HBV diagnostic, prognostic and therapeutic methods and products

Abstract

Provided herein are compositions, systems and methods for assessing and monitoring the stage and phase of disease, predicting the likelihood of disease progression, and predicting and monitoring the response to Hepatitis B virus infection.

Description

[Technical Field]

[0001] Related Application Information This application claims priority to U.S. Patent Application No. 63 / 406,830, filed September 15, 2022, the contents of which are incorporated herein by reference.

[0002] Sequence Listing The contents of the Electronic Sequence Listing entitled 40844_601-SQL-ST26.xml (Size: 57,344 bytes; Created: September 14, 2023) are incorporated herein by reference in their entirety.

[0003] Provided herein are compositions, systems and methods for assessing and monitoring the stage and phase of Hepatitis B Virus (HBV), as well as predicting and monitoring response to treatment, by determining the amount of Hepatitis B Core Antigen (HBcAg) and / or phosphorylated Hepatitis B Core Antigen (P-HBcAg) in a sample obtained from a subject. [Background technology]

[0004] Hepatitis is a general term meaning "inflammation of the liver" and has several etiologies. Viral etiologies are the most common and are caused by hepatitis viruses A, B, C, D, or E. Hepatitis B virus (HBV) is a serious and common infectious disease, particularly of the liver, affecting millions of people worldwide.

[0005] HBV is a hepatotropic DNA virus belonging to the Hepadnaviridae family. The complete length of the viral genome is approximately 3.2 kb and contains four open reading frames (ORFs), including a surface antigen (S gene), a core antigen (C gene), a DNA polymerase (P gene), and a gene with an undetermined function called the X gene.

[0006] Over 2 billion people alive today have been infected with HBV at some time during their lifetime, and of these, approximately 248 million remain chronically infected and are carriers of the virus. HBV infection can cause acute and chronic hepatitis B, which can ultimately lead to the development of chronic liver failure, cirrhosis, and hepatocellular carcinoma. Furthermore, HBV carriers can transmit the disease for many years.

[0007] HBV is transmitted by percutaneous or parenteral contact with infected body fluids or blood. The most common routes of infection are vertical transmission from mother to child and in adults via sexual intercourse or sharing of intravenous needles or ear-piercing devices. Many cases of acute HBV infection occur without a traceable route of transmission.

[0008] Individuals with chronic HBV infection ("carriers") have a 12- to 300-fold higher risk of developing hepatocellular carcinoma than non-carriers, and globally, HBV causes 60-80% of primary liver cancers worldwide. Approximately 25% of the more than 4 million acute clinical cases die each year from chronic active hepatitis, cirrhosis, or liver cancer due to HBV. As a result, HBV ranks second only to tobacco among known human carcinogens.

[0009] Although vaccines against HBV have been widely used for decades, the prevalence of HBV in the population remains high. Current therapies for chronic HBV infection have limited inhibitory effects on viral gene expression and replication in the majority of chronically infected patients. For example, lamivudine suppresses HBV replication in carriers, but this effect is reversed when treatment is discontinued. Furthermore, a major limitation of chronic lamivudine therapy is the emergence of viral resistance, which typically occurs six months after treatment. Resistance is usually associated with mutations in the highly conserved catalytic region of the HBV polymerase gene. Furthermore, current markers for HBV (e.g., HBsAg, anti-HBs, HBeAg, and anti-HBe) have limited ability to measure the effectiveness of antiviral treatment, for example, to measure the reduction in HBV DNA levels in subjects after treatment.

[0010] For these reasons, there remains a need for new treatment strategies for HBV infection and methods for selecting and monitoring appropriate therapy. [Brief explanation of the drawings]

[0011] [Figure 1] HBV seroconversion panel comparing levels of phosphorylated HBV core antigen (P-HBcAg), HBV core antigen (HBcAg), HBeAg, and HBcrAg with levels of HBV DNA over time. [Figure 2] Comparison of P-HBcAg and HBcAg levels with HBV DNA and HBV RNA levels after treatment of HBV infection is shown. HBcAg levels (black line) correspond to HBV DNA levels over the course of treatment. Phosphorylated P-HBcAg (blue line) correlates more strongly with HBV RNA levels compared to HBV DNA levels over the course of treatment. Some data shown are taken from Bazinet et al., Hepatology Communications 5;11 (2021) pp. 1873-1887. [Figure 3] Various sequences associated with P-HBcAg and / or HBcAg are shown. [Figure 4A] Figure 4A shows the analysis of P-HBcAg levels in clinical samples with a positive HBsAg test and a viral load of 103 to 109 HBV DNA copies / ml. Figure 4B shows the analysis of HBcAg levels in clinical samples with a positive HBsAg test and a viral load of 103 to 109 HBV DNA copies / ml. P-HBcAg was detectable in 84% of samples with a viral load of 105 cp / ml or greater. HBcAg was detectable in 94% of samples with a viral load of 106 cp / ml or greater. [Figure 4B]Figure 4A shows the analysis of P-HBcAg levels in clinical samples with a positive HBsAg test and a viral load of 103 to 109 HBV DNA copies / ml. Figure 4B shows the analysis of HBcAg levels in clinical samples with a positive HBsAg test and a viral load of 103 to 109 HBV DNA copies / ml. P-HBcAg was detectable in 84% of samples with a viral load of 105 cp / ml or greater. HBcAg was detectable in 94% of samples with a viral load of 106 cp / ml or greater. [Figure 5A] Figure 5A shows the consensus sequence for HBcAg. Figure 5B shows the consensus sequence for HBcAg genotype A. Figure 5C shows the consensus sequence for HBcAg genotype B. Figure 5D shows the consensus sequence for HBcAg genotype C. Figure 5E shows the consensus sequence for HBcAg genotype D. Figure 5F shows the consensus sequence for HBcAg genotype E. Figure 5G shows the consensus sequence for HBcAg genotype F. Figure 5H shows the consensus sequence for HBcAg genotype G. Figure 5I shows the consensus sequence for HBcAg genotype H. In each of Figures 5A-5I, an * (asterisk) indicates a position with a single, completely conserved residue. A : (colon) indicates conservation between groups with strongly similar properties, approximately equivalent to a score of greater than 0.5 in the Gonnet PAM 250 matrix. [Figure 5B]Figure 5A shows the consensus sequence for HBcAg. Figure 5B shows the consensus sequence for HBcAg genotype A. Figure 5C shows the consensus sequence for HBcAg genotype B. Figure 5D shows the consensus sequence for HBcAg genotype C. Figure 5E shows the consensus sequence for HBcAg genotype D. Figure 5F shows the consensus sequence for HBcAg genotype E. Figure 5G shows the consensus sequence for HBcAg genotype F. Figure 5H shows the consensus sequence for HBcAg genotype G. Figure 5I shows the consensus sequence for HBcAg genotype H. In each of Figures 5A-5I, an * (asterisk) indicates a position with a single, completely conserved residue. A : (colon) indicates conservation between groups with strongly similar properties, approximately equivalent to a score of greater than 0.5 in the Gonnet PAM 250 matrix. [Figure 5C] Figure 5A shows the consensus sequence for HBcAg. Figure 5B shows the consensus sequence for HBcAg genotype A. Figure 5C shows the consensus sequence for HBcAg genotype B. Figure 5D shows the consensus sequence for HBcAg genotype C. Figure 5E shows the consensus sequence for HBcAg genotype D. Figure 5F shows the consensus sequence for HBcAg genotype E. Figure 5G shows the consensus sequence for HBcAg genotype F. Figure 5H shows the consensus sequence for HBcAg genotype G. Figure 5I shows the consensus sequence for HBcAg genotype H. In each of Figures 5A-5I, an * (asterisk) indicates a position with a single, completely conserved residue. A : (colon) indicates conservation between groups with strongly similar properties, approximately equivalent to a score of greater than 0.5 in the Gonnet PAM 250 matrix. [Figure 5D]Figure 5A shows the consensus sequence for HBcAg. Figure 5B shows the consensus sequence for HBcAg genotype A. Figure 5C shows the consensus sequence for HBcAg genotype B. Figure 5D shows the consensus sequence for HBcAg genotype C. Figure 5E shows the consensus sequence for HBcAg genotype D. Figure 5F shows the consensus sequence for HBcAg genotype E. Figure 5G shows the consensus sequence for HBcAg genotype F. Figure 5H shows the consensus sequence for HBcAg genotype G. Figure 5I shows the consensus sequence for HBcAg genotype H. In each of Figures 5A-5I, an * (asterisk) indicates a position with a single, completely conserved residue. A : (colon) indicates conservation between groups with strongly similar properties, approximately equivalent to a score of greater than 0.5 in the Gonnet PAM 250 matrix. [Figure 5E] Figure 5A shows the consensus sequence for HBcAg. Figure 5B shows the consensus sequence for HBcAg genotype A. Figure 5C shows the consensus sequence for HBcAg genotype B. Figure 5D shows the consensus sequence for HBcAg genotype C. Figure 5E shows the consensus sequence for HBcAg genotype D. Figure 5F shows the consensus sequence for HBcAg genotype E. Figure 5G shows the consensus sequence for HBcAg genotype F. Figure 5H shows the consensus sequence for HBcAg genotype G. Figure 5I shows the consensus sequence for HBcAg genotype H. In each of Figures 5A-5I, an * (asterisk) indicates a position with a single, completely conserved residue. A : (colon) indicates conservation between groups with strongly similar properties, approximately equivalent to a score of greater than 0.5 in the Gonnet PAM 250 matrix. [Figure 5F]Figure 5A shows the consensus sequence for HBcAg. Figure 5B shows the consensus sequence for HBcAg genotype A. Figure 5C shows the consensus sequence for HBcAg genotype B. Figure 5D shows the consensus sequence for HBcAg genotype C. Figure 5E shows the consensus sequence for HBcAg genotype D. Figure 5F shows the consensus sequence for HBcAg genotype E. Figure 5G shows the consensus sequence for HBcAg genotype F. Figure 5H shows the consensus sequence for HBcAg genotype G. Figure 5I shows the consensus sequence for HBcAg genotype H. In each of Figures 5A-5I, an * (asterisk) indicates a position with a single, completely conserved residue. A : (colon) indicates conservation between groups with strongly similar properties, approximately equivalent to a score of greater than 0.5 in the Gonnet PAM 250 matrix. [Figure 5G] Figure 5A shows the consensus sequence for HBcAg. Figure 5B shows the consensus sequence for HBcAg genotype A. Figure 5C shows the consensus sequence for HBcAg genotype B. Figure 5D shows the consensus sequence for HBcAg genotype C. Figure 5E shows the consensus sequence for HBcAg genotype D. Figure 5F shows the consensus sequence for HBcAg genotype E. Figure 5G shows the consensus sequence for HBcAg genotype F. Figure 5H shows the consensus sequence for HBcAg genotype G. Figure 5I shows the consensus sequence for HBcAg genotype H. In each of Figures 5A-5I, an * (asterisk) indicates a position with a single, completely conserved residue. A : (colon) indicates conservation between groups with strongly similar properties, approximately equivalent to a score of greater than 0.5 in the Gonnet PAM 250 matrix. [Figure 5H]Figure 5A shows the consensus sequence for HBcAg. Figure 5B shows the consensus sequence for HBcAg genotype A. Figure 5C shows the consensus sequence for HBcAg genotype B. Figure 5D shows the consensus sequence for HBcAg genotype C. Figure 5E shows the consensus sequence for HBcAg genotype D. Figure 5F shows the consensus sequence for HBcAg genotype E. Figure 5G shows the consensus sequence for HBcAg genotype F. Figure 5H shows the consensus sequence for HBcAg genotype G. Figure 5I shows the consensus sequence for HBcAg genotype H. In each of Figures 5A-5I, an * (asterisk) indicates a position with a single, completely conserved residue. A : (colon) indicates conservation between groups with strongly similar properties, approximately equivalent to a score of greater than 0.5 in the Gonnet PAM 250 matrix. [Figure 5I] Figure 5A shows the consensus sequence for HBcAg. Figure 5B shows the consensus sequence for HBcAg genotype A. Figure 5C shows the consensus sequence for HBcAg genotype B. Figure 5D shows the consensus sequence for HBcAg genotype C. Figure 5E shows the consensus sequence for HBcAg genotype D. Figure 5F shows the consensus sequence for HBcAg genotype E. Figure 5G shows the consensus sequence for HBcAg genotype F. Figure 5H shows the consensus sequence for HBcAg genotype G. Figure 5I shows the consensus sequence for HBcAg genotype H. In each of Figures 5A-5I, an * (asterisk) indicates a position with a single, completely conserved residue. A : (colon) indicates conservation between groups with strongly similar properties, approximately equivalent to a score of greater than 0.5 in the Gonnet PAM 250 matrix.

[0012] STA NEQK NHQK NDEQ QHRK MILV MILF HY FYW A period (.) indicates conservation between groups with weakly similar properties, roughly equivalent to a score above 0 and below 0.5 in the Gonnet PAM 250 matrix.

[0013] CSA ATV SAG STNK STPA SGND SNDEQK NDEQHK NEQHRK FVLIM HFY Summary of the Invention [Means for solving the problem]

[0014] (Abstract) In some aspects, provided herein are methods for assessing and monitoring the stage or phase of chronic HBV infection in a subject or for monitoring the response to treatment for chronic HBV. In some embodiments, the methods comprise performing an assay to detect the presence or level of hepatitis B core antigen (HBcAg) and phosphorylated hepatitis B core antigen (P-HBcAg) in at least one sample obtained from a subject diagnosed with chronic HBV or receiving treatment for chronic HBV. In some embodiments, the assay comprises contacting the at least one sample with an antibody that specifically binds HBcAg and an antibody that specifically binds P-HBcAg. In some embodiments, the methods further comprise assessing and monitoring the stage or phase of chronic HBV infection or monitoring the response to treatment for chronic HBV based on the presence or level of HBcAg and P-HBcAg in the at least one sample.

[0015] In some embodiments, the subject is being evaluated and monitored for a stage or phase of chronic HB. In some embodiments, the method further includes providing the subject with treatment for chronic HBV based on the presence or level of HBcAg and P-HBcAg in the at least one sample.

[0016] In some embodiments, the subject is undergoing treatment for chronic HBV. In some embodiments, the method further comprises altering treatment for HBV based on the presence or level of HBcAg and P-HBcAg in the at least one sample.

[0017] In some aspects, methods for assessing and monitoring the stage or phase of chronic HBV infection are provided herein. In some embodiments, the methods include performing an assay to detect the presence or level of hepatitis B core antigen (HBcAg) in at least one sample obtained from a subject diagnosed with chronic HBV. In some embodiments, the assay includes contacting the at least one sample with an antibody that specifically binds to HBcAg. In some embodiments, the methods include determining the amount of infectious HBV particles in the at least one sample based on the presence or level of HBcAg in the at least one sample. In some embodiments, the amount of infectious HBV particles is determined regardless of whether the subject received treatment for HBV prior to performing the assay (e.g., an assay to detect the presence or level of HBcAg in at least one sample). In some embodiments, the methods further include providing treatment for chronic HBV to the subject if the amount of infectious HBV particles in the at least one sample equals or exceeds a threshold value.

[0018] In some aspects, provided herein are methods for monitoring a response to treatment for chronic hepatitis B (HBV) infection in a subject. In some embodiments, the method comprises performing an assay to detect the presence or level of hepatitis B core antigen (HBcAg) in at least one sample obtained from a subject receiving treatment for chronic HBV. In some embodiments, the assay comprises contacting the at least one sample with an antibody that specifically binds to HBcAg. In some embodiments, the method comprises determining the amount of infectious HBV particles in the at least one sample based on the presence or level of HBcAg in the at least one sample. In some embodiments, the method comprises using the amount of infectious HBV particles in the sample to determine whether treatment for chronic HBV is effective or ineffective in the subject. In some embodiments, the method comprises determining that the treatment is effective if the amount of infectious HBV particles in the at least one sample is less than a reference level. In some embodiments, the method comprises determining that the treatment is ineffective if the amount of infectious HBV particles in the at least one sample is greater than or equal to the reference level.

[0019] In some embodiments, a method for monitoring a response to treatment for chronic hepatitis B (HBV) infection in a subject comprises performing an assay to detect the presence or level of hepatitis B core antigen (HBcAg) and phosphorylated hepatitis B core antigen (P-HBcAg) in at least one sample obtained from the subject receiving treatment for chronic HBV. In some embodiments, the assay comprises contacting the at least one sample with an antibody that specifically binds HBcAg and an antibody that specifically binds P-HBcAg. In some embodiments, the method comprises determining the amount of infectious HBV particles in the at least one sample based on the presence or level of HBcAg in the at least one sample. In some embodiments, the method comprises determining the amount of non-infectious HBV particles in the at least one sample based on the presence or level of P-HBcAg in the at least one sample. In some embodiments, the method comprises determining the amount of infectious HBV particles in at least one sample based on the presence or level of HBcAg in the at least one sample, and determining the amount of non-infectious HBV particles in at least one sample based on the presence or level of P-HBcAg in the at least one sample. In some embodiments, the method comprises determining a response to a chronic HBV treatment based on the amount of infectious and / or non-infectious HBV particles in the at least one sample. For example, in some embodiments, determining the response to a treatment comprises determining whether the treatment is effective or ineffective in the subject. In some embodiments, the treatment is determined to be effective when the amount of infectious HBV particles in at least one sample is less than a reference level. In some embodiments, the treatment is determined to be effective when the amount of infectious HBV particles in at least one sample is less than the reference level and the amount of non-infectious HBV particles in at least one sample is less than the reference level. In some embodiments, the treatment is determined to be ineffective when the amount of infectious HBV particles in at least one sample is greater than or equal to the reference level.

[0020] In some embodiments, a method for monitoring a response to treatment for chronic hepatitis B (HBV) infection in a subject comprises performing an assay to detect the presence or level of hepatitis B core antigen (HBcAg) and phosphorylated hepatitis B core antigen (P-HBcAg) in at least two samples obtained from the subject receiving treatment for chronic HBV. In some embodiments, the at least two samples comprise a first sample obtained from the subject at a first time point before or after receiving treatment for chronic HBV and a second sample obtained from the subject at a second time point after the first time point. In some embodiments, the assay comprises contacting the at least two samples with an antibody that specifically binds HBcAg and an antibody that specifically binds P-HBcAg. In some embodiments, the method comprises determining the amount of infectious HBV particles in the at least two samples based on the presence or level of HBcAg and / or determining the amount of non-infectious HBV particles in the at least two samples based on the presence or level of P-HBcAg. In some embodiments, the method includes determining a response to a chronic HBV treatment based on the amount of infectious and / or non-infectious HBV particles in at least two samples. For example, determining the response to a treatment can include determining whether the treatment is effective or ineffective. In some embodiments, the treatment is determined to be effective if the amount of infectious HBV particles in the second sample is reduced by at least a certain absolute amount compared to the amount of infectious HBV particles in the first sample. In some embodiments, the treatment is determined to be effective if the amount of infectious HBV particles in the second sample is reduced by at least a certain absolute amount compared to the amount of infectious HBV particles in the first sample and the amount of non-infectious HBV particles in the second sample is reduced by at least a certain absolute amount compared to the amount of non-infectious HBV particles in the first sample. In some embodiments, the treatment is determined to be ineffective if the amount of infectious HBV particles in the second sample is not reduced by at least a certain absolute amount compared to the amount of infectious HBV particles in the first sample.

[0021] In some embodiments, the first sample is obtained from the subject within 24 hours after receiving treatment for chronic HBV. In some embodiments, the second sample is obtained from the subject 10-14 weeks after the first sample. In some embodiments, the first sample is obtained from the subject within 24 hours after receiving treatment for chronic HBV, and the second sample is obtained from the subject 10-14 weeks after the first sample.

[0022] In some embodiments, the methods of monitoring response to a chronic HBV treatment described herein further include modifying the chronic HBV treatment if the treatment is determined to be ineffective. In some embodiments, modifying the chronic HBV treatment includes providing the subject with an increased therapeutic dose, increasing the dosing frequency of the treatment, providing the subject with a second treatment, or any combination thereof.

[0023] In some embodiments, an antibody that specifically binds to HBcAg binds to an epitope comprising at least three amino acids of SEQ ID NO:2 or SEQ ID NO:25, and / or an antibody that specifically binds to P-HBcAg may bind to an epitope comprising at least three amino acids of SEQ ID NO:2, provided that at least one amino acid of SEQ ID NO:2 or SEQ ID NO:25 is phosphorylated.

[0024] In some embodiments, the treatment for chronic HBV may be an interferon, a nucleoside(t) analog, a nucleic acid, an immunomodulator, a core protein assembly inhibitor, a capsid assembly modulator (CAM), an HBsAg release inhibitor, an entry inhibitor, an interfering RNA, a DNA modifier, or a combination thereof. In some embodiments, the interferon is interferon alpha-2a or PEGylated interferon alpha-2a. In some embodiments, the nucleoside(t) analog is lamivudine, adefovir, tenofovir, telbivudine, or entecavir. In some embodiments, the nucleic acid is an siRNA, an antisense oligonucleotide, shRNA, or miRNA. In some embodiments, the core protein assembly inhibitor is NVR 3-1983, GLS4, or BAY 41-4109. In some embodiments, the CAM is JNJ-632, AT130, or BAY 41-4109. In some embodiments, the HBsAg release inhibitor is REP 9 AC. In some embodiments, the entry inhibitor is Myrcludex-B. In some embodiments, the treatment is a combination of any of the above treatments.

[0025] In some embodiments, performing an assay to measure the level of HBcAg in at least one sample comprises contacting at least one sample simultaneously or sequentially, in any order, with a hepatitis B core antigen (HBcAg) capture antibody that binds to an epitope on the C-terminus of HBcAg to form a capture antibody-HBcAg complex, and a detection antibody that binds to an epitope on HBcAg that is not bound by the HBcAg capture antibody, such that a capture antibody-HBcAg-detection antibody complex is formed. In some embodiments, performing the assay further comprises measuring the level of HBcAg in the sample based on a signal generated by a detectable label in the capture antibody-HBcAg-detection antibody complex.

[0026] In some embodiments, performing an assay to measure the level of P-HBcAg in a sample comprises contacting the sample simultaneously or sequentially, in any order, with a phosphorylated hepatitis B core antigen (P-HBcAg) capture antibody that binds to an epitope on the C-terminus of P-HBcAg to form a capture antibody-P-HBcAg complex, and a detection antibody that binds to an epitope on P-HBcAg that is not bound by the P-HBcAg capture antibody, such that a capture antibody-P-HBcAg-detection antibody complex is formed. In some embodiments, performing the assay further comprises measuring the level of P-HBcAg in the sample based on a signal generated by a detectable label in the capture antibody-P-HBcAg-detection antibody complex.

[0027] In some aspects, provided herein is the use of reagents for the detection of the presence or level of hepatitis B core antigen (HBcAg) and phosphorylated hepatitis B core antigen (P-HBcAg) in methods for assessing the stage or phase of hepatitis B (HBV) infection in a subject or in methods for monitoring response to treatment for chronic HBV.

[0028] In some aspects, provided herein are kits or systems comprising reagents for the detection of the presence, level or status of Hepatitis B Core Antigen (HBcAg) and phosphorylated Hepatitis B Core Antigen (P-HBcAg).

[0029] The section headings used in this section and throughout the disclosure herein are for organizational purposes only and are not intended to be limiting.

[0030] 1.Definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, various embodiments of the methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0031] As used herein, the term "algorithm" refers to a process or set of rules to be followed in a calculation or other problem-solving operation (e.g., by one or more computers, including one or more software programs, that analyze data from one or more markers and, optionally, one or more biometric data (e.g., history of intravenous drug use, chronic liver and / or kidney disease, employment history as a healthcare professional, age, sex, race, etc.) and include the necessary code for executing the algorithm). For example, analysis of HBV marker data performed using an algorithm may include analyzing (1) a single biomarker (e.g., hepatitis B core antigen (HBcAg), phosphorylated hepatitis B core antigen (P-HBcAg)), (2) a single marker with one or more biometric data, (3) a group of two or more markers (e.g., a group including hepatitis B core antigen (HBcAg) and phosphorylated hepatitis B core antigen (P-HBcAg), optionally including one or more additional markers), or (4) a group of two or more markers with one or more biometric data. A further approach can employ a multi-analyte algorithm (e.g., as described in U.S. Patent Publication No. 2016 / 0342757, incorporated herein by reference) rather than a single marker (with or without biometric markers) or a group of single markers (with or without biometric markers). Such algorithms can be used as part of the methods described herein to derive one or more values ​​reflective of disease state, stage, or phase, to predict the likelihood of disease progression, and / or to predict or monitor response to a therapy or treatment.

[0032] As used herein, the term "antibody" refers to an immunoglobulin molecule or an immunologically active portion thereof, i.e., an antigen-binding portion. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') fragments, which can be generated, for example, by treating antibodies with an enzyme such as pepsin. Examples of antibodies that can be used in the present disclosure include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, recombinant antibodies, single-chain Fvs ("scFvs"), affinity-matured antibodies, single-chain antibodies, single-domain antibodies, F(ab) fragments, F(ab') fragments, disulfide-linked Fvs ("sdFvs"), and anti-idiotypic ("anti-Id") antibodies, as well as functionally active epitope-binding fragments of any of the above. Antibodies may be of the class IgG, IgM, IgA, IgD, or IgE, or fragments or derivatives thereof. The antibody may be derivatized by the attachment of one or more chemical, peptide, or polypeptide moieties known in the art. The antibody may be conjugated with a chemical moiety.

[0033] As used herein, the term "antibody fragment" refers to a portion of an intact antibody that contains the antigen-binding site or variable region. The portion does not contain the heavy chain constant domain of the Fc region of the intact antibody (i.e., CH2, CH3, or CH4, depending on the antibody isotype). Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing three CDRs of a light chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing three CDRs of a heavy chain variable region.

[0034] "Area under the curve" or "AUC" refers to the area under the ROC curve. The AUC under the ROC curve is a measure of accuracy. An AUC of 1 represents a perfect test, and an AUC of 0.5 represents a meaningless test. A preferred AUC may be at least about 0.700, at least about 0.750, at least about 0.800, at least about 0.850, at least about 0.900, at least about 0.910, at least about 0.920, at least about 0.930, at least about 0.940, at least about 0.950, at least about 0.960, at least about 0.970, at least about 0.980, at least about 0.990, or at least about 0.995.

[0035] A "Receiver Operating Characteristic" curve or "ROC" curve is a graphical plot illustrating the performance of a binary classifier system as its discrimination threshold is varied. For example, an ROC curve can be a plot of the true positive rate against the false positive rate for different possible cutoff points of a diagnostic test. An ROC curve is constructed by plotting the proportion of true positives among positives (TPR = true positive rate) versus the proportion of false positives among negatives (FPR = false positive rate) at various threshold settings. TPR is also known as sensitivity, and FPR is 1 minus the specificity or true negative rate. ROC curves show a trade-off between sensitivity and specificity (increased sensitivity is accompanied by decreased specificity), with the test being more accurate as the curve approaches the left and upper boundaries of ROC space and less accurate as the curve approaches the 45-degree diagonal of ROC space. The slope of the tangent at the cutoff point gives the likelihood ratio (LR) of the test value, and the area under the curve is a measure of the test's accuracy.

[0036] The terms "bead" and "particle" are used interchangeably herein and refer to a substantially spherical solid support. One example of a bead or particle is a microparticle. Microparticles usable herein can be of any type known in the art. For example, the bead or particle can be a magnetic bead or particle. Magnetic beads / particles can be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic, or ferrofluid. Typical ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrO2, MnAs, MnBi, EuO, and NiO / Fe. Examples of ferrimagnetic materials include NiFe2O4, CoFe2O4, Fe3O4 (or FeO-Fe2O3). Beads can have a solid core that is magnetic and surrounded by one or more nonmagnetic layers. Alternatively, the magnetic portion can be a layer around the nonmagnetic core. The microparticles can be of any size that is believed to function in the methods described herein, for example, from about 0.75 to about 5 nm, or from about 1 to about 5 nm, or from about 1 to about 3 nm.

[0037] As used herein, the term "binding protein" refers to a monomeric or multimeric protein that binds to and forms a complex with a binding partner, such as a polypeptide, antigen, compound, or other molecule, or any type of substrate. A binding protein specifically binds to a binding partner. Binding proteins include antibodies, as well as antigen-binding fragments thereof and various other forms and derivatives thereof known in the art and described herein below, and other molecules containing one or more antigen-binding domains that bind to antigen molecules or specific sites (epitopes) on antigen molecules. Thus, binding proteins include, but are not limited to, tetrameric immunoglobulin antibodies, IgG molecules, IgG1 molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, affinity-matured antibodies, and fragments of any such antibodies that retain the ability to bind to antigen.

