HBV diagnosis, prognosis and treatment methods and products

Abstract

Provided herein are compositions, systems, and methods for assessing and monitoring disease stages and staging, predicting the likelihood of disease progression, and predicting and monitoring response to hepatitis B virus infection.

Description

[0001] Related application information

[0002] This application claims priority to U.S. application No. 63 / 406,830, filed on September 15, 2022, the contents of which are incorporated herein by reference.

[0003] Sequence Listing Statement

[0004] The contents of the electronic sequence listing entitled 40844_601-SQL-ST26.xml (size: 57,344 bytes; and creation date: September 14, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] Provided herein are compositions, systems, and methods for assessing and monitoring hepatitis B virus (HBV) stage and staging, and 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 Art

[0006] Hepatitis is a general term meaning "inflammation of the liver," and it has many causes. Viral causes are the most common and can be caused by hepatitis A, B, C, D, or E viruses. Hepatitis B virus (HBV), in particular, is a serious and common liver infection that affects millions of people worldwide.

[0007] HBV is a hepatotropic DNA virus belonging to the Hepadnaviridae family. The viral genome is approximately 3.2 kb in length and has four open reading frames (ORFs), including the surface antigen ("S gene"), the core antigen ("C gene"), the DNA polymerase ("P gene"), and a gene of undefined function, called the "X gene."

[0008] Today, more than 2 billion people are infected with HBV at some point in their lives, and approximately 248 million remain chronically infected and are carriers of the virus. HBV infection can cause acute and chronic hepatitis B and may ultimately lead to the development of chronic liver failure, cirrhosis, and hepatocellular carcinoma. Furthermore, HBV carriers may continue to transmit the disease for many years.

[0009] HBV is transmitted through percutaneous or parenteral contact with infected body fluids or blood. The most common route of infection is vertical transmission from mother to child and, among adults, through sexual intercourse or shared intravenous needles or ear piercing equipment. Many cases of acute HBV infection occur without a traceable route of infection.

[0010] Chronic HBV infection ("carriers") has a 12-300 times greater risk of developing hepatocellular carcinoma (HCC) than non-carriers. Globally, HBV causes 60-80% of primary liver cancer cases worldwide. Of the more than 4 million acute clinical cases annually, approximately 25% die from chronic active hepatitis, cirrhosis, or HBV-induced HCC. Therefore, HBV is second only to tobacco as a known human carcinogen.

[0011] Although HBV vaccines have been widely used for decades, the prevalence of HBV in the population remains high. Current therapies for chronic HBV infection have only limited inhibitory effects on viral gene expression and replication in most chronically infected patients. For example, lamivudine inhibits HBV replication in carriers, but the effect is reversible if treatment is stopped. In addition, the main limitation of chronic lamivudine therapy is the development of viral resistance, which usually develops after six months of treatment. Resistance is usually associated with mutations in the highly conserved catalytic region of the HBV polymerase gene. In addition, current HBV markers (e.g., HBsAg, anti-HBs, HBeAg, anti-HBe) have limited ability to measure the efficacy of antiviral treatment, such as the ability to measure the decline in HBV DNA levels in subjects after treatment.

[0012] For these reasons, there remains a need for new therapeutic options of action as well as methods for selecting and monitoring appropriate treatment for HBV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Shown are HBV seroconversion panels comparing the levels of phosphorylated HBV core antigen (P-HBcAg), HBV core antigen (HBcAg), HBeAg, and HBcrAg with the levels of HBV DNA over time.

[0014] Figure 2 The figure shows a comparison of P-HBcAg and HBcAg levels with HBV DNA and HBV RNA levels after treatment of HBV infection. HBcAg levels (black line) correspond to HBV DNA levels during treatment. During treatment, phosphorylated P-HBcAg (blue line) is more closely correlated with HBV RNA levels than with HBV DNA levels. Some of the data presented in the figure were obtained from Bazinet et al., Hepatology Communications 5; 11 (2021) 1873-1887.

[0015] Figure 3 Various sequences related to P-HBcAg and / or HBcAg are shown.

[0016] Figure 4A Shown tested positive for HBsAg and contained 10 3 to 10 9 Analysis of P-HBcAg levels in clinical samples with respect to viral load of HBV DNA copies / ml. Figure 4B Shown tested positive for HBsAg and contained 10 3 to 10 9 Analysis of HBcAg levels in clinical samples of HBV DNA copies / ml viral load. In 84% of cases with viral load ≥10 5 cp / ml samples, P-HBcAg can be detected. In 94% of the samples with viral load ≥10 6 HBcAg can be detected in samples with a concentration of cp / ml.

[0017] Figure 5A The consensus sequence of HBcAg is shown. Figure 5B The consensus sequence of HBcAg genotype A is shown. Figure 5C The consensus sequence of HBcAg genotype B is shown. Figure 5D The consensus sequence of HBcAg genotype C is shown. Figure 5E The consensus sequence of HBcAg genotype D is shown. Figure 5F The consensus sequence of HBcAg genotype E is shown. Figure 5G The consensus sequence of HBcAg genotype F is shown. Figure 5H The consensus sequence of HBcAg genotype G is shown. Figure 5I The consensus sequence of HBcAg genotype H is shown. Figures 5A-5I In each of the , * (asterisk) indicates a position with a single completely conserved residue. : (colon) indicates conservation between groups with highly similar properties, roughly equivalent to a score > 0.5 in the Gonnet PAM 250 matrix, as follows:

[0018] STA

[0019] NEQK

[0020] NHQK

[0021] NDEQ

[0022] QHRK

[0023] MILV

[0024] MILF

[0025] HY

[0026] FYW.

[0027] . (period) indicates conservation between groups with weakly similar properties as follows, roughly equivalent to scores = <0.5 and >0 in the Gonnet PAM250 matrix:

[0028] CSA

[0029] ATV

[0030] SAG

[0031] STNK

[0032] STPA

[0033] SGND

[0034] SNDEQK

[0035] NDEQHK

[0036] NEQHRK

[0037] FVLIM

[0038] HFY. DETAILED DESCRIPTION

[0039] In some aspects, provided herein is a method for evaluating and monitoring the stage or staging of chronic HBV infection or monitoring the response to chronic HBV treatment in a subject. In some embodiments, the method includes determining 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 chronic HBV treatment. In some embodiments, the determination includes contacting at least one sample with an antibody that specifically binds to HBcAg and an antibody that specifically binds to P-HBcAg. In some embodiments, the method also includes evaluating and monitoring the stage or staging of chronic HBV infection or monitoring the response to chronic HBV treatment based on the presence or level of HBcAg and P-HBcAg in at least one sample.

[0040] In some embodiments, the subject is being evaluated and monitored for the stage or staging of chronic HB.In some embodiments, the method further comprises providing chronic HBV treatment to the subject based on the presence or level of HBcAg and P-HBcAg in the at least one sample.

[0041] In some embodiments, the subject is receiving chronic HBV treatment.In some embodiments, the method further comprises changing the HBV treatment based on the presence or level of HBcAg and P-HBcAg in the at least one sample.

[0042] In some aspects, provided herein is a method for assessing and monitoring the stage or staging of chronic HBV infection. In some embodiments, the method includes performing a determination of 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 determination includes contacting at least one sample with an antibody that specifically binds to HBcAg. In some embodiments, the method includes determining the amount of infectious HBV particles in at least one sample based on the presence or level of HBcAg in at least one sample. In some embodiments, regardless of whether the subject has or has not received HBV treatment before performing the determination (e.g., detecting the presence or level of HBcAg in at least one sample), the amount of infectious HBV particles is determined. In some embodiments, the method also includes providing chronic HBV treatment to the subject when the amount of infectious HBV particles in at least one sample is equal to or higher than a threshold value.

[0043] In some aspects, provided herein is a method for monitoring a response to treatment of chronic hepatitis B (HBV) infection in a subject. In some embodiments, the method includes performing a determination of the presence or level of hepatitis B core antigen (HBcAg) in at least one sample obtained from a subject receiving chronic HBV treatment. In some embodiments, the determination includes contacting at least one sample with an antibody that specifically binds to HBcAg. In some embodiments, the method includes determining the amount of infectious HBV particles in at least one sample based on the presence or level of HBcAg in at least one sample. In some embodiments, the method includes determining whether chronic HBV treatment is effective or ineffective in a subject using the amount of infectious HBV particles in the sample. In some embodiments, the method includes determining that treatment is effective when the amount of infectious HBV particles in at least one sample is less than a reference level. In some embodiments, the method includes determining that treatment is ineffective when the amount of infectious HBV particles in at least one sample is greater than or equal to a reference level.

[0044] 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 a subject receiving chronic HBV treatment. In some embodiments, the assay comprises contacting at least one sample with an antibody that specifically binds to HBcAg and an antibody that specifically binds to P-HBcAg. 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 at least one sample. In some embodiments, the method comprises determining the amount of non-infectious HBV particles in at least one sample based on the presence or level of P-HBcAg in 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 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 at least one sample. 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 one sample. For example, in some embodiments, determining a response to treatment includes 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 a reference level and the amount of non-infectious HBV particles in at least one sample is less than a 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.

[0045] In some embodiments, a method for monitoring a response to treatment for chronic hepatitis B (HBV) infection in a subject comprises performing an assay for 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 receiving chronic HBV treatment. 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 chronic HBV treatment 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 to HBcAg and an antibody that specifically binds to 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 comprises determining a response to chronic HBV treatment based on the amount of infectious and / or non-infectious HBV particles in the at least two samples. For example, determining a response to treatment can include determining whether the treatment is effective or ineffective. In some embodiments, the treatment is determined to be effective when the amount of infectious HBV particles in the second sample is reduced by at least one absolute amount compared to the amount of infectious HBV particles in the first sample. In some embodiments, the treatment is determined to be effective when the amount of infectious HBV particles in the second sample is reduced by at least one 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 one 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 when the amount of infectious HBV particles in the second sample has not decreased by at least one absolute amount compared to the amount of infectious HBV particles in the first sample.

[0046] In some embodiments, a first sample is obtained from a subject within 24 hours of receiving chronic HBV treatment. In some embodiments, a second sample is obtained from a subject 10-14 weeks after the first sample. In some embodiments, a first sample is obtained from a subject within 24 hours of receiving chronic HBV treatment, and a second sample is obtained from a subject 10-14 weeks after the first sample.

[0047] In some embodiments, the method for monitoring the response to chronic HBV treatment described herein also includes changing the chronic HBV treatment when it is determined that the treatment is ineffective. In some embodiments, changing the chronic HBV treatment includes providing the subject with an increased dose of treatment, increasing the dosing frequency of treatment, providing the subject with a second treatment, or any combination thereof.

[0048] In some embodiments, the antibody that specifically binds to HBcAg can bind to an epitope comprising at least 3 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 3 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.

[0049] In some embodiments, chronic HBV treatment can be interferon, nucleoside (acid) analogs, nucleic acids, immunomodulators, core protein assembly inhibitors, capsid assembly regulators (CAMs), HBsAg release inhibitors, entry inhibitors, interferon RNA, DNA modification, or a combination thereof. In some embodiments, interferon is interferon α-2a or pegylated interferon α-2a. In some embodiments, nucleoside (acid) analogs are lamivudine, adefovir, tenofovir, telbivudine, or entecavir. In some embodiments, nucleic acids are siRNA, antisense oligonucleotides, shRNA, or miRNA. In some embodiments, core protein assembly inhibitors are NVR 3-1983, GLS4, or BAY 41-4109. In some embodiments, CAMs are JNJ-632, AT130, or BAY41-4109. In some embodiments, HBsAg release inhibitors are REP 9AC. In some embodiments, the entry inhibitor is Myrcludex-B. In some embodiments, the treatment is a combination of any of the treatments described above.

[0050] In some embodiments, performing an assay to measure the level of HBcAg in at least one sample comprises contacting the 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, thereby forming a capture antibody-HBcAg-detection antibody complex. 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.

[0051] 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, thereby forming a capture antibody-P-HBcAg-detection antibody complex. 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.

[0052] In some aspects, provided herein is a reagent for detecting the presence or level of hepatitis B core antigen (HBcAg) and phosphorylated hepatitis B core antigen (P-HBcAg) for use in a method for assessing the stage or staging of hepatitis B (HBV) infection in a subject or monitoring the response to chronic HBV treatment.

[0053] In some aspects, provided herein is a kit or system comprising reagents for detecting the presence, level, or status of hepatitis B core antigen (HBcAg) and phosphorylated hepatitis B core antigen (P-HBcAg).

[0054] The section headings as used in this section and throughout the disclosure herein are for organizational purposes only and are not intended to be limiting.

[0055] 1. Definition

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. In the event of conflict, this document (including definitions) shall prevail. Various embodiments of methods and materials are described below, but methods and materials similar to or equivalent to those described herein may be used in the practice or testing disclosed herein. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. Materials, methods and embodiments disclosed herein are merely illustrative and are not intended to be restrictive.

[0057] As used herein, the term "algorithm" refers to a process or set of rules to be followed in a computational or other problem-solving operation (such as performed by one or more computers that include one or more software programs that analyze data from one or more markers and optionally one or more biometric data (such as, for example, history of intravenous medication use, chronic liver disease and / or kidney disease, work history as a healthcare worker, age, sex, race, etc.), and includes the code required to execute the algorithm). For example, analysis of HBV marker data using an algorithm can 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 panel of two or more markers, e.g., a panel comprising hepatitis B core antigen (HBcAg) and phosphorylated hepatitis B core antigen (P-HBcAg), optionally including one or more additional markers; or (4) a panel of two or more markers with one or more biometric data. Additional methods may employ multi-analyte algorithms (such as those described in U.S. Patent Publication No. 2016 / 0342757, which is incorporated herein by reference) rather than a single marker (with or without biometric data) or a panel of single markers (with or without biometric data). Such algorithms may be used as part of the methods described herein to derive one or more values reflecting disease state, stage, or staging, thereby predicting the likelihood of disease progression and / or predicting or monitoring response to therapy or treatment.

[0058] As used herein, the term "antibody" refers to an immunoglobulin molecule or its immunologically active portion, that is, an antigen binding portion. Examples of immunologically active portions of immunoglobulin molecules include F (ab) and F (ab') 2 fragments, which can be generated by, for example, 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 Fv ("scFv"), affinity matured antibodies, single-chain antibodies, single-domain antibodies, F (ab) fragments, F (ab') fragments, disulfide-linked Fv ("sdFv") and anti-idiotype ("anti-Id") antibodies, and the like, and functionally active epitope binding fragments of any of the above. The antibody can be IgG, IgM, IgA, IgD or IgE class, or a fragment or derivative thereof. The antibody can be derivatized by connecting one or more chemical moieties, peptide moieties or polypeptide moieties known in the art. The antibody can be conjugated to a chemical moiety.

[0059] As used herein, "antibody fragment" refers to a portion of an intact antibody that contains the antigen binding site or variable region. The portion does not include the constant heavy chain domains (i.e., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. 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 the three CDRs of the light chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.

[0060] "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, while an AUC of 0.5 represents a meaningless test. Preferred AUCs can 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.

[0061] A "receiver operating characteristic" curve, or "ROC" curve, refers to a graph that illustrates the performance of a binary classifier system as its discrimination threshold is varied. For example, an ROC curve can be a graph of the true positive rate versus the false positive rate for different possible cutoff points for a diagnostic test. The curve is created by plotting the fraction of true positives in the positives (TPR = true positive rate) versus the fraction of false positives in the negatives (FPR = false positive rate) at different threshold settings. TPR is also called sensitivity, and FPR is one minus specificity or true negative rate. The ROC curve illustrates the trade-off between sensitivity and specificity (any increase in sensitivity is accompanied by a decrease in specificity); the more closely the curve follows the left boundary of the ROC space, and then the top boundary, the more accurate the test; the closer the curve is to the 45-degree diagonal of the ROC space, the less accurate the test; the slope of the tangent line at the cutoff point gives the likelihood ratio (LR) of the test value; and the area under the curve is a measure of textual accuracy.

[0062] "Beads" and "particles" are used interchangeably herein and refer to a substantially spherical solid support. An example of a bead or particle is a microparticle. The microparticles that can be used herein can be of any type known in the art. For example, the beads or particles can be magnetic beads or magnetic particles. The magnetic beads / particles can be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic, or ferrofluidic. 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 magnetic solid core portion and be surrounded by one or more non-magnetic layers. Alternatively, the magnetic portion can be a layer surrounding the non-magnetic core. The particles can have any size that works in the methods described herein, for example, about 0.75 to about 5 nm, or about 1 to about 5 nm, or about 1 to about 3 nm.

[0063] "Binding protein" is used herein to refer to a monomeric or multimeric protein that is bound to a binding partner and forms a complex therewith, such as a polypeptide, antigen, chemical compound or other molecule or substrate of any kind. The binding protein specifically binds to the binding partner. The binding protein includes antibodies, and antigen-binding fragments thereof and other various forms and derivatives known in the art and described below, and other molecules of the antigen-binding domain that contain one or more binding antigen molecules or specific sites (epitopes) on the antigen molecule. Therefore, binding proteins include but are not limited to antibodies, tetrameric immunoglobulins, 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 antigens.

[0064] "Bispecific antibodies" are used herein to refer to full-length antibodies produced by the following techniques: quadroma technology (also known as hybrid-hybridoma technology; see Milstein et al., Nature, 305(5934):537-540 (1983)); by chemical conjugation of two different monoclonal antibodies (see Staerz et al., Nature, 314(6012):628-631 (1985)); or by the knob-in-hole method or similar methods of introducing mutations in the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90(14):6444-6448 (1993)), which produce multiple different immunoglobulin species, only one of which is a functional bispecific antibody. A bispecific antibody binds one antigen (or epitope) on one of its two binding arms (a pair of HC / LC) and binds a different antigen (or epitope) on its second arm (the other pair of HC / LC). By this definition, a bispecific antibody has two distinct antigen-binding arms (in terms of both specificity and CDR sequences) and is monovalent for each antigen it binds.

[0065] "CDR" is used herein to refer to the "complementarity determining region" within the antibody variable structure sequence. There are three CDRs in each variable region of the heavy chain and light chain. For each variable region, starting from the N-terminus of the heavy chain or light chain, these regions are expressed as "CDR1", "CDR2" and "CDR3". The term "CDR group" as used herein refers to a group of three CDRs present in a single variable region that binds to an antigen. Therefore, an antigen binding site can include six CDRs, which include a CDR group from each of the heavy and light chain variable regions. A polypeptide comprising a single CDR (e.g., CDR1, CDR2 or CDR3) can be referred to as a "molecular recognition unit". Crystallographic analysis of antigen-antibody complexes has shown that the amino acid residues of CDR form extensive contacts with the bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Therefore, a molecular recognition unit may be primarily responsible for the specificity of the antigen binding site. Generally speaking, CDR residues are directly and most substantially involved in influencing antigen binding.

[0066] The precise 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 variable region of an antibody, 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)) found that despite the enormous diversity at the level of amino acid sequence, certain subportions within the Kabat CDRs adopt nearly identical peptide backbone conformations. These subportions are designated "L1," "L2," and "L3," or "H1," "H2," and H3, where "L" and "H" designate the light and heavy chain regions, respectively. These regions may be referred to as "Chothia CDRs" and have similarities to the Kabat CDRs. Boundaries of CDR overlap. Other boundaries that define CDRs that overlap with Kabat CDRs have been described by Padlan, FASEB J., 9: 133-139 (1995) and MacCallum, J. Mol. Biol, 262(5): 732-745 (1996). Still other CDR boundary definitions may not strictly follow one of the systems herein, but will still overlap with Kabat CDRs, but they may be shortened or lengthened based on predictions or experimental findings that particular residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding. The methods used herein can utilize CDRs defined according to any of these systems, but certain embodiments use CDRs defined by Kabat or Chothia.

