (Table 4). The Balanced energy weight score combines the Electrostatic- favoured, Hydrophobic-favoured, and VdW+Elec to give more favourable interacts and stable docking. Highlighted in blue is the preferred structure with the highest lowest energy weight score. The interaction was further analysed using PYMOL 2.0 to assess the binding interface and to check the distances between the residues, such as the hydrogen bonds, salt bridges, and hydrophobic contacts. The interaction of the predicted epitopes with the antibody was analysed as shown in Figure 9 which depicts the 3D structure in two different orientations of the antigen-antibody docking. The grey highlights the antibody and the brown and white highlight the antigen. The white marking shows the bound epitopes. Table 4: Balanced weight score

[0103] REFERENCES

[0104] Caviglia, G. P., Armandi, A., Rosso, C., Ribaldone, D. G., Pellicano, R., & Fagoonee, S. (2021). Hepatitis B Core-Related Antigen as Surrogate Biomarker of Intrahepatic Hepatitis B Virus Covalently-Closed-Circular DNA in Patients with Chronic Hepatitis B: A Meta-Analysis. Diagnostics (Basel), 11 (2).

[0105] Inoue, T., & Tanaka, Y. (2019). The Role of Hepatitis B Core-Related Antigen. Genes (Basel), 70(5).

[0106] Kimura, T., Rokuhara, A., Sakamoto, Y., Yagi, S., Tanaka, E., Kiyosawa, K., & Maki, N. (2002). Sensitive enzyme immunoassay for hepatitis B virus core-related antigens and their correlation to virus load. J Clin Microbiol, 40(2), 439-445.

[0107] Kramvis, A., Chang, K. M., Dandri, M., Farci, P., Glebe, D., Hu, J., Janssen, H. L. A., Lau, D. T. Y., Penicaud, C., Pollicino, T., Testoni, B., Van Bdmmel, F., Andrisani, O., Beumont-Mauviel, M., Block, T. M., Chan, H. L. Y., Cloherty, G. A., Delaney, W. E., Geretti, A. M., Gehring, A., Jackson, K., Lenz, O., Maini, M. K., Miller, V., Protzer, II., Yang, J. C., Yuen, M. F., Zoulim, F., & Revill, P. A. (2022). A roadmap for serum biomarkers for hepatitis B virus: current status and future outlook. Nat Rev Gastroenterol Hepatol, 19(11), 727-745.

[0108] Mak, L. Y., Wong, D. K., Cheung, K. S., Seto, W. K., Lai, C. L., & Yuen, M. F. (2018). Review article: hepatitis B core-related antigen (HBcrAg): an emerging marker for chronic hepatitis B virus infection. Aliment Pharmacol Ther, 47(1), 43-54.

[0109] Nkosi, N. A. (2025). Dissertation: Comparison of Hepatitis B Core-Related Antigen to Hepatitis B Virus Deoxyribonucleic Acid in Hepatitis B Surface Antigen Negative Specimens at Dr George Mukhari National Health Laboratory Services.

[0110] Raimondo, G., Locarnini, S., Pollicino, T., Levrero, M., Zoulim, F., & Lok, A. S. (2019). Update of the statements on biology and clinical impact of occult hepatitis B virus infection. J Hepatol, 71(2), 397-408.

[0111] Watanabe, T., Inoue, T., & Tanaka, Y. (2021). Hepatitis B Core-Related Antigen and New Therapies for Hepatitis B. Microorganisms, 9(10), 2083.

Claims

CLAIMS1 . A method of diagnosing Hepatitis B Virus (HBV) infection in a subject, comprising: a) providing a sample from the subject; b) contacting the sample with a selective agent which specifically binds to one or more hepatitis B core-related antigen (HBcrAg) epitopes; and c) detecting binding of the selective agent with the one or more HBcrAg epitopes, wherein binding of the selective agent with the one or more HBcrAg epitopes indicates HBV infection in the subject.

2. The method of claim 1 , wherein the one or more HBcrAg epitopes are B cell epitopes.

3. The method of claim 1 or 2, wherein the one or more HBcrAg epitopes are selected from HBV genotype A.

4. The method of any one of claims 1 to 3, wherein the one or more HBcrAg epitopes has an amino acid sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO:2, and SEQ ID NO:3.