[0038] As used herein, the term "bispecific antibody" is used to refer to full-length antibodies produced by quadroma technology (also called hybrid-hybridoma technology; see Milstein et al., Nature, 305(5934):537-540 (1983)), chemical conjugation of two different monoclonal antibodies (see Staerz et al., Nature, 314(6012):628-631 (1985)), or knob-into-hole (KIH) or similar techniques that introduce mutations within the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90(14):6444-6448 (1993)), resulting in multiple different immunoglobulin species, only one of which is the functional bispecific antibody. A bispecific antibody binds to one antigen (or epitope) in one of its two binding arms (one HC / LC pair) and to a different antigen (or epitope) in its second arm (a different HC / LC pair). By this definition, a bispecific antibody has two significantly different antigen-binding arms (both specificity and CDR sequences) and is monovalent for each antigen to which it binds.

[0039] As used herein, "CDR" refers to a "complementarity-determining region" within an antibody variable sequence. There are three CDRs in each of the heavy and light chain variable regions. Starting from the N-terminus of the heavy or light chain, these regions are designated "CDR1," "CDR2," and "CDR3" for each variable region. As used herein, the term "CDR set" refers to a group of three CDRs occurring in a single variable region that bind to an antigen. Thus, an antigen-binding site may contain six CDRs, including the CDR sets from each of the heavy and light chain variable regions. A polypeptide containing a single CDR (e.g., CDR1, CDR2, or CDR3) may be referred to as a "molecular recognition unit." Crystal structure analysis of antigen-antibody complexes supports the finding that amino acid residues in the CDRs form extensive contacts with the bound antigen, with the most extensive antigen contact occurring with the heavy chain CDR3. Thus, the molecular recognition units may be primarily responsible for the specificity of the antigen-binding site. In general, the CDR residues are directly and most substantially involved in influencing binding to antigen.

[0040] The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any antibody variable region, but also provides the precise residue boundaries that define the three CDRs. These CDRs may be referred to as "Kabat CDRs." Chothia and colleagues (Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); and Chothia et al., Nature, 342:877-883 (1989)) have described the Kabat CDRs in a numbered manner. It was found that certain subportions within the CDRs adopt nearly identical peptide backbone conformations despite the large diversity at the amino acid sequence level. These subportions were designated "L1," "L2," and "L3," or "H1," "H2," and "H3," with "L" and "H" designating the light chain and heavy chain regions, respectively. These regions are sometimes referred to as "Chothia CDRs," and they have boundaries that overlap with the Kabat CDRs. For other boundaries defining CDRs that overlap with the Kabat CDRs, see Padlan, FASEB, and others. J., 9:133-139 (1995); and MacCallum, J. Mol. Biol., 262(5):732-745 (1996). Still other CDR boundary definitions may not strictly adhere to the system defined herein and may be shorter or longer in light of predicted or experimental findings that a particular residue or group of residues, or even the entire CDR, does not significantly affect antigen binding, yet still overlap with the Kabat CDRs. While the methods used herein may use CDRs defined according to any of these systems, certain embodiments use the Kabat-defined CDRs or the Chothia-defined CDRs.

[0041] As used herein, "suitable" refers, in certain embodiments, to a level of one or more biomarkers measured and / or determined according to the methods described herein being less than or lower than one or more reference levels. However, in other embodiments, depending on the biomarkers measured or determined, the term "suitable" can also refer to a level of one or more biomarkers measured and / or determined according to the methods described herein being greater than or higher than one or more reference levels. Whether a "suitable" level is higher or lower than a reference level depends on whether there is an elevation or depression of a biomarker associated with HBV infection. In some embodiments, a "suitable" level indicates that the level of one or more biomarkers has decreased by at least a certain absolute amount from a first time point to a second time point. For example, a "suitable" level may indicate that the level of HBcAg and / or the level of P-HBcAg has decreased by at least a certain absolute amount from a first sample to a second sample collected after the first sample. Generally, a suitable level of a biomarker is one that indicates the absence of HBV infection, an improvement in the subject's health related to HBV infection, or that the subject will benefit from treatment (or continued treatment) with an HBV therapeutic agent (i.e., the treatment of HBV is effective in the subject). In some embodiments, a suitable level correlates with a clinical improvement in the infection. For example, a subject's clinical improvement can be determined by any number of different parameters (e.g., patient report, improvement in skin color (e.g., reduction in yellowness or jaundice), reduction in the amount of liver inflammation, etc.).

[0042] As used herein, "unfavorable" refers, in certain embodiments, to a level of one or more biomarkers measured and / or determined according to the methods described herein being higher or greater than the reference level when compared to one or more reference levels. However, in other embodiments, depending on the biomarkers measured or determined, the term "unfavorable" can also refer to a level of one or more biomarkers measured and / or determined according to the methods described herein being lower or less than the reference level. As noted above, whether an "unfavorable" level is higher or lower than a reference level depends on whether there is an elevation or depression of a biomarker associated with HBV infection. In some embodiments, an "unfavorable" level indicates that the level of one or more biomarkers did not decrease by at least a certain absolute amount from a first time point to a second time point. For example, an "unfavorable" level may indicate that the level of HBcAg and / or the level of P-HBcAg did not decrease by at least a certain absolute amount from a first sample to a second sample collected after the first sample. Generally, an unfavorable level of a biomarker is one that indicates the presence of HBV infection, a deterioration in the subject's health associated with HBV infection, or that the subject will not benefit from HBV treatment (i.e., HBV treatment is not effective in the subject). In some embodiments, an unfavorable level correlates with a clinical worsening in infection. For example, a worsening of a subject's condition can be determined by any number of different parameters (e.g., patient report, changes or worsening in skin color (e.g., increased yellowness or increased jaundice), increased liver inflammation, etc.).

[0043] As used herein, "identical" or "identity" in the context of two or more polypeptide or polynucleotide sequences can mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, and determining the number of positions in both sequences where identical residues occur to determine the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to determine the percentage of sequence identity. If the two sequences are of different length or the alignment results in one or more staggered ends, and the specified comparison region includes only a single sequence, the residues of the single sequence are included in the denominator but not the numerator of the calculation.

[0044] As used herein, "hepatitis surface antigen or HBsAg isoform" or "hepatitis surface antigen or HBsAg isoform" refers to one or more polypeptides encoded by the pre-S1, pre-S2, and / or S sections of the HBsAg gene, designated large (L), medium (M), and small (S) HBs. Each of the large, medium, and small HBs contains an S domain. The medium HBs protein (MHBs) has a 55 amino acid long N-terminal extension, i.e., the pre-S2 domain. The HBV large surface protein (LHBs) further has a 108 or 119 amino acid N-terminal extension, i.e., the pre-S1 domain. The HBsAg of infectious virions and subviral particles consists primarily of HBV small surface proteins (SHBs), with minor components including LHBs and MHBs. Isoforms contemplated herein include (i) large HBs only, (ii) medium HBs only, (iii) small HBs only, (iv) large and medium HBs, (v) large and small HBs, (vi) medium and small HBs, or (vii) large, medium and small HBs.

[0045] As used herein, "substantially identical" can mean that the first and second sequences are at least about 50% to about 99% identical over a range of about 8 to about 100 or more residues (including any range within about 8 to about 100 residues).

[0046] As used herein, the term "test sample" or "sample" generally refers to a biological material to be tested for and / or suspected of containing an analyte of interest, e.g., a marker described herein. A test sample can be derived from any biological source, such as a physiological fluid, including, but not limited to, whole blood, serum, plasma, interstitial fluid, saliva, lens fluid, cerebrospinal fluid, sweat, urine, milk, ascites fluid, mucous membranes, nasal fluid, sputum, synovial fluid, peritoneal fluid, vaginal fluid, menstrual fluid, urinary fluid, semen, etc. In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a plasma sample. In yet other embodiments, the sample is a serum sample. A test sample may be used directly from a biological source or after pretreatment to modify the characteristics of the sample. For example, such pretreatment may include preparing plasma from blood, diluting viscous fluids, etc. Pretreatment methods may also include filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of interfering components, addition of agents, dissolution, etc. Additionally, it may be beneficial to modify a solid test sample to form a liquid medium or to release the analyte.

[0047] The term "nucleic acid" refers to a polymer of nucleotides and, unless otherwise limited, includes known analogues of natural nucleotides that can function (e.g., hybridize) in a manner similar to naturally occurring nucleotides.

[0048] The term "nucleic acid" includes any form of DNA or RNA, including, for example, genomic DNA, complementary DNA (cDNA), which is usually obtained by reverse transcription or amplification of messenger RNA (mRNA) and is the DNA representation of mRNA, DNA molecules produced synthetically or by amplification, and mRNA.

[0049] The term "nucleic acid" encompasses double- or triple-stranded nucleic acids as well as single-stranded molecules. In a double- or triple-stranded nucleic acid, the nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded over the entire length of both strands).

[0050] The term "nucleic acid" also encompasses any chemical modification thereof, such as modification by methylation and / or capping. Modification of nucleic acids can include the addition of chemical groups that incorporate additional charge, polarity, hydrogen bonding, electrostatic interactions, and functionality into individual nucleic acid bases or into the nucleic acid as a whole. Such modifications can include modifications of the sugar at the 2' position, modifications of the pyrimidine at the 5 position, modifications of the purine at the 8 position, modifications at the exocyclic amine of cytosine, substitution of 5-bromouracil, backbone modifications, and base modifications such as isobasic isocytidine and isoguanidine.

[0051] As used herein, the term "target nucleotide sequence" refers to a molecule that includes the nucleotide sequence of a target nucleic acid (e.g., a nucleic acid to be detected in an assay), such as an amplification product obtained by amplifying the target nucleic acid or a cDNA produced upon reverse transcription of an RNA target nucleic acid.

[0052] As used herein, the term "complementary" refers to the ability for precise pairing between two nucleotides. That is, if a nucleotide at a given position in a nucleic acid can hydrogen bond with a nucleotide in another nucleic acid, the two nucleic acids are considered to be complementary to each other at that position. The complementarity between two single-stranded nucleic acid molecules can be "partial," in which only a portion of the nucleotides bind, or it can be complete, in which there is total complementarity between the single-stranded molecules. The degree of complementarity between nucleic acid strands significantly affects the efficiency and strength of hybridization between nucleic acid strands.

[0053] "Specific hybridization" refers to the binding of a nucleic acid to a target nucleotide sequence under defined stringency conditions without substantial binding to other nucleotide sequences present in the hybridization mixture. Those skilled in the art will recognize that sequence mismatches can be tolerated by relaxing the stringency of the hybridization conditions.

[0054] The term "oligonucleotide" is used to refer to a relatively short nucleic acid, generally less than 200 nucleotides, less than 100 nucleotides, and in some cases less than 50 nucleotides. Typically, an oligonucleotide is a single-stranded DNA molecule.

[0055] The term "primer" refers to an oligonucleotide that can hybridize (also called "anneal") to a nucleic acid and serve as a site for initiation of nucleotide (RNA or DNA) polymerization under appropriate conditions (i.e., in the presence of four different nucleoside triphosphates and a polymerization reagent, such as a DNA or RNA polymerase or reverse transcriptase), in an appropriate buffer, at a suitable temperature. While the appropriate length of a primer depends on the primer's intended use, primers are typically at least 7 nucleotides in length, more typically in the range of 10 to 30 nucleotides, or even more typically 15 to 30 nucleotides in length. Other primers may be somewhat longer, e.g., 30 to 50 nucleotides in length. In this context, "primer length" refers to the portion of an oligonucleotide or nucleic acid that hybridizes to a complementary "target" sequence and primes nucleotide synthesis. Short primer molecules generally require lower temperatures to form a sufficiently stable hybrid complex with the template. A primer need not reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with the template. The term "primer site" or "primer binding site" refers to the segment of a target nucleic acid to which a primer hybridizes.

[0056] A primer is said to be annealed to a nucleic acid if the primer, or a portion thereof, hybridizes to a nucleotide sequence in another nucleic acid. A statement that a primer hybridizes to a particular nucleotide sequence is not intended to imply that the primer hybridizes completely or exclusively to that nucleotide sequence.

[0057] The term "primer pair" refers to a set of primers comprising a 5' "upstream primer" or "forward primer" that hybridizes to the complement of the 5' end of a DNA sequence to be amplified, and a 3' "downstream primer" or "reverse primer" that hybridizes to the 3' end of the sequence to be amplified. As will be recognized by those of skill in the art, the terms "upstream" and "downstream" or "forward" and "reverse" are not intended to be limiting, but rather provide descriptive orientation in particular embodiments.

[0058] A "probe" is a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bond, typically through complementary base pairing, usually by hydrogen bond formation, thereby forming a duplex structure. A probe binds to or hybridizes to a "probe-binding site." Probes can, in some cases, be labeled with a detectable label that allows the probe to be easily detected once it hybridizes to its complementary target. Alternatively, however, a probe can be unlabeled and detectable by specific binding to a directly or indirectly labeled ligand. The size of a probe can vary significantly. Generally, probes are at least 7-15 nucleotides in length. Other probes are at least 20, 30, or 40 nucleotides in length. Still other probes are somewhat longer, at least 50, 60, 70, 80, or 90 nucleotides in length. Still other probes are even longer, at least 100, 150, 200 nucleotides, or longer. Probes can be within any range of lengths bounded by any of the above values ​​(e.g., 15-30 nucleotides in length).

[0059] A primer or probe may be perfectly complementary to a target nucleic acid sequence, or may be less than perfectly complementary. In certain embodiments, a primer has at least 65% identity, and often at least 75%, at least 85%, at least 90%, or at least 95%, 96%, 97%, 98%, or 99% identity, with the complement of the target nucleic acid sequence over a sequence of at least 7 nucleotides, more typically over a sequence ranging from 10 to 30 nucleotides, and often over a sequence of at least 14 to 25 nucleotides. It is understood that certain bases (e.g., the 3' base of a primer) are generally desirably perfectly complementary to the corresponding base of the target nucleic acid sequence. Primers and probes typically anneal to target sequences under stringent hybridization conditions.

[0060] "Amplification" encompasses any means by which at least a portion of at least one target nucleic acid is replicated in a template-dependent manner, typically linear or exponential, including, without limitation, a wide range of techniques for amplifying nucleic acid sequences. Illustrative means for carrying out the amplification step include ligase chain reaction (LCR), ligase detection reaction (LDR), ligation followed by Q-replicase amplification, PCR, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid strand-based amplification (NASBA), two-step multiplexed amplification, rolling circle amplification (RCA), and the like, including multiplex versions and combinations thereof, such as, but not limited to, OLA / PCR, PCR / OLA, LDR / PCR, PCR / PCR / LDR, PCR / LDR, LCR / PCR, PCR / LCR (also known as combinatorial chain reaction - CCR), and the like. Descriptions of these techniques can be found in, among other sources, Ausubel et al.; PCR Primer: A Laboratory Manual, Diffenbach (ed.), Cold Spring Harbor Press (1995); The Electronic Protocol Book, Chang Bioscience (2002); Msuih et al., J. Clin. Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley (ed.), Humana Press, Totowa, NJ (2002); Abramson et al., Curr Opin Biotechnol.1993 February;4(1):41-7; U.S. Patent No. 6,027,998; U.S. Patent No. 6,605,451; Barany et al., PCT Publication WO 97 / 31256; Wenz et al., PCT Publication WO 01 / 92579; Day et al., Genomics, 29(1):152-162 (1995); Ehrlich et al., Science 252:1643-50 (1991); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press (1990); Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenau et al., Infection 28:97-102 (2000); Belgrader, Barany, and Lubin, Development of a Multiplex Ligation Detection Reaction DNA Typing Assay, Sixth International Symposium on Human Identification, 1995 (available on the World Wide Web at promega.com / geneticidproc / ussymp6proc / blegrad.html); LCR Kit Instruction Manual, Cat. #200520, Rev. #050002, Stratagene, 2002; Barany, Proc. Natl. Acad. Sci. USA 88:188-93 (1991); Bi and Sambrook, Nucl. Acids Res. 25:2924-2951 (1997); Zirvi et al., Nucl. Acid Res. 27:e40i-viii (1999); Dean et al., Proc. Natl. Acad. Sci. USA 99:5261-66 (2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl. Acid Res.20:1691-96(1992); Polstra et al., BMC Inf.Dis.2:18-(2002); Lage et al., Genome Res.2003 February;13(2):294-307, and Landegren et al., Science 241:1077-80(1988), Demidov, V., Expert Rev Mol Diagn. 2002 Nov;2(6):542-8; Cook et al., J Microbiol Methods. 2003 May;53(2):165-74; Schweitzer et al., Curr Opin Biotechnol. 2001 Feb;12(1):21-7; U.S. Patent No. 5,830,711; U.S. Patent No. 6,027,889; U.S. Patent No. 5,686,243; PCT Publication No. WO 00 / 56927A3; and WO 98 / 03673A1.

[0061] As used herein, a "reference level" refers to an assay or cutoff value used to diagnose (a "diagnostic" cutoff), determine prognosis, or evaluate the effectiveness of a treatment, and is linked to or herein associated with various clinical parameters (e.g., the presence of a disease, e.g., determining that a subject has a disease (a "rule in") or determining that a subject does not have a disease (a "rule out"), disease stage, disease severity, disease progression, non-progression, or improvement, etc.). The present disclosure provides exemplary reference levels. However, it is well known that reference levels can vary depending on the properties of the immunoassay (e.g., in an immunoassay, the antibody employed, reaction conditions, sample purity, etc.) and that assays can be compared and standardized. Furthermore, it is well within the ordinary capabilities of one of ordinary skill in the art to adapt the disclosure herein to other assays and to derive assay-specific reference levels for other immunoassays based on the instructions provided by the present disclosure. While the exact values ​​of the reference levels may vary between assays, the findings described herein are generally applicable and should be able to be extrapolated to other assays.

[0062] As used herein, the term "single molecule detection" refers to the detection and / or measurement of a single molecule of an analyte in a test sample at extremely low levels of concentration (e.g., pg / mL or femtogram / mL levels). Many different single molecule analysis instruments or devices are known in the art, including nanopore and nanowell devices. Examples of nanopore devices are described in WO 2016 / 161402, which is hereby incorporated by reference in its entirety. Examples of nanowell devices are described in WO 2016 / 161400, which is hereby incorporated by reference in its entirety.

[0063] "Reagent" refers broadly to any substance used in a reaction other than the analyte (e.g., the nucleic acid or polypeptide being analyzed). Illustrative reagents for nucleic acid amplification reactions include, but are not limited to, buffers, metal ions, polymerases, reverse transcriptases, primers, template nucleic acids, nucleotides, labels, dyes, nucleases, etc. Reagents for enzymatic reactions include, for example, substrates, cofactors, buffers, metal ions, inhibitors, and activators. Reagents for immunoassays include, for example, antibodies specific for target markers, detection (e.g., labeled) antibodies, controls, buffers, etc.

[0064] The term "label," as used herein, refers to any atom or molecule that can be used to provide a detectable and / or quantifiable signal. In some cases, the label can be directly or indirectly attached to a nucleic acid or protein. Suitable labels that can be attached to a probe include, but are not limited to, radioisotopes, fluorescent dyes, chromophores, mass labels, electron-dense particles, magnetic particles, spin labels, chemiluminescent molecules, electrochemically active molecules, enzymes, cofactors, and enzyme substrates.

[0065] The term "dye," as used herein, generally refers to any organic or inorganic molecule that absorbs electromagnetic radiation at wavelengths of 340 nm or greater.

[0066] The term "fluorescent dye," as used herein, generally refers to any dye that emits long wavelength electromagnetic radiation by a fluorescence mechanism upon illumination by an electromagnetic radiation source such as a lamp, photodiode, or laser.

[0067] As used herein, the terms "comprise(s)," "include(s)," "having," "having," "can be," "containing," and variations thereof are open-ended transitional phrases, transitional terms, or transitional words that do not exclude additional acts or structures. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprise," "consist of," or "consist essentially of" the embodiments or elements presented herein, whether expressly stated or otherwise.

[0068] In this specification, to recite numerical ranges, each intervening number with the same precision is expressly contemplated. For example, for the range of 6 to 9, the numbers 7 and 8 are also contemplated in addition to 6 and 9, and for the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0069] As used herein, "absolute amount" refers to the absolute value of the change or difference between at least two assay results taken or sampled at different time points and linked to or herein associated with various clinical parameters (e.g., presence of disease, stage of disease, severity of disease, progression, non-progression, or improvement of disease, etc.) as well as reference levels. As used herein, "absolute value" refers to the magnitude of the actual number (e.g., the difference between two levels being compared (e.g., a level taken at a first time point and a level taken at a second time point) regardless of its sign, i.e., whether the number is positive or negative).

[0070] The present disclosure provides exemplary reference levels and absolute amounts (e.g., calculated by comparing reference levels at different time points). However, it is well known that reference levels and absolute amounts can vary depending on the properties of the immunoassay (e.g., the antibody employed, the reaction conditions, the purity of the sample, etc.) and that assays can be compared and standardized. Furthermore, it is well within the routine capabilities of one of ordinary skill in the art to adapt the disclosure herein to other immunoassays and to obtain immunoassay-specific reference levels and absolute amounts for other immunoassays based on the explanations provided by the present disclosure. While the exact values ​​of reference levels and absolute amounts may vary between assays, the findings described herein are generally applicable and can be extrapolated to other assays.

[0071] "Component," "component(s)," or "at least one component" generally refers to capture antibodies, detection or conjugates, calibrators, controls, sensitivity panels, containers, buffers, diluents, salts, enzymes, cofactors for enzymes, detection reagents, pretreatment reagents / solutions, substrates (e.g., in solution), stop solutions, and the like that may be included in a kit for assaying a test sample, such as a patient urine, whole blood, serum, or plasma sample, according to the methods described herein and other methods known in the art. Some components may be in solution for reconstitution or lyophilized for use in the assay.

[0072] As used herein, "control" generally refers to a reagent intended to assess the performance of a measurement system to ensure that it continues to produce results within acceptable boundaries (e.g., boundaries ranging from scales appropriate for research assays on the one hand to analytical boundaries established by quality specifications for commercially available assays on the other). To accomplish this, the control must be indicative of patient outcome and, optionally, somehow assess the impact of error on the measurement (e.g., error due to reagent stability, calibrator variability, instrument variability, etc.).

[0073] As used herein, "correlates to" refers to "compared to."

[0074] As used herein, a "derivative" of an antibody may refer to an antibody that has one or more modifications to its amino acid sequence compared to the original or parent antibody and may exhibit a modified domain structure. A derivative may not only adopt an amino acid sequence capable of specifically binding to a target (antigen), but may also adopt the typical domain organization found in a natural antibody. Typical examples of antibody derivatives are antibodies coupled to other polypeptides, rearranged antibody domains, or antibody fragments. A derivative may also contain at least one additional compound, e.g., a protein domain, linked by covalent or non-covalent bonds. Linkage may be based on gene fusion according to methods known in the art. Additional domains present in an antibody-containing fusion protein may be linked by a flexible linker, advantageously a peptide linker, comprising multiple hydrophilic, peptide-bonded amino acids of sufficient length to span the distance between the C-terminus of the additional protein domain and the N-terminus of the antibody, or vice versa. The antibody may have a conformation suitable for biological activity or may be linked to an effector molecule that selectively binds, for example, a solid support, a biologically active substance (e.g., a cytokine or growth hormone), a chemical agent, a peptide, a protein, or a drug.

[0075] As used herein, "hepatitis B core related antigen (HBcrAg)" refers to antigenic reactivity resulting from modified hepatitis B e antigen (HBeAg), HBV core antigen (HBcAg), and artificial core-related protein (p22cr).

[0076] "Phosphorylated Hepatitis B Core Antigen," "phosphorylated HBcAg," or P-HBcAg are all used interchangeably herein and all refer to Hepatitis B Core Antigen (HBcAg) phosphorylated at one or more amino acids. For example, phosphorylated Hepatitis B Core Antigen can have the sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, provided that the sequence is phosphorylated at one or more amino acids.

[0077] As used interchangeably herein, "subject" and "patient" refer to any vertebrate, including, but not limited to, mammals and humans. In some embodiments, the subject can be human or non-human. The subject or patient may be undergoing some form of treatment. As used herein, "mammal" refers to any member of the class Mammalia, including, without limitation, humans and non-human primates, such as chimpanzees and other ape and monkey species; farm animals, such as cows, sheep, pigs, goats, llamas, camels, and horses; livestock, such as dogs and cats; and laboratory animals, including rodents, such as mice, rats, rabbits, and guinea pigs. The term does not denote a particular age or sex; that is, both male and female adult and newborn subjects, as well as fetuses, are intended to be included within the scope of this term.

[0078] As used herein, the terms "treating," "treating," and "treatment" are used interchangeably to describe preventing, alleviating, or inhibiting the progression of a disease and / or injury, or one or more symptoms of such a disease to which such terms apply. Depending on the subject's condition, the term also refers to preventing a disease, including preventing the onset of a disease or preventing symptoms associated with a disease. Treatment can be performed acutely or chronically. The acute phase of HBV infection generally lasts from about 4 weeks to about 6 months after infection. Meanwhile, the chronic phase of HBV infection generally includes a period after the acute phase has ended. In some cases, treating or monitoring chronic HBV infection involves performing assays on samples obtained from about 24 weeks after the subject's infection. The term also refers to reducing the severity of a disease or symptoms associated with such a disease before the onset of the disease. Such prevention or reduction of disease severity before onset refers to administration of the pharmaceutical composition to a subject who is not affected by the disease at the time of administration. "Preventing" also refers to preventing the recurrence of a disease or one or more symptoms associated with such a disease. "Treatment" and "therapeutically" refer to the act of treating, as "treating" is defined above.

[0079] As used herein, the term "variant" is used to describe a peptide or polypeptide that differs in amino acid sequence due to an amino acid insertion, deletion, or conservative substitution but retains at least one biological activity. "SNP" refers to a variant that is a single nucleotide polymorphism. Representative examples of "biological activity" include the ability to be bound by a specific antibody or the ability to stimulate an immune response. As used herein, the term "variant" is also used to describe a protein with an amino acid sequence that is substantially identical to a reference protein, with the amino acid sequence retaining at least one biological activity. Conservative amino acid substitutions, i.e., replacing an amino acid with a different amino acid with similar properties (e.g., hydrophilicity, degree of charge, and distribution of charged regions), are recognized in the art as typically involving minor changes. As understood in the art, these minor changes can be identified, in part, by examining the hydrophobicity index of an amino acid (Kyte et al., J. Mol. Biol. 157:105-132 (1982)). The hydrophobicity index of an amino acid is based on its hydrophobicity and charge. It is known in the art that amino acids with similar hydrophilicity indices may be substituted and still retain protein function. In one embodiment, amino acids with hydrophilicity indices of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that result in proteins that retain biological function. Consideration of the hydrophilicity of amino acids in the context of a peptide allows for calculation of the maximum local average hydrophilicity of the peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity (U.S. Pat. No. 4,554,101, incorporated herein by reference). Substitution with amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, such as immunogenicity as understood in the art. Substitutions can be made with amino acids with hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the specific side chain of the amino acid.Consistent with this observation is the understanding that amino acid substitutions that are compatible with biological function depend on the relative similarity of the amino acids, including the side chains of those amino acids, as manifested by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0080] As used herein, the term "vector" is used to describe a nucleic acid molecule capable of carrying another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Since plasmids are the most commonly used form of vector, the terms "plasmid" and "vector" can be used interchangeably. However, other forms of expression vectors, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions, can also be used. In this regard, RNA forms of vectors (including viral RNA vectors) can also be used in the context of the present disclosure.

[0081] Unless otherwise specified, technical and scientific terms used herein shall have the same meaning as commonly understood by those skilled in the art. For example, the terminology used in connection with, and techniques relating to, cell and tissue culture methods, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well-known terminology and techniques commonly used in the art. The meaning and scope of terms shall be clear, but in the unlikely event of any potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular.

[0082] 2. Detection of HBcAg and P-HBcAg In some aspects, the present disclosure relates to assays for determining the presence, level, or status of hepatitis B core antigen (HBcAg) and / or phosphorylated hepatitis B core antigen (P-HBcAg) in a sample obtained from a subject. HBcAg and / or P-HBcAg are also referred to herein as "markers" or "protein markers." In some embodiments, the present disclosure provides assays for determining the presence or level of HBcAg in a sample obtained from a subject. In some embodiments, the present disclosure provides assays for determining the presence or level of P-HBcAg in a sample obtained from a subject. In some embodiments, the present disclosure provides assays for determining the presence or level of HBcAg and P-HBcAg in at least one sample obtained from a subject.

[0083] In some embodiments, HBcAg and / or P-HBcAg are detected in at least one sample obtained from a subject, and one or more additional markers are detected. HBcAg and P-HBcAg are considered protein markers. In some embodiments, additional biomarkers described herein that are detected in addition to HBcAg and / or P-HBcAg are also considered protein markers. For example, additional protein markers include hepatitis B e antigen (HBeAg), hepatitis B surface antigen (HBsAg), isoforms of HBsAg, hepatitis B core-related antigen (HBcrAg), anti-hepatitis B surface antigen antibody (anti-HBs), anti-hepatitis B e antigen antibody (anti-HBe), anti-hepatitis surface antigen antibody (anti-HBs), and complexes formed between HBsAg and anti-HBs (also referred to as "HBsAg immune complexes"). In some embodiments, the additional marker is an enzyme marker. In some embodiments, the enzyme marker is selected from AST and ALT. AST and / or ALT are generally detected using an enzymatic assay in which AST or ALT acts on a substrate to produce a detectable product (e.g., a colorimetric product). In some embodiments, commercially available products are utilized (e.g., available from Abcam, Cambridge, MA, or other sources). In some embodiments, the Architect clinical chemistry analyzer system (Abbott, Abbott Park, IL) is utilized.