[0067] As used herein, "favorable" in some aspects means that when the level of one or more biomarkers measured and / or determined according to the methods described herein is less than or below the reference level when compared to one or more reference levels. However, in other aspects, depending on the biomarkers being measured or determined, the term "favorable" may mean that the level of one or more biomarkers measured and / or determined according to the methods described herein is higher or higher than one or more reference levels. Whether a "favorable" level is higher or lower than the reference level depends on whether the biomarkers rise or fall in the case of HBV infection. In some embodiments, a "favorable" level indicates that from a first time point to a second time point, the level of one or more biomarkers decreases by at least one absolute amount. For example, a "favorable" level may indicate that from a first sample to a second sample collected after the first sample, the HBcAg level and / or P-HBcAg level decreases by at least one absolute amount. In general, a favorable level of a biomarker is a level that indicates the absence of HBV infection, an improvement in the health status of the subject regarding HBV infection, or that the subject benefits from treatment (or continued treatment) with an HBV therapeutic agent (i.e., HBV treatment is effective in the subject). In some embodiments, a favorable level is associated with clinical improvement in infection. For example, clinical improvement of a subject can be determined by any number of parameters (e.g., patient report, improvement in skin color (e.g., less yellow or less jaundiced), reduction in the amount of liver inflammation, etc.).

[0068] As used herein, "unfavorable" in some aspects means that when the level of one or more biomarkers measured and / or determined according to the methods described herein is compared to one or more reference levels, it is higher or greater than the reference level. However, in other aspects, depending on the biomarkers being measured or determined, the term "unfavorable" may mean that the level of one or more biomarkers measured and / or determined according to the methods described herein is lower or less than one or more reference levels. As described above, whether an "unfavorable" level is higher or lower compared to a reference level depends on whether the biomarker is rising or falling in the case of HBV infection. In some embodiments, an "unfavorable" level indicates that the level of one or more biomarkers has not decreased by at least one absolute amount from a first time point to a second time point. For example, an "unfavorable" level may indicate that the HBcAg level and / or P-HBcAg level has not decreased by at least one absolute amount from a first sample to a second sample collected after the first sample. In general, an unfavorable level of a biomarker is a level that indicates the presence of HBV infection, a worsening of the subject's health status regarding HBV infection, or a subject's lack of benefit from HBV treatment (i.e., HBV treatment is ineffective in the subject). In some embodiments, an unfavorable level is associated with clinical worsening of the infection. For example, worsening of a subject's condition can be determined by any number of parameters (e.g., patient report, change or worsening of skin color (e.g., increased yellow color or jaundice), increased liver inflammation, etc.).

[0069] 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 identical residues over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the sequences over a specified region, determining the number of positions at which the identical residue occurs in both sequences to obtain 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 obtain the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of the single sequence are included in the denominator of the calculation but not the numerator.

[0070] As used herein, "hepatitis surface antigen or isoform of HBsAg" or "hepatitis surface antigen or HBsAg isoform" refers to one or more polypeptides encoded by the pre-S1, pre-S2 and / or S segments of the HBsAg gene, which are referred to as large (L), medium (M) and small (S) HBs. Large, medium and small HBs all contain an S domain. The medium HBs protein (MHBs) has a 55 amino acid long N-terminal extension, the pre-S2 domain. The HBV large surface protein (LHBs) has an additional 108 or 119 amino acid N-terminal extension, the pre-S1 domain. The HBsAg of infectious virions and subviral particles is primarily composed of the HBV small surface protein (SHB), with LHB and MHB being minor components. Isoforms contemplated herein may include (i) large HBs only; (ii) medium HBs only; (iii) small HBs only; (iv) large HBs and medium HBs; (v) large HBs and small HBs; (vi) medium HBs and small HBs; or (vii) large HBs, medium HBs, and small HBs.

[0071] As used herein, "substantially identical" can mean that the first and second sequences are at least about 50% to about 99% identical over a region of about 8 to about 100 or more residues, including any range within about 8 to about 100 residues.

[0072] As used herein, the term "test sample" or "sample" generally refers to a biological material that is tested and / or suspected of containing an analyte of interest (such as a marker as 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, mucus, nasal fluid, sputum, synovial fluid, peritoneal fluid, vaginal fluid, menstruation, amniotic fluid, semen, etc. In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a plasma sample. In other embodiments, the sample is a serum sample. The test sample can be used directly after obtaining it from a biological source or after pretreatment to change the characteristics of the sample. For example, such pretreatment can include preparing plasma from blood, diluting a viscous fluid, etc. The pretreatment method can also involve filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of interfering components, addition of reagents, lysis, etc. In addition, it may also be beneficial to modify a solid test sample to form a liquid medium or release an analyte.

[0073] The term "nucleic acid" refers to a polymer of nucleotides and, unless otherwise limited, includes known analogs of natural nucleotides that can function in a manner similar to (eg, hybridize) naturally occurring nucleotides.

[0074] The term nucleic acid includes any form of DNA or RNA, including, for example, genomic DNA, complementary DNA (cDNA) (which is a DNA representation of mRNA, typically obtained by reverse transcription of messenger RNA (mRNA) or by amplification), DNA molecules produced synthetically or by amplification, and mRNA.

[0075] The term nucleic acid encompasses double-stranded or triple-stranded nucleic acids as well as single-stranded molecules.In a double-stranded or triple-stranded nucleic acid, the nucleic acid strands need not be coextensive (ie, a double-stranded nucleic acid need not be double-stranded along the entire length of both strands).

[0076] The term nucleic acid also encompasses any chemical modification thereof (such as by methylation and / or by capping). Nucleic acid modifications can include the addition of chemical groups that incorporate extra charge, polarizability, hydrogen bonding, electrostatic interactions, and functionality into a single nucleic acid base or the entire nucleic acid. Such modifications can include base modifications such as 2'-sugar modifications, 5-pyrimidine modifications, 8-purine modifications, modifications at the amine outside the cytosine ring, substitutions of 5-bromo-uracil, backbone modifications, unusual base pairing combinations such as the isobases isocytidine and isoguanidine, and the like.

[0077] As used herein, the term "target nucleotide sequence" refers to a molecule comprising the nucleotide sequence of a target nucleic acid (e.g., a nucleic acid to be detected in an assay), such as, for example, an amplification product obtained by amplifying a target nucleic acid or a cDNA generated following reverse transcription of an RNA target nucleic acid.

[0078] As used herein, the term "complementarity" refers to the ability of two nucleotides to pair precisely, i.e., if a nucleotide at a given position in a nucleic acid is capable of hydrogen bonding with a nucleotide in another nucleic acid, then the two nucleic acids are considered complementary to each other at that position. Complementarity between two single-stranded nucleic acid molecules may be "partial," in which only some of the nucleotides bind, or may be complete, in which case there is complete complementarity between the single-stranded molecules. The degree of complementarity between nucleic acid chains has a significant impact on the efficiency and strength of hybridization between nucleic acid chains.

[0079] "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 relaxing the stringency of hybridization conditions allows for tolerance of sequence mismatches.

[0080] The term "oligonucleotide" is used to refer to relatively short nucleic acids, typically shorter than 200 nucleotides, shorter than 100 nucleotides, and in some cases shorter than 50 nucleotides. Typically, an oligonucleotide is a single-stranded DNA molecule.

[0081] The term "primer" refers to an oligonucleotide that can hybridize to nucleic acids (also referred to as "annealing") and serve as a starting point for nucleotide (RNA or DNA) polymerization under appropriate conditions (i.e., in the presence of four different nucleoside triphosphates and polymerizing agents, such as DNA or RNA polymerase or reverse transcriptase), at an appropriate buffer and an appropriate temperature. The appropriate length of a primer depends on the intended use of the primer, but the primer is typically at least 7 nucleotides in length, more typically within the range of 10 to 30 nucleotides or even more typically 15 to 30 nucleotides. Other primers can be longer to a certain extent, for example, 30 to 50 nucleotides long. In this context, "primer length" refers to an oligonucleotide or nucleic acid portion that hybridizes with a complementary "target" sequence and initiates nucleotide synthesis. Short primer molecules generally require colder temperatures to form sufficiently stable hybrid complexes with templates. Primers do not necessarily reflect the exact sequence of the template, but must be fully complementary to hybridize with the template. The term "primer site" or "primer binding site" refers to the target nucleic acid segment of the primer hybridization.

[0082] If a primer or a portion thereof hybridizes to a nucleotide sequence within another nucleic acid, then the primer is said to anneal to the nucleic acid.Reference 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.

[0083] The term "primer pair" refers to a set of primers comprising a 5' "upstream primer" or "forward primer" that hybridizes to a complementary sequence at 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. Those skilled in the art will recognize that the terms "upstream" and "downstream" or "forward" and "reverse" are not intended to be limiting, but rather to provide illustrative orientation in specific embodiments.

[0084] The term "probe" is a nucleic acid that can bind to a target nucleic acid of a complementary sequence by one or more types of chemical bonds, generally by complementary base pairing, usually by hydrogen bond formation, to form a duplex structure. The probe is combined with or hybridized to a "probe binding site". The probe can be labeled with a detectable marker to allow easy detection of the probe, and in some cases, when the probe has hybridized with a complementary target. However, alternatively, the probe can be unlabeled, but can be detected by specific binding directly or indirectly with a labeled ligand. The size of the probe can be significantly different. Typically, the length of the probe is at least 7 to 15 nucleotides. The length of other probes is at least 20, 30 or 40 nucleotides. Still other probes are slightly longer, with a length of at least 50, 60, 70, 80 or 90 nucleotides. Still other probes are longer, and a length of at least 100, 150, 200 or more nucleotides. The probe can also be any length (e.g., a length of 15-30 nucleotides) within any range defined by any of the above values.

[0085] Primer or probe can be fully complementary with target nucleic acid sequence, or can be not fully complementary.In certain embodiments, the complementary sequence of primer and target nucleic acid sequence is on the sequence of at least 7 nucleotide, more usually on the sequence in the range of 10-30 nucleotide, and often on the sequence of at least 14-25 nucleotide, there is at least 65% homogeneity, and more often there is at least 75% homogeneity, at least 85% homogeneity, at least 90% homogeneity or at least 95%, 96%, 97%, 98% or 99% homogeneity.Should be understood that some base (for example, 3 ' base of primer) is usually expected to be fully complementary with the corresponding base of target nucleic acid sequence.Primer and probe are annealed with target sequence under stringent hybridization conditions conventionally.

[0086] "Amplification" encompasses any means of reproducing at least a portion of at least one target nucleic acid, typically in a template-dependent manner, including but not limited to a wide range of techniques for linear or exponential amplification of nucleic acid sequences. Illustrative means for performing the amplification step include ligase chain reaction (LCR), ligase detection reaction (LDR), ligation-post-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 multiplex amplification, rolling circle amplification (RCA), and the like, including multiplex formats 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 combined chain reaction - CCR), and the like. Descriptions of such 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.Feb. 1993; 4(1):41-7; U.S. Pat. No. 6,027,998; U.S. Pat. No. 6,605,451; Barany et al., PCT Publication No. WO 97 / 31256; Wenz et al., PCT Publication No. WO Publication No. 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 Feb;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 / 56927 A3; and WO 98 / 03673 A1.

[0087] As used herein, "reference level" refers to an assay or cutoff value that is used to assess diagnosis ("diagnostic" cutoff), prognosis, or therapeutic efficacy and is associated or correlated with various clinical parameters (e.g., the presence of disease, such as, for example, ruling a subject as having the disease ("in") or ruling a subject as not having the disease ("out"), disease stage, disease severity, progression, non-progression, or improvement of the disease, etc.). The present disclosure provides exemplary reference levels. However, it is well known that reference levels can vary depending on the nature of the immunoassay (e.g., such as the immunoassay, the antibodies used, the reaction conditions, the purity of the sample, etc.), and assays can be compared and standardized. Based on the description provided in the present disclosure, it is further within the ability of one of ordinary skill in the art to adapt the disclosure herein to other assays to obtain assay-specific reference levels for those other immunoassays. Although the precise values of the reference levels may vary between assays, the findings as described herein should be generally applicable and capable of being extrapolated to other assays.

[0088] As used herein, the term "single molecule detection" refers to the detection and / or measurement of single molecules of an analyte in a test sample at very low concentration levels (such as pg / mL or femtogram / mL levels). Many different single molecule analyzers or devices are known in the art and include nanopore and nanowell devices. Examples of nanopore devices are described in International Patent Publication No. WO 2016 / 161402, which is hereby incorporated by reference in its entirety. Examples of nanowell devices are described in International Patent Publication No. WO 2016 / 161400, which is hereby incorporated by reference in its entirety.

[0089] "Reagent" generally refers to any agent 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, and the like. 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, and the like.

[0090] As used herein, the term "label" 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 the probe include, but are not limited to, radioisotopes, fluorophores, chromophores, mass labels, electron-dense particles, magnetic particles, spin labels, molecules that emit chemiluminescence, electrochemically active molecules, enzymes, cofactors, and enzyme substrates.

[0091] The term "dye" as used herein generally refers to any organic or inorganic molecule that absorbs electromagnetic radiation having a wavelength greater than or equal to 340 nm.

[0092] The term "fluorescent dye" as used herein generally refers to any dye that emits longer wavelength electromagnetic radiation by a fluorescence mechanism upon illumination by an electromagnetic radiation source, such as a lamp, photodiode, or laser.

[0093] As used herein, the terms "comprise," "include," "have," "may," "contain," and variations thereof are intended to be open-ended conjunctions, terms, or words that do not exclude the possibility of additional actions or structures. Unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" include plural references. The present disclosure also encompasses other embodiments that "comprise," "consist of," and "consist essentially of" the embodiments or elements presented herein, whether or not explicitly set forth.

[0094] For the recitation of numerical ranges herein, each intervening number therebetween is expressly contemplated with equal precision. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range of 6.0-7.0, 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.

[0095] As used herein, "absolute amount" refers to the absolute value of the change or difference between at least two determination results obtained or sampled at different time points, and which, similar to a reference level, has been associated or correlated herein with various clinical parameters (e.g., presence of disease, stage of disease, severity of disease, progression, non-progression, or improvement of disease, etc.). As used herein, "absolute value" refers to a real number magnitude (e.g., such as the difference between two comparative levels (e.g., a level collected at a first time point and a level collected at a second time point)) regardless of its sign (i.e., whether it is positive or negative).

[0096] 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 antibodies used, reaction conditions, sample purity, etc.), and the assays can be compared and standardized. Based on the description provided by the present disclosure, it is also entirely within the capabilities of those of ordinary skill in the art to adapt the disclosure herein to other immunoassays to obtain immunoassay-specific reference levels and absolute amounts for those other immunoassays. Although the precise values of reference levels and absolute amounts may vary between assays, the findings described herein should be generally applicable and can be extrapolated to other assays.

[0097] "Components," "components," or "at least one component" generally refer to capture antibodies, detectors or conjugates, calibrators, controls, sensitivity panels, containers, buffers, diluents, salts, enzymes, enzyme cofactors, detection reagents, pretreatment reagents / solutions, substrates (e.g., as solutions), stop solutions, etc. that can 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 or lyophilized for reconstitution for use in the assay.

[0098] As used herein, a "control" generally refers to a reagent whose purpose is to evaluate the performance of a measurement system to ensure that it continues to produce results within acceptable boundaries (e.g., boundaries ranging from measurements suitable for research use assays on one end to analytical boundaries established by quality specifications for commercial assays on the other end). To achieve this, the control should be indicative of patient results and, optionally, should be evaluated in some way for the effect of errors (e.g., errors caused by reagent stability, calibrator variability, instrument variability, etc.) on the measurement results.

[0099] As used herein, "associated with" means compared to.

[0100] As used herein, the "derivative" of an antibody can refer to an antibody having one or more modifications to its amino acid sequence compared to a true or parent antibody and exhibiting a modified domain structure. Derivatives can still be configured to adopt the typical domains found in natural antibodies, as well as amino acid sequences that can specifically bind to a target (antigen). Typical examples of antibody derivatives are antibodies coupled to other polypeptides, rearranged antibody domains, or antibody fragments. Derivatives can also include at least one other compound, such as a protein domain, which is connected by covalent or non-covalent bonds. According to methods known in the art, linkage can be based on genetic fusion. The other domains present in the fusion protein comprising the antibody can be connected by a flexible linker, advantageously a peptide linker, wherein the peptide linker comprises multiple hydrophilic peptide-bonded amino acids, and its length is sufficient to span the distance between the C-terminus of the other protein domain and the N-terminus of the antibody, or vice versa. Antibodies can be connected to effector molecules that have a conformation suitable for biological activity or selective binding, such as solid supports, biologically active substances (e.g., cytokines or growth hormones), chemical reagents, peptides, proteins, or drugs.

[0101] As used herein, "hepatitis B core-related antigen (HBcrAg)" refers to the antigenic reactivity generated by denatured hepatitis B e antigen (HBeAg), HBV core antigen (HBcAg) and artificial core-related protein (p22cr).

[0102] "Phosphorylated hepatitis B core antigen," "phosphorylated HBcAg," or P-HBcAg are all used interchangeably herein and refer to hepatitis B core antigen (HBcAg) that is phosphorylated at one or more amino acids. For example, the phosphorylated hepatitis B core antigen can have the sequence 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.

[0103] As used herein, "subject" and "patient" refer interchangeably 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 can receive various forms of treatment. As used herein, "mammal" refers to any member of the class Mammalia, including but not limited to humans and non-human primates, such as chimpanzees and other ape and monkey species; livestock, such as cattle, sheep, pigs, goats, llamas, camels and horses; domestic mammals, such as dogs and cats; laboratory animals, including rodents, such as mice, rats, rabbits and guinea pigs, etc. The terms do not indicate a particular age or sex. Thus, adult and infant subjects, as well as male or female fetuses, are intended to be included within the scope of the terms.

[0104] "Treat / treating / treatment" are each used interchangeably herein to describe reversing, alleviating or inhibiting the progression of the disease and / or injury to which the term applies, or one or more symptoms of the disease. Depending on the subject's condition, the term also refers to preventing the disease, and includes preventing the onset of the disease or preventing symptoms associated with the disease. Treatment can be carried out in an acute or chronic manner. The acute phase of HBV infection typically lasts from about 4 weeks to about 6 months after infection, while the chronic phase of HBV infection typically includes a period of time after the end of the acute phase. In some cases, treating or monitoring chronic HBV infection includes testing a sample obtained about 24 weeks after the subject is infected. The term also refers to reducing the severity of the disease or symptoms associated with the disease before being affected by the disease. This prevention of disease or reduction of disease severity before being affected refers to not administering the pharmaceutical composition to the subject at the time of administration when the subject is affected by the disease. "Prevention" also refers to preventing the recurrence of the disease or one or more symptoms associated with the disease. "Treatment" and "therapeutic" refer to the act of treating, as "treatment" is defined above.

[0105] "Variant" is used herein to describe a peptide or polypeptide that differs in amino acid sequence due to insertion, deletion or conservative substitution of amino acids, but retains at least one biologically active peptide or polypeptide. "SNP" refers to a variant of a single nucleotide polymorphism. Representative examples of "biological activity" include the ability to be bound by a specific antibody or to promote an immune response. Variant is also used herein to describe a protein whose amino acid sequence is substantially identical to a reference protein whose amino acid sequence retains at least one biologically active protein. Conservative substitution of amino acids, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree, and distribution of charged regions), is generally recognized in the art as generally involving minor changes. As understood in the art, these minor changes can be identified, in part, by considering the hydropathic index of an amino acid. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on considerations of its hydrophobicity and charge. As known in the art, amino acids with similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids with a hydropathic index of ± 2 are substituted. Amino acid hydrophilicity can also be used to reveal the replacement of proteins that will produce retention biological function. Considering the hydrophilicity of amino acid in the context of peptides allows calculation of the local average hydrophilicity of the maximum peptide, which is a useful measure that has been reported to be well associated with antigenicity and immunogenicity. U.S. Patent number 4,554,101 is incorporated herein by reference in its entirety. As known in the art, amino acid replacements with similar hydrophilicity values can produce peptides that retain biological activity (e.g., immunogenicity). Replacement can be performed with amino acids with hydrophilicity values within ± 2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acid are affected by the specific side chain of the amino acid. Consistent with the observations, amino acid replacements compatible with biological function are understood to depend on amino acid, including the relative similarity of those amino acid side chains, as disclosed by hydrophobicity, hydrophilicity, charge, size and other characteristics.

[0106] "Vector" is used herein to describe a nucleic acid molecule that can transport another nucleic acid to which it has been connected. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which another DNA segment can be connected. Another type of vector is a viral vector, in which another DNA segment can be connected to a viral genome. Certain vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors and additional mammalian vectors comprising a bacterial origin of replication). Other vectors (e.g., non-additive mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicate together with the host genome. In addition, certain vectors can direct the expression of the genes to which they are operably connected. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). Generally speaking, expression vectors useful in recombinant DNA technology are typically in the form of plasmids. "Plasmid" and "vector" are used interchangeably because plasmids are the most commonly used vector forms. However, other forms of expression vectors with equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), can be used. In this regard, RNA versions of vectors (including RNA viral vectors) may also be used in the context of the present disclosure.