5. The method of any one of claims 1 to 4, wherein the HBV infection is occult hepatitis B (OBI).

6. The method of any one of claims 1 to 5, wherein the subject has previously tested negative for HBV using an HBsAg assay.

7. The method of any one of claims 1 to 6, wherein the sample is selected from the group consisting of blood, serum, plasma, and / or liver tissue extract.

8. The method of claim 7, wherein the sample is a serum sample or a plasma sample.

9. The method of any one of claims 1 to 8, wherein the selective agent is an antibody and wherein the antibody binds to the HBcrAg epitope to form an HBcrAg-antibody complex.

10. The method of claim 9, wherein the antibody is a capture antibody and / or a detection antibody.

11. The method of claim 10, wherein the detection antibody is conjugated covalently or non-covalently to a detection label.

12. The method of claim 11, wherein the detection label is selected from the group consisting of colourimetric labels, fluorescent labels, chemiluminescent labels, biotin, phosphor-based labels, thermal-based labels, enzymatic labels, gold nanoparticles, silver nanoparticles and magnetic beads.

13. The method of any one of claims 9 to 12, wherein the antibody is a polyclonal antibody.

14. The method of any one of claims 9 to 13, wherein the antibody is a full-length antibody or fragment thereof that binds to the HBcrAg epitope.

15. The method of any one of claims 1 to 14, wherein the method is a point-of-care method of diagnosing HBV.

16. The method of claim 15, wherein binding of the antibody to the HBcrAg epitope is detected within 20 minutes.

17. A diagnostic device for detecting Hepatitis B Virus (HBV) infection in a sample of a subject, comprising: a) a selective agent which specifically binds to one or more HBcrAg epitope; and b) an indicator for detecting binding of the selective agent with the one or more HBcrAg epitopes, wherein binding of the selective agent with the one or more HBcrAg epitopes indicates HBV infection in the subject.

18. The diagnostic device of claim 17, wherein the one or more HBcrAg epitopes are B cell epitopes.

19. The diagnostic device of claim 17 or 18, wherein the one or more HBcrAg epitopes are selected from HBV genotype A.

20. The diagnostic device of any one of claims 17 to 19, wherein the one or more HBcrAg epitopes has an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

21. The diagnostic device of any one of claims 17 to 20, wherein the HBV infection is occult hepatitis B (OBI).

22. The diagnostic device of any one of claims 17 to 21, wherein the subject has previously tested negative for HBV using an HBsAg assay.

23. The diagnostic device of any one of claims 17 to 22, wherein the diagnostic device comprises a zone for loading the sample.

24. The diagnostic device of any one of claims 17 to 23, wherein the sample is selected from the group consisting of blood, serum, plasma, and / or liver tissue extract.

25. The diagnostic device of claim 24, wherein the sample is a serum sample or a plasma sample.

26. The diagnostic device of any one of claims 17 to 25, wherein the selective agent is an antibody and wherein the antibody binds to the HBcrAg epitope to form an HBcrAg- antibody complex.

27. The diagnostic device of claim 26, wherein the antibody is a capture antibody and / or a detection antibody.

28. The diagnostic device of claim 27, wherein the detection antibody is conjugated covalently or non-covalently to a detection label.

29. The diagnostic device of claim 28, wherein the detection label is selected from the group consisting of colourimetric labels, fluorescent labels, chemiluminescent labels, biotin, phosphor-based labels, thermal-based labels, enzymatic labels, gold nanoparticles, silver nanoparticles and magnetic beads.

30. The diagnostic device of any one of claims 26 to 29, wherein the antibody is a polyclonal antibody.

31. The diagnostic device of any one of claims 26 to 30, wherein the antibody is a full- length antibody or fragment thereof that binds to the HBcrAg epitope.

32. The diagnostic device of any one of claims 17 to 31, wherein the device is a point- of-care device for diagnosing HBV.

33. The diagnostic device of claim 32, wherein binding of the antibody to the HBcrAg epitope is detected within 20 minutes.

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