[0084] In some embodiments, assays for the detection of protein markers (eg, HBcAg and / or P-HBcAg) are quantitative or qualitative (eg, detection of the marker or a specific variant of the marker).

[0085] Assays for the detection of contemplated protein markers include immunoassays (e.g., sandwich and competitive immunoassays), clinical chemistry assays, and enzyme assays. An assay for determining a protein marker (e.g., HBcAg and / or P-HBcAg) in a sample obtained from a subject may include (a) preparing a test sample obtained from the subject and (b) determining the concentration, presence, or status of one or more markers in the test sample. A specific type of assay that can be performed for the determination is an immunoassay. Immunoassays can be performed using any format known in the art, such as, but not limited to, a sandwich format, a competitive inhibition format (including forward and reverse competitive inhibition assays), or a fluorescence polarization format. As described above, the immunoassay is a sandwich-format assay. Specifically, in one embodiment of the present disclosure, at least two antibodies are employed to separate and quantify each of the markers in the test sample. More specifically, at least two antibodies bind to specific epitopes of the markers to form an immune complex referred to as a "sandwich." Generally, in immunoassays, one or more antibodies can be used to capture markers in a test sample (these antibodies are often referred to as "capture" antibodies), and one or more antibodies can be used to bind a detectable (i.e., quantifiable) label to the sandwich (these antibodies are often referred to as "detection antibodies" or "conjugates"). In a sandwich assay, for example, both antibodies that bind to the marker are not diminished by binding to their respective binding sites of any other antibody in the assay. In other words, antibodies should be selected so that one or more first antibodies contacted with a test sample or test sample extract suspected of containing a marker do not bind to all or part of the binding site recognized by a second or subsequent antibody, thereby interfering with the ability of one or more second detection antibodies to bind to the marker.

[0086] In some embodiments, commercially available antibodies known in the art are utilized.

[0087] A sample to be tested for a marker (e.g., suspected of containing a marker) can be contacted with at least one capture antibody and at least one detection antibody (second detection antibody or third detection antibody) simultaneously or sequentially, in any order. For example, the test sample can be contacted first with at least one capture antibody and then (sequentially) with at least one detection antibody. Alternatively, the test sample can be contacted first with at least one detection antibody and then (sequentially) with at least one capture antibody. In yet another alternative, the test sample can be contacted with the capture antibody and the detection antibody simultaneously.

[0088] In a sandwich assay format, a test sample suspected of containing a marker is first contacted with at least one first capture antibody under conditions that allow the formation of a first antibody-marker complex. If more than one capture antibody is used, multiple first capture antibody-marker complexes are formed. In a sandwich assay, an antibody, such as at least one capture antibody, is used in a molar excess over the maximum amount of marker expected in the test sample.

[0089] Optionally, prior to contacting the test sample with the at least one capture antibody (e.g., the first capture antibody), the at least one capture antibody can be bound to a solid support or solid phase that facilitates separation of the first antibody-marker from the test sample. Any solid support known in the art can be used, including, but not limited to, solid supports made from polymeric materials in the form of reaction tray wells, test tubes, or beads (e.g., polystyrene beads, magnetic beads), nitrocellulose strips, membranes, microparticles (e.g., latex particles, sheepskin, and DURACYTES® (Abbott Laboratories, Abbott Park, IL; DURACYTES® are red blood cells "fixed" with pyruvaldehyde and formaldehyde)).

[0090] The solid phase can also comprise any suitable porous material with sufficient porosity to allow access by the detection antibody and suitable surface affinity for binding to the antigen. Microporous structures are commonly used, although materials with a gel structure in the hydrated state are also used. Such useful solid supports include, but are not limited to, nitrocellulose and nylon. Such porous solid supports are in the form of sheets with thicknesses of about 0.01 to 0.5 mm, including about 0.1 mm. Pore sizes vary within wide limits and may range from about 0.025 to about 15 microns, particularly about 0.15 to about 15 microns. The surface of such supports can be activated by chemical processes that covalently link the antigen or antibody to the support. However, irreversible binding of the antigen or antibody is typically achieved by adsorption to the porous material via poorly understood hydrophobic forces.

[0091] Antibodies can be bound to a solid support or solid phase by adsorption, covalent bonding using chemical coupling agents, or other means known in the art, provided that the binding does not interfere with the antibody's ability to bind to the marker. Alternatively, antibodies can be bound to microparticles pre-coated with streptavidin or biotin (e.g., using Power-Bind™-SA-MP streptavidin-coated microparticles available from Seradyn, Indianapolis, Indiana, along with antibodies biotinylated using means known in the art). Alternatively, antibodies can be bound using microparticles pre-coated with anti-species-specific monoclonal antibodies. Furthermore, if necessary, solid supports can be derivatized to allow reactivity with various functional groups on the antibody. Such derivatization requires the use of certain coupling agents, such as, but not limited to, maleic anhydride, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0092] After the test sample to be tested and / or suspected of containing a marker is contacted with at least one capture antibody (e.g., a first capture antibody), the mixture is incubated to allow for the formation of a first antibody (or multiple antibodies)-marker complex. Incubation can be carried out at a pH of about 4.5 to about 10.0, at a temperature of about 2°C to about 45°C, for a time period ranging from at least about 1 minute to about 18 hours, including about 1 to 20 minutes, or about 2 to 6 minutes. The immunoassays described herein can be carried out in one step (meaning that the test sample, at least one capture antibody, and at least one detection antibody are all added sequentially or simultaneously to a reaction vessel), or in two or more steps, e.g., two-step, three-step, etc.

[0093] After the (first or multiple) capture antibody-marker complexes are formed, the complexes are contacted with at least one detection antibody (under conditions that allow the formation of (first or multiple) capture antibody-marker-(second or multiple) antibody detection complexes). The at least one detection antibody can be the second, third, fourth, etc. antibody used in an immunoassay. If the capture antibody-complexes are contacted with two or more detection antibodies, (first or multiple) capture antibody-marker-detection antibody complexes are formed. As with the capture antibody (e.g., the first capture antibody), if at least a second (subsequent) detection antibody is contacted with the capture antibody-marker complex, an incubation period under similar conditions as above is required for the formation of (first or multiple) capture antibody-marker (second or multiple different markers)-detection antibody complexes. In some embodiments, at least one detection antibody comprises a detectable label. The detectable label can be attached to at least one detection antibody (e.g., second detection antibody) before, simultaneously with, or after the formation of the (first or multiple) capture antibody-marker-(second or multiple) detection antibody complex. Any detectable label known in the art can be used. For example, the detectable label can be a radioactive label, e.g., 3 H, 125 I, 35 S, 14 C. 32 P, 33P, enzyme labels, such as horseradish peroxidase, alkaline phosphatase, glucose 6-phosphate dehydrogenase, etc.; chemiluminescent labels, such as acridinium (e.g., acridinium ester, acridinium SPSP (N10-(3-sulfopropyl)-N-(3-sulfopropyl), etc.), luminol, isoluminol, thioesters, sulfonamides, phenanthridinium esters, etc.); fluorescent labels, such as fluorescein (5-fluorescein, 6-carbohydrate); The label may be a fluorophore, such as 3', 6-difluoromethanesulfonyl fluorescein, 3', 6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachlorofluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, rhodamine, phycobiliprotein, R-phycoerythrin, quantum dots (zinc sulfide-capped cadmium selenide), thermometric labels, or immunopolymerase chain reaction labels. Introductions to labels, labeling procedures, and detection of labels can be found in Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, NY (1997) and Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996). The latter is a comprehensive handbook and catalog published by Molecular Probes, Inc., Eugene, Oregon.

[0094] The detectable label can be attached to the antibody directly or through a linking agent. An example of a linking agent that can be used is EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), commercially available from Sigma-Aldrich, St. Louis, MO. Other linking agents that can be used are known in the art. Methods for attaching a detectable label to an antibody are known in the art. Additionally, many detectable labels can be purchased or synthesized that already contain a terminal group that facilitates attachment of the detectable label to an antibody, such as N10-(3-sulfopropyl)-N-(3-carboxypropyl)-acridinium-9-carboxamide, otherwise known as CPSP-acridinium ester, or N10-(3-sulfopropyl)-N-(3-sulfopropyl)-acridinium-9-carboxamide, otherwise known as SPSP-acridinium ester.

[0095] The (first or multiple) capture antibody-marker-(second or multiple) detection antibody complexes can be, but need not be, separated from the remainder of the test sample before quantifying the label. For example, if at least one capture antibody (e.g., the first capture antibody) is bound to a solid support or solid phase, such as, but not limited to, a well in a reaction tray, beads, or microparticles, separation can be achieved by removing the fluid (of the test sample) from contact with the solid support. Alternatively, if at least the first capture antibody is bound to a solid support, it can be simultaneously contacted with a sample containing a marker and at least one second detection antibody to form a first (multiple) antibody-marker-antibody complex, followed by removing the fluid (test sample) from contact with the solid support. If at least one first capture antibody is not bound to a solid support, the (first or multiple) capture antibody-marker (second or multiple)-detection antibody complexes do not need to be removed from the test sample for quantifying the amount of label.

[0096] After the labeled capture antibody-marker complex (e.g., the first capture antibody-marker complex) is formed, the amount of label in the complex is quantified using techniques known in the art. For example, if an enzyme label is used, the labeled complex is reacted with a substrate for the label that gives a quantifiable reaction, such as color development. If the label is radioactive, the label is quantified using a scintillation counter. If the label is fluorescent, the label is quantified by stimulating the label with one color of light (known as the "excitation wavelength") and detecting another color emitted by the label in response to the stimulation (known as the "emission wavelength"). If the label is chemiluminescent, the label is quantified by detecting the emitted light visually or with a luminometer, X-ray film, high-speed photographic film, a CCD camera, or the like. Once the amount of label in the complex is quantified, the concentration of the marker in the test sample is determined by using a standard curve generated using serial dilutions of known concentrations of the marker. In addition to using serial dilutions of the marker, standard curves can be generated gravimetrically, by mass spectrometry, and other techniques known in the art.

[0097] The methods and kits described herein encompass other reagents and methods for performing immunoassays. Various buffers, such as those known in the art and / or that can be easily prepared or optimized for use, for example, for washing, as conjugate diluents, and / or as calibrator diluents, are included. An exemplary conjugate diluent is ARCHITECT® Diluent (Abbott Laboratories, Abbott Park, IL), which contains 2-(N-morpholino)ethanesulfonic acid (MES), additional salts, a protein blocker, an antimicrobial agent, and a surfactant. An exemplary calibrator diluent is ARCHITECT® Calibrator Diluent (Abbott Laboratories, Abbott Park, IL), which contains a buffer containing MES, additional salts, a protein blocker, and an antimicrobial agent.

[0098] Furthermore, as previously mentioned, the present methods and kits are optionally adapted for use in automated or semi-automated systems. Compared to non-automated systems (e.g., ELISAs), some of the differences between automated or semi-automated systems include the substrate to which the capture antibody is bound (which can affect sandwich formation and analyte reactivity) and the length and timing of the capture, detection, and / or optional wash steps. Non-automated formats, such as ELISAs, may involve relatively long incubation times with the capture reagent (e.g., about 2 hours), while automated or semi-automated formats (e.g., ARCHITECT®) may have relatively short incubation times (e.g., 18 minutes for ARCHITECT®). Similarly, non-automated formats, such as ELISAs, may incubate the detection antibody, such as the conjugate reagent (Pb264), for relatively long incubation times (e.g., about 2 hours), while automated or semi-automated formats (e.g., ARCHITECT®) may have relatively short incubation times (e.g., about 4 minutes for ARCHITECT®).

[0099] The markers of the present disclosure (e.g., HBcAg and P-HBcAg) can be used in diagnostic tests to assess, determine, and / or quantify (used interchangeably herein) the HBV status in a patient. For example, HBcAg and / or P-HBcAg can be used in diagnostic tests to assess, determine, and / or quantify the HBV status in a patient. The phrase "HBV status" includes any identifiable manifestation of the status, including the absence of HBV. For example, HBV status includes, without limitation, the presence or absence of HBV infection in a patient, the stage or severity of HBV infection, the progression of HBV infection (e.g., progression of HBV infection over time), the effectiveness or response to treatment of HBV infection (e.g., clinical follow-up after treatment and surveillance of the infection), and the type of HBV infection. Based on this status, further procedures, including further diagnostic tests or treatment procedures or regimens, are indicated.

[0100] The ability of a diagnostic test to correctly predict a condition is commonly measured as the sensitivity of the assay, the specificity of the assay, or the area under the receiver operating characteristic ("ROC") curve. Sensitivity is the percentage of true positives predicted by the test as positive, and specificity is the percentage of true negatives predicted as negative by the test. The ROC curve provides the sensitivity of the test as a function of 1 minus specificity. The larger the area under the ROC curve, the stronger the predictive value of the test. Other useful measures of a test's usefulness are the positive predictive value and the negative predictive value. The positive predictive value is the percentage of people who test positive and who actually are positive. The negative predictive value is the percentage of people who test negative and who actually are negative.

[0101] Analysis of the data described in this disclosure and clinical data from cohorts of HBV and control patients has led to the identification of HBV biomarkers that can be used individually or in various combinations with each other or with other biomarkers in the form of panels to diagnose and / or assess HBV infection in subjects. An HBV biomarker panel can include any one of the HBV biomarkers disclosed herein. In some embodiments, the HBV biomarker panel includes HBcAg. In some embodiments, the HBV biomarker panel includes P-HBcAg. In some embodiments, the HBV biomarker panel includes HBcAg and P-HBcAg. The HBV biomarker panel can also include non-HBV biomarkers (e.g., assay control biomarkers) and biomarkers previously identified as associated with HBV. In some embodiments, the biomarker panels of the present disclosure can demonstrate statistical differences in different HBV states. Diagnostic tests using these biomarkers can demonstrate an ROC of at least 0.6, at least about 0.7, at least about 0.8, or at least about 0.9.

[0102] HBV biomarkers are differentially present / expressed depending on the HBV type or subclass (e.g., HBV signature); therefore, a panel of two or more HBV biomarkers may be useful in aiding in the determination of HBV status. In some embodiments, biomarkers are measured in patient samples using the methods described herein, e.g., compared to predefined biomarker levels, and correlated with HBV status. In some embodiments, measurements are compared to an associated diagnostic amount, cutoff, reference level, or multivalent model score that distinguishes a positive HBV status (e.g., seroconversion) from a negative HBV status (e.g., seroclearance). A diagnostic amount represents the measured amount of a biomarker above or below which a patient is classified as having a particular HBV status. For example, a measured amount or level above a diagnostic cutoff or reference level may provide a diagnosis of HBV if the biomarker is unfavorable (e.g., increased) compared to a control subject (e.g., a subject who did not sustain HBV infection). Furthermore, if a biomarker is present during HBV infection and undetectable in controls, any detectable measurable amount can provide a diagnosis of HBV infection. Alternatively, if a biomarker is favorable (e.g., decreased) during HBV infection, any measurable amount below the diagnostic cutoff or reference level can provide a diagnosis of non-HBV infection. Furthermore, if a biomarker is absent during HBV infection and detectable in controls, any detectable measurable amount can provide a diagnosis of non-HBV infection. As is well understood in the art, adjusting the particular diagnostic cutoff or reference level used in an assay can increase the sensitivity or specificity of a diagnostic assay according to the preference of the diagnostician. In some embodiments, a particular diagnostic cutoff or reference level can be determined, for example, by measuring the amount of a biomarker in a statistically significant number of samples from patients with various HBV infection states and drawing a cutoff to meet the desired level of specificity and sensitivity.

[0103] Furthermore, in certain embodiments, the values ​​measured for the markers of the biomarker panel are mathematically combined, and the combined value is correlated with the underlying diagnostic question. Biomarker values ​​may be combined by any appropriate state-of-the-art mathematical method. Well-known mathematical methods for correlating marker combinations with disease states employ methods such as discriminant analysis (DA) (e.g., linear, quadratic, and regularized DA), discriminant function analysis (DFA), kernel methods (e.g., SVM), multidimensional scaling (MDS), non-parametric methods (e.g., k-nearest neighbor classifiers), partial least squares (PLS), tree-based methods (e.g., logistic regression, CART, random forests, boosting / bagging), generalized linear models (e.g., logistic regression), principal component-based methods (e.g., SIMCA), generalized additive models, fuzzy logic-based methods, neural networks, and genetic algorithm-based methods. Those skilled in the art will have no problem selecting an appropriate method for evaluating the biomarker combinations of the present disclosure. In one embodiment of the present disclosure, the method used to correlate a combination of biomarkers (e.g., to diagnose HBV status) is selected from DA (e.g., linear, quadratic, regularized discriminant analysis), DFA, Kernel methods (e.g., SVM), MDS, non-parametric methods (e.g., k-nearest neighbor classifiers), PLS (partial least squares), tree-based methods (e.g., logistic regression, CART, random forests, boosting / bagging), or generalized linear models (e.g., logistic regression), and principal component analysis.For more information on these statistical methods, see the following references: Ruczinski et al., 12 J. OF COMPUTATIONAL AND GRAPHICAL STATISTICS 475-511 (2003); Friedman, J.H., 84 J. OF THE AMERICAN STATISTICAL ASSOCIATION 165-75 (1989); Hastie, Trevor, Tibshirani, Robert, Friedman, Jerome, The Elements of Statistical Learning, Springer Series in Statistics (2001); Breiman, L., Friedman, J.H., Olshen, R.A., Stone, C.J., Classification and regression trees, California: Wadsworth (1984); Breiman, L., 45 MACHINE LEARNING 5-32 (2001); Pepe, M.S., The Statistical Evaluation of Medical Tests for Classification and Prediction, Oxford Statistical Science Series, 28 (2003); and Duda, RO, Hart, PE, Stork, DG, Pattern Classification, Wiley Interscience, 2nd ed. (2001).

[0104] 3. Monitoring and treatment of chronic HBV infection In some embodiments, the presence or level of HBcAg and / or P-HBcAg is used to assess or monitor the stage or phase of chronic HBV infection in a subject. For example, in some embodiments, the presence or level of HBcAg and / or P-HBcAg in a sample obtained from a subject diagnosed with HBV is used to assess the stage or phase of chronic HBV infection in a subject. The subject may have been diagnosed with and / or be receiving treatment for any HBV genotype, including, but not limited to, HBV genotype A, HBV genotype B, HBV genotype C, HBV genotype D, HBV genotype E, HBV genotype F, HBV genotype G, HBV genotype H, HBV genotype I, or HBV genotype J. In some embodiments, the level of HBcAg and / or P-HBcAg in the sample is determined to be favorable or unfavorable. In some embodiments, an unfavorable level or amount of HBcAg and / or P-HBcAg in the sample indicates that HBV infection is active in the subject. In some embodiments, an unfavorable level indicates that treatment for HBV should be provided to the subject. In contrast, a favorable level or amount of HBcAg and / or P-HBcAg in the sample indicates that the HBV infection is improving or inactive in the subject.

[0105] In some embodiments, an unfavorable level indicates that the level of HBcAg and / or P-HBcAg is greater than or equal to a threshold level. For example, in some embodiments, the stage or phase of HBV infection is determined based on whether the level or amount of HBcAg in a sample obtained from a subject is greater than or equal to a reference level of HBcAg (e.g., the level in a control sample obtained from a subject not affected by HBV). As another example, in some embodiments, the stage or phase of HBV infection is determined based on whether the level or amount of P-HBcAg in a sample obtained from a subject is greater than or equal to a reference level of P-HBcAg (e.g., the level in a control sample obtained from a subject not affected by HBV).

[0106] In some embodiments, an unfavorable level indicates that the level of HBcAg and / or P-HBcAg did not decrease, by at least some absolute amount, from a first sample to a second sample obtained from the subject at a separate time. For example, in some embodiments, the methods described herein include obtaining a first sample from the subject at a first time point, either before or after receiving treatment for chronic HBV, and obtaining a second sample from the subject at a second time point after the first sample was obtained. An unfavorable level may indicate that the level of HBcAg and / or P-HBcAg in the sample did not decrease, by at least some absolute amount, from the first time point to the second time point.

[0107] In some embodiments, a "suitable level" indicates that the level of HBcAg and / or P-HBcAg is below a threshold level. In some embodiments, a "suitable level" indicates that the level of HBcAg and / or P-HBcAg has decreased by at least some absolute amount from a first sample to a second sample obtained from the subject.

[0108] In some embodiments, the stage or phase of HBV infection is determined based on whether the level or amount of HBcAg and / or P-HBcAg in a sample obtained from the subject is greater than or equal to a control level of HBcAg and / or a control level of P-HBcAg (e.g., the level in a control sample obtained from a subject not suffering from HBV). In some embodiments, the method includes providing treatment for HBV to the subject.

[0109] In some embodiments, one or more additional markers are measured to assess or monitor the stage or phase of HBV infection, including additional protein markers or enzymatic markers described above.

[0110] In some embodiments, a method for assessing and monitoring the stage or phase of chronic HBV infection comprises performing an assay to detect the presence or level of HBcAg and / or P-HBcAg in at least one sample obtained from a subject diagnosed with or receiving treatment for chronic HBV. In some embodiments, the method further comprises determining the amount of infectious and / or non-infectious HBV particles in the sample. As used herein, the term "infectious HBV particles" refers to HBV particles containing HBV DNA. In contrast, the terms "non-infectious HBV particles" or "empty HBV particles" are used interchangeably herein and refer to HBV particles that do not contain HBV DNA. Non-infectious or empty HBV particles may still contain HBV RNA (e.g., pgRNA). In some embodiments, the amount of infectious and / or non-infectious HBV particles in a sample is used to assess the stage or phase of chronic HBV infection in a subject.

[0111] As demonstrated in the accompanying figures and examples, the level of HBcAg is shown herein to correlate with the level of HBV DNA in a given sample. Accordingly, in some embodiments, a method for assessing and monitoring the stage or phase of HBV infection comprises performing an assay to detect the presence or level of HBcAg in at least one sample obtained from a subject, and determining the level or amount of infectious HBV particles in the at least one sample based on the presence or level of HBcAg. Thus, provided herein is a method for assessing and monitoring the stage or phase of HBV infection in a subject without the need to perform a nucleic acid test to measure HBV DNA in the subject. Furthermore, the method can be performed regardless of whether the subject has been treated for chronic HBV, since the amount of HBcAg is shown herein to correlate with the amount of HBV DNA in a given sample regardless of treatment status (i.e., whether the subject has been treated for chronic HBV). Thus, the methods for assessing and monitoring the stage or phase of HBV infection described herein are advantageous in that, rather than performing one assay for HBcAg and a separate assay for HBV nucleic acid (e.g., HBV DNA), the methods can be performed efficiently and accurately by using a single assay to assess the number of infectious HBV particles in a sample.

[0112] As demonstrated in the accompanying figures and examples, the level of P-HBcAg is shown herein to correlate with the level of HBV DNA or the level of HBV RNA (e.g., HBV pgRNA) in a given sample depending on the subject's treatment status. In particular, the level of P-HBcAg is shown herein to correlate with the level of HBV DNA in a sample obtained from a subject who has not received treatment for HBV. Accordingly, in some embodiments, a method for assessing and monitoring the stage or phase of HBV infection comprises performing an assay to detect the presence or level of P-HBcAg in at least one sample obtained from a subject who has not received treatment for HBV, and determining the amount of infectious HBV particles in the at least one sample based on the presence or level of P-HBcAg. Thus, in some embodiments, a method for assessing and monitoring the stage or phase of HBV infection in a subject is provided herein without requiring the administration of a nucleic acid test to measure HBV DNA in the subject, and thus the method can be performed efficiently and accurately using only a single test rather than separate tests for P-HBcAg and HBV DNA.

[0113] In some embodiments, the methods described herein further include selecting an appropriate chronic HBV treatment for the subject. In some embodiments, the methods for assessing and monitoring the stage or phase of chronic HBV infection include providing chronic HBV treatment to the subject. For example, in some embodiments, the methods described herein include providing chronic HBV treatment to the subject if the HBcAg level and / or P-HBcAg level are determined to be unfavorable. In some embodiments, the methods include providing chronic HBV treatment to the subject if the amount of infectious HBV particles in the sample is equal to or exceeds a threshold value. In some embodiments, the methods include providing chronic HBV treatment to the subject if the amount of infectious HBV particles in the sample does not decrease by at least a certain absolute amount from a first sample to a second sample obtained from the subject. Suitable treatments for chronic HBV infection are described herein and include, but are not limited to, nucleoside analogs (e.g., lamivudine, adefovir, tenofovir, telbivudine, or entecavir), nucleic acids (e.g., siRNA, antisense oligonucleotides, shRNA, or miRNA), immunomodulators (e.g., interferon alpha-2a or pegylated interferon alpha-2a), core protein assembly inhibitors (e.g., NVR 3-1983, GLS4, or BAY 41-4109), capsid assembly modulators (CAMs) (e.g., JNJ-632, AT130, or BAY41-4109), HBsAg release inhibitors (e.g., REP 9 AC), entry inhibitors (e.g., Myrcludex-B), DNA modifying agents (e.g., CRISPR-based DNA editing agents, TALENs, ZNFs, etc.), or combinations thereof.

[0114] In some embodiments, the presence, level, or status of the markers is used to optimize HBV treatment. For example, if a subject is found to have markers indicative of a more aggressive, aggressive, or drug-resistant infection, a more aggressive treatment or different drug administration may be administered. Conversely, if a subject is found to have markers indicative of a less aggressive or aggressive infection, a less aggressive treatment or monitoring (e.g., monitoring of the infection without specific pharmaceutical treatment) may be selected.

[0115] In some embodiments, a subject is monitored at multiple time points to assess the HBV status. In some embodiments, multiple samples are obtained from the subject at various time points to monitor the chronic HBV infection in the subject. For example, in some embodiments, a first sample is obtained from the subject at a first time point, and additional samples are collected daily, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, every 13 weeks, every 14 weeks, every 15 weeks, every 16 weeks, every 5 months, every 6 months, every year, or less frequently to continuously monitor the stage or phase of chronic HBV infection in the subject.

[0116] In some embodiments, monitoring is continued and / or repeated until subject (1) achieves a suitable level of HBcAg for a sufficient period of time. In some embodiments, monitoring or determining treatment methods (e.g., detecting a marker) is continued and / or repeated until subject (2) achieves a suitable level of P-HBcAg for a sufficient period of time. In some embodiments, monitoring or determining treatment methods (e.g., detecting a marker) is continued and / or repeated until subject (3) has less than the reference amount of infectious HBV particles for a sufficient period of time. In some embodiments, monitoring or determining treatment methods (e.g., detecting a marker) is continued and / or repeated until the subject achieves two or more of (1) through (3) for a sufficient period of time. In some embodiments, a sufficient period of time is about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27 weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks, about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, The duration is about 45 weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2 years, about 3.0 years, about 3.5 years, about 4.0 years, about 4.5 years, about 5.0 years, about 5.5 years, about 6.0 years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0 years, about 8.5 years, about 9.0 years, about 9.5 years, about 10.0 years, about 11.0 years, about 12.0 years, about 13.0 years, about 14.0 years, about 15.0 years, about 16.0 years, about 17.0 years, about 18.0 years, about 19.0 years, or about 20.0 years.

[0117] 4. Monitoring response to HBV treatment In some embodiments, the assays of the present disclosure can be used to monitor the response of a subject undergoing treatment for HBV. "Chronic HBV treatment" is also referred to herein as "anti-HBV drug," "HBV therapeutic drug," "HBV treatment," "HBV therapy," "HBV therapy," etc. For example, the assays of the present disclosure can be used to monitor drug resistance in a subject undergoing treatment for HBV. For example, it is known that the emergence of resistant strains with amino acid substitutions in the tyrosine-methionine-aspartic acid-aspartic acid (YMDD) motif of reverse transcriptase can be a serious problem for subjects undergoing lamivudine therapy (see, e.g., Hatakeyama, T. et al., Hepatology, 45(5):1179-1186 (2007)). The assays of the present disclosure can be used to monitor and / or predict the early emergence of mutations and / or drug resistance during HBV treatment.

[0118] In some embodiments, the presence or level of HBcAg and / or P-HBcAg is used to monitor the response to treatment of chronic HBV infection in a subject. For example, in some embodiments, the presence or level of HBcAg and / or P-HBcAg in a sample obtained from a subject diagnosed with chronic HBV is used to monitor the responsiveness of a subject to treatment of chronic HBV.

[0119] In some embodiments, the level of HBcAg and / or P-HBcAg in the sample is determined to be suitable or unsuitable. In some embodiments, an unsuitable level or amount of HBcAg and / or P-HBcAg in the sample indicates that treatment of chronic HBV in the subject is not effective. In contrast, a suitable level or amount of HBcAg and / or P-HBcAg in the sample indicates that treatment of chronic HBV in the subject is effective (e.g., HBV infection is improving).