[0107] Unless otherwise defined herein, the scientific and technical terms used in conjunction with the present disclosure will have the meanings commonly understood by those of ordinary skill in the art. For example, any nomenclature used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein, and the techniques thereof, are those well known and commonly used in the art. The meaning and scope of the terms should be clear; however, if there is any implicit ambiguity, the definitions provided herein take precedence over any dictionary or external definition. Further, unless the context otherwise requires, singular terms should include the plural, and plural terms should include the singular.

[0108] 2. Detection of HBcAg and P-HBcAg

[0109] 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.

[0110] 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 can be 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 antibodies (anti-HBs), anti-hepatitis B e antigen antibodies (anti-HBe), anti-hepatitis surface antigen antibodies (anti-HBs), and complexes formed between HBsAg and anti-HBs (also referred to herein as "HBsAg immune complexes"). In some embodiments, the additional markers are enzyme markers. In some embodiments, the enzyme markers are selected from AST and ALT. AST and / or ALT are typically detected using an enzymatic assay in which AST or ALT acts on a substrate to generate 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 analysis system (Abbott, Abbott Park, IL) is utilized.

[0111] In some embodiments, the assay for detecting a protein marker (eg, HBcAg and / or P-HBcAg) is quantitative or qualitative (eg, detecting a marker or a specific variant of a marker).

[0112] Assays for detecting protein markers under consideration include immunoassays (such as sandwich and competitive immunoassays), clinical chemistry assays, and enzyme assays. Assays for determining protein markers (e.g., HBcAg and / or P-HBcAg) in a test sample obtained from a subject may include the following steps: (a) providing 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 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 or reverse competitive inhibition assays), or a fluorescence polarization format. As described above, the immunoassay is a sandwich format. Specifically, in one aspect of the present disclosure, at least two antibodies are used to separate and quantify each marker in the test sample. More specifically, at least two antibodies bind to certain epitopes of the marker to form an immune complex known as a "sandwich." Typically, in an immunoassay, one or more antibodies can be used to capture the marker in the 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"). For example, in a sandwich assay, the binding of the two antibodies to the marker is not diminished by the binding of any other antibody in the assay to its corresponding binding site. In other words, the antibodies should be selected such that the one or more first antibodies contacted with the test sample or test sample extract suspected of containing the marker do not bind to all or part of the binding site recognized by the second or subsequent antibody, thereby interfering with the ability of the one or more second detection antibodies to bind to the marker.

[0113] In some embodiments, commercially available antibodies well known in the art are utilized.

[0114] The sample being tested (e.g., suspected of containing) the marker can be contacted with at least one capture antibody and at least one detection antibody (which is a second detection antibody or a third detection antibody) simultaneously or sequentially and in any order. For example, the test sample can be first contacted with at least one capture antibody and then (sequentially) with at least one detection antibody. Alternatively, the test sample can be first contacted with at least one detection antibody and then (sequentially) with at least one capture antibody. In another alternative, the test sample can be contacted with the capture antibody and the detection antibody simultaneously.

[0115] 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 formation of a first antibody-marker complex. If more than one capture antibody is used, a first multiple capture antibody-marker complex is formed. In a sandwich assay, antibodies (such as at least one capture antibody) are used in a molar excess relative to the maximum amount of marker expected in the test sample.

[0116] 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 may be bound to a solid support or solid phase, which facilitates separation of the first antibody-marker from the test sample. Any solid support known in the art may be used, including but not limited to solid supports made of polymeric materials in the form of wells of a reaction plate, test tubes or beads (e.g., polystyrene beads, magnetic beads), nitrocellulose strips, membranes, microparticles (e.g., latex particles, sheep and (Abbott Laboratories, Abbott Park, IL; These are red blood cells that have been "fixed" with methylglyoxal and formaldehyde).

[0117] The solid phase can also comprise any suitable porous material, the porosity of the porous material being enough to allow the detection antibody to enter and the surface affinity being suitable for binding antigen. Microporous structure is usually used, but the material with gel structure in hydrated state can also be used. This available solid support includes but is not limited to nitrocellulose and nylon. This type of porous solid support is in the form of a sheet with a thickness of about 0.01 to 0.5 mm (including about 0.1 mm). The pore size can vary over a wide range and can be about 0.025 to about 15 microns, especially about 0.15 to about 15 microns. The surface of this type of support can be activated by a chemical process, thereby causing the covalent bonding of antigen or antibody to the support. However, the irreversible binding of antigen or antibody is usually obtained by the adsorption of the little-known hydrophobic force on the porous material.

[0118] The antibody (or antibodies) can be bound to the solid support or solid phase by adsorption, by covalent bonding using chemical coupling agents, or by other means known in the art, provided that such binding does not interfere with the ability of the antibody to bind to the marker. Alternatively, the antibody can be bound to a solid support or solid phase that has been previously treated with streptavidin or biotin (e.g., using Power-Bind® available from Seradyn, Indianapolis, Indiana). TMThe SA-MP streptavidin is bound to microparticles coated with an antibody that has been biotinylated using methods known in the art. Alternatively, the antibody (or antibodies) can be bound to microparticles that have been previously coated with an anti-species-specific monoclonal antibody. In addition, if desired, the solid support can be derivatized to allow reaction with various functional groups on the antibody. This 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.

[0119] After contacting the test sample that is tested and / or suspected of containing the marker with at least one capture antibody (e.g., a first capture antibody), the mixture is incubated to form a first antibody (or multiple antibodies)-marker complex. The incubation can be carried out at a pH of about 4.5 to about 10.0 and a temperature of about 2°C to about 45°C, and for a period of at least about one (1) minute to about eighteen (18) hours, including about 1 to 20 minutes, and also including about 2-6 minutes. The immunoassays described herein can be performed in one step (meaning that the test sample, at least one capture antibody, and at least one detection antibody are all added to the reaction vessel sequentially or simultaneously) or in multiple steps, such as two steps, three steps, etc.

[0120] After the (first or multiple) capture antibody-marker complex is formed, the complex is then contacted with at least one detection antibody (under conditions that allow the formation of the (first or multiple) capture antibody-marker-(second or multiple) antibody detection complex). The at least one detection antibody can be the second, third, fourth, etc. antibody used in the immunoassay. If the capture antibody-complex is contacted with more than one detection antibody, a (first or multiple) capture antibody-marker detection antibody complex is formed. As with the use of a capture antibody (e.g., a first capture antibody), when at least a second (and subsequent) detection antibody is contacted with the capture antibody-marker complex, it is necessary to incubate for a period of time under conditions similar to those described above in order to form a (first or multiple) capture antibody-marker (second or multiple different markers) detection antibody complex. In some embodiments, at least one detection antibody contains a detectable label. The detectable label can be combined with at least one detection antibody (e.g., a 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, such as 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, thioester, sulfonamide, phenanthridinium ester, etc.); fluorescent labels such as fluorescein (5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachlorofluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, etc.), rhodamine, phycobiliprotein, R-phycoerythrin, quantum dots (zinc sulfide-capped cadmium selenide), thermometric labels or immunopolymerase chain reaction labels. An introduction to labels, labeling procedures, and label detection is found in Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, NY (1997) and in Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996), a combined handbook and catalog published by Molecular Probes, Inc., Eugene, Oregon.

[0121] Detectable label can be directly or by coupling agent and antibody combination.The example of spendable coupling agent is EDAC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), it is commercially available from Sigma-Aldrich, St.Louis, MO.Other spendable coupling agents are known in the art.The method for making detectable label be combined with antibody is known in the art.In addition, can buy or synthesize many detectable labels, these labels contain the end group that is convenient to detectable label and antibody coupling, such as N10-(3-sulfopropyl)-N-(3-carboxypropyl)-acridine-9-methane amide (also referred to as CPSP-acridinium ester) or N10-(3-sulfopropyl)-N-(3-sulfopropyl)-acridine-9-methane amide (also referred to as SPSP-acridinium ester).

[0122] Before the label is quantified, the (first or multiple) capture antibody-marker-(second or multiple) detection antibody complex can be, but need not be, separated from the remainder of the test sample. For example, if at least one capture antibody (e.g., a first capture antibody) is bound to a solid support or solid phase (such as, but not limited to, a well, a bead, or a microparticle of a reaction disk), 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 the marker and at least one second detection antibody to form a first (multiple) antibody-marker antibody complex, and the fluid (test sample) is then removed from contact with the solid support. If the at least one first capture antibody is not bound to the solid support, it is not necessary to remove the (first or multiple) capture antibody-marker (second or multiple) detection antibody complex from the test sample to quantify the amount of label.

[0123] After forming the capture antibody-marker complex (for example, the first capture antibody-marker complex) of the label, 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 the substrate of the label, and the substrate produces a quantifiable reaction such as color development. If the label is a radioactive label, a scintillation counter is used to quantify the label. If the label is a fluorescent label, the label is quantified by stimulating the label with a color of light (which is called "excitation wavelength") and detecting another color (which is called "emission wavelength") emitted by the label in response to the stimulus. If the label is a chemiluminescent label, the label is quantified by visual inspection or by detecting the light emitted using a photometer, x-ray film, high-speed photographic film, CCD camera, etc. Once the amount of label in the complex has been quantified, the concentration of the marker in the test sample is determined by using a standard curve, which is generated using a serial dilution of the marker of known concentration. In addition to using the serial dilution of the marker, a standard curve can be generated gravimetrically, by mass spectrometry, and by other techniques known in the art.

[0124] Other reagents and methods for performing immunoassays are encompassed by the methods and kits described herein. For example, various buffers are encompassed, such as those known in the art and / or that can be readily prepared or optimized, for example, for washing, for use as conjugate diluents, microparticle diluents, and / or for use as calibrant diluents. Exemplary conjugate diluents are Diluent (Abbott Laboratories, Abbott Park, IL) containing 2-(N-morpholino)ethanesulfonic acid (MES), another salt, a protein blocking agent, an antimicrobial agent, and a detergent. An exemplary calibration diluent is Calibration diluent (Abbott Laboratories, Abbott Park, IL) contains a buffer containing MES, additional salts, a protein blocking agent, and an antimicrobial agent.

[0125] In addition, as previously mentioned, the methods and kits are optionally adapted for use in automated or semi-automated systems. Some differences between automated or semi-automated systems and non-automated systems (e.g., ELISA) include the substrate to which the capture antibody is attached (which can affect sandwich formation and analyte reactivity), as well as the length and timing of the capture, detection, and / or any optional washing steps. Non-automated formats (such as ELISA) may include relatively long incubation times (e.g., about 2 hours) with the sample and capture reagent, while automated or semi-automated formats (e.g., ) may have a relatively short incubation time (e.g., for Similarly, non-automated formats (such as ELISA) may incubate detection antibodies (such as conjugated reagent (Pb264)) for relatively long incubation times (e.g., about 2 hours), while automated or semi-automated formats (e.g., ) may have a relatively short incubation time (e.g., for about 4 minutes).

[0126] Markers of the present disclosure (e.g., HBcAg and P-HBcAg) can be used in diagnostic tests to assess, determine, and / or limit (interchangeably used herein) the HBV status of a patient. For example, HBcAg and / or P-HBcAg can be used in diagnostic tests to assess, determine, and / or limit the HBV status of a patient. The phrase "HBV status" includes any distinguishable manifestation of a condition, including not having HBV. For example, HBV status includes, but is not limited to, whether there is HBV infection in the patient, the stage or severity of HBV infection, the progression of HBV infection (e.g., HBV infection over time), the effectiveness or response of HBV infection treatment (e.g., clinical follow-up and infection monitoring after treatment), and the type of HBV infection. Based on this state, further procedures may be indicated, including additional diagnostic tests or treatment procedures or regimens.

[0127] The ability of a diagnostic test to correctly predict a condition is typically 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 as positive by the test, while 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 - specificity. The larger the area under the ROC curve, the more powerful the predictive value of the test. Other useful measures of test effectiveness are the positive predictive value and the negative predictive value. The positive predictive value is the percentage of people who test positive who actually test positive. The negative predictive value is the percentage of people who test negative who actually test negative.

[0128] Analysis of the data described in this disclosure and clinical data from HBV patients and control patient cohorts results in the identification of HBV biomarkers to diagnose and / or evaluate HBV infection in a subject. The biomarkers can be used alone or in combination with each other and in combination with other biomarkers in the form of a set. The HBV biomarker set may include any HBV biomarker disclosed herein. In some embodiments, the HBV biomarker set includes HBcAg. In some embodiments, the HBV biomarker set includes P-HBcAg. In some embodiments, the HBV biomarker set includes HBcAg and P-HBcAg. The HBV biomarker set may also include non-HBV biomarkers (e.g., assay control biomarkers) and biomarkers previously determined to be associated with HBV. In some embodiments, the biomarker set of the present disclosure may show statistical differences in different HBV states. Diagnostic tests using these biomarkers may show an ROC of at least 0.6, at least about 0.7, at least about 0.8, or at least about 0.9.

[0129] HBV biomarkers can be present / expressed differently according to the type or subclass of HBV (e.g., HBV characteristics), and therefore, more than one set of HBV biomarkers may be helpful in determining HBV status. In some embodiments, biomarkers in patient samples are measured using the methods described herein and compared with, for example, predefined biomarker levels and associated with HBV status. In some embodiments, the measurement results can then be compared with relevant diagnostic amounts, cutoff values, reference levels, or multivariate model scores to distinguish positive HBV status (e.g., seroconversion) from negative HBV status (e.g., seroclearance). The diagnostic amount represents the amount of the measurement of the biomarker, and above or below the amount, the patient is classified as having a specific HBV status. For example, if the biomarker is unfavorable (e.g., increased) compared to a control subject (e.g., a subject not suffering from HBV infection), the amount or level of measurement above the diagnostic cutoff value or reference level can provide a diagnosis of HBV. In addition, if the biomarker is present during HBV infection and undetectable in the control, any detectable amount of measurement can provide a diagnosis of HBV infection. Alternatively, if biomarker is favorable (for example, reduced) during HBV infection, the amount of measurement equal to or lower than diagnostic cutoff value or reference level can provide the diagnosis of non-HBV infection. In addition, if biomarker does not exist during HBV infection and is detectable in the control, the amount of any detectable measurement can provide the diagnosis of non-HBV infection. As is well understood in the art, by adjusting the specific diagnostic cutoff value or reference level used in the determination, the sensitivity or specificity of the diagnostic determination can be increased according to the preference of the diagnostician. In some embodiments, specific diagnostic cutoff value or reference level can be, for example, by measuring the amount of biomarker in the statistically significant number of samples of patients with different HBV infection states, and drawing the cutoff value suitable for required specificity and sensitivity level to determine.

[0130] In addition, in certain embodiments, the values measured for the markers of the biomarker set are mathematically combined, and the combined values are associated with potential diagnostic questions. Biomarker values can be combined by any appropriate mathematical method of the prior art. Well-known mathematical methods for associating marker combinations with disease states are used, such as the following methods: discriminant analysis (DA) (e.g., linear, quadratic, regularized DA), discriminant function analysis (DFA), kernel methods (e.g., SVM), multidimensional scaling (MDS), nonparametric methods (e.g., k- nearest neighbor classifiers), PLS (partial least squares), tree-based methods (e.g., logistic regression, CART, random forest method (Random Forest Method), boosting / bagging methods (Boosting / Bagging Method)), generalized linear models (e.g., logistic regression), principal component-based methods (e.g., SIMCA), generalized additive models, fuzzy logic-based methods, neural networks, and methods based on genetic algorithms. Technicians have no problem selecting appropriate methods for evaluating the biomarker combinations of the present disclosure. In one embodiment of the present disclosure, the method used in the association biomarker combination (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 classifier), PLS (partial least squares), tree-based methods (e.g., logistic regression, CART, random forest method, boosting method) or generalized linear models (e.g., logistic regression) and principal component analysis.Details on these statistical methods are found in the following references: Ruczinski et al., 12 J. OF COMPUTATIONAL AND GRAPHICAL STATISTICS 475-511 (2003); Friedman, JH, 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, JH, Olshen, RA, Stone, CJ Classification and regression trees, California: Wadsworth (1984); Breiman, L., 45 MACHINE LEARNING 5-32 (2001); Pepe, MS, 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 edition (2001).

[0131] 3. Monitoring and treatment of chronic hepatitis B infection

[0132] In some embodiments, the presence or level of HBcAg and / or P-HBcAg is used to assess or monitor the stage or staging of a subject's chronic HBV infection. 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 staging of the subject's chronic HBV infection. The subject can be diagnosed with any HBV genotype and / or receive any treatment for 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, it is determined whether the level of HBcAg and / or P-HBcAg in the sample is favorable or unfavorable. In some embodiments, the 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, unfavorable levels indicate that HBV treatment should be provided to the subject. In contrast, favorable levels and amounts of HBcAg and / or P-HBcAg in a sample indicate that the subject's HBV infection is improving or is inactive.

[0133] In some embodiments, the level of unfavorable level indicates that HBcAg and / or P-HBcAg is greater than or equal to a threshold level. For example, in some embodiments, the stage or staging of HBV infection is based on whether the level or amount of HBcAg in the sample obtained from the subject is greater than or equal to a reference level of HBcAg (for example, the level in a control sample obtained from a subject who is not troubled by HBV) is determined. For example, in some embodiments, the stage or staging of HBV infection is based on whether the level or amount of P-HBcAg in the sample obtained from the subject is greater than or equal to a reference level of P-HBcAg (for example, the level in a control sample obtained from a subject who is not troubled by HBV) is determined.

[0134] In some embodiments, an unfavorable level indicates that the level of HBcAg and / or P-HBcAg does not decrease by at least one absolute amount from a first sample obtained from a subject to a second sample at a separate time point. For example, in some embodiments, the methods described herein include obtaining a first sample from a subject at a first time point before or after receiving chronic HBV treatment, and obtaining a second sample from the subject at a second time point after obtaining the first sample. An unfavorable level can indicate that the level of HBcAg and / or P-HBcAg in the sample does not decrease by at least one absolute amount from the first time point to the second time point.

[0135] In some embodiments, "favorable levels" indicate that the level of HBcAg and / or P-HBcAg is less than a threshold level. In some embodiments, "favorable levels" indicate that the level of HBcAg and / or P-HBcAg decreases by at least one absolute amount from a first sample obtained from a subject to a second sample.

[0136] In some embodiments, the stage or staging of HBV infection is determined based on whether the level or amount of HBcAg and / or P-HBcAg in a sample obtained from a subject is greater than or equal to a control level of HBcAg and / or a control level of P-HBcAg (e.g., a level in a control sample obtained from a subject not suffering from HBV). In some embodiments, the method includes providing HBV treatment to the subject.

[0137] In some embodiments, one or more additional markers are measured to assess or monitor the stage or staging of HBV infection, including additional protein markers or enzyme markers as described above.

[0138] In some aspects, the method for evaluating and monitoring the stage or staging of chronic HBV infection includes 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 chronic HBV or receiving chronic HBV treatment. In some aspects, the method also includes 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 term "non-infectious HBV particles" or "empty HBV particles" are interchangeably used herein to 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 the sample is used to assess the stage or staging of a subject's chronic HBV infection.

[0139] As shown in the accompanying drawings and examples, the level of HBcAg is shown herein as being associated with the level of HBV DNA in a given sample. Therefore, in some embodiments, the method for assessing and monitoring the stage or staging of HBV infection includes performing a determination for detecting 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 at least one sample based on the presence or level of HBcAg. Therefore, a method for assessing and monitoring the stage or staging of HBV infection of a subject is provided herein, and the method does not need to implement nucleic acid testing to measure the HBV DNA of the subject. In addition, regardless of whether the subject has received chronic HBV treatment, this method can be performed because the amount of HBcAg is shown herein as being associated with the amount of HBV DNA in a given sample, regardless of treatment status (that is, regardless of whether the subject has received chronic HBV treatment). Therefore, the advantage of the method for assessing and monitoring the stage or staging of HBV infection as described herein is that the method can be performed efficiently and accurately by using a single determination to evaluate the number of infectious HBV particles in the sample, rather than performing a determination of HBcAg once and performing another determination of HBV nucleic acid (e.g., HBV DNA).