[0120] In some embodiments, an unfavorable level indicates that the level of HBcAg and / or P-HBcAg is greater than or equal to a threshold level. For example, in some embodiments, responsiveness to HBV treatment is determined based on whether the level or amount of HBcAg in a sample obtained from a subject is greater than or equal to a reference level of HBcAg (e.g., the level in a control sample obtained from a subject not suffering from HBV). As another example, in some embodiments, responsiveness to HBV treatment is determined based on whether the level or amount of P-HBcAg in a sample obtained from a subject is greater than or equal to a reference level of P-HBcAg (e.g., the level in a control sample obtained from a subject not suffering from HBV).

[0121] In some embodiments, an unfavorable level may indicate that the level of HBcAg and / or P-HBcAg did not decrease, by at least some absolute amount, from a first sample to a second sample obtained from the subject at a separate time. For example, in some embodiments, the methods described herein include obtaining a first sample from the subject at a first time point before or after receiving treatment for chronic HBV, and obtaining a second sample from the subject at a second time point after the first sample was obtained. The second sample is obtained after the subject has received at least one dose of treatment for chronic HBV. An unfavorable level may indicate that the level of HBcAg and / or P-HBcAg in the sample did not decrease, by at least some absolute amount, from the first time point to the second time point.

[0122] In some embodiments, a "suitable level" indicates that the level of HBcAg and / or P-HBcAg is below a threshold level. In some embodiments, a "suitable level" indicates that the level of HBcAg and / or P-HBcAg has decreased by at least some absolute amount from a first sample to a second sample obtained from the subject.

[0123] In some embodiments, the second time point is about 1 day to about 1 year after the first time point. In some embodiments, the second time point is more than 1 year after the first time point. In some embodiments, the second time point is about 1 week to about 40 weeks after the first time point. In some embodiments, the second time point is about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, or about 14 weeks after the first time point. In some embodiments, the first time point is within 24 hours of receiving treatment for chronic HBV, and the second time point is about 1 week to about 40 weeks after the first time point. For example, in some embodiments, the first time point is within 24 hours of receiving treatment for chronic HBV, and the second time point is about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, or about 14 weeks after the first time point. In some embodiments, the first time point is within 24 hours of receiving treatment for chronic HBV, and the second time point is about 12 weeks (about 3 months) after the first time point.

[0124] In some embodiments, one or more additional markers are measured to monitor responsiveness to treatment of chronic HBV infection in a subject, including additional protein or enzymatic markers described above.

[0125] Various HBV treatments affect the secretion of HBV-derived particles differently. HBV particles containing HBV DNA encapsulated in HBcAg are secreted from infected cells when cccDNA is transcribed into HBV RNA (e.g., pgRNA), and HBV RNA (e.g., pgRNA) is reverse-transcribed into HBV DNA (e.g., rcDNA). Certain HBV treatments, such as the use of nucleoside(t) analogs, inhibit the reverse transcription of HBV RNA to HBV DNA, thereby reducing the amount of HBV particles containing HBV DNA encapsulated in HBcAg (also referred to herein as "infectious" HBV particles). Thus, in some embodiments, the methods described herein include detecting the presence or amount of HBcAg in a sample obtained from a subject to determine whether a treatment aimed at disrupting the reverse transcription of pgRNA to rcDNA is effective in the subject. For example, in some embodiments, the presence or amount of HBcAg in a sample obtained from a subject can be assessed to determine whether treatment with a nucleoside(t) analog is effective. In some embodiments, the presence or amount of P-HBcAg is not affected by such treatment because pgRNA is still translated and P-HBcAg is released from cells as "empty" particles (i.e., particles that do not contain HBV DNA). These "empty" particles can be detected by measuring phosphorylated HBcAg (P-HBcAg). Furthermore, reducing the level of HBV RNA in cells can reduce the level of HBV DNA produced because less template RNA is available for reverse transcription. Thus, in some embodiments, a treatment that interferes with the production of HBV RNA (e.g., CAM) reduces the amount of both P-HBcAg and HBcAg produced. Therefore, to assess or predict whether such a treatment (e.g., CAM) is effective in a subject, the level of P-HBcAg or the levels of P-HBcAg and HBcAg can be obtained in a sample obtained from the subject.

[0126] In some embodiments, the methods described herein can be used to assess the effectiveness of a CAM treatment in a subject. For example, in some embodiments, the methods described herein include detecting the presence or amount of P-HBcAg in a sample obtained from a subject receiving CAM treatment to determine whether the treatment is effective. In some embodiments, the methods include determining that the treatment is effective if the level of P-HBcAg in the sample is less than a reference level of P-HBcAg. In some embodiments, the methods include determining that the treatment is effective if the level of P-HBcAg in the sample is less than the reference level of P-HBcAg and the level of HBcAg in the sample is less than the reference level of HBcAg. In some embodiments, the methods include determining that the treatment is not effective if the level of P-HBcAg in the sample is greater than or equal to the reference level of P-HBcAg.

[0127] As demonstrated in the accompanying figures and examples, it has been shown herein that the amount of HBcAg correlates with the amount of HBV DNA in a given sample after a subject has undergone treatment for chronic HBV. Accordingly, in some embodiments, a method for monitoring a subject's response to treatment for chronic HBV comprises performing an assay to detect the presence or level of HBcAg in at least one sample obtained from the subject after treatment, and determining the amount of infectious HBV particles in the at least one sample based on the presence or level of HBcAg. Accordingly, in some embodiments, a method for monitoring a subject's response to treatment for chronic HBV infection is provided herein without the need to perform a nucleic acid test to measure HBV DNA to accurately monitor the response to HBV DNA treatment. That is, the methods for monitoring a subject's response to treatment for chronic HBV infection described herein are advantageous in that they can be performed efficiently and accurately by using a single assay to assess the number of infectious HBV particles in a sample, rather than performing one assay for HBcAg and a separate assay for HBV nucleic acid (e.g., HBV DNA).

[0128] As described above, the amount of P-HBcAg is herein shown to correlate with the amount of HBV DNA or the amount of HBV RNA (e.g., HBV pgRNA) in a given sample depending on the subject's treatment status. In some embodiments, the amount of P-HBcAg is herein shown to correlate with the amount of HBV RNA (e.g., pgRNA) in a sample when the subject is treated for HBV. In some embodiments, the treatment interferes with the transcription of cccDNA into HBV RNA (e.g., pgRNA). HBV RNA (e.g., pgRNA) is released from cells in "empty" particles, also referred to as "non-infectious" HBV particles. Such particles can be assessed by determining the level of P-HBcAg in a sample. Thus, in some embodiments, a method for monitoring response to HBV treatment comprises performing an assay to detect the presence or level of P-HBcAg in at least one sample obtained from a subject treated for HBV, and determining the amount of non-infectious HBV particles in the at least one sample based on the presence or level of P-HBcAg. In some embodiments, the treatment is an agent that interferes with reverse transcription of pgRNA. Suitable treatments include, for example, capsid assembly modulators / inhibitors (CAMs). CAMs indirectly inhibit reverse transcription by preventing capsid formation. Reverse transcription of pgRNA occurs inside the capsid. Thus, CAMs indirectly inhibit reverse transcription. Thus, the method for monitoring response to chronic HBV treatment is advantageous in that, rather than performing one assay for P-HBcAg and another assay for HBV nucleic acid (e.g., HBV RNA), the method can be efficiently and accurately performed by using a single assay that evaluates the number of non-infectious HBV particles in a sample. In some embodiments, a reduction in the amount of non-infectious HBV particles indicates that HBV treatment (e.g., CAM) is effective in a subject.

[0129] In some embodiments, the methods described herein include determining the amount of infectious and non-infectious particles in a sample. In some embodiments, the subject is receiving a treatment that affects the reverse transcription of HBV RNA (e.g., pgRNA) into HBV DNA. For example, some HBV treatments, such as nucleoside inhibitors, inhibit the reverse transcription of pgRNA, thus reducing the amount of HBV DNA (e.g., infectious particles) released from cells. However, such HBV treatments do not interfere with the process of transcription of cccDNA into pgRNA; therefore, these treatments do not reduce the amount of HBV RNA (e.g., pgRNA) secreted from cells into "empty" or "non-infectious" particles. Therefore, while the level of HBcAg (and therefore the amount of infectious particles) is reduced, the level of P-HBcAg (and therefore the amount of non-infectious particles) should not be affected by such treatment.

[0130] In some embodiments, the method for monitoring response to HBV treatment further includes determining whether the HBV treatment is effective or ineffective in the subject. In some embodiments, the treatment is determined to be ineffective if the HBcAg and / or P-HBcAg levels are not favorable. For example, in some embodiments, the treatment is determined to be ineffective if the HBcAg and / or P-HBcAg levels do not decrease by at least a certain absolute amount from a first sample to a second sample obtained from the subject.

[0131] In some embodiments, a treatment is determined to be ineffective if the amount of infectious HBV particles is greater than or equal to a threshold value. In some embodiments, a treatment is determined to be ineffective if the amount of infectious HBV particles does not decrease by at least a certain absolute amount from a first sample to a second sample collected from the subject. For example, in some embodiments, a first sample is collected from the subject at a first time point within 24 hours after receiving treatment for HBV, and a second sample is collected from the subject at a second time point after the first sample. In some embodiments, the second time point is about 10 weeks to about 14 weeks (e.g., about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks) after the first time point. In some embodiments, a treatment is determined to be ineffective if the amount of infectious HBV particles does not decrease by at least a certain absolute amount from the first sample to the second sample.

[0132] In some embodiments, treatment is determined to be effective if the level of HBcAg and / or P-HBcAg is suitable. In some embodiments, treatment is determined to be effective if the amount of infectious HBV particles is reduced by at least a certain absolute amount from a first sample to a second sample collected from the subject. For example, in some embodiments, the first sample is collected from the subject at a first time point within 24 hours after receiving treatment for HBV, and the second sample is collected from the subject at a second time point after the first sample. In some embodiments, the second time point is about 10 to about 14 weeks (e.g., about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks) after the first time point. In some embodiments, treatment is determined to be effective if the amount of infectious HBV particles is reduced by at least a certain absolute amount from the first sample to the second sample.

[0133] In some embodiments, the method for monitoring a subject's response to a chronic HBV treatment further includes modifying the chronic HBV treatment if the treatment is determined to be ineffective. In some embodiments, modifying the chronic HBV treatment includes providing the subject with an increased dose of the treatment, increasing the dosing frequency of the treatment, providing the subject with a second treatment, or any combination thereof. For example, in some embodiments, modifying the HBV treatment includes providing the patient with an increased dose of the HBV treatment. In some embodiments, modifying the treatment includes increasing the dosing frequency of the HBV treatment. In some embodiments, modifying the treatment includes increasing the dose and dosing frequency of the HBV treatment. In some embodiments, modifying the treatment includes providing the subject with a second HBV treatment. The second HBV treatment may include any suitable HBV treatment (e.g., an HBV therapeutic agent) described herein. The second HBV treatment may be provided to the subject as an alternative treatment or as an additional treatment. For example, in some embodiments, the HBV treatment the subject was previously receiving (e.g., the treatment determined to be ineffective) may be discontinued, and the subject may be given the second HBV treatment instead. As another example, in some embodiments, a second HBV therapy is administered in addition to an HBV therapy that the subject is already receiving, hi some embodiments, the second HBV therapy is administered in addition to increasing the dose and / or dosing frequency of an HBV therapy that the subject is already receiving.

[0134] 5. Method for measuring HBcAg and / or P-HBcAg In the above methods, the level of an HBV biomarker (e.g., HBcAg, P-HBcAg) can be measured by any means, such as antibody-dependent methods, such as immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis, protein immunostaining, electrophoretic analysis, protein assays, competitive binding assays, functional protein assays, or chromatographic or spectroscopic methods, such as high-performance liquid chromatography (HPLC), mass spectrometry, or liquid chromatography-mass spectrometry (LC / MS) or capillary electrophoresis (CE)-MS, or by direct injection or any pre-separation step coupled to MS. Assays can also be adapted to clinical chemistry formats known to those skilled in the art.

[0135] Immunoassay In some embodiments, measuring the level of the HBV biomarker comprises contacting the sample with a first specific binding member and a second specific binding member, hi some embodiments, the first specific binding member is a capture antibody and the second specific binding member is a detection antibody. In some embodiments, the step of measuring the level of an HBV biomarker includes contacting the sample simultaneously or sequentially, in any order, with (1) a capture antibody (e.g., an HBV biomarker capture antibody) that binds to an epitope on the HBV biomarker or HBV biomarker fragment to form a capture antibody-HBV biomarker antigen complex (e.g., an HBV biomarker capture antibody-HBV biomarker antigen complex), and (2) a detection antibody (e.g., an HBV biomarker detection antibody) that comprises a detectable label and binds to an epitope on the HBV biomarker not bound by the capture antibody to form an HBV biomarker antigen-detection antibody complex (e.g., an HBV biomarker antigen-HBV biomarker detection antibody complex), to form a capture antibody-HBV biomarker antigen-detection antibody complex (e.g., an HBV biomarker capture antibody-HBV biomarker antigen-HBV biomarker detection antibody complex), and measuring the amount or concentration of the HBV biomarker in the sample based on a signal generated by the detectable label in the capture antibody-HBV biomarker antigen-detection antibody complex.

[0136] In some embodiments, the HBV biomarker is HBcAg. In some embodiments, the methods described herein include performing an assay to detect the presence, level, or status of HBcAg in at least one sample obtained from a subject. In some embodiments, the biomarker is P-HBcAg. In some embodiments, the methods described herein include performing an assay to detect the presence, level, or status of P-HBcAg in at least one sample obtained from a subject. In some embodiments, the presence, level, or status of HBcAg and P-HBcAg are measured in a sample. In some embodiments, the presence, level, or status of HBcAg and P-HBcAg are measured in different samples obtained from the subject. The samples are obtained at the same time point or at different time points.

[0137] In some embodiments, detecting the presence, level, or status of HBcAg in a sample comprises contacting the sample with an antibody that specifically binds to human HBcAg (e.g., an HBcAg capture antibody) to form a capture antibody-HBcAg complex. In some embodiments, the HBcAg capture antibody binds to an epitope on the C-terminus of HBcAg. HBV is phylogenetically classified into 10 genotypes, namely, A to J. The sequence of human HBcAg genotype A comprises the following amino acids:

[0138] [ka]

[0139] The sequences of HBcAg for HBV genotypes A to J are provided below, with the bolded amino acids indicating the C-terminus for each sequence.

[0140] Genotype A:

[0141] [ka]

[0142] Genotype AC terminus:

[0143] [ka]

[0144] Genotype A (amino acids 1-149 of SEQ ID NO: 1)

[0145] [ka]

[0146] Genotype B:

[0147] [ka]

[0148] Genotype BC terminal:

[0149] [ka]

[0150] Genotype C:

[0151] [ka]

[0152] Genotype CC terminal:

[0153] [ka]

[0154] Genotype D:

[0155] [ka]

[0156] Genotype DC terminal:

[0157] [ka]

[0158] Genotype E:

[0159] [ka]

[0160] Genotype EC end: RRRGRSPRRRTPSPRRRRSQSPRRRRSQSPASQC (SEQ ID NO: 16)

[0161] Genotype F:

[0162] [ka]

[0163] Genotype FC end: RRRGRSPRRRTPSPRRRRSQSPRRRRSQSPASQC (SEQ ID NO: 17)

[0164] Genotype G:

[0165] [ka]

[0166] Genotype GC terminal:

[0167] [ka]

[0168] Genotype H:

[0169] [ka]

[0170] Genotype HC terminal:

[0171] [ka]

[0172] Genotype I:

[0173] [ka]

[0174] Genotype IC terminus:

[0175] [ka]

[0176] Genotype J:

[0177] [ka]

[0178] Genotype JC terminus:

[0179] [ka]

[0180] The following table contains the consensus sequences for the above HBcAgs, including genotypes A to J.

[0181] [Table 1] TIFF2025533470000023.tif165168

[0182] The methods described herein can be used to detect HBcAg or phosphorylated HBcAg for any one of the above HBV genotypes (e.g., HBV genotype A, HBV genotype B, HBV genotype C, HBV genotype D, HBV genotype E, HBV genotype F, HBV genotype G, HBV genotype H, HBV genotype I, HBV genotype J).

[0183] In some embodiments, the methods described herein comprise contacting a sample with an HbcAg capture antibody that binds to an epitope on SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40. In some embodiments, the methods comprise contacting a sample with an HbcAg capture antibody that binds to an epitope on the C-terminus of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40. In rare cases, some patients affected by one of the above genotypes express an HbcAg with one or more mutations compared to the above sequences. Thus, in some embodiments, the methods comprise contacting a sample with a capture antibody and / or a detection antibody that binds to an epitope having at least 95% sequence identity to the amino acid sequence provided herein. In some embodiments, the method comprises contacting the sample with an HbcAg capture antibody that binds to an epitope on the C-terminus of an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40. In some embodiments, the method comprises contacting the sample with an HBcAg capture antibody that binds to an epitope on SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39. In some embodiments, the HbcAg capture antibody binds to an epitope that is about 3 to about 36 amino acids (e.g., contiguous amino acids) in length. In some embodiments, the HbcAg capture antibody binds to an epitope that is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35 or about 36 consecutive amino acids in length.

[0184] In further embodiments, the methods described herein comprise contacting a sample with an HbcAg capture antibody that binds to an epitope on SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In some embodiments, the methods comprise contacting a sample with an HbcAg capture antibody that binds to an epitope on the C-terminus of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In rare cases, some patients affected by one of the above genotypes express an HbcAg with one or more mutations compared to the above sequences. Thus, in some embodiments, the methods comprise contacting a sample with a capture antibody and / or a detection antibody that binds to an epitope having at least 95% sequence identity to the amino acid sequence provided herein. In some embodiments, the method comprises contacting a sample with an HbcAg capture antibody that binds to an epitope on the C-terminus of an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the method comprises contacting a sample with an HbcAg capture antibody that binds to an epitope on SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. In some embodiments, the HbcAg capture antibody binds to an epitope that is about 3 to about 36 amino acids (e.g., contiguous amino acids) in length. In some embodiments, the HbcAg capture antibody binds to an epitope that is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35 or about 36 consecutive amino acids in length.

[0185] In some embodiments, the HbcAg capture antibody binds to an epitope of about 3 to about 36 consecutive amino acids in length on SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39. In some embodiments, the HbcAg capture antibody binds to an epitope about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, or about 36 consecutive amino acids in length on SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39. In some embodiments, the HbcAg capture antibody binds to a discontinuous epitope on the C-terminus of HbcAg. For example, in some embodiments, the HbcAg capture antibody binds to multiple binding sites on SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39, each binding site comprising at least three consecutive amino acids (e.g., at least three, at least four, at least five, at least six, at least seven consecutive amino acids), and each binding site being separated by at least one amino acid (e.g., at least one, at least two, at least three).

[0186] In a further embodiment, the HbcAg capture antibody binds to an epitope of about 3 to about 36 consecutive amino acids in length on SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21. In some embodiments, the HbcAg capture antibody binds to an epitope about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, or about 36 consecutive amino acids in length on SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. In some embodiments, the HbcAg capture antibody binds to a discontinuous epitope on the C-terminus of HbcAg. For example, in some embodiments, the HbcAg capture antibody binds to multiple binding sites on SEQ ID NO:2, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, each binding site comprising at least three consecutive amino acids (e.g., at least three, at least four, at least five, at least six, at least seven consecutive amino acids), and each binding site being separated by at least one amino acid (e.g., at least one, at least two, at least three).

[0187] The C-terminus of human HbcAg genotype A is represented by amino acids 150-185 of SEQ ID NO: 1. Amino acids 150-185 of SEQ ID NO: 1 are represented by SEQ ID NO: 2. In some embodiments, the HbcAg capture antibody binds to an epitope about 3 to about 36 amino acids (e.g., consecutive amino acids) in length above SEQ ID NO: 2 or the consensus sequence represented by SEQ ID NO: 25. In some embodiments, the HbcAg capture antibody binds to an epitope of about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35 or about 36 consecutive amino acids on SEQ ID NO: 2 or SEQ ID NO: 25. In some embodiments, the HbcAg capture antibody binds to multiple binding sites on SEQ ID NO:2 or SEQ ID NO:25, each binding site comprising at least three consecutive amino acids (e.g., at least three, at least four, at least five, at least six, at least seven consecutive amino acids), and each binding site is separated by at least one amino acid (e.g., at least one, at least two, at least three).

[0188] In some embodiments, detecting the presence, level, or status of HbcAg in a sample further comprises contacting the sample with a detection antibody that binds to an epitope on HbcAg that is not bound by the HbcAg capture antibody, thereby forming a capture antibody-HbcAg-detection antibody complex. In some embodiments, the HbcAg detection antibody binds to an epitope contained in SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40. In some embodiments, the HbcAg detection antibody binds to an epitope contained in an amino acid sequence having at least 95% sequence identity to SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40. In some embodiments, the HbcAg detection antibody binds to an epitope of about 3 to about 50 consecutive amino acids (about 3 to about 40 consecutive amino acids, about 3 to about 35 consecutive amino acids, about 3 to about 30 consecutive amino acids, about 3 to about 25 consecutive amino acids, about 3 to about 20 consecutive amino acids, or about 3 to about 15 consecutive amino acids).

[0189] In further embodiments, detecting the presence, level, or status of HbcAg in a sample further comprises contacting the sample with a detection antibody that binds to an epitope on HbcAg that is not bound by the HbcAg capture antibody, thereby forming a capture antibody-HbcAg-detection antibody complex. In some embodiments, the HbcAg detection antibody binds to an epitope contained within amino acids 1-149 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In some embodiments, the HbcAg detection antibody binds to an epitope contained within amino acids 1-149 of an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In some embodiments, the HbcAg detection antibody binds to an epitope contained within amino acids 1-161 of SEQ ID NO:9. In some embodiments, the HbcAg detection antibody binds to an epitope contained within amino acids 1-161 of an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 9. In some embodiments, the HbcAg detection antibody binds to an epitope of about 3 to about 50 consecutive amino acids (about 3 to about 40 consecutive amino acids, about 3 to about 35 consecutive amino acids, about 3 to about 30 consecutive amino acids, about 3 to about 25 consecutive amino acids, about 3 to about 20 consecutive amino acids, or about 3 to about 15 consecutive amino acids).

[0190] In some embodiments, the HbcAg detection antibody binds to an epitope of about 3 to about 50 consecutive amino acids (about 3 to about 40 consecutive amino acids, about 3 to about 35 consecutive amino acids, about 3 to about 30 consecutive amino acids, about 3 to about 25 consecutive amino acids, about 3 to about 20 consecutive amino acids, or about 3 to about 15 consecutive amino acids) in SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40. In some embodiments, the HbcAg detection antibody binds to a discontinuous epitope. In some embodiments, the HbcAg detection antibody binds to a discontinuous epitope in SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40. In some embodiments, the HbcAg detection antibody binds to a larger number of binding sites, each of which is separated by at least two amino acids. For example, in some embodiments, the HbcAg detection antibody binds to a larger number of binding sites, each of which comprises at least two consecutive amino acids, and each of which is separated by at least two amino acids. In some embodiments, each of which comprises at least two consecutive amino acids (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids), and each of which is separated by at least 10 amino acids. In some embodiments, each of which is separated by at least 20 amino acids (e.g., at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acids).

[0191] In further embodiments, the HbcAg detection antibody binds to an epitope of about 3 to about 50 consecutive amino acids (about 3 to about 40 consecutive amino acids, about 3 to about 35 consecutive amino acids, about 3 to about 30 consecutive amino acids, about 3 to about 25 consecutive amino acids, about 3 to about 20 consecutive amino acids, or about 3 to about 15 consecutive amino acids) within amino acids 1-149 of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, or within amino acids 1-161 of SEQ ID NO: 9. In some embodiments, the HbcAg detection antibody binds to a discontinuous epitope. In some embodiments, the HbcAg detection antibody binds to a discontinuous epitope on SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the HbcAg detection antibody binds to a larger number of binding sites, each of which is separated by at least two amino acids. For example, in some embodiments, the HbcAg detection antibody binds to a larger number of binding sites, each of which comprises at least two consecutive amino acids, and each of which is separated by at least two amino acids. In some embodiments, each of which comprises at least two consecutive amino acids (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids), and each of which is separated by at least 10 amino acids. In some embodiments, each of which is separated by at least 20 amino acids (e.g., at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acids).

[0192] In yet another embodiment, the detection antibody binds to an epitope contained within amino acids 1-149 of SEQ ID NO: 1. Amino acids 1-149 of SEQ ID NO: 1 are represented by SEQ ID NO: 3. In some embodiments, the HbcAg detection antibody binds to an epitope of about 3 to about 50 amino acids on SEQ ID NO: 3. In some embodiments, the HbcAg detection antibody binds to an epitope of about 3 to about 50 consecutive amino acids on SEQ ID NO: 3. In some embodiments, the detection antibody binds to an epitope of about 3 to about 40 consecutive amino acids, about 3 to about 35 consecutive amino acids, about 3 to about 30 consecutive amino acids, about 3 to about 25 consecutive amino acids, about 3 to about 20 consecutive amino acids, or about 3 to about 15 consecutive amino acids on SEQ ID NO: 3. In some embodiments, the HbcAg detection antibody binds to an epitope of about 3 to about 15 amino acids (e.g., consecutive amino acids) on SEQ ID NO: 3. For example, in some embodiments, the HbcAg detection antibody binds to an epitope of about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 consecutive amino acids on SEQ ID NO: 3. In some embodiments, the HbcAg detection antibody binds to a non-consecutive epitope on SEQ ID NO: 3. For example, in some embodiments, the HbcAg detection antibody binds to a larger number of binding sites, each of which is separated by at least two amino acids. For example, in some embodiments, the HbcAg detection antibody binds to a larger number of binding sites, each of which comprises at least two consecutive amino acids, each of which is separated by at least two amino acids. In some embodiments, each of which comprises at least two consecutive amino acids (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids), each of which is separated by at least 10 amino acids. In some embodiments, each binding site is separated by at least 20 amino acids (eg, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 amino acids).

[0193] In some embodiments, the HbcAg capture antibody binds to an epitope on HbcAg other than the C-terminus. In some embodiments, the HbcAg capture antibody binds to an epitope contained in SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40. In some embodiments, the HbcAg capture antibody binds to an epitope contained in SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40. In some embodiments, the HbcAg capture antibody binds to an epitope of about 3 to about 50 consecutive amino acids (about 3 to about 40 consecutive amino acids, about 3 to about 35 consecutive amino acids, about 3 to about 30 consecutive amino acids, about 3 to about 25 consecutive amino acids, about 3 to about 20 consecutive amino acids, or about 3 to about 15 consecutive amino acids).

[0194] In still other embodiments, the HbcAg capture antibody binds to an epitope on HbcAg other than the C-terminus. In some embodiments, the HbcAg capture antibody binds to an epitope contained within amino acids 1-149 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In some embodiments, the HbcAg capture antibody binds to an epitope contained within amino acids 1-161 of SEQ ID NO:9. In some embodiments, the HbcAg capture antibody binds to an epitope contained within amino acids 1-149 of an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In some embodiments, the HbcAg capture antibody binds to an epitope contained within amino acids 1-161 of an amino acid sequence having at least 95% sequence identity to SEQ ID NO:9. In some embodiments, the HbcAg capture antibody binds to an epitope of about 3 to about 50 consecutive amino acids (about 3 to about 40 consecutive amino acids, about 3 to about 35 consecutive amino acids, about 3 to about 30 consecutive amino acids, about 3 to about 25 consecutive amino acids, about 3 to about 20 consecutive amino acids, or about 3 to about 15 consecutive amino acids).

[0195] In some embodiments, the HbcAg capture antibody binds to an epitope of about 3 to about 50 consecutive amino acids (about 3 to about 40 consecutive amino acids, about 3 to about 35 consecutive amino acids, about 3 to about 30 consecutive amino acids, about 3 to about 25 consecutive amino acids, about 3 to about 20 consecutive amino acids, or about 3 to about 15 consecutive amino acids) in SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40. In some embodiments, the HbcAg capture antibody binds to a discontinuous epitope in SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40. In some embodiments, the HbcAg capture antibody binds to a greater number of binding sites, with each binding site separated by at least two amino acids. For example, in some embodiments, the HbcAg capture antibody binds to multiple binding sites, each binding site comprising at least two consecutive amino acids, and each binding site is separated by at least two amino acids. In some embodiments, each binding site comprises at least two consecutive amino acids (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids), and each binding site is separated by at least 10 amino acids. In some embodiments, each binding site is separated by at least 20 amino acids (e.g., at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acids).

[0196] In still other embodiments, the HbcAg capture antibody binds to an epitope of about 3 to about 50 consecutive amino acids (about 3 to about 40 consecutive amino acids, about 3 to about 35 consecutive amino acids, about 3 to about 30 consecutive amino acids, about 3 to about 25 consecutive amino acids, about 3 to about 20 consecutive amino acids, or about 3 to about 15 consecutive amino acids) within amino acids 1-149 of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, or within amino acids 1-161 of SEQ ID NO: 9. In some embodiments, the HbcAg capture antibody binds to a discontinuous epitope. In some embodiments, the HbcAg capture antibody binds to a discontinuous epitope on SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the HbcAg capture antibody binds to a larger number of binding sites, each of which is separated by at least two amino acids. For example, in some embodiments, the HbcAg capture antibody binds to a larger number of binding sites, each of which comprises at least two consecutive amino acids, and each of which is separated by at least two amino acids. In some embodiments, each of which comprises at least two consecutive amino acids (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids), and each of which is separated by at least 10 amino acids. In some embodiments, each of which is separated by at least 20 amino acids (e.g., at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acids).

[0197] In some embodiments, the HBcAg capture antibody binds to an epitope contained within amino acids 1-149 of SEQ ID NO: 1. Amino acids 1-149 of SEQ ID NO: 1 are set forth in SEQ ID NO: 3. In some embodiments, the HBcAg capture antibody binds to an epitope of about 3 to about 50 amino acids on SEQ ID NO: 3. In some embodiments, the HBcAg capture antibody binds to an epitope of about 3 to about 40 consecutive amino acids, about 3 to about 35 consecutive amino acids, about 3 to about 30 consecutive amino acids, about 3 to about 25 consecutive amino acids, about 3 to about 20 consecutive amino acids, or about 3 to about 15 consecutive amino acids on SEQ ID NO: 3. In some embodiments, the HBcAg capture antibody binds to an epitope of about 3 to about 15 amino acids (e.g., consecutive amino acids) on SEQ ID NO: 3. In some embodiments, the HBcAg capture antibody binds to an epitope of about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 consecutive amino acids on SEQ ID NO: 3. In some embodiments, the HBcAg capture antibody binds to a non-contiguous epitope. For example, in some embodiments, the HBcAg capture antibody binds to a larger number of binding sites, each of which is separated by at least two amino acids. For example, in some embodiments, the capture antibody binds to a larger number of binding sites, each of which comprises at least two consecutive amino acids, each of which is separated by at least two amino acids. In some embodiments, each of which comprises at least two consecutive amino acids (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids), each of which is separated by at least 10 amino acids. In some embodiments, each binding site is separated by at least 20 amino acids (eg, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 amino acids).

[0198] In some embodiments, the HBcAg detection antibody binds to an epitope on HBcAg that is not bound by the capture antibody, thereby forming a capture antibody-HBcAg-detection antibody complex. In some embodiments, the HBcAg detection antibody binds to an epitope on the C-terminus of HBcAg. In some embodiments, the HBcAg detection antibody binds to an epitope on the C-terminus of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40. In some embodiments, the HBcAg detection antibody binds to an epitope on the C-terminus of an amino acid sequence having at least 95% sequence identity to SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40. In some embodiments, the HBcAg detection antibody binds to an epitope on SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40. In some embodiments, the HBcAg detection antibody binds to an epitope about 3 to about 36 amino acids (e.g., consecutive amino acids) in length. In some embodiments, the HBcAg detection antibody binds to an epitope about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, or about 36 consecutive amino acids in length.

[0199] In yet other embodiments, the HBcAg detection antibody binds to an epitope on HBcAg that is not bound by the capture antibody, thereby forming a capture antibody-HBcAg-detection antibody complex. In some embodiments, the HBcAg detection antibody binds to an epitope on the C-terminus of HBcAg. In some embodiments, the HBcAg detection antibody binds to an epitope on the C-terminus of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In some embodiments, the HBcAg detection antibody binds to an epitope on the C-terminus of an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In some embodiments, the HBcAg detection antibody binds to an epitope on SEQ ID NO:2, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21. In some embodiments, the HBcAg detection antibody binds to an epitope that is about 3 to about 36 amino acids (e.g., consecutive amino acids) in length. In some embodiments, the HBcAg detection antibody binds to an epitope that is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, or about 36 consecutive amino acids in length. In yet another embodiment, the HBcAg detection antibody capture antibody specifically binds to an epitope within amino acids 150-185 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, and the HBcAg detection antibody specifically binds to an epitope within amino acids 1-149 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.In some aspects of this embodiment, the HBcAg capture antibody and the HBcAg detection antibody can specifically bind to overlapping epitopes within the amino acids of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. For example, the HBcAg capture antibody can bind to an epitope within amino acids 145-155 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, the HBcAg detection antibody can bind to an epitope within amino acids 139-149 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, etc.

[0200] In yet other embodiments, the HBcAg capture antibody specifically binds to an epitope within amino acids 1-149 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, and the HBcAg detection antibody specifically binds to an epitope within amino acids 150-185 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In some aspects of this embodiment, the HBcAg capture antibody and the HBcAg detection antibody can specifically bind to overlapping epitopes within amino acids of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. For example, the HbcAg capture antibody can bind to an epitope within amino acids 139-149 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, and the HBcAg detection antibody can bind to an epitope within amino acids 145-155 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, etc.

[0201] In some embodiments, the HBcAg detection antibody binds to an epitope having a length of about 3 to about 36 amino acids (e.g., consecutive amino acids) on SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39. In some embodiments, the HBcAg detection antibody binds to an epitope of about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, or about 36 consecutive amino acids on SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39. In some embodiments, the HBcAg detection antibody binds to a discontinuous epitope. In some embodiments, the HBcAg detection antibody binds to a discontinuous epitope on the C-terminus of HBcAg. For example, in some embodiments, the HBcAg detection antibody binds to multiple binding sites, each of which comprises at least three consecutive amino acids (e.g., at least three, at least four, at least five, at least six, at least seven consecutive amino acids), and each of which is separated by at least one amino acid (e.g., at least one, at least two, at least three). In some embodiments, the HBcAg detection antibody binds to multiple binding sites on SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39, each of which comprises at least three consecutive amino acids (e.g., at least three, at least four, at least five, at least six, at least seven consecutive amino acids), and each of which is separated by at least one amino acid (e.g., at least one, at least two, at least three).

[0202] In yet another embodiment, the HBcAg detection antibody binds to an epitope having a length of about 3 to about 36 amino acids (e.g., consecutive amino acids) on SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. In some embodiments, the HBcAg detection antibody binds to an epitope of about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, or about 36 consecutive amino acids on SEQ ID NO:2, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21. In some embodiments, the HBcAg detection antibody binds to a discontinuous epitope. In some embodiments, the HBcAg detection antibody binds to a discontinuous epitope on the C-terminus of HBcAg. For example, in some embodiments, the HBcAg detection antibody binds to multiple binding sites, each of which comprises at least three consecutive amino acids (e.g., at least three, at least four, at least five, at least six, at least seven consecutive amino acids), and each of which is separated by at least one amino acid (e.g., at least one, at least two, at least three). In some embodiments, the HBcAg detection antibody binds to multiple binding sites on SEQ ID NO:2, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, and each of which comprises at least three consecutive amino acids (e.g., at least three, at least four, at least five, at least six, at least seven consecutive amino acids), and each of which is separated by at least one amino acid (e.g., at least one, at least two, at least three).

[0203] In some embodiments, the HBcAg detection antibody binds to an epitope of about 3 to about 36 consecutive amino acids on SEQ ID NO: 2. In some embodiments, the HBcAg detection antibody binds to an epitope of about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, or about 36 consecutive amino acids in length on SEQ ID NO: 2. In some embodiments, the HBcAg detection antibody binds to multiple binding sites on SEQ ID NO:2, each binding site comprising at least three consecutive amino acids (e.g., at least three, at least four, at least five, at least six, at least seven consecutive amino acids), and each binding site is separated by at least one amino acid (e.g., at least one, at least two, at least three).

[0204] In some embodiments, the detection antibody further comprises a detectable label. Suitable detectable labels are described herein. In some embodiments, the signal generated by the detectable label in the capture antibody-HBcAg-detection antibody complex is indicative of the presence and / or amount (e.g., level) of HBcAg in the sample.

[0205] In some embodiments, detecting the presence, level, or status of human P-HBcAg in a sample comprises contacting the sample with an antibody that specifically binds to P-HBcAg (e.g., a P-HBcAg capture antibody) to form a capture antibody-P-HBcAg complex. In some embodiments, the P-HBcAg capture antibody binds to an epitope on the C-terminus of P-HBcAg. In some embodiments, the P-HBcAg capture antibody binds to an epitope on the C-terminus of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40. In some embodiments, the P-HBcAg capture antibody binds to an epitope on the C-terminus of an amino acid sequence having at least 95% sequence identity to SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40. In some embodiments, at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, or seven amino acids are phosphorylated.

[0206] In still other embodiments, detecting the presence, level, or status of human P-HBcAg in a sample comprises contacting the sample with an antibody that specifically binds to P-HBcAg (e.g., a P-HBcAg capture antibody) to form a capture antibody-P-HBcAg complex. In some embodiments, the P-HBcAg capture antibody binds to an epitope on the C-terminus of P-HBcAg. In some embodiments, the P-HBcAg capture antibody binds to an epitope on the C-terminus of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In some embodiments, the P-HBcAg capture antibody binds to an epitope on the C-terminus of an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In some embodiments, at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, or seven amino acids are phosphorylated.

[0207] In some embodiments, the P-HBcAg capture antibody binds to an epitope on SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39. In some embodiments, the P-HBcAg capture antibody binds to an epitope that is about 3 to about 36 amino acids (e.g., contiguous) in length. In some embodiments, the P-HBcAg capture antibody binds to an epitope about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, or about 36 amino acids (e.g., contiguous) in length, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, or seven amino acids are phosphorylated.

[0208] In still other embodiments, the P-HBcAg capture antibody binds to an epitope on SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. In some embodiments, the P-HBcAg capture antibody binds to an epitope that is about 3 to about 36 amino acids (e.g., contiguous) in length. In some embodiments, the P-HBcAg capture antibody binds to an epitope about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, or about 36 amino acids (e.g., contiguous) in length, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, or seven amino acids are phosphorylated.

[0209] In some embodiments, the P-HBcAg capture antibody binds to an epitope of about 3 to about 36 amino acids (e.g., contiguous) in length on SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, or seven amino acids are phosphorylated. In some embodiments, the P-HBcAg capture antibody binds to an epitope about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, or about 36 amino acids (e.g., consecutive) in length on SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, or seven amino acids are phosphorylated. In some embodiments, the P-HBcAg capture antibody binds to a discontinuous epitope on the C-terminus of P-HBcAg. In some embodiments, the P-HBcAg capture antibody binds to multiple binding sites, each binding site comprising at least three consecutive amino acids (e.g., at least three, at least four, at least five, at least six, or at least seven consecutive amino acids), each binding site being separated by at least one amino acid (e.g., at least one, at least two, or at least three), and at least one, at least two, at least three, at least four, at least five, at least six, or seven amino acids being phosphorylated.For example, in some embodiments, a P-HBcAg capture antibody binds to multiple binding sites on SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39, each binding site comprising at least three consecutive amino acids (e.g., at least three, at least four, at least five, at least six, at least seven consecutive amino acids), each binding site being separated by at least one amino acid (e.g., at least one, at least two, at least three) and at least one, at least two, at least three, at least four, at least five, at least six, or seven amino acids being phosphorylated.

[0210] In yet other embodiments, the P-HBcAg capture antibody binds to an epitope of about 3 to about 36 amino acids (e.g., contiguous) in length on SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, or seven amino acids are phosphorylated. In some embodiments, the P-HBcAg capture antibody binds to an epitope about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, or about 36 amino acids (e.g., consecutive) in length on SEQ ID NO:2, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, or seven amino acids are phosphorylated. In some embodiments, the P-HBcAg capture antibody binds to a discontinuous epitope on the C-terminus of P-HBcAg. In some embodiments, the P-HBcAg capture antibody binds to multiple binding sites, each binding site comprising at least three consecutive amino acids (e.g., at least three, at least four, at least five, at least six, or at least seven consecutive amino acids), each binding site being separated by at least one amino acid (e.g., at least one, at least two, or at least three), and at least one, at least two, at least three, at least four, at least five, at least six, or seven amino acids being phosphorylated.For example, in some embodiments, a P-HBcAg capture antibody binds to multiple binding sites on SEQ ID NO:2, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, each binding site comprising at least three consecutive amino acids (e.g., at least three, at least four, at least five, at least six, at least seven consecutive amino acids), each binding site being separated by at least one amino acid (e.g., at least one, at least two, at least three) and at least one, at least two, at least three, at least four, at least five, at least six, or seven amino acids being phosphorylated.

[0211] The C-terminus of P-HBcAg genotype A is represented by amino acids 150-185 of SEQ ID NO: 1. Amino acids 150-185 of SEQ ID NO: 1 are represented by SEQ ID NO: 2. In some embodiments, the P-HBcAg capture antibody binds to an epitope about 3 to about 36 amino acids (e.g., contiguous) in length on SEQ ID NO: 2, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, or seven amino acids are phosphorylated. In some embodiments, the P-HBcAg capture antibody binds to an epitope of about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, or about 36 amino acids (e.g., consecutive) in length on SEQ ID NO: 2, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, or seven amino acids are phosphorylated.

[0212] In some embodiments, detecting the presence, level or status of P-HBcAg in a sample further comprises contacting the sample with a detection antibody that binds to an epitope on P-HBcAg that is not bound by the P-HBcAg capture antibody, thereby forming a capture antibody-P-HBcAg-detection antibody complex.

[0213] In some embodiments, the P-HBcAg detection antibody binds to an epitope on HBcAg other than the C-terminus. In some embodiments, the P-HBcAg detection antibody binds to an epitope contained in SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40. In some embodiments, the P-HBcAg detection antibody binds to an epitope contained in SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40. In some embodiments, the P-HBcAg detection antibody binds to an epitope of about 3 to about 50 consecutive amino acids (about 3 to about 40 consecutive amino acids, about 3 to about 35 consecutive amino acids, about 3 to about 30 consecutive amino acids, about 3 to about 25 consecutive amino acids, about 3 to about 20 consecutive amino acids, or about 3 to about 15 consecutive amino acids).

[0214] In still other embodiments, the P-HBcAg detection antibody binds to an epitope on HBcAg other than the C-terminus. In some embodiments, the P-HBcAg detection antibody binds to an epitope contained within amino acids 1-149 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In some embodiments, the P-HBcAg detection antibody binds to an epitope contained within amino acids 1-161 of SEQ ID NO:9. In some embodiments, the P-HBcAg detection antibody binds to an epitope contained within amino acids 1-149 of an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In some embodiments, the P-HBcAg detection antibody binds to an epitope contained within amino acids 1-161 of an amino acid sequence having at least 95% sequence identity to SEQ ID NO:9. In some embodiments, the P-HBcAg detection antibody binds to an epitope of about 3 to about 50 consecutive amino acids (about 3 to about 40 consecutive amino acids (about 3 to about 35 consecutive amino acids, about 3 to about 30 consecutive amino acids, about 3 to about 25 consecutive amino acids, about 3 to about 20 consecutive amino acids, or about 3 to about 15 consecutive amino acids).

[0215] In some embodiments, the P-HBcAg detection antibody binds to an epitope of about 3 to about 50 consecutive amino acids (about 3 to about 40 consecutive amino acids, about 3 to about 35 consecutive amino acids, about 3 to about 30 consecutive amino acids, about 3 to about 25 consecutive amino acids, about 3 to about 20 consecutive amino acids, or about 3 to about 15 consecutive amino acids) in SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40. In some embodiments, the P-HBcAg detection antibody binds to a discontinuous epitope. In some embodiments, the P-HBcAg detection antibody binds to a discontinuous epitope in SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40. In some embodiments, the P-HBcAg detection antibody binds to a larger number of binding sites, each of which is separated by at least two amino acids. For example, in some embodiments, the P-HBcAg detection antibody binds to a larger number of binding sites, each of which comprises at least two consecutive amino acids, and each of which is separated by at least two amino acids. In some embodiments, each of which comprises at least two consecutive amino acids (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids), and each of which is separated by at least 10 amino acids. In some embodiments, each of which is separated by at least 20 amino acids (e.g., at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acids).

[0216] In still other embodiments, the P-HBcAg detection antibody binds to an epitope of about 3 to about 50 consecutive amino acids (about 3 to about 40 consecutive amino acids, about 3 to about 35 consecutive amino acids, about 3 to about 30 consecutive amino acids, about 3 to about 25 consecutive amino acids, about 3 to about 20 consecutive amino acids, or about 3 to about 15 consecutive amino acids) within amino acids 1-149 of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, or within amino acids 1-161 of SEQ ID NO: 9. In some embodiments, the P-HBcAg detection antibody binds to a discontinuous epitope. In some embodiments, the P-HBcAg detection antibody binds to a discontinuous epitope on SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the P-HBcAg detection antibody binds to a larger number of binding sites, each of which is separated by at least two amino acids. For example, in some embodiments, the P-HBcAg detection antibody binds to a larger number of binding sites, each of which comprises at least two consecutive amino acids, and each of which is separated by at least two amino acids. In some embodiments, each of which comprises at least two consecutive amino acids (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids), and each of which is separated by at least 10 amino acids. In some embodiments, each of which is separated by at least 20 amino acids (e.g., at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acids).

[0217] In some embodiments, the detection antibody binds to an epitope contained within amino acids 1-149 of SEQ ID NO: 1. Amino acids 1-149 of SEQ ID NO: 1 are represented by SEQ ID NO: 3. In some embodiments, the P-HBcAg detection antibody binds to an epitope of about 3 to about 50 amino acids on SEQ ID NO: 3. In some embodiments, the P-HBcAg detection antibody binds to an epitope of about 3 to about 40 consecutive amino acids, about 3 to about 35 consecutive amino acids, about 3 to about 30 consecutive amino acids, about 3 to about 25 consecutive amino acids, about 3 to about 20 consecutive amino acids, or about 3 to about 15 consecutive amino acids on SEQ ID NO: 3. In some embodiments, the P-HBcAg detection antibody binds to an epitope of about 3 to about 15 amino acids (e.g., consecutive amino acids) in length on SEQ ID NO: 3. In some embodiments, the P-HBcAg detection antibody binds to an epitope of about 3 to about 15 amino acids (e.g., consecutive) in length on SEQ ID NO: 3. For example, in some embodiments, the P-HBcAg detection antibody binds to an epitope of about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 consecutive amino acids on SEQ ID NO: 3. In some embodiments, the P-HBcAg detection antibody binds to a non-contiguous epitope. For example, in some embodiments, the P-HBcAg detection antibody binds to a larger number of binding sites, each of which is separated by at least two amino acids. For example, in some embodiments, the detection antibody binds to a larger number of binding sites, each of which comprises at least two consecutive amino acids, each of which is separated by at least two amino acids. In some embodiments, each of which comprises at least two consecutive amino acids (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids), each of which is separated by at least 10 amino acids.In some embodiments, each binding site is separated by at least 20 amino acids (eg, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 amino acids).

[0218] In some embodiments, the P-HBcAg capture antibody binds to an epitope on HBcAg other than the C-terminus. In some embodiments, the P-HBcAg capture antibody binds to an epitope contained within SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40. In some embodiments, the P-HBcAg capture antibody binds to an epitope contained within an amino acid sequence having at least 95% sequence identity to SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40. In some embodiments, the P-HBcAg capture antibody binds to an epitope of about 3 to about 50 consecutive amino acids (about 3 to about 40 consecutive amino acids, about 3 to about 35 consecutive amino acids, about 3 to about 30 consecutive amino acids, about 3 to about 25 consecutive amino acids, about 3 to about 20 consecutive amino acids, or about 3 to about 15 consecutive amino acids). In further embodiments, the P-HBcAg capture antibody binds to an epitope on HBcAg other than the C-terminus. In some embodiments, the P-HBcAg capture antibody binds to an epitope contained within amino acids 1-149 of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the P-HBcAg capture antibody binds to an epitope contained within amino acids 1-161 of SEQ ID NO: 9. In some embodiments, the P-HBcAg capture antibody binds to an epitope contained within amino acids 1-149 of an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the P-HBcAg capture antibody binds to an epitope contained within amino acids 1-161 of an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 9.In some embodiments, the P-HBcAg capture antibody binds to an epitope of about 3 to about 50 consecutive amino acids (about 3 to about 40 consecutive amino acids, about 3 to about 35 consecutive amino acids, about 3 to about 30 consecutive amino acids, about 3 to about 25 consecutive amino acids, about 3 to about 20 consecutive amino acids, or about 3 to about 15 consecutive amino acids).

[0219] In some embodiments, the P-HBcAg capture antibody binds to an epitope of about 3 to about 50 consecutive amino acids (about 3 to about 40 consecutive amino acids, about 3 to about 35 consecutive amino acids, about 3 to about 30 consecutive amino acids, about 3 to about 25 consecutive amino acids, about 3 to about 20 consecutive amino acids, or about 3 to about 15 consecutive amino acids) in SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40. In some embodiments, the P-HBcAg capture antibody binds to a discontinuous epitope. In some embodiments, the P-HBcAg capture antibody binds to a discontinuous epitope in SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40. In some embodiments, the P-HBcAg capture antibody binds to a larger number of binding sites, with each binding site separated by at least two amino acids. For example, in some embodiments, the P-HBcAg detection antibody binds to a larger number of binding sites, with each binding site comprising at least two consecutive amino acids and each binding site separated by at least two amino acids. In some embodiments, each binding site comprises at least two consecutive amino acids (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids), with each binding site separated by at least 10 amino acids. In some embodiments, each binding site is separated by at least 20 amino acids (e.g., at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acids).

[0220] In further embodiments, the P-HBcAg capture antibody binds to an epitope of about 3 to about 50 consecutive amino acids (about 3 to about 40 consecutive amino acids, about 3 to about 35 consecutive amino acids, about 3 to about 30 consecutive amino acids, about 3 to about 25 consecutive amino acids, about 3 to about 20 consecutive amino acids, or about 3 to about 15 consecutive amino acids) within amino acids 1-149 of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, or within amino acids 1-161 of SEQ ID NO: 9. In some embodiments, the P-HBcAg capture antibody binds to a discontinuous epitope. In some embodiments, the P-HBcAg capture antibody binds to a discontinuous epitope on SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the P-HBcAg capture antibody binds to a larger number of binding sites, with each binding site separated by at least two amino acids. For example, in some embodiments, the P-HBcAg detection antibody binds to a larger number of binding sites, with each binding site comprising at least two consecutive amino acids and each binding site separated by at least two amino acids. In some embodiments, each binding site comprises at least two consecutive amino acids (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids), with each binding site separated by at least 10 amino acids. In some embodiments, each binding site is separated by at least 20 amino acids (e.g., at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acids).

[0221] In some embodiments, the P-HBcAg capture antibody binds to an epitope contained within amino acids 1-149 of SEQ ID NO: 1. In some embodiments, the P-HBcAg capture antibody binds to an epitope of about 3 to about 50 amino acids on SEQ ID NO: 3. In some embodiments, the P-HBcAg capture antibody binds to an epitope of about 3 to about 40 consecutive amino acids, about 3 to about 35 consecutive amino acids, about 3 to about 30 consecutive amino acids, about 3 to about 25 consecutive amino acids, about 3 to about 20 consecutive amino acids, or about 3 to about 15 consecutive amino acids on SEQ ID NO: 3. In some embodiments, the P-HBcAg capture antibody binds to an epitope of about 3 to about 15 amino acids (e.g., consecutive amino acids) on SEQ ID NO: 3. In some embodiments, the P-HBcAg capture antibody binds to an epitope of about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 consecutive amino acids on SEQ ID NO: 3. In some embodiments, the P-HBcAg capture antibody binds to a non-contiguous epitope. For example, in some embodiments, the P-HBcAg capture antibody binds to a larger number of binding sites, each of which is separated by at least two amino acids. For example, in some embodiments, the capture antibody binds to a larger number of binding sites, each of which comprises at least two consecutive amino acids, each of which is separated by at least two amino acids. In some embodiments, each of which comprises at least two consecutive amino acids (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids), each of which is separated by at least 10 amino acids. In some embodiments, each binding site is separated by at least 20 amino acids (eg, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 amino acids).

[0222] In some embodiments, the P-HBcAg detection antibody binds to an epitope on P-HBcAg that is not bound by the capture antibody, thereby forming a capture antibody-P-HBcAg-detection antibody complex. In some embodiments, the P-HBcAg detection antibody binds to an epitope on the C-terminus of P-HBcAg. In some embodiments, the P-HBcAg detection antibody binds to an epitope on the C-terminus of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40. In some embodiments, the P-HBcAg detection antibody binds to an epitope on the C-terminus of an amino acid sequence having at least 95% sequence identity to SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40. In some embodiments, the P-HBcAg detection antibody binds to an epitope on SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39. In some embodiments, the P-HBcAg detection antibody binds to an epitope that is about 3 to about 36 amino acids (e.g., contiguous) in length. In some embodiments, the P-HBcAg detection antibody binds to an epitope about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, or about 36 amino acids (e.g., contiguous) in length, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, or seven amino acids are phosphorylated.

[0223] In yet other embodiments, the P-HBcAg detection antibody capture antibody specifically binds to an epitope within amino acids 150-185 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, and the P-HBcAg detection antibody specifically binds to an epitope within amino acids 1-149 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In some aspects of this embodiment, the P-HBcAg capture antibody and the P-HBcAg detection antibody can specifically bind to overlapping epitopes within the amino acids of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. For example, the P-HBcAg capture antibody can bind to an epitope within amino acids 145-155 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, and the P-HBcAg detection antibody can bind to an epitope within amino acids 139-149 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, etc.

[0224] In yet other embodiments, the P-HBcAg capture antibody specifically binds to an epitope within amino acids 1-149 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, and the P-HBcAg detection antibody specifically binds to an epitope within amino acids 150-185 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In some aspects of this embodiment, the P-HbcAg capture antibody and the P-HBcAg detection antibody can specifically bind to overlapping epitopes within amino acids of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. For example, the P-HbcAg capture antibody can bind to an epitope within amino acids 139-149 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, and the P-HBcAg detection antibody can bind to an epitope within amino acids 145-155 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, etc.

[0225] In further embodiments, the P-HBcAg detection antibody binds to an epitope on P-HBcAg that is not bound by the capture antibody, thereby forming a capture antibody-P-HBcAg-detection antibody complex. In some embodiments, the P-HBcAg detection antibody binds to an epitope on the C-terminus of P-HBcAg. In some embodiments, the P-HBcAg detection antibody binds to an epitope on the C-terminus of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the P-HBcAg detection antibody binds to an epitope on the C-terminus of an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the P-HBcAg detection antibody binds to an epitope on SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. In some embodiments, the P-HBcAg detection antibody binds to an epitope that is about 3 to about 36 amino acids (e.g., contiguous) in length. In some embodiments, the P-HBcAg detection antibody binds to an epitope about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, or about 36 amino acids (e.g., contiguous) in length, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, or seven amino acids are phosphorylated.

[0226] In some embodiments, the P-HBcAg detection antibody binds to an epitope of about 3 to about 36 amino acids (e.g., contiguous) in length on SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, or seven amino acids are phosphorylated. In some embodiments, the P-HBcAg detection antibody binds to an epitope about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, or about 36 amino acids (e.g., contiguous) in length on SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, or seven amino acids are phosphorylated. In some embodiments, the P-HBcAg detection antibody binds to a discontinuous epitope on the C-terminus of P-HBcAg. In some embodiments, the P-HBcAg detection antibody binds to multiple binding sites, each of which comprises at least three consecutive amino acids (e.g., at least three, at least four, at least five, at least six, or at least seven consecutive amino acids), each of which is separated by at least one amino acid (e.g., at least one, at least two, or at least three), and at least one, at least two, at least three, at least four, at least five, at least six, or seven amino acids are phosphorylated.For example, in some embodiments, a P-HBcAg detection antibody binds to multiple binding sites on SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39, each binding site comprising at least three consecutive amino acids (e.g., at least three, at least four, at least five, at least six, at least seven consecutive amino acids), each binding site being separated by at least one amino acid (e.g., at least one, at least two, at least three) and at least one, at least two, at least three, at least four, at least five, at least six, or seven amino acids being phosphorylated.

[0227] In a further embodiment, the P-HBcAg detection antibody binds to an epitope of about 3 to about 36 amino acids (e.g., contiguous) in length on SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, or seven amino acids are phosphorylated. In some embodiments, the P-HBcAg detection antibody binds to an epitope about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, or about 36 amino acids (e.g., contiguous) in length on SEQ ID NO:2, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, or seven amino acids are phosphorylated. In some embodiments, the P-HBcAg detection antibody binds to a discontinuous epitope on the C-terminus of P-HBcAg. In some embodiments, the P-HBcAg detection antibody binds to multiple binding sites, each of which comprises at least three consecutive amino acids (e.g., at least three, at least four, at least five, at least six, or at least seven consecutive amino acids), each of which is separated by at least one amino acid (e.g., at least one, at least two, or at least three), and at least one, at least two, at least three, at least four, at least five, at least six, or seven amino acids are phosphorylated.For example, in some embodiments, a P-HBcAg detection antibody binds to multiple binding sites on SEQ ID NO:2, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, each binding site comprising at least three consecutive amino acids (e.g., at least three, at least four, at least five, at least six, at least seven consecutive amino acids), each binding site being separated by at least one amino acid (e.g., at least one, at least two, at least three) and at least one, at least two, at least three, at least four, at least five, at least six, or seven amino acids being phosphorylated.