[0140] As shown in the drawings and examples, the level of P-HBcAg is shown herein as being associated with the HBV DNA level or HBV RNA (for example, HBV pgRNA) level in a given sample, which depends on the treatment status of the subject. Specifically, the level of P-HBcAg is shown herein as being associated with the level of HBV DNA in a sample obtained from a subject who has never received HBV treatment. Therefore, in some embodiments, the method for assessing and monitoring the stage or staging of HBV infection includes detecting the presence or level of P-HBcAg in at least one sample obtained from a subject who has never received HBV treatment, and determining the amount of infectious HBV particles in at least one sample based on the presence or level of P-HBcAg. Therefore, in some embodiments herein, a method for assessing and monitoring the stage or staging of HBV infection of a subject is provided, the method does not need to perform nucleic acid testing to measure the HBV DNA of the subject, and therefore the method can be effectively and accurately performed using only a single test, rather than using a separate test for P-HBcAg and HBV DNA.

[0141] In some embodiments, the methods described herein also include selecting an appropriate chronic HBV treatment method for the subject. In some embodiments, the method for assessing and monitoring the stage or staging of chronic HBV infection includes providing chronic HBV treatment to the subject. For example, in some embodiments, the methods described herein include providing chronic HBV treatment to the subject when determining that the level of HBcAg and / or the level of P-HBcAg is unfavorable. In some embodiments, the method includes providing chronic HBV treatment to the subject when the amount of infectious HBV particles in the sample is equal to or higher than a threshold value. In some embodiments, the method includes providing chronic HBV treatment to the subject when the amount of infectious HBV particles of the same dose does not decrease by at least one absolute amount from a first sample obtained from the subject to a second sample. Described herein are treatments suitable for chronic HBV infection, and include, but are not limited to, nucleoside (acid) analogs (e.g., lamivudine, adefovir, tenofovir, telbivudine, or entecavir), nucleic acids (e.g., siRNA, antisense oligonucleotides, shRNA, or miRNA), immunomodulators (e.g., interferon α-2a or pegylated interferon α-2a), core protein assembly inhibitors (e.g., NVR 3-1983, GLS4, or BAY41-4109), capsid assembly modulators (CAMs) (e.g., JNJ-632, AT130, or BAY41-4109), HBsAg release inhibitors (e.g., REP 9AC), entry inhibitors (e.g., Myrcludex-B), DNA modifying agents (e.g., CRISPR-based DNA editing agents, TALENs, ZNFs, etc.), or combinations thereof.

[0142] In some embodiments, the presence, level or state of a marker are used to optimize HBV treatment. For example, if it is found that the subject has a marker indicating a more toxic or aggressive or drug-resistant infection, more active treatment or different medicines can be administered. On the contrary, if it is found that the subject has a marker indicating a less toxic or aggressive infection, less active treatment or monitoring (for example, monitoring infection without specific drug therapy) can be selected.

[0143] In some embodiments, subject is monitored at multiple time points to evaluate HBV status.In some embodiments, multiple samples are obtained from subject at different time points to monitor the chronic HBV infection of subject.For example, in some embodiments, the first sample is obtained from subject at the first time point, and every day, every two 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 with lower frequency collection other sample, with the stage or the staging of the chronic HBV infection of continuous monitoring subject.

[0144] In some embodiments, monitoring is continued and / or repeated until the subject (1) obtains a favorable HBcAg level for a sufficient period of time. In some embodiments, monitoring or determining a treatment method (e.g., detection of a marker) is continued and / or repeated until the subject (2) obtains a favorable HBcAg level for a sufficient period of time. In some embodiments, monitoring or determining a treatment method (e.g., detection of a marker) is continued and / or repeated until the subject (3) has less than a reference amount of infectious HBV particles for a sufficient period of time. In some embodiments, monitoring or determining a treatment method (e.g., detection of a marker) is continued and / or repeated until the subject completes two or more of (1)-(3) for a sufficient period of time. In some embodiments, the 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, 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 53 weeks, about 54 weeks, about 55 weeks, about 56 weeks, about 57 weeks, about 58 weeks, about 59 weeks, about 60 weeks, about 61 weeks, about 62 weeks, about 63 weeks, about 64 weeks, about 65 weeks, about 66 weeks, about 67 weeks, about 68 weeks, about 69 weeks, about 70 weeks, about 71 weeks, about 72 weeks, about 73 weeks, about 74 weeks, about 75 weeks, about 76 weeks, about 77 weeks, about 78 weeks, about 79 weeks, about 80 weeks , about 4 weeks, 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 2.5 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.

[0145] 4. Monitoring response to HBV treatment

[0146] In some embodiments, the assay of the present disclosure can be used to monitor the response of subjects receiving HBV treatment. "Chronic HBV treatment" may also be referred to herein as "anti-HBV agent", "HBV therapeutic agent", "HBV treatment (HBV treatment)", "HBV treatment (treatment for HBV)", "HBV therapy" and the like. By way of example, the assay of the present disclosure can be used to monitor drug resistance in subjects receiving HBV treatment. For example, it is known that the emergence of drug-resistant strains with amino acid substitutions in the tyrosine-methionine-aspartic acid-aspartic acid (YMDD) motif of reverse transcriptase may be a serious problem for subjects receiving lamivudine therapy (see, for example, Hatakeyama, T. et al., Hepatology, 45 (5): 1179-1186 (2007)). The assay of the present disclosure can be used to monitor and / or predict the early emergence of mutants and / or drug resistance during HBV therapy.

[0147] In some embodiments, the presence or level of HBcAg and / or P-HBcAg is used to monitor the response of a subject to treatment for chronic HBV infection. 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 the subject to treatment for chronic HBV.

[0148] In some embodiments, it is determined whether the level of HBcAg and / or P-HBcAg in the sample is favorable or unfavorable. In some embodiments, an unfavorable level or amount of HBcAg and / or P-HBcAg in the sample indicates that the chronic HBV treatment of the subject is ineffective. In contrast, a favorable level or amount of HBcAg and / or P-HBcAg in the sample indicates that the chronic HBV treatment of the subject is effective (e.g., HBV infection is improving).

[0149] In some embodiments, the level of unfavorable level indicates that HBcAg and / or P-HBcAg is greater than or equal to the threshold level. For example, in some embodiments, the responsiveness of HBV treatment is based on whether the level or amount of HBcAg in the sample obtained from the subject is greater than or equal to the reference level of HBcAg (for example, the level in the control sample obtained from a subject who is not troubled by HBV) is determined. For example, in some embodiments, the responsiveness of HBV treatment is based on whether the level or amount of P-HBcAg in the sample obtained from the subject is greater than or equal to the reference level of P-HBcAg (for example, the level in the control sample obtained from a subject who is not troubled by HBV) is determined.

[0150] In some embodiments, the level of an unfavorable level indication HBcAg and / or P-HBcAg does not decrease by at least one absolute amount from a first sample to a second sample obtained from a subject at a separate time point. For example, in some embodiments, the methods described herein include obtaining a first sample from a subject at a first time point before or after receiving chronic HBV treatment, and obtaining a second sample from a subject at a second time point after obtaining the first sample. The second sample is obtained after the subject receives at least one dose of chronic HBV treatment. An unfavorable level can indicate that the level of HBcAg and / or P-HBcAg in the sample does not decrease by at least one absolute amount from the first time point to the second time point.

[0151] In some embodiments, "favorable levels" indicate that the level of HBcAg and / or P-HBcAg is less than a threshold level. In some embodiments, "favorable levels" indicate that the level of HBcAg and / or P-HBcAg decreases by at least one absolute amount from a first sample obtained from a subject to a second sample.

[0152] 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 after receiving chronic HBV treatment, 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 after receiving chronic HBV treatment, 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 after receiving chronic HBV treatment, and the second time point is about 12 weeks (about 3 months) after the first time point.

[0153] In some embodiments, one or more additional markers are measured to monitor a subject's responsiveness to treatment for chronic HBV infection, including additional protein markers or enzyme markers as described above.

[0154] Various HBV treatments have different effects on the secretion of HBV-derived particles. 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), HBV particles containing HBV DNA encapsulated in HBcAg are secreted from infected cells. Certain HBV treatments, such as nucleoside (acid) analogs, are used to inhibit the reverse transcription of HBV RNA into HBV DNA, and thereby reduce the amount of HBV particles (also referred to herein as “infectious” HBV particles) containing HBV DNA encapsulated in HBcAg. Therefore, 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 intended to interrupt the reverse transcription of pgRNA into rcDNA is effective in a subject. For example, in some embodiments, the presence or amount of HBcAg in a sample obtained from a subject can be evaluated to determine whether a treatment using nucleoside (acid) analogs is effective. In some embodiments, the presence or amount of P-HBcAg is not affected by such treatment because the pgRNA is still translated and P-HBcAg is released from the cell as "empty" particles (i.e., particles that do not contain HBV DNA). These "empty" particles can be detected by measuring phosphorylated HBcAg (P-HBcAg). In addition, reducing the level of HBV RNA in the cell can also reduce the level of HBV DNA produced because less template RNA is available for reverse transcription. Therefore, in some embodiments, treatments that interrupt the production of HBV RNA (e.g., CAM) will reduce the amount of P-HBcAg and HBcAg produced. Therefore, the level of P-HBcAg or the levels of P-HBcAg and HBcAg can be obtained in a sample obtained from a subject to evaluate or predict whether such a treatment (e.g., CAM) is effective in the subject.

[0155] In some embodiments, the methods described herein can be used to evaluate the efficacy 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 treatment using a CAM to determine whether the treatment is effective. In some embodiments, the method includes determining that the treatment is effective when the level of P-HBcAg in the sample is less than a reference level of P-HBcAg. In some embodiments, the method includes determining that the treatment is effective when the level of P-HBcAg in the sample is less than a reference level of P-HBcAg and the level of HBcAg in the sample is less than a reference level of HBcAg. In some embodiments, the method includes determining that the treatment is ineffective when the level of P-HBcAg in the sample is greater than or equal to the reference level of P-HBcAg.

[0156] As shown in the drawings and examples, the amount of HBcAg is shown herein as being associated with the amount of HBV DNA in a given sample after the subject receives chronic HBV treatment. Therefore, in some embodiments, the method for monitoring the response of a subject to chronic HBV treatment includes measuring to detect the presence or level of HBcAg in at least one sample obtained from the subject after receiving treatment, and determining the amount of infectious HBV particles in at least one sample based on the presence or level of HBcAg. Therefore, in some embodiments herein, a method for monitoring the response of a subject to chronic HBV infection treatment is provided, and the method does not need to measure the nucleic acid test of HBV DNA to accurately monitor the response to treatment. Therefore, the advantage of the method for monitoring the response to chronic HBV infection treatment as described herein is that the method can be efficiently and accurately performed by using a single assay to evaluate the quantity of infectious HBV particles in the sample, rather than HBcAg being measured once and HBV nucleic acid (e.g., HBV DNA) being measured again.

[0157] As described above, the amount of P-HBcAg is shown herein as being associated with the amount of HBV DNA or HBV RNA (e.g., HBV pgRNA) in a given sample, which depends on the treatment status of the subject. In some embodiments, the amount of P-HBcAg is shown herein as being associated with the amount of HBV RNA (e.g., pgRNA) in a sample when the subject has received HBV treatment. In some embodiments, the treatment interrupts the transcription of cccDNA to HBV RNA (e.g., pgRNA). HBV RNA (e.g., pgRNA) is released from cells as "empty" particles (also referred to as "non-infectious" HBV particles). Such particles can be evaluated by determining the level of P-HBcAg in the sample. Therefore, in some embodiments, the method for monitoring the response to HBV treatment includes performing an assay to detect the presence or level of P-HBcAg in at least one sample obtained from a subject who has received HBV treatment, and determining the amount of non-infectious HBV particles in at least one sample based on the presence or level of P-HBcAg. In some embodiments, treatment is an agent that interrupts pgRNA reverse transcription. Suitable treatments include, for example, capsid assembly regulators / inhibitors (CAMs). CAMs indirectly inhibit reverse transcription by preventing the formation of capsids. Reverse transcription of pgRNA occurs inside the capsid. Therefore, CAMs indirectly inhibit reverse transcription. Therefore, an advantage of the method for monitoring responses to treatment of chronic HBV infection is that the method can be performed efficiently and accurately by using a single assay to evaluate the number of non-infectious HBV particles in a sample, rather than performing one assay on P-HBcAg and another assay on HBV nucleic acid (e.g., HBV RNA). In some embodiments, a reduction in the amount of non-infectious HBV particles indicates that the HBV treatment (e.g., CAM) is effective in the subject.

[0158] 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 has received treatment that affects the reverse transcription of HBV RNA (e.g., pgRNA) to HBV DNA. For example, some HBV treatments (such as nucleoside inhibitors) inhibit pgRNA reverse transcription and thus reduce the amount of HBV DNA (e.g., infectious particles) released from cells. However, such HBV treatments do not interfere with the process of cccDNA transcription into pgRNA, and therefore these treatments do not reduce the amount of HBV RNA (e.g., pgRNA) secreted from cells as "empty" or "non-infectious" particles. Therefore, the level of HBcAg (and therefore the amount of infectious particles) should be reduced, while the level of P-HBcAg (and therefore the amount of non-infectious particles) should not be affected by this treatment.

[0159] In some embodiments, the method for monitoring a response to HBV treatment further comprises determining whether the HBV treatment is effective in the subject. In some embodiments, the treatment is determined to be ineffective when the levels of HBcAg and / or P-HBcAg are unfavorable. For example, in some embodiments, the treatment is determined to be ineffective when the levels of HBcAg and / or P-HBcAg do not decrease by at least an absolute amount from a first sample to a second sample obtained from the subject.

[0160] In some embodiments, when the amount of infectious HBV particles is greater than or equal to a threshold value, it is determined that the treatment is ineffective. In some embodiments, when the amount of infectious HBV particles does not reduce at least one absolute amount from the first sample to the second sample collected from the subject, it is determined that the treatment is ineffective. For example, in some embodiments, the first sample is collected from the subject at a first time point within 24 hours of receiving HBV treatment, and the second sample is collected from the subject at a second time point after the first time point. 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, when the amount of infectious HBV particles does not reduce at least one absolute amount from the first sample to the second sample, it is determined that the treatment is ineffective.

[0161] In some embodiments, when the level of HBcAg and / or P-HBcAg is favorable, it is determined that the treatment is effective. In some embodiments, when the amount of infectious HBV particles from the first sample collected from the subject to the second sample is reduced by at least one absolute amount, it is determined that the treatment is effective. For example, in some embodiments, the first sample is collected from the subject at a first time point within 24 hours of receiving HBV treatment, and the second sample is collected from the subject at a second time point after the first time point. 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, when the amount of infectious HBV particles from the first sample to the second sample is reduced by at least one absolute amount, it is determined that the treatment is effective.

[0162] In some embodiments, the method for monitoring the response of a subject to chronic HBV treatment also includes changing the chronic HBV treatment when it is determined that the treatment is ineffective. In some embodiments, changing the chronic HBV treatment includes providing the subject with an increased dose of treatment, increasing the frequency of treatment, providing the subject with a second treatment, or any combination thereof. For example, in some embodiments, changing the HBV treatment includes providing the subject with an increased dose of HBV treatment. In some embodiments, changing the treatment includes increasing the frequency of treatment of HBV treatment. In some embodiments, changing the treatment includes increasing the dose and frequency of treatment of HBV treatment. In some embodiments, changing the treatment includes providing the subject with a second HBV treatment. The second HBV treatment may include any suitable HBV treatment described herein (e.g., HBV therapeutic agent). The subject may be provided with a second HBV treatment as an alternative treatment or additional treatment. For example, in some embodiments, the HBV treatment that the subject has been receiving (e.g., determining an ineffective treatment) is stopped, and a second HBV treatment is provided to the subject. As another example, in some embodiments, in addition to the HBV treatment that the subject has been receiving, a second HBV treatment is also given. In 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.

[0163] 5. Methods for measuring HBcAg and / or P-HBcAg

[0164] In the above methods, the level of HBV biomarkers (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 determination, competitive binding assays, functional protein determinations, or chromatography or spectroscopy such as high performance liquid chromatography (HPLC), mass spectrometry, or liquid chromatography-mass spectrometry (LC / MS) or capillary electrophoresis (CE)-MS, or direct infusion, or any separation front end coupled to MS. Additionally, the assay can be performed in a clinical chemistry format known to those skilled in the art.

[0165] a. Immunoassay

[0166] In some embodiments, measuring the level of HBV biomarkers comprises contacting the sample with a first specific binding member and a second specific binding member. In some embodiments, the first specific binding member is a capture antibody and the second specific binding member is a detection antibody. In some embodiments, measuring the level of HBV biomarkers comprises contacting the sample simultaneously or sequentially in any order with: (1) a capture antibody (e.g., HBV biomarker-capture antibody), which binds to an epitope on an HBV biomarker or an HBV biomarker fragment to form a capture antibody-HBV biomarker antigen complex (e.g., HBV biomarker-capture antibody-HBV biomarker antigen complex), and (2) a detection antibody (e.g., HBV biomarker-detection antibody), which comprises a detectable label and binds to an epitope on the HBV biomarker that is not detected. The capture antibody binds to an epitope to form an HBV biomarker antigen-detection antibody complex (e.g., HBV biomarker antigen-HBV biomarker-detection antibody complex), so that a capture antibody-HBV biomarker antigen-detection antibody complex (e.g., HBV biomarker-capture antibody-HBV biomarker antigen-HBV biomarker-detection antibody complex) is formed; and based on the signal generated by the detectable label in the capture antibody-HBV biomarker antigen-detection antibody complex, the amount or concentration of the HBV biomarker in the sample is measured.

[0167] 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 in a sample is measured. In some embodiments, the presence, level, or status of HBcAg and P-HBcAg in different samples obtained from a subject is measured. The samples can be obtained at the same time point or at different time points.

[0168] 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 divided into 10 genotypes A-J. The sequence of human HBcAg genotype A comprises the following amino acids: MDIDPYKEFG ATVELLSFLP SDFFPSVRDLLDTASALYREALESPEHCSP HHTALRQAIL CWGELMTLAT WVGNNLEDPA SRDLVVNYVNTNMGLKIRQLLWFHISCLTF GRETVLEYLV SFGVWIRTPP AYRPPNAPILSTLPETTVVR RRDRGRSPRR RTPSPRRRRSQSPRRRRSQS RESQC (SEQ ID NO: 1; Figure 3 ). HBcAg sequences of HBV genotypes AJ are provided below. The bold amino acids indicate the C-terminus of each corresponding sequence.

[0169] Genotype A:

[0170]

[0171] Genotype AC end: RRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC (SEQ ID NO: 2)

[0172] Genotype A (amino acids 1-149 of SEQ ID NO: 1):

[0173] MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNLEDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVV(SEQ ID NO:3)

[0174] Genotype B:

[0175]

[0176] Genotype BC end: RRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC (SEQ ID NO: 13)

[0177] Genotype C:

[0178]

[0179] Genotype CC end: RRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC (SEQ ID NO: 14)

[0180] Genotype D:

[0181]

[0182]

[0183] Genotype DC end: RRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC (SEQ ID NO: 15)

[0184] Genotype E:

[0185]

[0186] Genotype EC end: RRRGRSPRRRTPSPRRRRSQSPRRRRSQSPASQC (SEQ ID NO: 16)

[0187] Genotype F:

[0188]

[0189] Genotype FC end: RRRGRSPRRRTPSPRRRRSQSPRRRRSQSPASQC (SEQ ID NO: 17)

[0190] Genotype G:

[0191]

[0192] Genotype GC end: RRRRGRSPRRRTPSPRRRRSQSPRRRRSASPASQC (SEQ ID NO: 18)

[0193] Genotype H:

[0194]

[0195] Genotype HC end: RQRGRAPRRRTPSPRRRRSQSPRRRRSQSPASQC (SEQ ID NO: 19)

[0196] Genotype I:

[0197] MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALRQAIVCWGELMNLATWVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC(SEQ ID NO:11)

[0198] Genotype IC end: RRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC (SEQ ID NO: 20)

[0199] Genotype J:

[0200] MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALRQAVLCWGELMNLATWVGSNLEDPASRELVVSYVNINMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSPSSQC(SEQ ID NO:12)

[0201] Genotype JC end: RRRGRSPRRRTPSPRRRRSQSPRRRRSQSPSSQC (SEQ ID NO: 21)

[0202] The following table includes the consensus sequences of HBcAg above, including genotypes AJ.