[0228] In some embodiments, the P-HBcAg detection antibody binds to an epitope of about 3 to about 36 consecutive amino acids on SEQ ID NO: 2 or SEQ ID NO: 25, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, or seven amino acids are phosphorylated. In some embodiments, the P-HBcAg detection antibody binds to an epitope about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, or about 36 amino acids (e.g., consecutive) in length on SEQ ID NO: 2 or SEQ ID NO: 25, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, or seven amino acids are phosphorylated.

[0229] In some embodiments, the detection antibody further comprises a detectable label. Suitable detectable labels are described herein. In some embodiments, the signal generated by the detectable label in the capture antibody-P-HBcAg-detection antibody complex is indicative of the presence and / or amount (e.g., level) of P-HBcAg in the sample.

[0230] In some embodiments, the first specific binding member is immobilized on a solid support. In some embodiments, the second specific binding member is immobilized on a solid support. In some embodiments, the first specific binding member is an HBV biomarker antibody described below.

[0231] In some embodiments, the sample is diluted or undiluted. The sample may be about 1 to about 25 microliters, about 1 to about 24 microliters, about 1 to about 23 microliters, about 1 to about 22 microliters, about 1 to about 21 microliters, about 1 to about 20 microliters, about 1 to about 18 microliters, about 1 to about 17 microliters, about 1 to about 16 microliters, about 15 microliters, or about 1 microliter, about 2 microliters, about 3 microliters, about 4 microliters, about 5 microliters, about 6 microliters, about 7 microliters, about 8 microliters, about 9 microliters, about 10 microliters, about 11 microliters, about 12 microliters, about 13 microliters, about 14 microliters, about 15 microliters, about 16 microliters, about 17 microliters, about 18 microliters, about 19 microliters, about 20 microliters, about 21 microliters, about 22 microliters, about 23 microliters, about 24 microliters, or about 25 microliters. In some embodiments, the sample is about 1 to about 150 microliters or less, or about 1 to about 25 microliters or less.

[0232] Some devices other than polynt-of-care devices (e.g., Abbott Laboratories' ARCHITECT® device, Abbott Alinity's device, and other core laboratory devices) can measure HBV biomarker levels of approximately 4 pg / L in a sample with a CV of 10% or less. Other detection methods include or can be adapted for use with nanopore or nanowell devices. Examples of nanopore devices are described in WO 2016 / 161402, which is hereby incorporated by reference in its entirety. Examples of nanowell devices are described in WO 2016 / 161400, which is hereby incorporated by reference in its entirety.

[0233] Antibodies may be prepared by any of a variety of techniques, including techniques well known to those of skill in the art. Generally, antibodies may be produced by conventional techniques or by cell culture techniques, including the generation of monoclonal antibodies by transfecting antibody genes, heavy chains, and / or light chains into a suitable bacterial or mammalian cell host to enable the production of antibodies, which may be recombinant. The term "transfection" in its various forms is intended to encompass a wide variety of techniques commonly used for the introduction of foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and others. While antibodies can be expressed in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, including mammalian host cells, is specifically contemplated, since such eukaryotic cells (e.g., mammalian cells) are more likely than prokaryotic cells to assemble and secrete properly folded and immunologically active antibodies.

[0234] Exemplary mammalian host cells for expressing recombinant antibodies include Chinese hamster ovary (CHO) cells (including the dhfr-CHO cells described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)) used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982)), NS0 myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody within the host cell or secretion of the antibody into the culture medium in which the host cell is grown. The antibody may be recovered from the culture medium using standard protein purification methods.

[0235] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It is understood that variations on the above procedures can be implemented. For example, it may be desirable to transfect host cells with DNA encoding functional fragments of the antibody light and / or heavy chains. Recombinant DNA technology can also be used to remove some or all of the DNA encoding one or both of the light and heavy chains that is not necessary for binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also encompassed by antibodies. Additionally, bifunctional antibodies, in which one heavy and one light chain is an antibody (i.e., binds human troponin I) and the other heavy and light chains are specific for an antigen other than a human HBV biomarker, can also be produced by crosslinking an antibody to a second antibody via standard chemical crosslinking methods.

[0236] In some embodiments, a system for recombinantly expressing an antibody or antigen-binding portion thereof comprises a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain, which is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy chain gene and the antibody light chain gene are each operably linked to a CMV enhancer / AdMLP promoter regulatory element to drive high levels of gene transcription. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells transfected with the vector using methotrexate selection / amplification. Selected transformant host cells are cultured to allow expression of the antibody heavy and light chains, and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. Furthermore, a method for synthesizing a recombinant antibody is by culturing the host cells in an appropriate culture medium until the recombinant antibody is synthesized. The method may further comprise isolating the recombinant antibody from the culture medium.

[0237] Methods for preparing monoclonal antibodies include preparing immortal cell lines capable of producing antibodies with the desired specificity. Such cell lines can be generated from spleen cells obtained from immunized animals. Animals can be immunized with an HBV biomarker or a fragment and / or variant thereof. The peptide used to immunize the animal can include amino acids encoding a human Fc, such as a fragment crystallizable region or tail region of a human antibody. The spleen cells can then be immortalized, for example, by fusion with a myeloma cell fusion partner. Various fusion methods can be used. For example, spleen cells and myeloma cells can be combined with a non-ionic detergent for several minutes and then plated at low density on a selective medium that supports the growth of hybrid cells but not myeloma cells. One such technique uses hypoxanthine, aminopterin, and thymidine (HAT) selection. Another technique involves electrofusion. After sufficient time, usually about 1 to 2 weeks, hybrid colonies can be observed. Single colonies are selected and their culture supernatants are tested for binding activity to the polypeptide. Hybridomas with high reactivity and specificity can be used.

[0238] Monoclonal antibodies can be isolated from the supernatant of growing hybridoma colonies. In addition, various techniques can be used to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. The monoclonal antibody can then be collected from the ascites fluid or blood. Contaminants can be removed from the antibody by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. Affinity chromatography is an example of a method that can be used in the process of purifying the antibody.

[0239] The proteolytic enzyme papain preferentially cleaves IgG molecules into several fragments, two of which (F(ab) fragments) comprise covalently linked heterodimers, each of which contains an intact antigen-binding site. The enzyme pepsin can cleave IgG molecules into several fragments, including the F(ab')2 fragment, which contains both antigen-binding sites.

[0240] Fv fragments can be generated by preferential proteolytic cleavage of IgM, and in rare cases, IgG or IgA immunoglobulin molecules. Fv fragments can be derived using recombinant methods. Fv fragments comprise a noncovalent VH:VL heterodimer containing an antigen-binding site that retains much of the antigen recognition and binding capabilities of a native antibody molecule.

[0241] Each antibody, antibody fragment, or derivative may comprise a set of heavy and light chain complementarity determining regions ("CDRs") interposed between a set of heavy and light chain frameworks ("FRs") that provide support for the CDRs and define the spatial relationship of the CDRs to each other. A set of CDRs may contain three hypervariable regions, consisting of heavy chain V regions or light chain V regions.

[0242] Other suitable methods for generating or isolating antibodies with the required specificity can be used, including, but not limited to, recombinant antibody selection from peptide or protein libraries (e.g., but not limited to, display libraries such as bacteriophage libraries, ribosomal libraries, oligonucleotide libraries, RNA libraries, cDNA libraries, yeast libraries, etc.) using methods known in the art, which are commercially available from a variety of commercial sources, including Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinsreid / Planegg, Del.), Biovation (Aberdeen, Scotland, UK), and BioInvent (Lund, Sweden). See U.S. Patent Nos. 4,704,692; 5,723,323; 5,763,192; 5,814,476; 5,817,483; 5,824,514; and 5,976,862. An alternative method relies on immunization of transgenic animals capable of generating a repertoire of human antibodies (e.g., SCID mice; Nguyen et al. (1997), Microbiol. Immunol., 41:901-907; Sandhu et al. (1996), Crit. Rev. Biotechnol., 16:95-118; Eren et al. (1998), Immunol., 93:154-161), as known in the art and / or described herein.Such techniques include ribosome display (Hanes et al. (1997), Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes et al. (1998), Proc. Natl. Acad. Sci. USA, 95:14130-14135); single-cell antibody generation techniques (e.g., selected lymphocyte antibody method ("SLAM") (U.S. Patent No. 5,627,052; Wen et al. (1987), J. Immunol., 17:887-892; Babcook et al. (1996), Proc. Natl. Acad. Sci. USA, 93:7843-7848); gel microdroplets and flow cytometry (Powell et al. (1990), Biotechnol., 8:333-337; One Cell Systems (Cambridge, Mass; Gray et al. (1995), J. Imm. Meth., 182:155-163; Kenny et al. (1995), Bio / Technol., 13:787-790); B cell selection (Steenbakkers et al. (1994), Molec. Biol. Reports, 19:125-134 (1994)).

[0243] Affinity matured antibodies can be produced by any one of a number of procedures known in the art, for example, Marks et al., BioTechnology, 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues has been described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169:147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol., 154(7):3310-3319 (1995); Hawkins et al., J. Mol. Biol., 226:889-896 (1992). Selective mutagenesis positions and selective mutations at contact or hypermutation positions with activity-enhancing amino acid residues are described in U.S. Pat. No. 6,914,128 B1.

[0244] Antibody variants may also be prepared using delivery of a polynucleotide encoding the antibody to a suitable host, e.g., to provide transgenic animals or mammals, such as goats, cows, horses, sheep, and the like, which produce such antibodies in their milk. Such methods are known in the art and are described, for example, in U.S. Patent Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; and 5,304,489.

[0245] Antibody variants can also be prepared by delivering polynucleotides to transgenic plants and cultured plant cells (e.g., but not limited to, tobacco, corn, and duckweed) that produce such antibodies, specified portions, or variants in plant parts or cells cultured therefrom. For example, Cramer et al. (1999) Curr. Top. Microbiol. Immunol. 240:95-118 and references cited therein describe the production of transgenic tobacco leaves that express large amounts of recombinant proteins, e.g., using inducible promoters. Transgenic corn has been used to express mammalian proteins at commercial production levels with biological activity equivalent to mammalian proteins produced in other recombinant systems or purified from natural sources. See, e.g., Hood et al., Adv. Exp. Med. Biol. (1999) 464:127-147 and references cited therein. Antibody variants have also been produced in large quantities from transgenic plant seeds containing antibody fragments, such as single-chain antibodies (scFv), including tobacco seeds and potato tubers. See, e.g., Conrad et al. (1998), Plant Mol. Biol., 38:101-109 and references cited therein. Thus, antibodies can also be produced using transgenic plants according to known methods.

[0246] Derivatives of antibodies can be made, for example, by adding exogenous sequences to modify immunogenicity or to reduce, enhance, or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic. Generally, non-human sequences in the variable and constant regions are substituted with human or other amino acids, while some or all of the non-human or human CDR sequences are maintained.

[0247] Small antibody fragments may be diabodies having two antigen-binding sites, in which the fragment comprises a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) (VH VL) in the same polypeptide chain. See, e.g., EP 404,097; WO 93 / 11161 and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. See also U.S. Pat. No. 6,632,926 by Chen et al., which is incorporated herein by reference in its entirety and which also discloses antibody mutants in which one or more amino acids are inserted into the hypervariable region of a parent antibody, resulting in a binding affinity for a target antigen that is at least about two-fold stronger than the binding affinity of the parent antibody for that antigen.

[0248] The antibody may be a linear antibody. Procedures for making linear antibodies are known in the art and are described in Zapata et al. (1995), Protein Eng., 8(10):1057-1062. Briefly, these antibodies contain a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions. Linear antibodies may be bispecific or monospecific.

[0249] Antibodies can be recovered and purified from recombinant cell culture by known methods, including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification.

[0250] It may be useful to detectably label an antibody. Methods for conjugating antibodies to these agents are known in the art. By way of example only, antibodies may be labeled with a detectable moiety such as a radioactive atom, a chromophore, a fluorophore, or the like. Such labeled antibodies may be used for diagnostic methods in vivo or in an isolated test sample. Such labeled antibodies may be linked to a cytokine, a ligand, or another antibody. Suitable agents for coupling to antibodies to achieve an anti-tumor effect include cytokines, such as interleukin 2 (IL-2) and tumor necrosis factor (TNF); photosensitizing agents for use in photodynamic therapy, including aluminum(III) phthalocyanine tetrasulfonate, hematoporphyrin, and phthalocyanine; radionuclides, such as iodine-131 (131I), yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi), technetium-99m (99mTc), and iodine-131 (131I). ), rhenium-186 (186Re) and rhenium-188 (188Re); antibiotics, such as doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin, neocarzinostatin and carboplatin; bacterial, plant and other toxins, such as diphtheria toxin, pseudomonas exotoxin A, staphylococcal enterotoxin A, abrin-A toxin, ricin A (deglycosylated ricin A and native ricin A), TGF-alpha toxin, Chinese cobra (Naja atra) atra) and gelonin (a plant toxin); ribosome-inactivating proteins from plants, bacteria, and fungi, such as restrictocin (a ribosome-inactivating protein produced by Aspergillus restrictus), saporin (a ribosome-inactivating protein from Saponaria officinalis), and RNase; tyrosine kinase inhibitors; ly207702 (a difluorinated purine nucleoside); liposomes containing anticyst agents (e.g., toxins, antisense oligonucleotides encoding methotrexate, plasmids, etc.); and other antibodies or antibody fragments, such as F(ab).

[0251] The generation of antibodies through hybridoma technology, selected lymphocyte antibody method (SLAM), transgenic animals and the use of recombinant antibody libraries are described in more detail below.

[0252] As defined herein, a sample is "suspected" of containing HBV if the sample is obtained from a subject, preferably a human, suspected of being infected with HBV. A subject is suspected of being infected with HBV if the subject has an increased risk of HBV. Newborns born to mothers infected with HBV have a high risk of HBV infection. Other high risk factors for HBV infection include, for example, intravenous drug use, hemophilia, high-risk sexual activity, hemodialysis, needlestick injuries in medical personnel, and body punctures and tattoos.

[0253] The sample can be any suitable sample obtained from any suitable subject, typically a mammal (e.g., a human). Samples can be obtained from any biological source, such as cervical, vaginal, or anal swabs or brushes, or physiological fluids, including, but not limited to, whole blood, serum, plasma, interstitial fluid, saliva, ocular fluid, cerebrospinal fluid, sweat, urine, milk, ascites fluid, mucus, nasal fluid, sputum, synovial fluid, peritoneal fluid, vaginal fluid, menstrual, amniotic fluid, semen, and the like. Samples can be obtained from subjects using routine techniques known to those of skill in the art. The sample can be used directly from the biological source or after pretreatment to modify the characteristics of the sample. Such pretreatment can include, for example, preparation of plasma from blood, dilution of viscous liquids, filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of interfering components, addition of reagents, cell lysis, and the like.

[0254] Aptamer The disclosed methods include the use of aptamers to detect or identify one or more HBV biomarkers. Aptamers are suitable for use in developing probes with high affinity and selectivity for target molecules, such as HBV peptide biomarkers. Aptamers include single-stranded DNA (ssDNA), RNA, or modified nucleic acids, which have the ability to specifically bind to their targets, which range from small organic molecules to proteins and peptides. Target recognition is based on the tertiary structure formed by single-stranded oligonucleotides, as known in the art. In some embodiments, aptamers used to detect or identify one or more HBV biomarkers can be obtained by an in vitro selection process known as SELEX, in which aptamers are selected from a library of random synthetic DNA or RNA sequences by iteratively binding oligonucleotides to target molecules.

[0255] In some embodiments, the nucleic acids constituting the aptamer library mixture used to screen candidate HBV biomarker capture agents may be single-stranded DNA or RNA, with or without chemical modifications. Introduction of additional chemicals into DNA during the selection process can include, for example, the use of 5-alkyne-modified nucleobases (e.g., thymine). Furthermore, 5-C8-alkyne-modified nucleotide triphosphates, such as deoxythymidine, are commercially available or can be synthesized. Such 5-C8-alkyne-modified nucleobases can be introduced into DNA by PCR. Such modifications can be further derivatized by so-called bioorthogonal chemistry, for example, using Cu(I)-catalyzed 1,3-dipolar cycloaddition of the respective azides with alkynes. In addition to Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), the copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) reaction is also useful. In some embodiments involving cellular or biological systems, strain-promoted azide-alkyne cycloaddition can overcome toxicity issues associated with the use of Cu(I). Any number of desirable chemical modifications can be added to the oligonucleotide libraries used for screening purposes. Examples of such modifications include, but are not limited to, aliphatic, aromatic, charged, basic, acidic, heteroaromatic, saccharide, metal-containing, or peptide residues.

[0256] In some embodiments, the nucleobase to be modified to include an azide alkyne chemical group can include an ethynyl, propynyl, or butynyl-dU, dA, dC, or dG nucleotide. In other embodiments, the nucleobase to be modified to include an azide alkyne chemical group can be an ethynyl-dU nucleotide, an ethynyl-dA nucleotide, an ethynyl-dC nucleotide, or an ethynyl-dG nucleotide. Nucleotide aptamer libraries with these example modifications can be used in various SELEX-based selection methods to enhance the chemical diversity of DNA aptamer libraries. The starting mixture of nucleic acids, i.e., the candidate mixture, can be modified so that at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% of the components of the mixture are modified to include functionalization, for example, by click chemistry. Modification of less than 100% allows for increased diversity by allowing some positions in the oligonucleotides to be modified but others not, while 100% modification ensures consistency during the selection process. In some embodiments, different modifications are made at different positions of the oligonucleotides to further increase diversity.

[0257] Aptamers that recognize HBV can be used in various methods to detect the presence or level of one or more HBV biomarkers in a biological sample (e.g., a biological substance of interest, such as a protein, nucleic acid, or microvesicle). Aptamers can function as binders or capture agents to assess the presence or level of relevant HBV biomarkers. In various diagnostic and / or prognostic embodiments of the present disclosure, one or more aptamers can be configured in a ligand-target assay, where one or more aptamers are contacted with a selected biological sample such that the one or more aptamers associate with or bind to their target HBV biomarker molecules. Aptamers can also be used to identify a profile of multiple HBV biomarkers (a "biomarker" profile or signature) based on the biological sample being evaluated and the biomarkers detected. A biomarker profile of a biological sample can include the presence, level, or other characteristics of one or more biomarkers of interest that can be assessed, including, but not limited to, the presence, level, sequence, mutation, rearrangement, rearrangement, deletion, extragenic modification, methylation, post-translational modification, allele, activity, complex partner, stability, half-life, etc.

[0258] Biomarker profiles or signatures can be used to assess diagnostic and / or prognostic criteria, such as the presence of disease, staging of disease, monitoring of disease, stratifying disease, or surveillance for the detection, metastasis, recurrence, or progression of disease. For example, methods of the present disclosure can include methods for correlating HBV biomarker profiles with selected conditions or diseases. Biomarker profiles can also be used clinically to make decisions regarding treatment measures, including therapeutic interventions. Biomarker profiles based on aptamer detection, identification, and / or quantification can further be used clinically to make treatment decisions, including whether to alter the course of treatment, e.g., whether a different HBV therapeutic should be administered to a subject.

[0259] b. Assay Variations The disclosed methods for determining the presence or amount of an analyte of interest (e.g., an HBV biomarker) present in a sample can be as described herein. The methods can also be adapted to account for other methods for analyzing the analyte. Examples of well-known variations include, but are not limited to, sandwich immunoassays (e.g., monoclonal-monoclonal sandwich immunoassays, monoclonal-polyclonal sandwich immunoassays, including enzyme detection (enzyme immunoassays (EIA) or enzyme-linked immunosorbent assays (ELISA))), competitive inhibition immunoassays (e.g., forward and reverse), immunoassays such as enzyme multiplexed immunoassay technique (EMIT), competitive binding assays, bioluminescence resonance energy transfer (BRET), one-step antibody detection assays, homogeneous assays, heterogeneous assays, capture-on-the-fly assays, etc.

[0260] i. Immunoassay Analytes of interest and / or peptides or fragments thereof (e.g., HBV biomarkers and / or peptides or fragments thereof) can be analyzed using HBV biomarker antibodies in immunoassays. The presence or amount of an analyte (e.g., an HBV biomarker) can be determined by using the antibody to detect specific binding to the analyte. For example, an antibody or antibody fragment thereof can specifically bind to the analyte. If desired, one or more of the antibodies can be used in combination with one or more commercially available monoclonal / polyclonal antibodies. Such antibodies are available from companies such as R&D Systems, Inc. (Minneapolis, MN) and Enzo Life Sciences International, Inc. (Plymouth Meeting, PA).

[0261] The presence or amount of an analyte (e.g., an HBV biomarker) present in a body sample can be readily determined using immunoassays such as sandwich immunoassays (e.g., monoclonal-monoclonal sandwich immunoassays, monoclonal-polyclonal sandwich immunoassays, including radioisotope detection (radioimmunoassays (RIA))), and enzyme detection (enzyme immunoassays (EIA) or enzyme-linked immunosorbent assays (ELISA) (e.g., Quantikine ELISA assays, R&D Systems, Minneapolis, MN)). An example of a point-of-care device that can be used is i-STAT® (Abbott Laboratories, Abbott Park, IL). Other methods that can be used include chemiluminescent microparticle immunoassays, including, by way of example, methods using the ARCHITECT® automated analyzer (Abbott Laboratories, Abbott Park, IL). Other methods include, for example, mass spectrometry and immunohistochemistry (e.g., using thin sections from tissue biopsies) using anti-analyte (e.g., anti-HBV biomarker) antibodies (monoclonal, polyclonal, chimeric, humanized, human, etc.) or antibody fragments thereof directed against the analyte (e.g., HBV biomarker). Other detection methods include those described in, for example, U.S. Patent Nos. 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and 5,480,792, each of which is hereby incorporated by reference in its entirety. Specific immunological binding of an antibody to an analyte can be detected by direct labels, such as fluorescent or luminescent tags, metals and radionuclides, attached to the antibody, or by indirect labels, such as alkaline phosphatase or horseradish peroxidase.

[0262] The use of immobilized antibodies or antibody fragments thereof can be incorporated into immunoassays. Antibodies may be immobilized on a variety of supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (such as a microtiter well), a piece of solid substrate material, and the like. An assay strip can be prepared by coating an antibody or multiple antibodies in an array on a solid support. The strip can then be dipped into a test sample and rapidly processed through washing and detection steps to generate a measurable signal, such as a colored dot.

[0263] A homogeneous format may also be used. For example, after a test sample is obtained from a subject, a mixture is prepared. The mixture includes the test sample to be assessed for an analyte (e.g., an HBV biomarker) and a specific binding partner. The order in which the test sample and specific binding partner are added to form the mixture is not critical. The test sample is contacted with the specific binding partner simultaneously. In some embodiments, the specific binding partner and any HBV biomarker contained in the test sample can form a specific binding partner-analyte (e.g., HBV biomarker)-antigen complex. The specific binding partner may be an anti-analyte antibody (e.g., an anti-HBV biomarker antibody that binds to an epitope having an amino acid sequence comprising at least three consecutive (3) amino acids of an HBV biomarker). Additionally, the specific binding partner may be labeled with or include a detectable label as described above.

[0264] A heterogeneous format may also be used. For example, after a test sample is obtained from a subject, a first mixture is prepared. The mixture includes the test sample to be assessed for an analyte (e.g., an HBV biomarker) and a first specific binding partner, where the first specific binding partner and any HBV biomarker contained in the test sample form a first specific binding partner-analyte (e.g., HBV biomarker)-antigen complex. The first specific binding partner may be an anti-analyte antibody (e.g., an anti-HBV biomarker antibody that binds to an epitope having an amino acid sequence comprising at least three consecutive (3) amino acids of the HBV biomarker). The order in which the test sample and the first specific binding partner are added to form the mixture is not critical.

[0265] The first specific binding partner can be immobilized on a solid phase. The solid phase used in the immunoassay (for the specific binding partner) can be any solid phase known in the art, including, but not limited to, magnetic particles, beads, test tubes, microtiter plates, cuvettes, membranes, scaffold molecules, films, filter paper, disks, and chips. In embodiments in which the solid phase is a bead, the bead can be a magnetic bead or particle. The magnetic bead / particle can be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic, or ferrofluid. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrO2, MnAs, MnBi, EuO, and NiO / Fe. Examples of ferrimagnetic materials include NiFe2O4, CoFe2O4, Fe3O4 (or FeO-Fe2O3). The beads can have a solid core that is magnetic and surrounded by one or more nonmagnetic layers. Alternatively, the magnetic portion can be a layer around the nonmagnetic core. The solid support on which the first specific binding member is immobilized may be stored dry or in liquid. The magnetic beads may be exposed to a magnetic field either before or after contacting the sample with the magnetic beads on which the first specific binding member is immobilized.

[0266] After a mixture containing the first specific binding partner-analyte (e.g., HBV biomarker) antigen complex is formed, any unbound analyte (e.g., HBV biomarker) is removed from the complex using any technique known in the art. For example, unbound analyte can be removed by washing. However, it is desirable that the first specific binding partner be present in excess of any analyte present in the test sample so that all analytes present in the test sample are bound by the first specific binding partner.

[0267] After removing any analyte (e.g., HBV biomarker), a second specific binding partner is added to the mixture to form a first specific binding partner-analyte of interest (e.g., HBV biomarker)-second specific binding partner complex. The second specific binding partner can be an anti-analyte antibody (e.g., an HBV biomarker antibody that binds to an epitope having an amino acid sequence comprising at least three consecutive (3) amino acids of the HBV biomarker). Additionally, the second specific binding partner is labeled with or includes a detectable label as described above.

[0268] The use of immobilized antibodies or antibody fragments thereof can be incorporated into immunoassays. Antibodies may be immobilized on a variety of supports, such as magnetic or chromatographic matrix particles (such as magnetic beads), latex particles or surface-modified latex particles, polymers or polymer films, plastics or plastic films, planar substrates, the surface of assay plates (such as microtiter wells), pieces of solid substrate material, and the like. An assay strip can be prepared by coating an antibody or multiple antibodies in an array on a solid support. The strip can then be dipped into a test sample and rapidly processed through washing and detection steps to generate a measurable signal, such as a colored dot.

[0269] ii. Sandwich immunoassay Sandwich immunoassays measure the amount of antigen between two layers of antibodies (i.e., at least one capture antibody) and detection antibodies (i.e., at least one detection antibody). The capture antibody and detection antibody bind to different epitopes on the antigen, e.g., the analyte of interest, such as an HBV biomarker. It is desirable that the binding of the capture antibody to the epitope does not interfere with the binding of the detection antibody to the epitope. In sandwich immunoassays, either monoclonal or polyclonal antibodies can be used as the capture and detection antibodies.

[0270] Typically, at least two antibodies are used to separate and quantify an analyte (e.g., an HBV biomarker) in a test sample. More specifically, at least two antibodies bind to a specific epitope on the analyte, forming an immune complex called a "sandwich." One or more antibodies can be used to capture the analyte in the test sample (these antibodies are often referred to as a "capture" antibody or antibodies), and one or more antibodies are used to bind a detectable (i.e., quantifiable) label to the sandwich (these antibodies are often referred to as a "detection" antibody or antibodies). In a sandwich assay, it is desirable that the binding of an antibody to its epitope is not diminished by the binding of any other antibody in the assay to its respective epitope. Antibodies are selected so that one or more first antibodies contacted with a test sample suspected of containing the analyte bind all or part of the epitope recognized by a second or subsequent antibody, thereby not interfering with the ability of one or more second detection antibodies to bind the analyte.

[0271] The antibody can be used as the first antibody in the immunoassay. The antibody immunospecifically binds to an epitope on an analyte (e.g., an HBV biomarker). In addition to the antibody of the present disclosure, the immunoassay can include a second antibody that immunospecifically binds to an epitope not recognized or bound by the first antibody.

[0272] A test sample suspected of containing an analyte (e.g., an HBV biomarker) can be contacted simultaneously or sequentially with at least one first capture antibody (or antibodies) and at least one second detection antibody. In a sandwich assay format, the test sample suspected of containing the analyte is first contacted with at least one first capture antibody that specifically binds to a particular epitope under conditions that allow the formation of a first antibody-analyte antigen complex. If more than one capture antibody is used, a first multi-capture antibody-HBV biomarker antigen complex is formed. In a sandwich assay, antibodies, such as at least one capture antibody, are used in molar excess over the maximum amount of analyte expected in the test sample. For example, about 5 μg / mL to about 1 mg / mL of antibody per ml of microparticle coating buffer can be used.

[0273] iii. Single molecule detection The methods and kits described herein may also include single molecule counting. In certain embodiments, the method of analyzing an analyte may include assessing the analyte present in a sample. In certain embodiments, assessing may be used to determine the presence and / or concentration of the analyte in the sample. In certain embodiments, the method may be used to determine the presence and / or concentration of multiple different analytes present in a sample.