[0203]

[0204]

[0205]

[0206] The methods described herein can be used to detect HBcAg or phosphorylated HbcAg of any of the above-mentioned 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 or HBV genotype J).

[0207] 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 afflicted with one of the above genotypes express HbcAg that has one or more mutations compared to the above sequences. Thus, in some embodiments, the method comprises contacting the sample with a capture antibody and / or detection antibody that binds to an epitope having at least 95% sequence identity to an amino acid sequence provided herein. In some embodiments, the method comprises contacting the sample with an HBcAg capture antibody that binds to an epitope at 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 (eg, 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.

[0208] In additional 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 afflicted with one of the above genotypes express HbcAg that has 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 an 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: 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 the 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 in length (e.g., contiguous amino acids). 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.

[0209] In some embodiments, the 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 that is about 3 to about 36 consecutive amino acids in length. 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 of 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 non-contiguous 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, wherein each binding site comprises at least three consecutive amino acids (e.g., at least 3, at least 4, at least 5, at least 6, at least 7 consecutive amino acids) and wherein each binding site is separated by at least one (e.g., at least one, at least two, at least three) amino acid.

[0210] In additional embodiments, the 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 that is about 3 to about 36 consecutive amino acids in length. 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 of 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 non-contiguous 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, wherein each binding site comprises at least three consecutive amino acids (e.g., at least 3, at least 4, at least 5, at least 6, at least 7 consecutive amino acids) and wherein each binding site is separated by at least one (e.g., at least one, at least two, at least three) amino acid.

[0211] The AC terminus of the human HbcAg genotype 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 of about 3 to about 36 amino acids (e.g., contiguous amino acids) in length on 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, wherein each binding site comprises at least three consecutive amino acids (e.g., at least 3, at least 4, at least 5, at least 6, at least 7 consecutive amino acids) and wherein each binding site is separated by at least one (e.g., at least one, at least two, at least three) amino acid.

[0212] 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 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 HbcAg detection 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 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).

[0213] In other 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 with 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).

[0214] 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) 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 HbcAg detection antibody binds to a non-continuous epitope. In some embodiments, the HbcAg detection antibody binds to a non-contiguous 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 more binding sites, and each binding site is separated by at least 2 amino acids. For example, in some embodiments, the HbcAg detection antibody binds to multiple binding sites, wherein each binding site comprises at least 2 consecutive amino acids, and each binding site is separated by at least 2 amino acids. In some embodiments, each binding site comprises at least 2 consecutive amino acids (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 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, at least 50 amino acids).

[0215] In other 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 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 1-161 of SEQ ID NO: 9. In some embodiments, the HbcAg detection antibody binds to a non-continuous epitope. In some embodiments, the HbcAg detection antibody binds to a non-contiguous 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 more binding sites, and each binding site is separated by at least 2 amino acids. For example, in some embodiments, the HbcAg detection antibody binds to multiple binding sites, wherein each binding site comprises at least 2 consecutive amino acids, and wherein each binding site is separated by at least 2 amino acids. In some embodiments, each binding site comprises at least 2 consecutive amino acids (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 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, at least 50 amino acids).

[0216] In other aspects, 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-continuous epitope on SEQ ID NO: 3. For example, in some embodiments, the HbcAg detection antibody binds to more binding sites, and each binding site is separated by at least 2 amino acids. For example, in some embodiments, the HbcAg detection antibody binds to multiple binding sites, wherein each binding site comprises at least 2 consecutive amino acids, and wherein each binding site is separated by at least 2 amino acids. In some embodiments, each binding site comprises at least 2 consecutive amino acids (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 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, at least 50 amino acids).

[0217] In some embodiments, the HbcAg capture antibody binds to an epitope other than the C-terminus on HbcAg. In some embodiments, the 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 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 HbcAg capture antibody binds to an epitope of about 3 to about 50 contiguous amino acids (about 3 to about 40 contiguous amino acids, about 3 to about 35 contiguous amino acids, about 3 to about 30 contiguous amino acids, about 3 to about 25 contiguous amino acids, about 3 to about 20 contiguous amino acids, or about 3 to about 15 contiguous amino acids).

[0218] In other embodiments, the HbcAg capture antibody binds to an epitope other than the C-terminus of HbcAg. 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 contiguous amino acids (about 3 to about 40 contiguous amino acids, about 3 to about 35 contiguous amino acids, about 3 to about 30 contiguous amino acids, about 3 to about 25 contiguous amino acids, about 3 to about 20 contiguous amino acids, or about 3 to about 15 contiguous amino acids).

[0219] 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) 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 HbcAg capture antibody binds to a non-continuous epitope. In some embodiments, the HbcAg capture antibody binds to a non-contiguous 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 capture antibody binds to more binding sites, and each binding site is separated by at least 2 amino acids. For example, in some embodiments, the HbcAg capture antibody binds to multiple binding sites, wherein each binding site comprises at least 2 contiguous amino acids, and each binding site is separated by at least 2 amino acids. In some embodiments, each binding site comprises at least 2 contiguous amino acids (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 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, at least 50 amino acids).

[0220] In 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 non-continuous epitope. In some embodiments, the HbcAg capture antibody binds to a non-contiguous 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 more binding sites, and each binding site is separated by at least 2 amino acids. For example, in some embodiments, the HbcAg capture antibody binds to multiple binding sites, wherein each binding site comprises at least 2 contiguous amino acids, and each binding site is separated by at least 2 amino acids. In some embodiments, each binding site comprises at least 2 contiguous amino acids (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 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, at least 50 amino acids).

[0221] 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 more binding sites, and each binding site is separated by at least 2 amino acids. For example, in some embodiments, the capture antibody binds to multiple binding sites, wherein each binding site comprises at least 2 consecutive amino acids, and wherein each binding site is separated by at least 2 amino acids. In some embodiments, each binding site comprises at least 2 consecutive amino acids (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 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, at least 50 amino acids).

[0222] 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 that is about 3 to about 36 amino acids in length (e.g., contiguous amino acids). 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.

[0223] In 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 that has 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 in length (e.g., contiguous amino acids). 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.In other embodiments, 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 may 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 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 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 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 may 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.

[0225] In some embodiments, the 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 that is about 3 to about 36 amino acids in length (e.g., contiguous amino acids). 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 of 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 non-contiguous epitope. In some embodiments, the HBcAg detection antibody binds to a non-continuous epitope on the C-terminus of HBcAg. For example, in some embodiments, the HBcAg detection antibody binds to multiple binding sites, wherein each binding site comprises at least three consecutive amino acids (e.g., at least 3, at least 4, at least 5, at least 6, at least 7 consecutive amino acids) and wherein each binding site is separated by at least one (e.g., at least one, at least two, at least three) amino acids. 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, wherein each binding site comprises at least three consecutive amino acids (e.g., at least 3, at least 4, at least 5, at least 6, at least 7 consecutive amino acids) and wherein each binding site is separated by at least one (e.g., at least one, at least two, at least three) amino acids.

[0226] In other 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 that is about 3 to about 36 amino acids in length (e.g., contiguous amino acids). 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 of 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 non-contiguous epitope. In some embodiments, the HBcAg detection antibody binds to a non-continuous epitope on the C-terminus of HBcAg. For example, in some embodiments, the HBcAg detection antibody binds to multiple binding sites, wherein each binding site comprises at least three consecutive amino acids (e.g., at least 3, at least 4, at least 5, at least 6, at least 7 consecutive amino acids) and wherein each binding site is separated by at least one (e.g., at least one, at least two, at least three) amino acids. 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, wherein each binding site comprises at least three consecutive amino acids (e.g., at least 3, at least 4, at least 5, at least 6, at least 7 consecutive amino acids) and wherein each binding site is separated by at least one (e.g., at least one, at least two, at least three) amino acids.

[0227] 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 amino acids (e.g., consecutive amino acids) on SEQ ID NO: 2. In some embodiments, the HBcAg detection antibody binds to multiple binding sites on SEQ ID NO:2, wherein each binding site comprises at least three consecutive amino acids (e.g., at least 3, at least 4, at least 5, at least 6, at least 7 consecutive amino acids) and wherein each binding site is separated by at least one (e.g., at least one, at least two, at least three) amino acid.

[0228] 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 indicates the presence and / or amount (e.g., level) of HBcAg in the sample.

[0229] In some embodiments, detecting the presence, level, or status of 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.

[0230] In some other embodiments, detecting the presence, level, or status of 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.

[0231] 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 in length (e.g., contiguous amino acids). In some embodiments, the P-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 amino acids in length (e.g., contiguous amino acids), provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least 5 amino acids, at least 6 amino acids, or 7 amino acids are phosphorylated.

[0232] In 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 in length (e.g., contiguous amino acids). In some embodiments, the P-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 amino acids in length (e.g., contiguous amino acids), provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least 5 amino acids, at least 6 amino acids, or 7 amino acids are phosphorylated.

[0233] In some embodiments, the P-HBcAg capture antibody binds to an epitope of about 3 to about 36 amino acids (e.g., contiguous 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, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least 5 amino acids, at least 6 amino acids, or 7 amino acids are phosphorylated. In some embodiments, the P-HBcAg capture antibody binds to an epitope of about 3 to about 36 amino acids (e.g., contiguous 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, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least 5 amino acids, at least 6 amino acids, or 7 amino acids are phosphorylated. NO:39 in length 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., continuous amino acids), provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least 5 amino acids, at least 6 amino acids or 7 amino acids are phosphorylated. In some embodiments, the P-HBcAg capture antibody binds to a non-continuous epitope on the C-terminus of P-HBcAg. In some embodiments, the P-HBcAg capture antibody binds to multiple binding sites, wherein each binding site comprises at least three consecutive amino acids (e.g., at least 3, at least 4, at least 5, at least 6, at least 7 consecutive amino acids), and wherein each binding site is separated by at least one (e.g., at least one, at least two, at least three) amino acids, and wherein at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least 5 amino acids, at least 6 amino acids, or 7 amino acids are phosphorylated.For example, in some embodiments, the 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, wherein each binding site comprises at least three consecutive amino acids (e.g., at least 3, at least 4, at least 5, at least 6, at least 7 consecutive amino acids), and wherein each binding site is separated by at least one (e.g., at least one, at least two, at least three) amino acids, and wherein at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least 5 amino acids, at least 6 amino acids, or 7 amino acids are phosphorylated.

[0234] In other embodiments, the P-HBcAg capture antibody binds to an epitope of about 3 to about 36 amino acids (e.g., contiguous 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, 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 to about 36 amino acids (e.g., contiguous 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, 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. NO:21 in length 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., continuous amino acids), provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least 5 amino acids, at least 6 amino acids or 7 amino acids are phosphorylated. In some embodiments, the P-HBcAg capture antibody binds to a non-continuous epitope on the C-terminus of P-HBcAg. In some embodiments, the P-HBcAg capture antibody binds to multiple binding sites, wherein each binding site comprises at least three consecutive amino acids (e.g., at least 3, at least 4, at least 5, at least 6, at least 7 consecutive amino acids), and wherein each binding site is separated by at least one (e.g., at least one, at least two, at least three) amino acids, and wherein at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least 5 amino acids, at least 6 amino acids, or 7 amino acids are phosphorylated.For example, in some embodiments, the 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, wherein each binding site comprises at least three consecutive amino acids (e.g., at least 3, at least 4, at least 5, at least 6, at least 7 consecutive amino acids), and wherein each binding site is separated by at least one (e.g., at least one, at least two, at least three) amino acids, and wherein at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least 5 amino acids, at least 6 amino acids, or 7 amino acids are phosphorylated.

[0235] 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 of about 3 to about 36 amino acids (e.g., contiguous amino acids) 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 5 amino acids, at least 6 amino acids, or 7 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., contiguous amino acids) 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 5 amino acids, at least 6 amino acids, or 7 amino acids are phosphorylated.

[0236] 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.

[0237] In some embodiments, the P-HBcAg detection antibody binds to an epitope other than the C-terminus on HBcAg. In some embodiments, the P-HBcAg detection 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 detection 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 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).

[0238] In other embodiments, the P-HBcAg detection antibody binds to an epitope other than the C-terminus of HBcAg. 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 contiguous amino acids (about 3 to about 40 contiguous amino acids, about 3 to about 35 contiguous amino acids, about 3 to about 30 contiguous amino acids, about 3 to about 25 contiguous amino acids, about 3 to about 20 contiguous amino acids, or about 3 to about 15 contiguous amino acids).

[0239] 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) 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 detection antibody binds to a non-continuous epitope. In some embodiments, the P-HBcAg detection antibody binds to a non-contiguous 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 P-HBcAg detection antibody binds to more binding sites, and each binding site is separated by at least 2 amino acids. For example, in some embodiments, the P-HBcAg detection antibody binds to multiple binding sites, wherein each binding site comprises at least 2 consecutive amino acids, and each binding site is separated by at least 2 amino acids. In some embodiments, each binding site comprises at least 2 consecutive amino acids (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 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, at least 50 amino acids).

[0240] In 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 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 1-161 of SEQ ID NO: 9. In some embodiments, the P-HBcAg detection antibody binds to a non-continuous epitope. In some embodiments, the P-HBcAg detection antibody binds to a non-contiguous 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 more binding sites, and each binding site is separated by at least 2 amino acids. For example, in some embodiments, the P-HBcAg detection antibody binds to multiple binding sites, wherein each binding site comprises at least 2 consecutive amino acids, and each binding site is separated by at least 2 amino acids. In some embodiments, each binding site comprises at least 2 consecutive amino acids (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 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, at least 50 amino acids).

[0241] In some aspects, 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) 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. 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-continuous epitope. For example, in some embodiments, the P-HBcAg detection antibody binds to more binding sites, and each binding site is separated by at least 2 amino acids. For example, in some embodiments, the detection antibody binds to multiple binding sites, wherein each binding site comprises at least 2 consecutive amino acids, and each binding site is separated by at least 2 amino acids. In some embodiments, each binding site comprises at least 2 consecutive amino acids (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 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, at least 50 amino acids).

[0242] 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 other embodiments, the P-HBcAg capture antibody binds to an epitope other than the C-terminus on HBcAg. 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 contiguous amino acids (about 3 to about 40 contiguous amino acids, about 3 to about 35 contiguous amino acids, about 3 to about 30 contiguous amino acids, about 3 to about 25 contiguous amino acids, about 3 to about 20 contiguous amino acids, or about 3 to about 15 contiguous amino acids).

[0243] 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) 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 a non-continuous epitope. In some embodiments, the P-HBcAg capture antibody binds to a non-contiguous 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 P-HBcAg capture antibody binds to more binding sites, and each binding site is separated by at least 2 amino acids. For example, in some embodiments, the P-HBcAg detection antibody binds to multiple binding sites, wherein each binding site comprises at least 2 consecutive amino acids, and wherein each binding site is separated by at least 2 amino acids. In some embodiments, each binding site comprises at least 2 consecutive amino acids (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 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, at least 50 amino acids).

[0244] In additional 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 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 1-161 of SEQ ID NO: 9. In some embodiments, the P-HBcAg capture antibody binds to a non-continuous epitope. In some embodiments, the P-HBcAg capture antibody binds to a non-contiguous 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 more binding sites, and each binding site is separated by at least 2 amino acids. For example, in some embodiments, the P-HBcAg detection antibody binds to multiple binding sites, wherein each binding site comprises at least 2 consecutive amino acids, and each binding site is separated by at least 2 amino acids. In some embodiments, each binding site comprises at least 2 consecutive amino acids (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 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, at least 50 amino acids).

[0245] 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 more binding sites, and each binding site is separated by at least 2 amino acids. For example, in some embodiments, the capture antibody binds to multiple binding sites, wherein each binding site comprises at least 2 consecutive amino acids, and each binding site is separated by at least 2 amino acids. In some embodiments, each binding site comprises at least 2 consecutive amino acids (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 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, at least 50 amino acids).

[0246] 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 in length (e.g., contiguous amino acids). 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, 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 in length (e.g., contiguous amino acids), provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least 5 amino acids, at least 6 amino acids, or 7 amino acids are phosphorylated.

[0247] In 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.

[0248] In 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 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 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.

[0249] In other 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 in length (e.g., contiguous amino acids). 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, 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 in length (e.g., contiguous amino acids), provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least 5 amino acids, at least 6 amino acids, or 7 amino acids are phosphorylated.

[0250] In some embodiments, the P-HBcAg detection antibody binds to an epitope 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, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least 5 amino acids, at least 6 amino acids, or 7 amino acids are phosphorylated. In some embodiments, the P-HBcAg detection antibody binds to an epitope 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, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least 5 amino acids, at least 6 amino acids, or 7 amino acids are phosphorylated. NO:39 in length 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., continuous amino acids), provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least 5 amino acids, at least 6 amino acids or 7 amino acids are phosphorylated. In some embodiments, the P-HBcAg detection antibody binds to a non-continuous epitope on the C-terminus of P-HBcAg. In some embodiments, the P-HBcAg detection antibody binds to multiple binding sites, wherein each binding site comprises at least three consecutive amino acids (e.g., at least 3, at least 4, at least 5, at least 6, at least 7 consecutive amino acids), and wherein each binding site is separated by at least one (e.g., at least one, at least two, at least three) amino acid, and wherein at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least 5 amino acids, at least 6 amino acids, or 7 amino acids are phosphorylated.For example, in some embodiments, the 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, wherein each binding site comprises at least three consecutive amino acids (e.g., at least 3, at least 4, at least 5, at least 6, at least 7 consecutive amino acids), and wherein each binding site is separated by at least one (e.g., at least one, at least two, at least three) amino acids, and wherein at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least 5 amino acids, at least 6 amino acids, or 7 amino acids are phosphorylated.

[0251] In another embodiment, the P-HBcAg detection antibody binds to an epitope of about 3 to about 36 amino acids (e.g., contiguous 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, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least 5 amino acids, at least 6 amino acids, or 7 amino acids are phosphorylated. In some embodiments, the P-HBcAg detection antibody binds to an epitope of about 3 to about 36 amino acids (e.g., contiguous 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, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least 5 amino acids, at least 6 amino acids, or 7 amino acids are phosphorylated. NO:21 length 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., continuous amino acids) epitope binding, provided that at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least 5 amino acids, at least 6 amino acids or 7 amino acids are phosphorylated. In some embodiments, the P-HBcAg detection antibody binds to a non-continuous epitope on the C-terminus of P-HBcAg. In some embodiments, the P-HBcAg detection antibody binds to multiple binding sites, wherein each binding site comprises at least three consecutive amino acids (e.g., at least 3, at least 4, at least 5, at least 6, at least 7 consecutive amino acids), and wherein each binding site is separated by at least one (e.g., at least one, at least two, at least three) amino acid, and wherein at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least 5 amino acids, at least 6 amino acids, or 7 amino acids are phosphorylated.For example, in some embodiments, the 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, wherein each binding site comprises at least three consecutive amino acids (e.g., at least 3, at least 4, at least 5, at least 6, at least 7 consecutive amino acids), and wherein each binding site is separated by at least one (e.g., at least one, at least two, at least three) amino acids, and wherein at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least 5 amino acids, at least 6 amino acids, or 7 amino acids are phosphorylated.

[0252] 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 5 amino acids, at least 6 amino acids, or 7 amino acids are phosphorylated. 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, 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 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 5 amino acids, at least 6 amino acids or 7 amino acids are phosphorylated.

[0253] 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 indicates the presence and / or amount (e.g., level) of P-HBcAg in the sample.

[0254] In some embodiments, the first specific binding member is fixed on a solid support. In some embodiments, the second specific binding member is fixed on a solid support. In some embodiments, the first specific binding member is an HBV biomarker antibody as described below.

[0255] In some embodiments, the sample is diluted or undiluted. The sample can 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.

[0256] Some instruments other than point-of-care devices (such as Abbott Laboratories instruments) Abbott Alinity instruments and other core laboratory instruments) may be able to measure HBV biomarkers in samples at approximately 4 pg / L at 10% CV or less. Other detection methods include the use of or can be adapted for use on nanopore or nanowell devices. Examples of nanopore devices are described in International Patent Publication No. WO 2016 / 161402, which is hereby incorporated by reference in its entirety. Examples of nanowell devices are described in International Patent Publication No. WO 2016 / 161400, which is hereby incorporated by reference in its entirety.