[0274] Any device known in the art that allows for the detection of single molecules of one or more analytes of interest can be used in the systems described herein. For example, the device can be a microfluidic device, a digital microfluidic device (DMF), a surface acoustic wave-based microfluidic device (SAW), an integrated DMF and analyte detection device, an integrated SAW and analyte detection device, or a robotics-based assay processing unit. Examples of other devices that can be used include the Quanterix SIMOA™ (Lexington, MA), Singulex's Single Molecule Counting (SMC™) technology (Alameda, CA; see, e.g., U.S. Pat. No. 9,239,284, the contents of which are incorporated herein by reference), and the like.

[0275] Other methods of detection include the use of, or can be adapted for use in, nanopore or nanowell devices. Examples of nanopore devices are described in WO 2016 / 161402, which is hereby incorporated by reference in its entirety. Examples of nanowell devices are described in WO 2016 / 161400, which is hereby incorporated by reference in its entirety.

[0276] The methods and kits described herein may include mass spectrometry using DIA-MS, DDA-MS, or SRM / MRM-MS or PRM-MS. In certain embodiments, the method of analyzing an analyte may include evaluating a sample for the presence of the analyte. In certain embodiments, evaluating a sample for the presence of the analyte can be used to determine the presence and / or concentration of the analyte or fragment in the sample. In certain embodiments, the method can also be used to determine the presence and / or concentration of different analytes or fragments of analytes present in the sample. Quantitation can be performed using an internal control protein or peptide fragment.

[0277] 6. Sample As used herein, the terms "sample," "test sample," and "biological sample" refer to a bodily fluid sample containing or suspected of containing an HBV biomarker. The sample may be derived from any suitable source. In some cases, the sample may comprise a liquid, a flowable particulate solid, or a fluid suspension of solid particles. In some cases, the sample may be processed prior to the analysis described herein. For example, the sample may be separated or purified from its source prior to analysis. However, in certain embodiments, an unprocessed sample containing an HBV biomarker may be assayed directly. In one example, the source containing the HBV biomarker is human biological material (e.g., bodily fluid, blood, e.g., whole blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, tears, lymph, amniotic fluid, interstitial fluid, lung lavage fluid, cerebrospinal fluid, feces, tissue, organ, etc.). The tissue may include, but is not limited to, skeletal muscle tissue, liver tissue, lung tissue, kidney tissue, cardiac muscle tissue, brain tissue, bone marrow, cervical tissue, skin, etc. The sample can be a liquid sample or a liquid extract of a solid sample. In certain cases, the source of the sample can be an organ or tissue, such as a biopsy sample, which can be solubilized by tissue disruption / cell lysis.

[0278] A wide range of bodily fluid sample volumes can be analyzed. In some exemplary embodiments, the sample volume can be about 0.5 nL, about 1 nL, about 3 nL, about 0.01 μL, about 0.1 μL, about 1 μL, about 5 μL, about 10 μL, about 100 μL, about 1 mL, about 5 mL, about 10 mL, or others. In some cases, the volume of the bodily fluid sample is between about 0.01 μL and about 10 mL, between about 0.01 μL and about 1 mL, between about 0.01 μL and about 100 μL, or between about 0.1 μL and about 10 μL.

[0279] In some cases, the body fluid sample may be diluted before use in the assay. For example, in embodiments where the source containing the HBV biomarker is a human biological fluid (e.g., blood, serum), the body fluid may be diluted with an appropriate solvent (e.g., a buffer solution, e.g., a PBS buffer solution). The body fluid sample may be diluted about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold or more before use. In other cases, the body fluid sample is not diluted before use in the assay.

[0280] In some cases, the sample may undergo pre-analysis treatment. Pre-analysis treatment may provide additional functionality, such as non-specific protein removal and / or effective but inexpensive mixing functionality. Common methods of pre-analysis treatment may include the use of electrokinetic trapping, AC electrokinetics, surface acoustic waves, isotachophoresis, dielectrophoresis, electrophoresis, or other pre-concentration techniques known in the art. In some cases, the body fluid sample may be concentrated before use in an assay. For example, in embodiments where the source containing the HBV biomarker is a human biological fluid (e.g., blood, serum), the body fluid may be concentrated by precipitation, evaporation, filtration, centrifugation, or a combination thereof. The body fluid sample may be concentrated about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold, or more, before use.

[0281] It may be desirable to include a control. The control may be analyzed simultaneously with the subject-derived sample. Results obtained from the subject sample can be compared with results obtained from the control sample. A standard curve may be provided against which the assay results for the sample can be compared. Such a standard curve presents the marker level as a function of assay units, i.e., fluorescent signal intensity if a fluorescent label is used. Samples from multiple donors can be used to provide standard curves for reference levels of HBV biomarkers in normal healthy tissues, as well as for "at-risk" levels of HBV biomarkers in tissues from donors likely to have one or more of the characteristics set forth above.

[0282] In light of the above, therefore, there is provided a method for determining the presence, amount, or concentration of an HBV biomarker in a test sample. The method comprises, for example, assaying the test sample for the HBV biomarker by an immunoassay comprising using at least one capture antibody that binds to an epitope on the HBV biomarker and at least one detection antibody that binds to an epitope on the HBV biomarker different from the epitope on the capture antibody, and optionally comprising a detectable label, and comparing a signal generated by the detectable label as a direct or indirect indicator of the presence, amount, or concentration of the HBV biomarker in the test sample with a signal generated in a calibrator as a direct or indirect indicator of the presence, amount, or concentration of the HBV biomarker. The calibrator is optionally, and in some embodiments, part of a series of calibrators, each of which differs in concentration of the HBV biomarker from the other calibrators in the series.

[0283] 7. Kits and Systems In some embodiments, the present disclosure further provides kits and systems for detecting the presence, level, or status of HBcAg and / or P-HBcAg in a sample. In some embodiments, provided herein are kits or systems comprising reagents for detecting the presence, level, or status of HBcAg. In some embodiments, provided herein are kits or systems comprising reagents for detecting the presence, level, or status of P-HBcAg. In some embodiments, provided herein are kits or systems comprising reagents for detecting the presence, level, or status of HBcAg and P-HBcAg. In some embodiments, the kits or systems find use in multiplex and / or automated analytical methods. Exemplary reagents include, but are not limited to, nucleic acid primers, nucleic acid probes, antibodies, colorimetric reagents, enzymes, buffers, etc.

[0284] Optionally, the kit may include at least one calibrator or control. Any calibrator or control may be included in the kit.

[0285] That is, the present disclosure further provides kits for diagnostics and quality control comprising one or more antibodies or other detection reagents. Optionally, the assays, kits, and kit components of the present disclosure are optimized for use in commercially available platforms (e.g., immunoassays on Prism®, AxSYM®, ARCHITECT®, and EIA (bead) platforms from Abbott Laboratories, Abbott Park, IL, as well as other commercially available and / or in vitro diagnostic assays). Furthermore, the assays, kits, and kit components can be adapted for other formats, such as electrochemical or other handheld or point-of-care assay systems. The present disclosure can be applied, for example, to the commercially available Abbott Point of Care (i-STAT®, Abbott Laboratories, Abbott Park, IL) electrochemical immunoassay system. Immunosensors and methods for operating them in disposable test devices are described, for example, in U.S. Patent Applications Nos. 20030170881, 20040018577, 20050054078, and 20060160164, which are incorporated by reference herein. Further background regarding the manufacture of electrochemical and other types of immunosensors can be found in U.S. Patent No. 5,063,081, which is incorporated by reference for its teachings in this regard.

[0286] Optionally, the kit includes quality control reagents (e.g., sensitivity panels, calibrators, and positive controls), the preparation of which is well known in the art and described, for example, on the package inserts of various immunodiagnostic or nucleic acid products.

[0287] In another embodiment, the present disclosure provides quality control kits comprising one or more antibodies described herein for use as a sensitivity panel to assess assay performance characteristics and / or to quantify and monitor the integrity of antigens or nucleic acids used in the assay.

[0288] The kit may optionally include other reagents necessary to perform the diagnostic assay or to facilitate quality control evaluation, such as buffers, salts, enzymes, enzyme cofactors, substrates, detection reagents, and the like. Other components, such as buffers and solutions for isolating and / or processing the test sample (e.g., pretreatment reagents), may also be included in the kit. In addition, the kit may include one or more other controls. One or more of the components of the kit may be lyophilized, and the kit may further include reagents suitable for reconstituting the lyophilized components.

[0289] The various components of the kit are optionally provided in suitable containers. As mentioned above, one or more of the containers may be microtiter plates. The kit may further include containers for holding or storing samples (e.g., containers or cartridges for blood or urine samples). Where appropriate, the kit may also optionally include reaction vessels, mixing vessels, and reagents or other components that facilitate preparation of test samples. The kit may also include one or more devices to aid in obtaining test samples, such as syringes, pipettes, forceps, measuring spoons, etc.

[0290] The kit may optionally further comprise instructions for use, which may be provided in paper form or in computer readable form such as a disc, CD, DVD, or the like.

[0291] The disclosure described herein can also be adapted for use in a variety of automated and semi-automated systems (including systems in which the solid phase comprises microparticles) and other platforms, such as those described in U.S. Patent Nos. 5,089,424 and 5,006,309, and commercially available from Abbott Laboratories (Abbott Park, Ill.), including, but not limited to, Abbott's ARCHITECT®, AxSYM®, IMX, PRISM®, and Quantum II instruments. Additionally, the present disclosure is optionally adaptable for Abbott Laboratories' commercially available Point of Care (i-STAT™) electrochemical immunoassay system for performing sandwich immunoassays. Immunosensors and methods for their fabrication and operation in disposable test devices are described, for example, in U.S. Pat. No. 5,063,081, U.S. Patent Application Publication No. 2003 / 0170881, U.S. Patent Application Publication No. 2004 / 0018577, U.S. Patent Application Publication No. 2005 / 0054078, and U.S. Patent Application Publication No. 2006 / 0160164, which are incorporated by reference in their entireties for their teachings regarding same.

[0292] 8. HBV Treatment Drugs For any of the methods described herein, the method may further include identifying a treatment for chronic HBV infection and administering the treatment to the subject. For example, in some embodiments, the method includes detecting the presence, level, or status of HBcAg and / or P-HBcAg in a sample obtained from a subject diagnosed with HBV, selecting an appropriate treatment based on the presence, level, or status of HBcAg and / or P-HBcAg in the sample, and providing the treatment to the subject. In some embodiments, the subject is receiving treatment for chronic HBV. In some embodiments, the subject is receiving treatment for chronic HBV, and the methods described herein are employed to monitor responsiveness to the treatment.

[0293] For any of the methods described herein, the treatment may be any suitable HBV treatment, including those described in more detail below.

[0294] The treatment is an agent that affects the reverse transcription of HBV RNA (e.g., pgRNA) into HBV DNA. For example, some HBV treatments, such as nucleoside inhibitors, inhibit the reverse transcription of pgRNA, thus reducing the amount of HBV DNA (e.g., infectious particles) released from cells. Therefore, by measuring HBcAg, which is an indicator of the amount of infectious DNA particles in a sample, it is possible to determine whether such a treatment is effective or ineffective in a subject. However, such HBV treatments do not interfere with the process of transcribing cccDNA into pgRNA, and therefore, these treatments do not reduce the amount of HBV RNA (e.g., pgRNA) secreted from cells in "empty" or "non-infectious" particles. Therefore, the measurement of P-HBcAg, which correlates with non-infectious HBV particles, should not be affected by such treatments.

[0295] Therapeutic agents used to treat HBV include any of the following, as further described herein. Generally, the purpose and / or ultimate goal of such therapeutic agents is to silence and / or eliminate covalently closed circular DNA (cccDNA). cccDNA is a DNA structure that occurs in the cell nucleus during HBV proliferation. cccDNA can form stable microchromosomes in the nucleus of infected cells. cccDNA can serve as a template for viral replication and enable the production of viral antigens. Chronic HBV infection is characterized by the persistence of cccDNA microchromosomes in the nuclei of infected subjects' hepatocytes. Current HBV treatments are unable to eliminate cccDNA microchromosomes from host cells; therefore, elimination of cccDNA is considered a "functional cure" in subjects with HBV. Therefore, various therapeutic agents useful for silencing and / or eliminating cccDNA can be used in the methods described herein. In some embodiments, the HBV treatment includes tenofovir disoproxil fumarate, emtricitabine (Truvada®), adefovir, clevudine, ABX-203, lamivudine, PEG-IFN alpha, ABX-203, adefovir, PEG-IFN alpha, and GBV-015. DNA polymerase inhibitors include, for example, besifovir, entecavir (Baraclude®), adefovir (Hepsera®), tenofovir disoproxil fumarate (Viread®), tenofovir alafenamide, tenofovir, tenofovir disoproxil, tenofovir alafenamide fumarate, tenofovir alafenamide hemifumarate, tenofovir dipivoxil, tenofovir dipivoxil fumarate, tenofovir octadecyloxyethyl ester, telbivudine (Tyzeka®), pradefovir, clevudine, emtricitabine (Emtriva®), ribavirin, lamivudine (Epivir-HBV®), phosphazides, famciclovir, SNC-019754, FMCA, fusolin, AGX-1009, and metacavir.

[0296] In some embodiments, the HBV therapeutic agent comprises an immunomodulator, such as lintatolimod, imidole hydrochloride, Ingaron, DermaVir, Plaquenil (hydroxychloroquine), Proleukin, hydroxyurea, mycophenolate mofetil (MPA) and its ester derivative mycophenolate mofetil (MMF), WF-10, ribavirin, IL-12, the polymer polyethyleneimine (PEI), Gepon, VGV-1, MOR-22, BMS-936559, and IR-103. In some embodiments, the HBV treatment comprises a toll-like receptor 7 modulator, such as GS-9620, GSK-2245035, imiquimod, resiquimod, DSR-6434, DSP-3025, IMO-4200, MCT-465, 3M-051, SB-9922, 3M-052, Limtop, TMX-30X, TMX-202, RG-7863, and RG-7795. In some embodiments, the HBV treatment comprises a nucleic acid polymer (NAP), such as REP2139.

[0297] Toll-like receptor 8 modulators include motolimod, resiquimod, 3M-051, 3M-052, MCT-465, IMO-4200, VTX-763, and VTX-1463. Toll-like receptor 3 modulators include lintatolimod, polyICLC, MCT-465, MCT-475, Riboxxon, Riboxxim, and ND-1.1. Interferon alpha receptor ligands include interferon alpha-2b (Intron A (registered trademark), pegylated interferon alfa-2a (Pegasys®), interferon alfa 1b (Hapgen®), Veldona, Infradure, Roferon-A, YPEG-interferon alfa-2a (YPEG-rhIFN alfa-2a), P-1101, Algeron, Alfarona, Ingaron (interferon gamma), rSIFN-co (recombinant super compound interferon), Y pegylated interferon alfa-2b (YPEG -rhIFN alpha-2b), MOR-22, pegylated interferon alpha-2b (PEG-Intron®), Bioferon, Novaferon, Inmutag (IFN), Multiferon®, interferon alpha-nl (Humoferon®), interferon beta-1a (Avonex®), Shaferon, interferon alpha-2b (AXXO), Alfaferone, interferon alpha-2b (BioGeneric Pharma), interferon alpha-2 (CJ), Laferonum, VIPEG, BLAUFERON-B, BLAUFERON-A, Intermax Alpha, Realdiron, Lanstion, Pegaferon, PDferon-B, interferon alpha-2b (IFN,Laboratories Bioprofarma), alpha interferon a 2b, Kalferon, Pegnano, Feronsure, PegiHep, interferon alpha 2b (Zydus-Cadila), Optipeg A, Realfa 2B, Reliferon, interferon alpha-2b (Amega), interferon alpha 2b (Virchow), pegylated interferon alpha-2b (Amega), Reaferon-EC, Proquiferon, Uniferon, Urifron, interferon alpha-2b (Changchun Institute of Biological Products), Anterferon, Shanferon, Layfferon, Shang Sheng Lei Tai, INTEFEN, SINOGEN, Fukangtai, Pegstat, rHSA-IFN alpha-2b, and Interapo (Interapa).

[0298] Hyaluronidase inhibitors include astodrimer. HBsAg inhibitors include HBF-0259, PBHBV-001, PBHBV-2-15, PBHBV-2-1, REP 9AC, REP-9C, and REP 9AC. Toll-like receptor 9 modulators include CYT003. Cyclophilin inhibitors include OCB-030, SCY-635, and NVP-018. HBV prophylactic vaccines include Hexaxim, Heplisav, Mosquirix, DTwP-HBV vaccine, Bio-Hep-B, D / T / P / HBV / M (LBVP-0101; LBVW-0101), DTwP-Hepb-Hib-IPV vaccine, Heberpenta L, DTwP-HepB-Hib, V-419, CVI-HBV-001, Tetrabhay, hepatitis B prophylactic vaccine (Advax Super D), Hepatrol-07, GSK-223192A, Engerix B®, recombinant hepatitis B vaccine (intramuscular, Kangtai Biological Products), recombinant hepatitis B vaccine (Hansenual polymorphic yeast, intramuscular, Hualan Biological Engineering), Bimmugen, Euforavac, Eutravac, anrix-DTaP-IPV-Hep B, Infanrix-DTaP-IPV-Hep B-Hib, Pentabio Vaksin DTP-HB-Hib, Comvac 4, Twinrix, Euvax-B, Tritanrix HB, Infanrix Hep B, Comvax, DTP-Hib-HBV Vaccine, DTP-HBV Vaccine, Yi Tai, Heberbiovac HB, Trivac HB, GerVax, DTwP-Hep B-Hib Vaccine, Bilive, Hepavax-Gene, SUPERVAX, Comvac5, Shanvac-B, Hebsulin, Recombivax HB, Revac B mcf, Revac B+, Fendrix, DTwP-HepB-Hib, DNA-001, Shan6, rhHBsAG Vaccine and DTaP-rHB-Hib Vaccine.

[0299] HBV therapeutic vaccines include HBsAG-HBIG conjugate, Bio-Hep-B, NASVAC, abi-HB (intravenous), ABX-203, Tetrabhay, GX-110E, GS-4774, peptide vaccine (Epsilon PA-44), Hepatrol-07, NASVAC (NASTERAP), IMP-321, BEVAC, Revac B mcf, Revac B+, MGN-1333, KW-2, CVI-HBV-002, AltraHepB, VGX-6200, FP-02, TG-1050, NU-500, HBV ax, im / TriGrid / antigen vaccine, Mega-CD40L-adjuvanted vaccine, HepB-v, NO-1800, recombinant VLP-based therapeutic vaccine (HBV infection, VLP Biotech), AdTG-17909, and AdTG-17910. Includes AdTG-18202, ChronVac-B and Lm HBV.

[0300] HBV viral entry inhibitors include Myrcludex B. Antisense oligonucleotides targeting viral mRNA include ISIS-HBVRx. Interfering RNAs, including short interfering RNAs (siRNAs), can be used. For example, siRNAs that can be used include TKM-HBV (TKM-HepB), ALN-HBV, SR-008, ddRNAi, and ARC-520. Endonuclease modulators include PGN-514. Inhibitors of ribonucleotide reductase include Trimidox. Inhibitors of hepatitis B virus E antigen include wogonin. HBV antibodies targeting the hepatitis B virus surface antigen include GC-1102, XTL-17, XTL-19, XTL-001, K-003, and fully human monoclonal antibody therapy (Hepatitis B virus infection, Humabs BioMed). HBV antibodies, including monoclonal and polyclonal antibodies, include Zutectra, Shang Sheng Gan Di, Uman Big (Hepatitis B Hyperimmune), Omri-Hep-B, Nabi-HB, Hepatect CP, HepaGam B, igantibe, Niuliva, CT-P24, Hepatitis B Immune Globulin (intravenous, pH 4, HBV infection, Shanghai RAAS Blood Products), and Fovepta (BT-088). CCR2 chemokine antagonists include propagermanium. Thymosin agonists include thymalfasin. Cytokines include recombinant IL-7, CYT-107, interleukin-2 (IL-2, Immunex), recombinant human interleukin-2 (Shenzhen Neptunus), and celmoleukin. Nucleoprotein inhibitors (HBV core or capsid protein inhibitors) include NVR-1221, NVR-3778, BAY41-4109, morphotiazine mesylate, and DVR-23. Stimulators of retinoic acid-inducible gene 1 include SB-9200, SB-40, SB-44, ORI-7246, ORI-9350, ORI-7537, ORI-9020, ORI-9198, and ORI-7170. (28) Stimulators of NOD2 selected from the group consisting of SB-9200.Recombinant thymosin alpha-1 includes NL-004 and PEGylated thymosin alpha-1.

[0301] Hepatitis B virus replication inhibitors include isothiafluridine, IQP-HBV, RM-5038, and Xingantie. PI3K inhibitors include idelalisib, AZD-8186, buparlisib, CLR-457, pictilisib, neratinib, rigosertib, rigosertib sodium, EN-3342, TGR-1202, alpelisib, duvelisib, UCB-5857, taselisib, XL-765, gedatricisib, VS-5584, copanlisib, CAI orotate, perifosine, RG-7666, GSK-2636771, DS-7423, (32) A cccDNA inhibitor selected from the group consisting of BSBI-25, including panulisib, GSK-2269557, GSK-2126458, CUDC-907, PQR-309, INCB-040093, pilaralisib, BAY-1082439, pukitinib mesylate, SAR-245409, AMG-319, RP-6530, ZSTK-474, MLN-1117, SF-1126, RV-1729, sonolicib, LY-3023414, SAR-260301, and CLR-1401.

[0302] PD-L1 inhibitors include MEDI-0680, RG-7446, durvalumab, KY-1003, KD-033, MSB-0010718C, TSR-042, ALN-PDL, STI-A1014, and BMS-936559. PD-1 inhibitors include nivolumab, pembrolizumab, pidilizumab, BGB-108, and mDX-400. BTK inhibitors include ACP-196, dasatinib, ibrutinib, PRN-1008, SNS-062, ONO-4059, BGB-3111, MSC-2364447, X-022, spebrutinib, TP-4207, HM-71224, KBP-7536, and AC-0025.

[0303] Other drugs for treating HBV include gentiopicrin (gentiopicroside), nitazoxanide, birinapant, NOV-205 (Molixan; BAM-205), oligodeoxynucleotides, mivotilate, feron, levamisole, Ka Shu Ning, Alloferon, WS-007, Y-101 (Ti Fen Tai), rSIFN-co, PEG-IIFNm, KW-3, BP-Inter-014, oleanolic acid, HepB-nRNA, cTP-5 (rTP-5), HSK-II-2, HEISCO-106-1, HEISCO-106, Hepbama, IBPB-006IA, Hepuyinfen, DasKloster0014-01, Jiangantai (Ganxikang), picroside, and GA5. Contains NM-HBV, DasKloster-0039, Heplantay, IMB-2613, TCM-800B, reduced glutathione and ZH-2N.

[0304] In some embodiments, the HBV therapeutic agent comprises a capsid assembly modulator (CAM). A capsid assembly modulator refers to an agent that disrupts encapsidation of pregenomic RNA, causing nucleocapsid disintegration and thereby disrupting multiple steps in HBV replication. In some embodiments, the CAM is JNJ-632, AT130, or BAY41-4109.

[0305] In some embodiments, an HBV therapeutic agent can be combined with one, two, three, four, or more additional therapeutic agents. In certain embodiments, a compound disclosed herein, or a pharmaceutically acceptable salt thereof, is combined with two additional therapeutic agents. In other embodiments, a compound disclosed herein, or a pharmaceutically acceptable salt thereof, is combined with three additional therapeutic agents. In further embodiments, a compound disclosed herein, or a pharmaceutically acceptable salt thereof, is combined with four additional therapeutic agents. The one, two, three, four, or more additional therapeutic agents can be different therapeutic agents selected from the same class of therapeutic agents and / or can be selected from different classes of therapeutic agents.

[0306] In one embodiment, the HBV therapeutic agent is an immunomodulator, a toll-like receptor modulator (modulators of tlrl, tlr2, tlr3, tlr4, tlr5, tlr6, tlr7, tlr8, tlr9, tlrl0, tlrl1, tlrl2, and tlrl3), an interferon alpha receptor ligand, a hyaluronidase inhibitor, recombinant IL-7, an HBsAg inhibitor, a compound targeting HBcAg, a cyclophilin inhibitor, an HBV therapeutic vaccine, an HBV prophylactic vaccine, an HBV viral entry inhibitor, an NTCP inhibitor, a viral mRNA inhibitor, a HBV ... Targeted antisense oligonucleotides, short interfering RNA (siRNA), miRNA gene therapy, endonuclease modulators, inhibitors of ribonucleotide reductase, Hepatitis B virus E antigen inhibitors, recombinant scavenger receptor A (SRA) proteins, src kinase inhibitors, HBx inhibitors, cccDNA inhibitors, short synthetic hairpin RNA (sshRNA), HBV antibodies, including HBV antibodies and bispecific antibodies targeting the surface antigen of the Hepatitis B virus, and "antibody-like" therapeutic proteins (e.g., DARTs®, Duobod®). ies®, Bites®, XmAbs®, TandAbs®, Fab derivatives), CCR2 chemokine antagonists, thymosin agonists, cytokines, nucleoprotein inhibitors (HBV core or capsid protein inhibitors), stimulators of retinoic acid-inducible gene 1, stimulators of NOD2, stimulators of NODI, arginase-1 inhibitors, STING agonists, PI3K inhibitors, lymphotoxin beta receptor activators, natural killer cell receptor 2B4 inhibitors, lymphocyte activation gene 3 inhibitors, CD160 inhibitors, cytotoxic T lymphocyte-associated protein 4 inhibitors, CD137 inhibitors, killer cell lectin-like receptor subfamily G member 1 inhibitors, TIM-3 inhibitors, B and T lymphocyte attenuation gene inhibitors, CD305 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, PEG-interferon lambda, recombinant thymosin alpha-1, BTK inhibitors, TIGIT modulators, CD47 modulators, SIRP alpha modulators, ICOS modulators, CD27 modulators, CD70 modulators, OX40 modulators,Modulators of NKG2D, modulators of Tim-4, modulators of B7-H4, modulators of B7-H3, modulators of NKG2A, modulators of GITR, modulators of CD160, modulators of HEVEM, modulators of CD161, modulators of Axl, modulators of Mer, modulators of Tyro, genetic modifiers or editors such as CRISPR (including CRISPR Cas9), zinc finger nucleases or synthetic nucleases (TALEN), and inhibitors of hepatitis B virus replication.

[0307] In one embodiment, HBV therapeutic agents include HBsAg inhibitors, HBV therapeutic vaccines, HBV antibodies including HBV antibodies and bispecific antibodies targeting hepatitis B virus surface antigens and "antibody-like" therapeutic proteins (e.g., DARTs®, Duobodies®, Bites®, XmAbs®, TandAbs®, Fab derivatives), cyclophilin inhibitors, stimulators of retinoic acid-inducible gene 1, PD-1 inhibitors, PD-L1 inhibitors, arginase 1 inhibitors, PI3K inhibitors, and stimulators of NOD2.

[0308] In one embodiment, HBV therapeutic agents include HBV viral entry inhibitors, NTCP inhibitors, HBx inhibitors, cccDNA inhibitors, HBV antibodies targeting hepatitis B virus surface antigens, short interfering RNA (siRNA), miRNA gene therapy drugs, short synthetic hairpin RNA (sshRNA), and nucleoprotein inhibitors (HBV core or capsid protein inhibitors).

[0309] In one embodiment, HBV therapeutics include immunomodulators, toll-like receptor modulators (modulators of tlrl, tlr2, tlr3, tlr4, tlr5, tlr6, tlr7, tlr8, tlr9, tlr10, tlrl1, tlrl2 and tlrl3), HBsAg inhibitors, HBV therapeutic vaccines, HBV antibodies including HBV antibodies and bispecific antibodies targeting the surface antigen of the Hepatitis B virus, and "antibody-like" therapeutic proteins (e.g., DARTs®, Duobodies®, Bites®, XmAbs®, TandAbs®). trademark), Fab derivatives), cyclophilin inhibitors, stimulators of retinoic acid-inducible gene 1, PD-1 inhibitors, PD-L1 inhibitors, arginase 1 inhibitors, PI3K inhibitors and stimulators of NOD2, as well as one or two additional therapeutic agents selected from the group consisting of HBV viral entry inhibitors, NTCP inhibitors, HBx inhibitors, cccDNA inhibitors, HBV antibodies targeting hepatitis B virus surface antigens, short interfering RNA (siRNA), miRNA gene therapy drugs, short synthetic hairpin RNA (sshRNA) and nucleoprotein inhibitors (HBV core or capsid protein inhibitors).