[0257] Antibody can be prepared by any one of multiple technologies, and described technology includes those technologies well known to those skilled in the art.In general, antibody can be generated by cell culture technology, and described technology includes by conventional techniques or by antibody gene, heavy chain and / or light chain transfection into suitable bacterium or mammalian cell host to generate monoclonal antibody, to realize the production of antibody, wherein antibody can be recombinant.Various forms of term " transfection " are intended to contain the extensive multiple technologies that are generally used for foreign DNA to be introduced into protokaryotic or eukaryotic host cell, such as electroporation, calcium phosphate precipitation, DEAE-polydextrose transfection etc. Although can be in protokaryotic or eukaryotic host cell expression antibody, it is particularly contemplated that in eukaryotic cell expression antibody, and include mammalian host cell, because such eukaryotic cell (for example, mammalian cell) is more likely to express than prokaryotic cell and assemble and secrete the antibody that suitably folds and has immunocompetent.

[0258] Exemplary mammalian host cells for expressing recombinant antibodies include: Chinese hamster ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216-4220 (1980)), which are used with a DHFR selective marker, for example as described in Kaufman and Sharp, J. Mol. Biol., 159: 601-621 (1982); NSO 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 sufficient period of time to allow the antibody to be expressed in the host cell, or by secreting the antibody into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0259] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It should be understood that the above program can be modified. For example, it may be desirable to transfect host cells with DNA encoding the functional fragments of the light chain and / or heavy chain of the antibody. Recombinant DNA technology can also be used to remove part or all of the DNA encoding one or both of the light chain or heavy chain that is unnecessary for binding to the antigen of interest. The antibody also encompasses molecules expressed by such truncated DNA molecules. In addition, bifunctional antibodies can be produced, in which a heavy chain and a light chain are antibodies (i.e., in combination with human troponin I), and the other heavy chain and light chain are specific for antigens other than human HBV biomarkers by cross-linking the antibody with a second antibody by standard chemical cross-linking methods.

[0260] In some embodiments, the system for recombinant expression of an antibody or its antigen-binding portion thereof includes 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 and light chain genes are each operably linked to a CMV enhancer / AdMLP promoter regulatory element to drive high-level transcription of the gene. The recombinant expression vector also carries the DHFR gene, which allows the use of methotrexate selection / amplification to select CHO cells transfected with the vector. Selected transformant host cells are cultivated to express the antibody heavy chain and light chain, and the complete antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select the transformant, cultivate the host cells, and recover the antibody from the culture medium. In addition, the method for synthesizing recombinant antibodies can be by culturing host cells in a suitable culture medium until the recombinant antibody is synthesized. The method can also include separating the recombinant antibody from the culture medium.

[0261] The method for preparing monoclonal antibodies involves preparing immortalized cell lines capable of producing antibodies with the desired specificity. Such cell lines can be produced by splenocytes obtained from immunized animals. Animals can be immunized with HBV biomarkers or fragments and / or variants thereof. The peptide used to immunize the animal can contain amino acids encoding human Fc (e.g., a crystallizable fragment region or tail region of a human antibody). The splenocytes can then be immortalized, for example, by fusion with a myeloma cell fusion partner. A variety of fusion techniques can be used. For example, splenocytes and myeloma cells can be mixed with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells rather than myeloma cells. One such technique uses hypoxanthine, aminopterin, thymidine (HAT) selection. Another technique includes electrofusion. After a sufficient time (usually about 1 to 2 weeks), colonies of the hybrid are 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.

[0262] Monoclonal antibodies can be isolated from the supernatant of the hybridoma colonies grown. In addition, various techniques can be used to increase output, such as by injecting the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host (such as a mouse). Monoclonal antibodies can then be harvested from ascites 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 for purifying antibodies.

[0263] The proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, two of which (the F(ab) fragments) each contain a covalent heterodimer with an intact antigen-binding site. Pepsin is able to cleave IgG molecules to provide several fragments, including a F(ab')2 fragment containing two antigen-binding sites.

[0264] Fv fragments can be produced by preferably proteolytic cleavage of IgM, and in rare cases by proteolytic cleavage of IgG or IgA immunoglobulin molecules. Fv fragments can be derived using recombinant technology. Fv fragments include non-covalent VH:VL heterodimers that contain an antigen binding site that retains many of the antigen recognition and binding capabilities of a native antibody molecule.

[0265] Antibodies, antibody fragments or derivatives may comprise heavy and light chain complementarity determining regions ("CDRs"), respectively, inserted between heavy and light chain framework ("FR") groups, which provide support for the CDRs and define the spatial relationship between the CDRs. The CDR group may comprise three hypervariable regions of the heavy or light chain V region.

[0266] Other suitable methods for producing or isolating antibodies with the necessary specificity may be used, including but not limited to methods for selecting recombinant antibodies from peptide or protein libraries (e.g., but not limited to phage, ribosome, oligonucleotide, RNA, cDNA, yeast, etc. display libraries); for example, such as those available from various commercial suppliers such as Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinsreid / Planegg, Del.), Biovation (Aberdeen, Scotland, UK), BioInvent (Lund, Sweden) using methods known in the art. See U.S. Patent Nos. 4,704,692, 5,723,323, 5,763,192, 5,814,476, 5,817,483, 5,824,514, 5,976,862. Alternative approaches rely on immunizing transgenic animals capable of producing 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, but are not limited to, 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 production techniques (e.g., the selected lymphocyte antibody method ("SLAM") (U.S. Pat. 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 droplet 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)).

[0267] Affinity matured antibodies can be produced by any of a variety of procedures known in the art. For example, see Marks et al., BioTechnology, 10:779-783 (1992) for description of affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described in 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 at selective mutagenesis positions and with activity-enhancing amino acid residues at contact or hypermutation positions are described in US Pat. No. 6,914,128 Bl.

[0268] Antibody variants can also be prepared by delivering a polynucleotide encoding the antibody to a suitable host, such as to provide a transgenic animal or mammal that produces such antibodies in its milk, such as goats, cows, horses, sheep, etc. These 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.

[0269] Antibody variants can also be prepared by delivering polynucleotides to provide transgenic plants and cultivated plant cells (such as, but not limited to, tobacco, corn, and duckweed), which produce such antibodies, specific parts, or variants in plant parts or in cells cultivated therefrom. For example, Cramer et al. (1999) Curr.Top.Microbiol.Immunol.240:95-118 and references cited therein describe, for example, transgenic tobacco leaves expressing a large amount of recombinant proteins using an inducible promoter. Transgenic corn has been used to express mammalian proteins at commercial production levels, and its biological activity is the same as that produced in other recombinant systems or purified from natural sources. See, for example, 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 (including tobacco seeds and potato tubers) that contain antibody fragments, such as single-chain antibodies (scFv). See, e.g., Conrad et al. (1998) Plant Mol. Biol. 38: 101-109 and references cited therein. Thus, transgenic plants can also be used to produce antibodies according to known methods.

[0270] Antibody derivatives can be produced, 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, some or all of the non-human or human CDR sequences are retained, while the non-human sequences of the variable and constant regions are replaced by human or other amino acids.

[0271] Small antibody fragments can be diabodies with two antigen-binding sites, wherein the fragment comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH VL). See, for example, 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 of the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. See also U.S. Patent No. 6,632,926 to Chen et al., which is hereby incorporated by reference in its entirety and discloses antibody variants having one or more amino acids inserted into the hypervariable region of a parent antibody and having a binding affinity for the target antigen that is at least about twice as strong as the binding affinity of the parent antibody for the antigen.

[0272] The antibody can be a linear antibody. Procedures for preparing linear antibodies are known in the art and described in Zapata et al. (1995) Protein Eng. 8(10): 1057-1062. In short, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0273] Antibodies can be recovered and purified from recombinant cell cultures 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.

[0274] Detectably labeled antibodies may be useful. Methods for conjugating antibodies to these reagents are known in the art. For illustrative purposes only, antibodies can be labeled with a detectable moiety, such as a radioactive atom, a chromophore, or a fluorophore. Such labeled antibodies can be used in diagnostic techniques in vivo or in isolated test samples. They can be linked to cytokines, ligands, and another antibody. Suitable agents for conjugation to antibodies to achieve anti-tumor effects include cytokines, such as interleukin-2 (IL-2) and tumor necrosis factor (TNF); photosensitizers for photodynamic therapy, including aluminum (III) phthalocyanine aluminum tetrasulfonate, hematoporphyrin, and phthalocyanine; radionuclides, such as iodine-131 (131I), yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi), technetium-99m (99mTc), rhenium-186 (186Re), and rhenium-188 (188Re); antibiotics, such as adriamycin, doxorubicin, daunorubicin, methotrexate, daunorubicin, neocarbazine, and carboplatin; bacterial, plant, and other toxins, such as diphtheria toxin;

[0014] Examples of the present invention include toxins, Pseudomonas exotoxin A, Staphylococcal enterotoxin A, Abrin-A toxin, ricin A (deglycosylated ricin A and native ricin A), TGF-α toxin, cytotoxins from the Chinese cobra (naja elaeagnus), and gelonin (a plant toxin); ribosome-inactivating proteins from plants, bacteria, and fungi, such as restrictocin (a ribosome-inactivating protein produced by Aspergillus restrictocin), saporin (a ribosome-inactivating protein from soapwort), and RNases; tyrosine kinase inhibitors; ly207702 (a difluoropurine nucleoside); liposomes containing anti-cystic drugs (e.g., antisense oligonucleotides, toxin-encoding plasmids, methotrexate, etc.); and other antibodies or antibody fragments, such as F(ab).

[0275] Antibody production by using hybridoma technology, the selected lymphocyte antibody method (SLAM), transgenic animals, and recombinant antibody libraries is described in more detail below.

[0276] If the sample is obtained from a subject (preferably a human) suspected of being infected with HBV, then the sample as defined herein is "suspected" to contain HBV. If the subject has an increased risk of HBV, then the subject is suspected of being infected with HBV. Infants born to mothers infected with HBV are at high risk of HBV infection. Other high-risk factors for HBV infection include, for example, intravenous drug use, hemophilia, high-risk sexual behavior, hemodialysis, needlestick injuries to healthcare workers, and body piercings and tattoos.

[0277] Sample can be available from any suitable experimenter, normally any suitable sample of mammal (for example people).Sample can be available from any biological source, such as cervix, vagina or anus swab or brush or physiological fluid, include but not limited to whole blood, serum, blood plasma, interstitial fluid, saliva, eye lens fluid, cerebrospinal fluid, sweat, urine, milk, ascites fluid, mucus, nasal fluid, sputum, synovial fluid, peritoneal fluid, vaginal fluid, menstruation, amniotic fluid, semen etc.Sample can use conventional techniques well known by persons skilled in the art available from experimenter, and sample can be available from biological source and directly use or use after pre-treatment to change the feature of sample.This pre-treatment can comprise for example preparing blood plasma, diluting viscous fluid, filtering, precipitation, diluting, diluting, mixing, concentrating, making interfering component inactivation, adding reagent, cracking etc. by blood.

[0278] a. Aptamer

[0279] The method disclosed herein includes the use of aptamers to detect or identify one or more HBV biomarkers. Aptamers are suitable for 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 (from small organic molecules to proteins and peptides). The basis of target recognition is the tertiary structure formed by single-stranded oligonucleotides, as known in the art. In some embodiments, aptamers for detecting or identifying one or more HBV biomarkers can be obtained by an in vitro selection process called SELEX, in which aptamers are selected from a random sequence library of synthetic DNA or RNA by repeated binding of oligonucleotides to target molecules.

[0280] In some embodiments, the nucleic acid constituting the aptamer library mixture for screening candidate HBV biomarker capture agents can be single-stranded DNA or RNA with or without chemical modification. During the selection process, the introduction of additional chemical entities into DNA can include, for example, the use of 5-alkyne-modified nucleobases (e.g., thymine). In addition, 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 using so-called bio-orthogonal chemistry, such as using Cu(I)-catalyzed 1,3-dipolar cycloadditions of the corresponding azide and alkyne. In addition to Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), copper-free strain-promoted azide-alkyne cycloaddition (strain-promoted azide-alkyne cycloaddition, SPAAC) reactions are also available. In some embodiments involving cells or life systems, strain-promoted azide-alkyne cycloadditions can overcome the toxicity issues associated with the use of Cu(I). Any number of desired chemical modifications can be added to the oligonucleotide library for screening purposes. Examples of such modifications include, but are not limited to, aliphatic residues, aromatic residues, charged residues, basic residues, acidic residues, heteroaromatic residues, carbohydrate residues, metal-containing residues, or peptide residues.

[0281] In some embodiments, the nucleobase to be modified to contain 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 contain an azide-alkyne chemical group can be an ethynyl-dU nucleotide, or an ethynyl-dA nucleotide, an ethynyl-dC nucleotide, or an ethynyl-dG nucleotide. Nucleotide aptamer libraries with these exemplary modifications can be used in various SELEX-based selection methods to enhance the chemical diversity of DNA aptamer libraries. Initiation or candidate's nucleic acid mixture can be modified so that in the mixture 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% member is modified to comprise the functionalization introduced by, for example, click chemistry. Being less than 100% modification can realize the diversity of enhancement by allowing certain position in oligonucleotide to be modified while other positions are not modified, and 100% modification guarantees the consistency during the screening process. In some embodiments, different modifications are carried out at different positions of oligonucleotide to further enhance diversity.

[0282] HBV biomarker recognition aptamers can be used to detect the presence or level of one or more HBV biomarkers in a biological sample (e.g., a biological entity of interest, such as a protein, nucleic acid, or microvesicle). An aptamer can be used as a binding agent or capture agent to assess the presence or level of a homologous HBV biomarker. In various embodiments of the present disclosure for diagnosis and / or prognosis, one or more aptamers can be configured in a ligand-target-based assay, wherein one or more aptamers can be contacted with a selected biological sample to allow one or more aptamers to associate or bind with its target HBV biomarker molecule. An aptamer can also be used to identify the profiles ("biomarker" profiles or features) of a variety of HBV biomarkers based on the biological sample assessed and the biomarkers detected. The biomarker profiles of a biological sample can include the presence, level, or other features of one or more biomarkers of interest that can be assessed, including but not limited to presence, level, sequence, mutation, rearrangement, translocation, deletion, epigenetic modification, methylation, post-translational modification, allele, activity, complex partner, stability, half-life, etc.

[0283] Biomarker profiles or features can be used to evaluate diagnostic and / or prognostic criteria, such as the presence of disease, disease staging, disease monitoring, disease stratification, or monitoring of detection, metastasis, or recurrence or progression of disease. For example, the method of the present disclosure may include a method for associating an HBV biomarker profile with a selected symptom or disease. Biomarker profiles can also be used clinically to make decisions about treatment modalities (including therapeutic interventions). Biomarker profiles based on aptamer detection, identification, and / or quantification can be further used clinically to make treatment decisions, including whether to change the course of treatment, such as administering different HBV therapeutic agents to a subject.

[0284] b. Determination of variants

[0285] The disclosed method for determining the presence or amount of an analyte of interest (e.g., HBV biomarker) present in a sample can be as described herein. The method can also be adjusted according to other methods for analyzing analytes. Examples of well-known variations include, but are not limited to, immunoassays, such as sandwich immunoassays (e.g., monoclonal-monoclonal sandwich immunoassays, monoclonal-polyclonal sandwich immunoassays, including enzyme detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay) (ELISA), competitive inhibition immunoassays (e.g., forward and reverse), enzyme amplified immunoassay technology (EMIT), competitive binding assays, bioluminescence resonance energy transfer (BRET), one-step antibody detection assays, homogeneous assays, heterogeneous assays, instant capture (capture on the fly) assays, etc.

[0286] i. Immunoassay

[0287] HBV biomarker antibodies can be used in immunoassays to analyze analytes of interest and / or peptides or fragments thereof (e.g., HBV biomarkers and / or peptides or fragments thereof). The presence or quantity of an analyte (e.g., HBV biomarker) can be determined using antibodies and detecting specific binding to the analyte. For example, an antibody or an antibody fragment thereof can specifically bind to the analyte. If desired, one or more antibodies can be used in combination with one or more commercially available monoclonal / polyclonal antibodies. Such antibodies can be purchased from companies such as R&D Systems, Inc. (Minneapolis, MN) and EnzoLife Sciences International, Inc. (Plymouth Meeting, PA).

[0288] 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 (radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) (e.g., Quantikine ELISA assay, R&D Systems, Minneapolis, MN)). Examples of point-of-care devices that can be used are i- (Abbott, Laboratories, Abbott Park, IL). Other methods that can be used include, for example, chemiluminescent microparticle immunoassays, including those employing The method of automatic analyzer (Abbott Laboratories, Abbott Park, IL).Other methods include, for example, mass spectrometry and immunohistochemistry (for example, with sections from tissue biopsy), using anti-analyte (for example, anti-HBV biomarker) antibodies (monoclonal, polyclonal, chimeric, humanized, people, etc.) or their antibody fragments for analytes (for example, HBV biomarkers).Other detection methods include those described in 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. Specific immunological binding of the antibody to the analyte can be detected via direct labels such as fluorescent or luminescent tags, metals and radionuclides attached to the antibody, or via indirect labels such as alkaline phosphatase or horseradish peroxidase.

[0289] The use of fixed antibodies or antibody fragments thereof can be incorporated into immunoassays. Antibodies can be fixed on a variety of supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (such as a microtiter well), a solid substrate material sheet, etc. An assay strip can be prepared by coating the antibody or multiple antibodies in an array on a solid support. This strip can then be immersed in a test sample and rapidly processed to produce a measurable signal, such as a color-developed spot, by washing and detection steps.

[0290] A homogeneous form can be used. For example, a mixture is prepared after obtaining a test sample from a subject. The mixture contains a test sample and a specific binding partner of an analyte to be evaluated (e.g., an HBV biomarker). The order in which the test sample and the specific binding partner are added to form the mixture is not critical. The test sample is contacted with the specific binding partner at the same time. 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., an HBV biomarker)-antigen complex. The specific binding partner can be an anti-analyte antibody, for example, an anti-HBV biomarker antibody that binds to an epitope having an amino acid sequence of at least three (3) consecutive amino acids comprising an HBV biomarker. In addition, the specific binding partner can be labeled with a detectable label as described above or contain the detectable label.

[0291] Heterogeneous forms can be used. For example, after obtaining a test sample from a subject, a first mixture is prepared. The mixture contains a test sample of an analyte to be evaluated (e.g., an HBV biomarker) and a first specific binding partner, wherein 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 can be an anti-analyte antibody, for example, an anti-HBV biomarker antibody that binds to an epitope having an amino acid sequence of at least three (3) consecutive amino acids comprising an HBV biomarker. The order in which the test sample and the first specific binding partner are added to form the mixture is not critical.

[0292] The first specific binding partner can be fixed 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, such as but not limited to magnetic particles, beads, test tubes, microtiter plates, colorimetric tubes, membranes, scaffold molecules, films, filter paper, discs and chips. In those embodiments where the solid phase is a bead, the bead can be a magnetic bead or a magnetic particle. The magnetic bead / particle can be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic or ferrofluid. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrO , MnAs, MnBi, EuO and NiO / Fe. The example of ferrimagnetic materials includes NiFe 2 O 4 , CoFe 2 O 4 , Fe 3 O 4 (or FeO 4 ), Fe 3 O 4 (or FeO 4 ). . The beads may have a magnetic solid core and be surrounded by one or more non-magnetic layers. Alternatively, the magnetic portion may be a layer surrounding the non-magnetic core. The solid support having the first specific binding member fixed thereon may be stored in a dry form or in a liquid. The magnetic beads may be subjected to a magnetic field before or after contact with a sample having the magnetic beads having the first specific binding member fixed thereon.

[0293] After forming a mixture containing a first specific binding partner-analyte (e.g., HBV biomarker) antigen complex, 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 an amount exceeding any analyte present in the test sample so that all analytes present in the test sample are bound by the first specific binding partner.

[0294] After removing any unbound 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, for example, an HBV biomarker antibody that binds to an epitope having an amino acid sequence comprising at least three (3) consecutive amino acids of an HBV biomarker. In addition, the second specific binding partner is labeled with or contains a detectable label as described above.

[0295] The use of fixed antibodies or antibody fragments thereof can be incorporated into immunoassays. Antibodies can be fixed 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), solid substrate material sheets, etc. Assay strips can be prepared by coating the antibody or multiple antibodies in an array on a solid support. This strip can then be immersed in the test sample and rapidly processed to produce a measurable signal, such as a color-developed spot, by washing and detection steps.

[0296] ii. Sandwich immunoassay

[0297] Sandwich immunoassays measure the amount of antigen between two layers of an antibody (i.e., at least one capture antibody) and a detection antibody (i.e., at least one detection antibody). The capture antibody and the detection antibody bind to different epitopes on the antigen (e.g., an analyte of interest, such as an HBV biomarker). Desirably, the binding of the capture antibody to the epitope does not interfere with the binding of the detection antibody to the epitope. Both monoclonal and polyclonal antibodies can be used as the capture and detection antibodies in a sandwich immunoassay.

[0298] In general, at least two antibodies are used to separate and quantify the analyte (e.g., HBV biomarker) in the test sample. More specifically, at least two antibodies bind to certain epitopes of the analyte to form an immune complex known as a "sandwich". One or more antibodies can be used to capture the analyte in the 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"). In a sandwich assay, the binding of an antibody to its epitope is ideally not weakened by the binding of any other antibody to its corresponding epitope in the assay. Antibodies are selected so that one or more first antibodies that are in contact with a test sample suspected of containing an analyte do not bind to all or part of the epitope recognized by the second or subsequent antibodies, thereby interfering with the ability of one or more second detection antibodies to bind to the analyte.

[0299] The antibody can be used as a first antibody in the immunoassay. The antibody immunospecifically binds to an epitope on the analyte (e.g., an HBV marker). In addition to the disclosed antibodies, the immunoassay can include a second antibody that immunospecifically binds to an epitope not recognized or bound by the first antibody.

[0300] A test sample suspected of containing an analyte (e.g., an HBV biomarker) can be contacted with at least one first capture antibody (or multiple first capture antibodies) and at least one second detection antibody simultaneously or sequentially. In a sandwich assay format, a test sample suspected of containing an analyte is first contacted with at least one first capture antibody that specifically binds to a specific epitope under conditions that allow the formation of a first antibody-analyte antigen complex. If more than one capture antibody is used, a first multiple capture antibody-HBV biomarker antigen complex is formed. In a sandwich assay, antibodies (such as at least one capture antibody) are used in a molar excess relative to the maximum amount of analyte expected in the test sample. For example, about 5 μg / mL to about 1 mg / mL of antibody per milliliter of microparticle coating buffer can be used.

[0301] iii. Single molecule detection

[0302] The methods and kits described herein can also involve single molecule counting. In certain embodiments, the methods for analyte analysis can involve evaluating an analyte present in a sample. In certain embodiments, the evaluation can be used to determine the presence and / or concentration of an analyte in a sample. In certain embodiments, the methods can also be used to determine the presence and / or concentration of a plurality of different analytes present in a sample.

[0303] Any device known in the art that allows 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 robot-based assay processing unit. Examples of other devices that can be used include Quanterix SIMOA TM (Lexington, MA), Singulex Single Molecule Counting (SMC TM ) technology (Alameda, CA, see, for example, U.S. Patent No. 9,239,284, the contents of which are incorporated herein by reference), etc.

[0304] Other detection methods include the use of or can be adapted for use on nanopore devices or nanowell devices. Examples of nanopore devices are described in International Patent Publication No. WO 2016 / 161402, which is hereby incorporated by reference in its entirety. Examples of nanowell devices are described in International Patent Publication No. WO 2016 / 161400, which is hereby incorporated by reference in its entirety.

[0305] As described herein, the methods and kits can involve mass spectrometry using DIA-MS, DDA-MS, or SRM / MRM-MS or PRM-MS. In certain embodiments, the methods for analyte analysis can involve assessing the presence of an analyte in a sample. In certain embodiments, assessing the presence of an analyte in a sample can be used to determine the presence and / or concentration of an analyte or fragment in the sample. In certain embodiments, the methods can also be used to determine the presence and / or concentration of a plurality of different analytes or analyte fragments present in a sample. Internal control proteins or peptide fragments can be used for quantification.

[0306] 6. Samples

[0307] As used herein, "sample", "test sample", "biological sample" refers to a fluid sample containing or suspected of containing HBV biomarkers. The sample can be derived from any suitable source. In some cases, the sample can comprise a liquid, a flowing particulate solid, or a fluid suspension of solid particles. In some cases, the sample can be processed before the analysis described herein. For example, the sample can be separated or purified from its source before analysis; however, in certain embodiments, an untreated sample containing HBV biomarkers can be directly measured. In one example, the source containing the HBV biomarker is a human substance (e.g., body fluid, blood (such as 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.). Tissues can include, but are not limited to, skeletal muscle tissue, liver tissue, lung tissue, kidney tissue, myocardial 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 some cases, the source of the sample may be an organ or tissue, such as a biopsy sample, which may be lysed by tissue dissociation / cell lysis.

[0308] A range of volumes of fluid samples 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, etc. In some cases, the volume of the 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.

[0309] In some cases, fluid sample can be diluted before being used in measuring.For example, in the embodiment that the source containing HBV biomarker is human body fluid (for example, blood, serum), fluid can be diluted with suitable solvent (for example, buffer, such as PBS buffer).Before use, fluid sample can be diluted about 1 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 10 times, about 100 times or more times.In other cases, fluid sample is not diluted before being used in measuring.

[0310] In some cases, sample can be subjected to pre-analysis treatment.Pre-analysis treatment can provide extra functionality, such as non-specific protein removal and / or the mixed functionality that can be realized effectively but cheaply.The general method of pre-analysis treatment can include using electrokinetic capture, AC electrokinetics, surface acoustic wave, isotachophoresis, dielectrophoresis, electrophoresis or other pre-concentration techniques as known in the art. In some cases, fluid sample can be concentrated before being used for measuring. For example, in the embodiment that the source containing HBV biomarker is human body fluid (for example, blood, serum), fluid can be concentrated by precipitation, evaporation, filtration, centrifugation or its combination. Before use, fluid sample can be concentrated about 1 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 10 times, about 100 times or more times.

[0311] It may be desirable to include a control. The control can be analyzed simultaneously with the sample from the subject as described above. The results obtained from the subject sample can be compared with the results obtained from the control sample. A standard curve can be provided, and the assay results of the sample can be compared with it. If a fluorescent marker is used, such a standard curve presents the marker level as a function of the assay unit, i.e., the intensity of the fluorescent signal. Using samples taken from multiple donors, a reference level for HBV biomarkers in normal healthy tissues can be provided, as well as a standard curve for the "risky" level of HBV biomarkers in tissues taken from donors who may have one or more of the characteristics set forth above.

[0312] Therefore, in view of the above, a method for determining the presence, amount or concentration of HBV biomarkers in a test sample is provided. The method includes determining the HBV biomarkers of the test sample by an immunoassay, wherein the immunoassay is, for example, using at least one capture antibody that binds to an epitope on an HBV biomarker and an epitope that binds to an epitope on an HBV biomarker that is different from the epitope of the capture antibody and optionally comprising at least one detection antibody of a detectable label, and including comparing a signal generated by a detectable label as a direct or indirect indication of the presence, amount or concentration of the HBV biomarker in the test sample with a signal generated as a direct or indirect indication of the presence, amount or concentration of the HBV biomarker in the calibrator. Optionally and in some embodiments, the calibrator is part of a series of calibrators, wherein each calibrator differs from the other calibrators in the series in the concentration of the HBV biomarker.

[0313] 7. Kits and Systems

[0314] In some embodiments, the present disclosure also provides a kit and system for detecting the presence, level or status of HBcAg and / or P-HBcAg in a sample. In some embodiments, a kit or system comprising a reagent for detecting the presence, level or status of HBcAg is provided herein. In some embodiments, a kit or system comprising a reagent for detecting the presence, level or status of P-HBcAg is provided herein. In some embodiments, a kit or system comprising a reagent for detecting the presence, level or status of HBcAg and P-HBcAg is provided herein. In some embodiments, the kit or system can be used for multiple 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.

[0315] Optionally, the kit may further comprise at least one calibrator or control.Any calibrator or control may be included in the kit.

[0316] Thus, the present disclosure also provides diagnostic and quality control kits comprising one or more antibodies or other detection reagents. Optionally, the assays, kits, and kit components of the present disclosure are directed to use on a commercial platform (e.g., at Abbott Laboratories, Abbott Park, IL). and EIA (Bead) platforms, as well as other commercial and / or in vitro diagnostic assays. In addition, the assays, kits, and kit components can be used in other formats, such as on electrochemical or other handheld or point-of-care assay systems. The present disclosure is applicable, for example, to commercial Abbott point-of-care (i- Abbott Laboratories, Abbott Park, IL) electrochemical immunoassay system. Immunosensors and methods of operating them in disposable test devices are described, for example, in U.S. Patent Applications 20030170881, 20040018577, 20050054078, and 20060160164, which are incorporated herein by reference. Additional background information regarding the manufacture of electrochemical and other types of immunosensors is found in U.S. Patent 5,063,081, which is incorporated by reference for its teachings related to the foregoing.

[0317] Optionally, the kit includes quality control reagents (eg, sensitivity panels, calibrators, and positive controls). The preparation of quality control reagents is well known in the art and is described, for example, in various immunodiagnostic or nucleic acid product inserts.

[0318] In another embodiment, the present disclosure provides a quality control kit comprising one or more antibodies described herein for use as a sensitivity panel for evaluating assay performance characteristics and / or quantifying and monitoring the integrity of antigens or nucleic acids used in an assay.

[0319] The kit may optionally include other reagents required for performing diagnostic assays or facilitating quality control evaluations, such as buffers, salts, enzymes, enzyme cofactors, substrates, detection reagents, and the like. Other components (such as buffers and solutions) for isolating and / or treating the test sample may also be included in the kit (e.g., pretreatment reagents). The kit may additionally include one or more other controls. One or more components of the kit may be lyophilized, and the kit may further include reagents suitable for reconstituting the lyophilized components.

[0320] The various components of test kit are optionally provided in suitable containers. As noted above, one or more containers can be microtiter plates. Test kit can also include a container for accommodating or storing a sample (for example, a container or box for a blood or urine sample). When appropriate, test kit can also optionally contain a reaction vessel, a mixing vessel and other components that are conducive to preparing a reagent or a test sample. Test kit can also include one or more instruments for helping to obtain a test sample, such as a syringe, a pipette, pliers, a measuring spoon, etc.

[0321] The kit may also optionally include instructions for use, which may be provided in paper form or in computer-readable form (such as a compact disc, CD, DVD, etc.).

[0322] The disclosure described herein can also be applied to a variety of automated and semi-automated systems (including systems in which the solid phase comprises microparticles), as described, for example, in U.S. Patent Nos. 5,089,424 and 5,006,309, and as commercially sold, for example, by Abbott Laboratories (Abbott Park, IL), including but not limited to Abbott's IMX, and Quantum II instruments and other platforms. In addition, the present disclosure is optionally applicable to Abbott Laboratories commercial point-of-care (i-STAT TM ) electrochemical immunoassay system for performing sandwich immunoassays. Immunosensors and methods of making and operating them in disposable test devices are described, for example, in U.S. Patent No. 5,063,081, U.S. Patent Application No. 2003 / 0170881, U.S. Patent Application No. 2004 / 0018577, U.S. Patent Application No. 2005 / 0054078, and U.S. Patent Application No. 2006 / 0160164, which are incorporated by reference in their entireties for their teachings regarding the foregoing.

[0323] 8. HBV treatment

[0324] For any of the methods described herein, the method may also 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 treatment to the subject. In some embodiments, the subject has already received treatment for chronic HBV. In some embodiments, the subject has already received treatment for chronic HBV, and the methods described herein are used to monitor responsiveness to treatment.

[0325] For any of the methods described herein, the treatment can be any suitable HBV treatment, including the treatments described in detail below.

[0326] The treatment is an agent that affects the reverse transcription of HBV RNA (e.g., pgRNA) to HBV DNA. For example, some HBV treatments (such as nucleoside inhibitors) inhibit pgRNA reverse transcription and therefore reduce the amount of HBV DNA (e.g., infectious particles) released from cells. Therefore, it is possible to determine whether such treatment is effective or ineffective in a subject by measuring HBcAg (which indicates the amount of infectious DNA particles in the sample). However, such HBV treatments do not interfere with the process of cccDNA being transcribed into pgRNA, and therefore these treatments do not reduce the amount of HBV RNA (e.g., pgRNA) in the form of "empty" or "non-infectious" particles secreted from cells. Therefore, the measurement of P-HBcAg associated with non-infectious HBV particles should not be affected by such treatment.

[0327] Therapeutic agents for treating HBV include any of the following, as further described herein. In general, the purpose and / or ultimate goal of such therapeutic agents is to silence covalently closed circular DNA (cccDNA) and / or eliminate cccDNA. cccDNA is a DNA structure produced in the cell nucleus during HBV propagation. cccDNA is able to form stable minichromosomes within the nucleus of infected cells. cccDNA can be used as a template for viral replication, thereby allowing the production of viral antigens. Chronic HBV infection is characterized in that cccDNA minichromosomes persist in the nuclei of hepatocytes of infected subjects. Current HBV treatments are unable to eliminate cccDNA minichromosomes from host cells, and therefore eliminating cccDNA is considered a "functional cure" for subjects suffering from HBV. Therefore, a variety of therapeutic agents that can be used to silence and / or eliminate cccDNA can be used in the methods described herein. In some embodiments, HBV therapeutic agents include tenofovir disoproxil fumarate, emtricitabine, Adefovir, clevudine, ABX-203, lamivudine, PEG-IFNα, ABX-203, adefovir, PEG-IFNα, and GBV-015. In some embodiments, HBV therapeutic agents include HBV DNA polymerase inhibitors such as besifovir, entecavir, Adefovir Tenofovir disoproxil fumarate tenofovir alafenamide, tenofovir, tenofovir disoproxil, tenofovir alafenamide fumarate, tenofovir alafenamide hemifumarate, tenofovir dipivoxil, tenofovir disoproxil fumarate, tenofovir octadecyloxyethylester, telbivudine Pradefovir, clavudine, emtricitabine Ribavirin, lamivudine (Epivir- ), phosphazide, famciclovir, SNC-019754, FMCA, fusolin, AGX-1009, and metacavir.

[0328] In some embodiments, HBV therapeutics include immunomodulators such as rintatolimod, imidol hydrochloride, ingaron, dermaVir, plaquenil (hydroxychloroquine), proleukin, hydroxyurea, mycophenolate mofetil (MPA) and its ester derivative mycophenolate mofetil (MMF), WF-10, ribavirin, IL-12, polymer polyethyleneimine (PEI), Gepon, VGV-1, MOR-22, BMS-936559, and IR-103. In some embodiments, HBV therapeutic agents include Toll-like receptor 7 modulators, 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, HBV therapeutic agents include nucleic acid polymers (NAPs), such as REP 2139.

[0329] 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 retasimad, poly-ICLC, MCT-465, MCT-475, Riboxxon, Riboxxim, and ND-1.1. Interferon alpha receptor ligands include interferon alpha-2b (Intron ), pegylated interferon α-2a Interferon αlb Veldona, Infradure, Roferon-A, YPEG-interferon alpha-2a (YPEG-rhIFNα-2a), P-1101, Algeron, Alfarona, Ingaron (interferon gamma), rSIFN-co (recombinant super compound interferon), Y-pegylated interferon alpha-2b (YPEG-rhIFNα-2b), MOR-22, pegylated interferon alpha-2b (PEG- ), Bioferon, Novaferon, Inmutag(IFN), Interferon α-nl Interferon beta-1a 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), alfainterferona 2b, Kalferon, Pegnano, Feronsure, PegiHep, interferon alpha 2b (Zydus-Cadila), Optipeg A, Realfa 2B, 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, Layfferon, Shang Sheng Lei Tai, INTEFEN, SINOGEN, Fukangtai, Pegstat, rHSA-IFNα-2b, and Interapo (Interapa).

[0330] Hyaluronidase inhibitors include astodrimers. 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 preventive 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 preventive vaccine (Advax Super D), Hepatrol-07, GSK-223192A, and Engerix Recombinant hepatitis B vaccine (intramuscular, Kangtai Biological Products), recombinant hepatitis B vaccine (Hansenual polymorpha 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-HepB-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.

[0331] HBV therapeutic vaccines include HBsAG-HBIG complex, Bio-Hep-B, NASVAC, abi-HB (intravenous), ABX-203, Tetrabhay, GX-110E, GS-4774, peptide vaccine (εPA-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 vaccines (HBV infection, VLP Biotech), AdTG-17909, AdTG-17910AdTG-18202, Chron Vac-B and Lm HBV.

[0332] HBV viral entry inhibitors include Myrcludex B. Antisense oligonucleotides targeting viral mRNA include ISIS-HBVRx. Interfering RNA, including short interfering RNA (siRNA), can be used. For example, siRNA that can be used include TKM-HBV (TKM-HepB), ALN-HBV, SR-008, ddRNAi, and ARC-520. Endonuclease regulators include PGN-514. Ribonucleotide reductase inhibitors include Trimidox. Hepatitis B virus E antigen inhibitors include wogonin. HBV antibodies targeting 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 include monoclonal and polyclonal antibodies, including 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, BAY 41-4109, morphothiadine mesilate, 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) NOD2 stimulators selected from the group consisting of SB-9200. Recombinant thymosin α-1 includes NL-004 and pegylated thymosin α-1.

[0333] Hepatitis B virus replication inhibitors include isothiafludine, 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, and Gemma. Gedatolisib, VS-5584, copanlisib, CAI orotate, perifosine, RG-7666, GSK-2636771, DS-7423, panulisib, GSK-2269557, GSK-2126458, CUDC-907, PQR-309, INCB-040093, pilaralisib, BAY-1082439, puquitinib mesylate mesylate), SAR-245409, AMG-319, RP-6530, ZSTK-474, MLN-1117, SF-1126, RV-1729, sonolisib, LY-3023414, SAR-260301 and CLR-1401; (32) cccDNA inhibitors selected from the group consisting of BSBI-25.

[0334] 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.

[0335] Other drugs used to treat HBV include gentiopicrin (gentiopicroside), nitazoxanide, birinapant, NOV-205 (Molixan; BAM-205), Oligotide, 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, and lecithin. acid), HepB-nRNA, cTP-5(rTP-5), HSK-II-2, HEISCO-106-1, HEISCO-106, Hepbama, IBPB-006IA, Hepuyinfen, DasKloster0014-01, Jiangantai(Ganxikang), picroside, GA5 NM-HBV, DasKloster-0039, hepulantai, IMB-2613, TCM-800B, reduced glutathione, and ZH-2N.

[0336] In some embodiments, HBV therapeutic agents include capsid assembly modulators (CAMs). Capsid assembly modulators are agents that interrupt the encapsidation of pregenomic RNA and can cause nucleocapsid disassembly, thereby interrupting multiple steps of HBV replication. In some embodiments, the CAM is JNJ-632, AT130, or BAY41-4109.

[0337] In some embodiments, HBV therapeutic agents can be combined with one, two, three, four or more additional therapeutic agents. In other embodiments, a compound disclosed herein or a pharmaceutically acceptable salt thereof is combined with two additional therapeutic agents. In certain embodiments, a compound disclosed herein or a pharmaceutically acceptable salt thereof is combined with three additional therapeutic agents. In another embodiment, a compound disclosed herein or a pharmaceutically acceptable salt thereof is combined with four additional therapeutic agents. One, two, three, four or more additional therapeutic agents can be different therapeutic agents selected from the same class of therapeutic agents, and / or they can be selected from different classes of therapeutic agents.