[0310] In one embodiment, the HBV treatment is adefovir (Hepsera®), tenofovir disoproxil fumarate plus emtricitabine (Truvada®), tenofovir disoproxil fumarate (Viread®), entecavir (Baraclude®), lamivudine (Epivir-HBV®), tenofovir alafenamide, tenofovir, tenofovir disoproxil, tenofovir alafenamide fumarate, tenofovir alafenamide hemifumarate, telbivudine (Tyzeka®), Clevudine®, emtricitabine (Emtriva®), pegylated interferon alfa-2b (PEG-Intron®), Multiferon®, interferon alfa 1b (Hapgen®), interferon alfa-2b (Intron®), A (registered trademark), pegylated interferon alfa-2a (Pegasys (registered trademark), interferon alfa-nl (Humoferon (registered trademark), ribavirin, interferon beta-1a (Avonex (registered trademark), Bioferon, Ingaron, Inmutag (IFN), Algeron, Roferon-A, Oligoide, Zutectra, Shaferon, interferon alfa-2b (AXXO), Alfaferone, interferon alfa-2b (BioGeneric Pharma), Feron, interferon alfa 2 (CJ), BEVAC, Laferonum, VIPEG, BLAUFERON-B, BLAUFERON-A, Intermax Alpha, Realdiron, Lanstion, Pegaferon, PDferon-B, interferon alfa-2b (IFN, Laboratorios Bioprofarma), alpha interferon 2b, Kalferon, Pegnano, Feronsure, PegiHep, interferon alpha 2b (Zydus-Cadila), Optipeg A, Realfa2B, Reliferon, interferon alfa-2b (Amega), interferon alfa-2b (Virchow), pegylated interferon alfa-2b (Amega), Reaferon-EC, Proquiferon, Uniferon, Urifron, interferon alfa-2b (Changchun Institute of Biological Products), Anterferon, Shanferon, MOR-22, interleukin-2 (IL-2, Immunex), recombinant human interleukin-2 (Shenzhen Neptunus), Layfferon, Ka Shu Ning, Shang Sheng Lei Tai, INTEFEN, SINOGEN, Fukangtai, Alloferon, and celmoleukin.

[0311] In some embodiments, a combination of one or more HBV therapeutic agents ("cocktails") can be used. Such combinations can be administered simultaneously or sequentially as part of a treatment, optionally alternating over time with various combinations of HBV therapeutic agents. In some embodiments, a treating physician will develop or design an individualized treatment regimen for a subject (meaning a treatment regimen specific to that subject or patient) using one or more HBV therapeutic agents based on clinical parameters, cutoffs, publications, or a combination thereof. In some embodiments, a treating physician will use an algorithm designed to evaluate data related to HBV treatment, as disclosed herein, as part of developing an individualized treatment regimen for a subject. In some embodiments, the administered treatment is part of a clinical trial. Treatment administered as part of a clinical trial includes a treatment regimen designed or developed for one or more subjects or patients by a clinician or physician based on clinical parameters (e.g., clinical parameters obtained from previous clinical trials), cutoffs (e.g., clinical parameters obtained from previous clinical trials), patient profiles, publications, or any combination thereof.

[0312] Embodiments of the present disclosure also include methods of treatment that combine the diagnostic methods described herein with literature-based treatments, protocols, analyses, and combinations thereof to establish an individualized treatment plan (e.g., a personalized treatment regimen) for a subject in need of HBV treatment. In some embodiments, the treatment methods of the present disclosure use one or more published mathematical models that reflect actual clinical data, allowing a prescribing physician to develop an individualized / personalized dosing regimen without the loss of data and / or model resolution that results from distilling the actual clinical data into a relatively coarse, stratified set of recommendations for an "average" or "typical" patient. Generally, the methods include building a mathematical model developed from clinical data collected from patients administered a particular medication (e.g., an HBV treatment), processing the model to create a composite model enriched with patient data, and determining a patient-specific dosing regimen as a function of patient-specific observed response data processed in conjunction with data from the mathematical model. In some embodiments, the methods involve Bayesian averaging, Bayesian updating, and Bayesian prediction techniques to develop patient-specific dosing regimens in response to a general mathematical model and observed patient-specific responses that reflect not only patient-specific characteristics accounted for as patient factors that are covariates in the model, but also "inter-subject variability" that cannot be accounted for within the model itself and that distinguishes a particular patient from the typical patient that the model reflects. Examples of such models are described in U.S. Patent Publication No. 2014 / 0351197, the contents of which are incorporated herein by reference.

[0313] Typical models also account for the expected impact of patient-specific characteristics, such as diagnostic test results, on response and quantify unexplained variability that cannot be attributed solely to patient characteristics. In such models, patient characteristics are reflected as covariates of patient factors in the mathematical model. That is, mathematical models are typically mathematical functions that account for the underlying clinical data and the associated variability observed in patient populations. Although models and functions are referred to herein in a general and non-limiting manner as "mathematical" models and functions, these mathematical functions include terms that account for the variation of individual patients from the "average" or typical patient, allowing the model to explain or predict various outcomes for a given dose, making the model a statistical function rather than just a mathematical function. It will be recognized that many suitable mathematical models already exist and are used for purposes such as drug product development. Examples of suitable mathematical models that describe response profiles for patient populations and account for covariates of patient factors include pharmacokinetic (PK) models, pharmacodynamic (PD) models, and exposure / response models, which are well known to those skilled in the art. Such mathematical models are typically published or available from drug manufacturers, peer-reviewed literature, and the FDA or other regulatory agencies. Alternatively, suitable mathematical models may be prepared through original research.

[0314] Embodiments of the present disclosure also contemplate continuing treatment in subjects currently receiving one or more HBV therapeutic agents to prevent or reduce the risk of DNA reactivation and / or recurrence. In other embodiments, the assays of the present disclosure can be used to predict or determine whether a subject whose treatment has been stopped (e.g., due to seroclearance) is at risk of recurrence.

[0315] All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which this disclosure pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0316] The disclosure adequately described herein may be practiced in the absence of any element or limitation not specifically disclosed herein. That is, for example, in each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced by either of the other two terms. The terms and expressions employed are used as terms of description and not of limitation, and no intention is intended in the use of such terms and expressions to exclude any equivalents of the features shown and described or portions thereof, recognizing that various modifications are possible within the scope of the present disclosure as claimed. That is, while the present disclosure includes various embodiments and optional features, it should be understood that those skilled in the art may resort to modifications and variations of the concepts disclosed herein, and that such modifications and variations are considered to be within the scope of the present disclosure as defined by the appended claims. [Example]

[0317] [Example 1] Described herein is the development and use of two fully automated assays for the specific analysis of phosphorylated HBcAg levels (P-HBcAg, representing non-infectious or "empty" particles) and non-phosphorylated HBcAg levels (representing HBV DNA-containing particles ("infectious" particles)) in patients with active infection at a single time point in longitudinal seroconversion panels and in patients undergoing HBV treatment.

[0318] The assay developed is a chemiluminescent microparticle immunoassay (CMIA) on a fully automated platform that uses specific monoclonal antibodies to capture and detect phosphorylated or non-phosphorylated HBcAg in the circulation of HBV-infected patients.

[0319] method: Detection of P-HBcAg / HBcAg: Patient samples were analyzed in a fully automated manner on the ARCHITECT i2000SR instrument. HBcAg was captured and detected by monoclonal antibodies directed against phosphorylated or non-phosphorylated HBcAg. Detection was achieved by antibodies labeled with a luminescent molecule.

[0320] Other HBV markers: HBV DNA viral load was determined using the Abbott HBV Realtime assay with an input volume of 0.2 ml according to the accompanying instructions. Serology testing was performed on an Abbott ARCHITECT for HBeAg. HBcrAg levels were analyzed on a LUMIPULSE G1200 instrument.

[0321] result: Table 1 shows the limits of detection (LOD) and limits of quantitation (LOQ) of the HBcAg assay, which were determined in triplicate using recombinant P-HBcAg / HBcAg.

[0322] [Table 2]

[0323] HBeAg cross-reactivity was assessed in triplicate with recombinant HBeAg at concentrations up to 10 μg / ml. No cross-reactivity was observed for both P-HBcAg / HBcAg assay formats. The linearity of the P-HBcAg / HBcAg assay was assessed in triplicate with recombinant antigen. Linearity of detection was 5-10 μg / ml for P-HBcAg. 4 pg / ml(R 2 =0.999) and 10 for HBcAg 1 ~10 5 pg / ml(R 2 =0.991).

[0324] Figure 4 shows that approximately 10 3 ~10 9Analysis of P-HBcAg / HBcAg levels in clinical samples containing viral load in HBV DNA copies / ml. P-HBcAg is 10 5 HBcAg could be detected in 84% of samples containing viral loads of 10 cp / ml or greater. 6 It was detected in 94% of samples with viral loads of cp / ml or greater.

[0325] Figure 1 shows an HBV seroconversion panel comparing phosphorylated HBV core antigen (P-HBcAg), HBV core antigen (HBcAg), HBeAg, and HBcrAg levels with HBV DNA levels over time. As shown in Figure 1, P-HBcAg and HBcAg levels correlated well with HBV DNA levels and could detect acute HBV infection, whereas HBcrAg levels appeared to track HBeAg levels more closely than HBcAg / P-HBcAg levels after HBV DNA reached peak levels.

[0326] HBV-positive subjects received antiviral treatment with nucleoside(t) analogs, interferon, or HBsAg inhibitors. P-HBcAg, HBV DNA, HBcAg, and HBV RNA levels were measured in samples obtained from the subjects at 1- to 4-week intervals during treatment, beginning on the first day of treatment and ending 146 weeks after treatment. As shown in Figure 2, HBcAg levels (black line) correspond to HBV DNA levels over the course of treatment. Therefore, this data demonstrates that non-phosphorylated HBcAg (present in infectious HBV particles, black line) can indicate the effectiveness of nucleoside analogs (i.e., a decrease in HBcAg levels) just at the beginning of treatment. Furthermore, phosphorylated P-HBcAg (present in non-infectious particles, blue line) can measure cccDNA transcriptional activity (i.e., non-infectious empty particles are still secreted despite undetectable HBV DNA in the blood).

[0327] For completeness, various aspects of this disclosure are presented in the following numbered clauses.

[0328] Clause 1. A method of assessing and monitoring the stage or phase of chronic hepatitis B (HBV) infection in a subject or monitoring response to treatment of chronic HBV, comprising: a) performing an assay to detect the presence or level of hepatitis B core antigen (HBcAg) and phosphorylated hepatitis B core antigen (P-HBcAg) in at least one sample obtained from a subject diagnosed with or receiving treatment for chronic HBV, the assay comprising contacting the at least one sample with an antibody that specifically binds HBcAg and an antibody that specifically binds P-HBcAg; and b) assessing and monitoring the stage or phase of chronic HBV infection or monitoring the response to treatment of chronic HBV based on the presence or level of HBcAg and P-HBcAg in at least one sample. A method comprising:

[0329] Clause 2. The method of clause 1, wherein the subject is being evaluated and monitored for a stage or phase of chronic HBV, and further comprising providing the subject with treatment for chronic HBV based on the presence or level of HBcAg and P-HBcAg in at least one sample.

[0330] Clause 3. The method of clause 1, wherein the subject is undergoing treatment for chronic HBV, and further comprising altering the HBV treatment based on the presence or level of HBcAg and P-HBcAg in the at least one sample.

[0331] Clause 4. A method for assessing and monitoring a stage or phase of chronic hepatitis B (HBV) infection, comprising: a) performing an assay to detect the presence or level of hepatitis B core antigen (HBcAg) in at least one sample obtained from a subject diagnosed with chronic HBV, the assay comprising contacting the at least one sample with an antibody that specifically binds to HBcAg; and b) determining the amount of infectious HBV particles in the at least one sample based on the presence or level of HBcAg in the at least one sample, wherein the amount of infectious HBV particles is determined regardless of whether the subject has been treated for HBV prior to performing the assay of step a). A method comprising:

[0332] Clause 5. The method of clause 4, further comprising providing treatment for chronic HBV to the subject if the amount of infectious HBV in at least one sample equals or exceeds a threshold value.

[0333] Clause 6. A method for assessing and monitoring the stage or phase of chronic Hepatitis B (HBV) infection, comprising: a) performing an assay to detect the presence or level of hepatitis B core antigen (HBcAg) in at least one sample obtained from a subject diagnosed with chronic HBV, the assay comprising contacting the at least one sample with an antibody that specifically binds to HBcAg; b) determining the amount of infectious HBV particles in the at least one sample based on the presence or level of HBcAg in the at least one sample, wherein the amount of infectious HBV particles is determined regardless of whether the subject has been treated for HBV prior to performing the assay of step a); and c) providing treatment for chronic HBV to the subject if the amount of infectious HBV particles in at least one sample equals or exceeds a threshold value. A method comprising:

[0334] Clause 7. A method for monitoring response to treatment of chronic hepatitis B (HBV) infection in a subject, comprising: a) performing an assay to detect the presence or level of hepatitis B core antigen (HBcAg) in at least one sample obtained from a subject undergoing treatment for chronic HBV, the assay comprising contacting the at least one sample with an antibody that specifically binds to HBcAg; b) determining the amount of infectious HBV particles in the at least one sample based on the presence or level of HBcAg in the at least one sample; and c) determining that the treatment is effective if the amount of infectious HBV particles in at least one sample is below the reference level; or d) determining that the treatment is ineffective if the amount of infectious HBV particles in at least one sample is greater than or equal to the reference level. A method comprising:

[0335] Clause 8. A method for monitoring response to treatment of chronic hepatitis B (HBV) infection in a subject, comprising: a) performing an assay to detect the presence or level of hepatitis B core antigen (HBcAg) and phosphorylated hepatitis B core antigen (P-HBcAg) in at least one sample obtained from a subject undergoing treatment for chronic HBV, the assay comprising contacting the at least one sample with an antibody that specifically binds HBcAg and an antibody that specifically binds P-HBcAg; b) determining the amount of infectious HBV particles in the at least one sample based on the presence or level of HBcAg in the at least one sample; c) determining the amount of non-infectious HBV particles in the at least one sample based on the presence or level of P-HBcAg in the at least one sample; and d) determining the response to treatment of chronic HBV based on the amount of infectious and / or non-infectious HBV particles in at least one sample. A method comprising:

[0336] Clause 9.a) The treatment is determined to be effective if the amount of infectious HBV particles in at least one sample is below the reference level; or b) the treatment is determined to be effective if the amount of infectious HBV particles in at least one sample is below the reference level and the amount of non-infectious HBV particles in at least one sample is below the reference level, or c) the treatment is determined to be ineffective if the amount of infectious HBV particles in at least one sample is greater than or equal to the reference level; The method described in clause 8.

[0337] Clause 10. A method for monitoring response to treatment of chronic hepatitis B (HBV) infection in a subject, comprising: a) performing an assay to detect the presence or level of hepatitis B core antigen (HBcAg) and phosphorylated hepatitis B core antigen (P-HBcAg) in at least two samples obtained from a subject undergoing treatment for chronic HBV, wherein the at least two samples include a first sample obtained from the subject at a first time point before or after receiving treatment for chronic HBV and a second sample obtained from the subject at a second time point after the first time point, and the assay comprises contacting the at least two samples with an antibody that specifically binds HBcAg and an antibody that specifically binds P-HBcAg; b) determining the amount of infectious HBV particles in at least two samples based on the presence or level of HBcAg and / or determining the amount of non-infectious HBV particles in at least two samples based on the presence or level of P-HBcAg; and determining response to treatment of chronic HBV based on the amount of infectious and / or non-infectious HBV particles in the at least two samples. A method comprising:

[0338] Clause 11.a) The treatment is determined to be effective if the amount of infectious HBV particles in the second sample is reduced by at least a certain absolute amount compared to the amount of infectious HBV particles in the first sample; or b) the treatment is determined to be effective if the amount of infectious HBV particles in the second sample is reduced by at least an absolute amount compared to the amount of infectious HBV particles in the first sample and the amount of non-infectious HBV particles in the second sample is reduced by at least an absolute amount compared to the amount of non-infectious HBV particles in the first sample; or c) determining that the treatment is ineffective if the amount of infectious HBV particles in the second sample is not reduced by at least a certain absolute amount compared to the amount of infectious HBV particles in the first sample; The method described in clause 10.

[0339] Clause 12. The method of clause 11, wherein the first sample is obtained from the subject within 24 hours after receiving treatment for chronic HBV.

[0340] Clause 13. The method of clause 11 or 12, wherein the second sample is obtained from the subject 10 to 14 weeks after the first sample.

[0341] Clause 14. The method of clause 13, wherein the first sample is obtained from the subject within 24 hours after receiving treatment for chronic HBV, and the second sample is obtained from the subject 10 to 14 weeks after the first sample.

[0342] Clause 15. The method of clause 7, clause 9 or clause 11, further comprising altering treatment for chronic HBV if treatment is determined to be ineffective.

[0343] Clause 16. The method of clause 15, wherein altering the chronic HBV treatment comprises providing the subject with an increased therapeutic dose, increasing the dosing frequency of the treatment, providing the subject with a second treatment, or any combination thereof.

[0344] Clause 17. The method of any one of clauses 1 to 16, wherein (a) the antibody that specifically binds to HBcAg binds to an epitope comprising at least three amino acids of SEQ ID NO:2, and / or the antibody that specifically binds to P-HBcAg binds to an epitope comprising at least three amino acids of SEQ ID NO:2 or SEQ ID NO:25, provided that at least one amino acid of SEQ ID NO:2 or SEQ ID NO:25 is phosphorylated, and / or (b) the antibody that specifically binds to HBcAg binds to an epitope within amino acids 1-149 of SEQ ID NO:1, and / or the antibody that specifically binds to P-HBcAg binds to an epitope within amino acids 1-149 of SEQ ID NO:1, provided that at least one of amino acids 1-149 is phosphorylated.

[0345] Clause 18. The method of any one of clauses 1-17, wherein the treatment for chronic HBV is an interferon, a nucleoside(t)ide analog, a nucleic acid, an immunomodulator, a core protein assembly inhibitor, a capsid assembly modulator (CAM), an HBsAg release inhibitor, an entry inhibitor, an interfering RNA, a DNA modifier, or a combination thereof.

[0346] Clause 19.a) the interferon is interferon alpha-2a or pegylated interferon alpha-2a; b) the nucleoside analogue is lamivudine, adefovir, tenofovir, telbivudine or entecavir; c) the nucleic acid is an siRNA, an antisense oligonucleotide, an shRNA, or an miRNA; d) the core protein assembly inhibitor is NVR 3-1983, GLS4, or BAY 41-4109; e) CAM is JNJ-632, AT130 or BAY41-4109; f) the HBsAg release inhibitor is REP 9 AC; g) the entry inhibitor is Myrcludex-B, or h) The treatment is any combination of a) to g). The method described in clause 18.

[0347] Clause 20. Carrying out an assay for measuring the level of HBcAg in at least one sample comprises simultaneously or sequentially, in any order, transferring at least one sample to a capture antibody-HBcAg-detection antibody complex so that the capture antibody-HBcAg-detection antibody complex is formed. a) a hepatitis B core antigen (HBcAg) capture antibody that binds to an epitope on the C-terminus of HBcAg to form a capture antibody-HBcAg complex; and b) a detection antibody that binds to an epitope on HBcAg that is not bound by the HBcAg capture antibody; contacting the c) determining the level of HBcAg in the sample based on the signal generated by the detectable label in the capture antibody-HBcAg-detection antibody complex. 20. The method of any one of clauses 1 to 19, comprising:

[0348] Clause 21. Carrying out an assay for measuring the level of P-HBcAg in a sample comprises simultaneously or sequentially, in any order, adding to the sample: a) a phosphorylated hepatitis B core antigen (P-HBcAg) capture antibody that binds to an epitope on the C-terminus of P-HBcAg to form a capture antibody-P-HBcAg complex; and b) a detection antibody that binds to an epitope on P-HBcAg that is not bound by the P-HBcAg capture antibody; contacting the c) determining the level of P-HBcAg in the sample based on the signal generated by the detectable label in the capture antibody-P-HBcAg-detection antibody complex. 21. The method of any one of clauses 1 to 20, comprising:

[0349] Clause 22. Use of a reagent for the detection of the presence or level of hepatitis B core antigen (HBcAg) and phosphorylated hepatitis B core antigen (P-HBcAg) in a method for assessing the stage or phase of hepatitis B (HBV) infection in a subject or in a method for monitoring response to treatment of chronic HBV. Clause 23. A kit or system comprising reagents for the detection of the presence, level or status of Hepatitis B Core Antigen (HBcAg) and phosphorylated Hepatitis B Core Antigen (P-HBcAg).

Claims

1. 1. A method of assessing and monitoring the stage or phase of chronic hepatitis B (HBV) infection in a subject or monitoring response to treatment for chronic HBV, comprising: a) performing an assay to detect the presence or level of hepatitis B core antigen (HBcAg) and phosphorylated hepatitis B core antigen (P-HBcAg) in at least one sample obtained from a subject diagnosed with or undergoing treatment for chronic HBV, said assay comprising contacting said at least one sample with an antibody that specifically binds HBcAg and an antibody that specifically binds P-HBcAg; and b) assessing and monitoring the stage or phase of chronic HBV infection or monitoring the response to treatment of chronic HBV based on the presence or level of HBcAg and P-HBcAg in said at least one sample. A method comprising:

2. 10. The method of claim 1, wherein the subject is being evaluated and monitored for a stage or phase of chronic HBV, and further comprising providing the subject with treatment for chronic HBV based on the presence or level of HBcAg and P-HBcAg in the at least one sample.

3. 10. The method of claim 1, wherein the subject is undergoing treatment for chronic HBV, and further comprising altering HBV treatment based on the presence or level of HBcAg and P-HBcAg in the at least one sample.

4. 1. A method for assessing and monitoring a stage or phase of chronic hepatitis B (HBV) infection, comprising: a) performing an assay to detect the presence or level of hepatitis B core antigen (HBcAg) in at least one sample obtained from a subject diagnosed with chronic HBV, said assay comprising contacting said at least one sample with an antibody that specifically binds to HBcAg; b) determining the amount of infectious HBV particles in the at least one sample based on the presence or level of HBcAg in the at least one sample, wherein the amount of infectious HBV particles is determined regardless of whether the subject has been treated for HBV prior to performing the assay of step a); and c) providing the subject with treatment for chronic HBV if the amount of infectious HBV particles in the at least one sample equals or exceeds a threshold value. A method comprising:

5. 1. A method for monitoring response to treatment of chronic hepatitis B (HBV) infection in a subject, comprising: a) performing an assay to detect the presence or level of hepatitis B core antigen (HBcAg) in at least one sample obtained from a subject undergoing treatment for chronic HBV, said assay comprising contacting said at least one sample with an antibody that specifically binds to HBcAg; b) determining the amount of infectious HBV particles in said at least one sample based on the presence or level of HBcAg in said at least one sample; and c) determining that the treatment is effective if the amount of infectious HBV particles in said at least one sample is below a reference level; or d) determining that the treatment is ineffective if the amount of infectious HBV particles in said at least one sample is greater than or equal to a reference level; A method comprising:

6. 1. A method for monitoring response to treatment of chronic hepatitis B (HBV) infection in a subject, comprising: a) performing an assay to detect the presence or level of hepatitis B core antigen (HBcAg) and phosphorylated hepatitis B core antigen (P-HBcAg) in at least one sample obtained from a subject undergoing treatment for chronic HBV, said assay comprising contacting said at least one sample with an antibody that specifically binds HBcAg and an antibody that specifically binds P-HBcAg; b) determining the amount of infectious HBV particles in said at least one sample based on the presence or level of HBcAg in said at least one sample; c) determining the amount of non-infectious HBV particles in said at least one sample based on the presence or level of P-HBcAg in said at least one sample; and d) determining a response to treatment for chronic HBV based on the amount of infectious and / or non-infectious HBV particles in said at least one sample. A method comprising:

7. a) determining that the treatment is effective if the amount of infectious HBV particles in the at least one sample is less than a reference level; b) the treatment is determined to be effective if the amount of infectious HBV particles in the at least one sample is below a reference level and the amount of non-infectious HBV particles in the at least one sample is below a reference level, or c) determining that the treatment is ineffective if the amount of infectious HBV particles in the at least one sample is greater than or equal to a reference level; The method of claim 6.

8. 1. A method for monitoring response to treatment of chronic hepatitis B (HBV) infection in a subject, comprising: a) performing an assay to detect the presence or level of hepatitis B core antigen (HBcAg) and phosphorylated hepatitis B core antigen (P-HBcAg) in at least two samples obtained from a subject undergoing treatment for chronic HBV, wherein the at least two samples include a first sample obtained from the subject at a first time point before or after receiving treatment for chronic HBV and a second sample obtained from the subject at a second time point after the first time point, the assay comprising contacting the at least two samples with an antibody that specifically binds HBcAg and an antibody that specifically binds P-HBcAg; b) determining the amount of infectious HBV particles in the at least two samples based on the presence or level of HBcAg and / or determining the amount of non-infectious HBV particles in the at least two samples based on the presence or level of P-HBcAg; and c) determining the response to said treatment of chronic HBV based on the amount of infectious and / or non-infectious HBV particles in said at least two samples; A method comprising:

9. a) determining that the treatment is effective if the amount of infectious HBV particles in the second sample is reduced by at least a certain absolute amount compared to the amount of infectious HBV particles in the first sample; b) the treatment is determined to be effective if the amount of infectious HBV particles in the second sample is reduced by at least an absolute amount compared to the amount of infectious HBV particles in the first sample and the amount of non-infectious HBV particles in the second sample is reduced by at least an absolute amount compared to the amount of non-infectious HBV particles in the first sample; or c) determining that the treatment is ineffective if the amount of infectious HBV particles in the second sample is not reduced by at least a certain absolute amount compared to the amount of infectious HBV particles in the first sample; The method of claim 8.

10. 10. The method of claim 9, wherein the first sample is obtained from the subject within 24 hours after receiving treatment for chronic HBV, and the second sample is obtained from the subject 10 to 14 weeks after the first sample.

11. 10. The method of claim 5, 7 or 9, further comprising altering the chronic HBV treatment if the treatment is determined to be ineffective.

12. 12. The method of claim 11, wherein altering the chronic HBV treatment comprises providing the subject with an increased therapeutic dose, increasing the dosing frequency of the treatment, providing the subject with a second treatment, or any combination thereof.

13. The method according to any one of claims 1 to 12, wherein the antibody that specifically binds to HBcAg binds to an epitope comprising at least three amino acids of SEQ ID NO: 2 or SEQ ID NO: 25, and / or the antibody that specifically binds to P-HBcAg binds to an epitope comprising at least three amino acids of SEQ ID NO: 2 or SEQ ID NO: 25, provided that at least one amino acid of SEQ ID NO: 2 or SEQ ID NO: 25 is phosphorylated.

14. 14. The method of any one of claims 1 to 13, wherein the chronic HBV treatment is an interferon, a nucleoside(t)ide analog, a nucleic acid, an immunomodulator, a core protein assembly inhibitor, a capsid assembly modulator (CAM), an HBsAg release inhibitor, an entry inhibitor, an interfering RNA, a DNA or RNA modifier, or a combination thereof.

15. a) the interferon is interferon alpha-2a or pegylated interferon alpha-2a; b) the nucleoside analog is lamivudine, adefovir, tenofovir, telbivudine or entecavir; c) the nucleic acid is an siRNA, an antisense oligonucleotide, an shRNA, or an miRNA; d) the core protein assembly inhibitor is NVR 3-1983, GLS4, or BAY 41-4109; e) the CAM is JNJ-632, AT130 or BAY41-4109; f) the HBsAg release inhibitor is REP 9 AC; g) the entry inhibitor is Myrcludex-B, or h) the treatment is any combination of a) to g); 15. The method of claim 14.

16. Performing an assay for measuring the level of HBcAg in said at least one sample comprises simultaneously or sequentially, in any order, transferring said at least one sample to said at least one sample such that a capture antibody-HBcAg-detection antibody complex is formed. a) a Hepatitis B core antigen (HBcAg) capture antibody that binds to an epitope on the C-terminus of HBcAg to form a capture antibody-HBcAg complex; and b) a detection antibody that binds to an epitope on HBcAg that is not bound by the HBcAg capture antibody; contacting the c) determining the level of HBcAg in the sample based on the signal generated by the detectable label in the capture antibody-HBcAg-detection antibody complex. The method according to any one of claims 1 to 15, comprising:

17. 3. Performing an assay for measuring the level of P-HBcAg in said sample comprises simultaneously or sequentially, in any order, transferring said sample to a capture antibody-P-HBcAg-detection antibody complex so that a capture antibody-P-HBcAg-detection antibody complex is formed. a) a phosphorylated Hepatitis B core antigen (P-HBcAg) capture antibody that binds to an epitope on the C-terminus of P-HBcAg to form a capture antibody-P-HBcAg complex; and b) a detection antibody that binds to an epitope on P-HBcAg that is not bound by the P-HBcAg capture antibody; contacting the c) determining the level of P-HBcAg in the sample based on the signal generated by the detectable label in the capture antibody-P-HBcAg-detection antibody complex; The method according to any one of claims 1 to 16, comprising:

18. Use of a reagent for detecting the presence or level of hepatitis B core antigen (HBcAg) and phosphorylated hepatitis B core antigen (P-HBcAg) in a method for assessing the stage or phase of hepatitis B (HBV) infection in a subject or in a method for monitoring response to treatment for chronic HBV.

19. A kit or system comprising reagents for the detection of the presence, level or status of hepatitis B core antigen (HBcAg) and phosphorylated hepatitis B core antigen (P-HBcAg).

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