[0338] In one embodiment, HBV therapeutic agents include immunomodulators, toll-like receptor modulators (tlrl, tlr2, tlr3, tlr4, tlr5, tlr6, tlr7, tlr8, tlr9, tlrlO, tlrl1, tlrl2 and tlrl3 modulators), interferon alpha receptor ligands, hyaluronidase inhibitors, recombinant IL-7, HBsAg inhibitors, compounds targeting HBcAg, cyclophilin inhibitors, HBV therapeutic vaccines, HBV preventive vaccines, HBV viral entry inhibitors, NTCP inhibitors, antisense oligonucleotides targeting viral mRNA, short interfering RNA (siRNA), miRNA gene therapy agents, endonuclease modulators, ribonucleotide reductase inhibitors, hepatitis B virus E antigen inhibitors, recombinant scavenger receptor A (SRA) protein, src kinase inhibitors, HBx inhibitors, cccDNA inhibitors, short synthetic hairpin RNA (sshRNA), HBV antibodies (including HBV antibodies and bispecific antibodies targeting hepatitis B virus surface antigen) and "antibody-like" therapeutic proteins (such as Fab derivatives), CCR2 chemokine antagonists, thymosin agonists, cytokines, nucleoprotein inhibitors (HBV core or capsid protein inhibitors), retinoic acid-inducible gene 1 stimulators, NOD2 stimulators, NODI stimulators, 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 inhibitors, CD305 inhibitors, PD-1 inhibitors, PD-L1 inhibitors agents, PEG-interferon λ, recombinant thymosin α-1, BTK inhibitors, TIGIT modulators, CD47 modulators, SIRPα modulators, ICOS modulators, CD27 modulators, CD70 modulators, OX40 modulators, NKG2D modulators, Tim-4 modulators, B7-H4 modulators, B7-H3 modulators, NKG2A modulators, GITR modulators, CD160 modulators, HEVEM modulators, CD161 modulators, Axl modulators, Mer modulators, Tyro modulators, gene modifiers or editing agents such as CRISPR (including CRISPR Cas9)), zinc finger nucleases or synthetic nucleases (TALENs), and hepatitis B virus replication inhibitors.

[0339] 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 (such as Fab derivatives)), cyclophilin inhibitors, stimulators of retinoic acid-inducible gene 1, PD-1 inhibitors, PD-L1 inhibitors, arginase-1 inhibitors, PI3K inhibitors and NOD2 stimulators.

[0340] In one embodiment, HBV treatment includes 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 agents, short synthetic hairpin RNA (sshRNA) and nucleoprotein inhibitors (HBV core or capsid protein inhibitors).

[0341] In one embodiment, HBV therapeutic agents include immunomodulators, toll-like receptor modulators (tlr1, tlr2, tlr3, tlr4, tlr5, tlr6, tlr7, tlr8, tlr9, tlr10, tlr11, tlr12 and tlr13 modulators), HBsAg inhibitors, HBV therapeutic vaccines, HBV antibodies (including HBV antibodies and bispecific antibodies targeting hepatitis B virus surface antigens and "antibody-like" therapeutic proteins (such as Fab derivatives)), cyclophilin inhibitors, stimulators of retinoic acid-inducible gene 1, PD-1 inhibitors, PD-L1 inhibitors, arginase-1 inhibitors, PI3K inhibitors and NOD2 stimulators, and 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 agents, short synthetic hairpin RNA (sshRNA) and nucleoprotein inhibitors (HBV core or capsid protein inhibitors).

[0342] In one embodiment, the HBV therapeutic agent comprises adefovir Tenofovir disoproxil fumarate + emtricitabine Tenofovir disoproxil fumarate Entecavir Lamivudine (Epivir- ), tenofovir alafenamide, tenofovir, tenofovir disoproxil, tenofovir alafenamide fumarate, tenofovir alafenamide hemifumarate, telbivudine Emtricitabine Pegylated interferon α-2b (PEG- ), Interferon αlb Interferon alfa-2b Pegylated interferon alfa-2a Interferon α-nl Ribavirin, interferon beta-1a Bioferon, Ingaron, Inmutag (IFN), Algeron, Roferon-A, Oligotide, Zutectra, Shaferon, interferon alfa-2b (AXXO), Alfaferone, interferon alfa-2b (BioGeneric Pharma), Feron, interferon-alpha 2 (CJ), BEVAC, Laferonum, VIPEG, BLAUFERON-B, BLAUFERON-A, Intermax Alpha, Realdiron, Lanstion, Pegaferon, PDferon-B, interferon alfa-2b (IFN, Laboratorios Bioprofarma), alfainterferona 2b, Kalferon, Pignano, Feronsure, PegiHep, interferon alfa 2b (Zydus-Cadila), Optipeg A, Realfa 2B, 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, Kashunin, Shangshengleitai, INTEFEN, SINOGEN, Fukangtai, Alloferon, and Simoleukin.

[0343] 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 treatment, and can optionally be staggered with various combinations of HBV therapeutic agents over time. In some embodiments, the treating physician will develop or design a personalized treatment regimen (meaning a treatment regimen specific to the subject or patient) using one or more HBV therapeutic agents based on clinical parameters, cutoffs, publications, or a combination thereof. In some embodiments, the treating physician will use an algorithm (as disclosed herein) designed to evaluate data related to HBV treatment as part of the development of a personalized treatment regimen for the subject. In some embodiments, the treatment administered is part of a clinical trial. Treatment implemented as part of a clinical trial includes a treatment regimen designed or developed for one or more subjects or patients by a clinician or doctor based on clinical parameters (such as clinical parameters obtained from a previous clinical trial), cutoffs (such as cutoffs obtained from a previous clinical trial), patient profiles, publications, or any combination thereof.

[0344] Embodiments of the present disclosure also include treatment methods that combine the diagnostic methods described herein with literature-based treatments, regimens, analyses, and combinations thereof to establish personalized treatment plans (such as personalized treatment regimens) for subjects in need of HBV treatment. In some embodiments, the treatment methods of the present disclosure enable prescribing physicians to use one or more published mathematical models that reflect actual clinical data to develop personalized / personalized dosing regimens without losing the resolution of the data and / or model, which is generated by refining the actual clinical data into a relatively coarsely stratified set of recommendations for "average" or "typical" patients. In general, the method involves assembling a mathematical model developed from clinical data collected from patients who have been administered a specific drug (e.g., an HBV therapeutic agent), processing the model to create a composite model rich in patient data, and determining a patient-specific dosing regimen based on patient-specific observed response data processed in conjunction with the data of the mathematical model. In some embodiments, the method involves Bayesian averaging, Bayesian updating, and Bayesian prediction techniques to develop patient-specific dosing regimens that vary not only with the general mathematical model and patient-specific characteristics considered as covariate patient factors in the model, but also with observed patient-specific responses that are not considered by the model itself, and that reflect "inter-subject variability" that distinguishes a specific patient from the typical patient reflected by the model. Examples of such models are described in U.S. Patent Publication No. 2014 / 0351197, the contents of which are incorporated herein by reference.

[0345] Typical models also describe the expected impact of specific patient characteristics (such as the results of diagnostic tests) on response, and quantify the amount of unexplained variability that cannot be considered only by patient characteristics. In such models, patient characteristics are reflected as patient factor covariates within the mathematical model. Therefore, mathematical models are generally mathematical functions that describe basic clinical data and the associated variability observed in patient populations. These mathematical functions include terms that describe the variation between individual patients and "average" or typical patients, allowing the model to describe or predict the multiple outcomes of a given dose, and making the model not only a mathematical function, but also a statistical function, but these models and functions are referred to as "mathematical" models and functions in a general and non-restrictive manner herein. It should be understood that many suitable mathematical models already exist and are used for purposes such as drug product development. Examples of suitable mathematical models that describe the response characteristics of patient populations and consider patient factor covariates 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 generally published or otherwise obtained from drug manufacturers, peer-reviewed literature, and FDA or other regulatory agencies. Alternatively, suitable mathematical models can be prepared by original research.

[0346] Embodiments of the present disclosure also contemplate continued treatment in subjects currently receiving treatment with one or more HBV therapeutic agents to prevent or reduce the risk of DNA reactivation and / or relapse. In other embodiments, the assay of the present disclosure can be used to predict or determine whether a subject who has stopped treatment (e.g., due to seroclearance) is likely to have or has a potential relapse.

[0347] All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which the 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.

[0348] The disclosures illustratively described herein may, where appropriate, be implemented in the absence of any one or more elements, one or more limitations, that are not explicitly disclosed herein. Thus, for example, in each case herein, any one of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms. The terms and expressions that have been adopted are used as terms of illustration and are not restrictive, and it is not intended that any equivalents of the features shown and described herein, or a portion thereof, be excluded by the use of such terms and expressions, but it will be appreciated that different modifications within the scope of the present disclosure as claimed are possible. Therefore, it will be understood that although the present disclosure includes various embodiments and optional features, modifications and variations of the concepts disclosed herein may be adopted by those skilled in the art, and it will be understood that such modifications and variations are considered to be within the scope of the present invention as defined by the appended claims.

[0349] Example

[0350] Example 1

[0351] This article describes the development and use of two fully automated assays that specifically analyze phosphorylated HBcAg levels (P-HBcAg, indicating non-infectious or "empty" particles) and non-phosphorylated HBcAg levels (indicating particles containing HBV DNA ("non-infectious" particles)) in patients with active infection at a single time point, in longitudinal seroconversion panels, and in patients receiving HBV treatment.

[0352] The developed assay 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 circulating in HBV-infected patients.

[0353] method:

[0354] P-HBcAg / HBcAg Assay: Patient specimens are analyzed fully automatically on the ARCHITEC Ti2000SR instrument. HBcAg is captured and detected using monoclonal antibodies targeting either phosphorylated or non-phosphorylated HBcAg. Detection is achieved using antibodies labeled with a luminescent molecule.

[0355] Other HBV markers: HBV DNA viral load was determined using the Abbott HBV Realtime assay with a 0.2 ml input volume, following the package insert instructions. Serologic testing for HBeAg was performed on the Abbott ARCHITECT. HBcrAg levels were analyzed on the LUMIPULSE G1200 instrument.

[0356] result:

[0357] Table 1 shows the limit of detection (LOD) and limit of quantification (LOQ) of the HBcAg assay, which were determined in triplicate using recombinant P-HBcAg / HBcAg.

[0358] antigen LOD (pg / ml) LOQ (pg / ml) HBcAg 6 14 P-HBcAg 0.8 4

[0359] HBeAg cross-reactivity was evaluated in triplicate using recombinant HBeAg for concentrations up to 10 μg / ml. No cross-reactivity was observed for both P-HBcAg / HBcAg assay formats. P-HBcAg / HBcAg assay linearity was evaluated in triplicate using recombinant antigen. Linearity assays showed 5-10 4 pg / ml(R 2 =0.999) and for HBcAg showed 10 1 -10 5 pg / ml(R 2 =0.991).

[0360] Figure 4 shows that the samples tested positive for HBsAg and contained approximately 10 3 to 10 9 Analysis of P-HBcAg / HBcAg levels in clinical samples with a viral load of HBV DNA copies / ml. 5 P-HBcAg was detected in 84% of the samples with a viral load of ≥10 cp / ml. 6 HBcAg could be detected in 94% of the samples with cp / ml.

[0361] Figure 1 HBV seroconversion panels comparing the levels of phosphorylated HBV core antigen (P-HBcAg), HBV core antigen (HBcAg), HBeAg, and HBcrAg with the levels of HBV DNA over time are shown. Figure 1 As shown in the results, P-HBcAg and HBcAg levels correlated well with HBV DNA levels and could detect acute HBV infection, whereas after HBV DNA reached its peak level, HBcrAg levels seemed to be closer to HBeAg levels than to HBcAg / P-HBcAg levels.

[0362] HBV-positive subjects received antiviral treatment using nucleoside (acid) analogs, interferon, or HBsAg inhibitors. The levels of P-HBcAg, HBV DNA, HBcAg, and HBV RNA in samples obtained from the subjects were measured every 1-4 weeks during treatment, starting from the first day of treatment until the end of 146 weeks. Figure 2As shown, HBcAg levels (black line) correspond to HBV DNA levels during treatment. Therefore, this data demonstrates that non-phosphorylated HBcAg (present in infectious HBV particles, black line) can immediately demonstrate the efficacy of nucleotide analogs (i.e., reduced HBcAg levels) at the start of treatment. Furthermore, phosphorylated P-HBcAg (present in non-infectious particles, blue line) can measure the transcriptional activity of cccDNA (i.e., non-infectious empty particles are secreted despite undetectable HBV DNA in the blood).

[0363] For reasons of completeness, various aspects of the disclosure are listed in the following numbered clauses:

[0364] Clause 1: A method for assessing and monitoring the stage or staging of chronic hepatitis B (HBV) infection or monitoring response to chronic HBV treatment in a subject, the method comprising the steps of:

[0365] 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, wherein the assay comprises contacting the at least one sample with an antibody that specifically binds to HBcAg and an antibody that specifically binds to P-HBcAg; and

[0366] b) assessing and monitoring the stage or staging 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 the at least one sample.

[0367] Clause 2: The method of clause 1, wherein the stage or staging of chronic HBV in the subject is assessed and monitored, and wherein the method further comprises providing chronic HBV treatment to the subject based on the presence or level of HBcAg and P-HBcAg in the at least one sample.

[0368] Clause 3: The method of clause 1, wherein the subject is receiving chronic HBV treatment, and wherein the method further comprises changing the HBV treatment based on the presence or level of HBcAg and P-HBcAg in the at least one sample.

[0369] Clause 4: A method for assessing and monitoring the stage or staging of chronic hepatitis B (HBV) infection, the method comprising the steps of:

[0370] 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, wherein the assay comprises contacting the at least one sample with an antibody that specifically binds to HBcAg; and

[0371] 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, wherein the amount of infectious HBV particles is determined regardless of whether said subject has or has not received HBV treatment prior to performing said determining in step a).

[0372] Clause 5. The method of Clause 4, further comprising providing chronic HBV treatment to the subject when the amount of infectious HBV particles in the at least one sample is equal to or above a threshold value.

[0373] Clause 6: A method for assessing and monitoring the stage or staging of chronic hepatitis B (HBV) infection, the method comprising the steps of:

[0374] 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, wherein the assay comprises contacting the at least one sample with an antibody that specifically binds to HBcAg;

[0375] 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, wherein the amount of infectious HBV particles is determined regardless of whether said subject has or has not received HBV treatment prior to performing said determining of step a); and

[0376] c) providing chronic HBV treatment to the subject when the amount of infectious HBV particles in the at least one sample is equal to or above a threshold value.

[0377] Clause 7: A method of monitoring response to treatment of chronic hepatitis B (HBV) infection in a subject, the method comprising the steps of:

[0378] 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 receiving chronic HBV treatment, wherein the assay comprises contacting the at least one sample with an antibody that specifically binds to HBcAg;

[0379] 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

[0380] c) determining that the treatment is effective when the amount of infectious HBV particles in the at least one sample is less than a reference level; or

[0381] d) determining that the treatment is ineffective when the amount of infectious HBV particles in the at least one sample is greater than or equal to a reference level.

[0382] Clause 8: A method of monitoring response to treatment of chronic hepatitis B (HBV) infection in a subject, the method comprising the steps of:

[0383] 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 receiving chronic HBV treatment, wherein the assay comprises contacting the at least one sample with an antibody that specifically binds to HBcAg and an antibody that specifically binds to P-HBcAg;

[0384] 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;

[0385] 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

[0386] d) determining a response to said chronic HBV treatment based on the amount of infectious and / or non-infectious HBV particles in said at least one sample.

[0387] Clause 9: The method of clause 8, wherein:

[0388] a) determining that the treatment is effective when the amount of infectious HBV particles in the at least one sample is less than a reference level;

[0389] b) determining that the treatment is effective when the amount of infectious HBV particles in the at least one sample is less than a reference level and the amount of non-infectious HBV particles in the at least one sample is less than a reference level; or

[0390] c) determining that the treatment is ineffective when the amount of infectious HBV particles in the at least one sample is greater than or equal to a reference level.

[0391] Clause 10: A method of monitoring response to treatment of chronic hepatitis B (HBV) infection in a subject, the method comprising the steps of:

[0392] 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 receiving chronic HBV treatment, wherein the at least two samples include a first sample obtained from the subject at a first time point before or after receiving the chronic HBV treatment and a second sample obtained from the subject at a second time point after the first time point, and wherein the assay comprises contacting the at least two samples with an antibody that specifically binds to HBcAg and an antibody that specifically binds to P-HBcAg;

[0393] 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

[0394] Based on the amount of infectious and / or non-infectious HBV particles in the at least two samples, the response to the chronic HBV treatment is determined.

[0395] Clause 11: The method of clause 10, wherein:

[0396] a) determining that the treatment is effective when 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;

[0397] b) determining that the treatment is effective when 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 wherein 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

[0398] c) determining that the treatment is ineffective when the amount of infectious HBV particles in the second sample has not decreased by at least an absolute amount compared to the amount of infectious HBV particles in the first sample.

[0399] Clause 12: The method of Clause 11, wherein the first sample is obtained from the subject within 24 hours of receiving the chronic HBV treatment.

[0400] Clause 13. The method of Clause 11 or Clause 12, wherein the second sample is obtained from the subject 10-14 weeks after the first sample.

[0401] Clause 14: The method of Clause 13, wherein the first sample is obtained from the subject within 24 hours of receiving the chronic HBV treatment, and the second sample is obtained from the subject 10-14 weeks after the first sample.

[0402] Clause 15: The method of Clause 7, Clause 9 or Clause 11, further comprising changing the chronic HBV treatment when the treatment is determined to be ineffective.

[0403] Clause 16: The method of Clause 15, wherein changing the chronic HBV treatment comprises 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.

[0404] Clause 17: A method as described in any of the preceding clauses, wherein: (a) the antibody that specifically binds to HBcAg binds to an epitope comprising at least 3 amino acids of SEQ ID NO: 2, and / or the antibody that specifically binds to P-HBcAg binds to an epitope comprising at least 3 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.

[0405] Clause 18: A method as described in any of the preceding clauses, wherein the chronic HBV treatment is interferon, nucleoside (acid) analogs, nucleic acids, immunomodulators, core protein assembly inhibitors, capsid assembly regulators (CAMs), HBsAg release inhibitors, entry inhibitors, interfering RNA, DNA modifying agents or a combination thereof.

[0406] Clause 19: The method of clause 18, wherein:

[0407] a) the interferon is interferon α-2a or pegylated interferon α-2a;

[0408] b) the nucleoside (acid) analogue is lamivudine, adefovir, tenofovir, telbivudine or entecavir;

[0409] c) the nucleic acid is siRNA, antisense oligonucleotide, shRNA or miRNA;

[0410] d) the core protein assembly inhibitor is NVR 3-1983, GLS4 or BAY 41-4109;

[0411] e) the CAM is JNJ-632, AT130 or BAY41-4109;

[0412] f) the HBsAg release inhibitor is REP 9AC;

[0413] g) the entry inhibitor is Myrcludex-B; or

[0414] h) Any combination of a)-g).

[0415] Clause 20: The method of any preceding clause, wherein performing the assay to measure the level of HBcAg in the at least one sample comprises contacting the at least one sample simultaneously or sequentially in any order with:

[0416] 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

[0417] b) 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; and

[0418] c) measuring the level of HBcAg in the sample based on the signal generated by the detectable label in the capture antibody-HBcAg-detection antibody complex.

[0419] Clause 21: The method of any preceding clause, wherein performing the assay to measure the level of P-HBcAg in the sample comprises contacting the sample simultaneously or sequentially in any order with:

[0420] 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

[0421] b) 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; and

[0422] c) measuring 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.

[0423] Item 22: 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 staging of hepatitis B (HBV) infection in a subject or monitoring the response to chronic HBV treatment.

[0424] Clause 23: A kit or system comprising reagents for detecting the presence, level or status of hepatitis B core antigen (HBcAg) and phosphorylated hepatitis B core antigen (P-HBcAg).

Claims

1. A method for assessing and monitoring the stage or staging of chronic hepatitis B (HBV) infection or monitoring response to chronic HBV treatment in a subject, the method comprising the steps of: 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, wherein the assay comprises contacting the at least one sample with an antibody that specifically binds to HBcAg and an antibody that specifically binds to P-HBcAg; and b) assessing and monitoring the stage or staging 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 the at least one sample.

2. The method of claim 1, wherein the stage or staging of chronic HBV in the subject is assessed and monitored, and wherein the method further comprises providing chronic HBV treatment to the subject based on the presence or level of HBcAg and P-HBcAg in the at least one sample.

3. The method of claim 1, wherein the subject is receiving chronic HBV treatment, and wherein the method further comprises changing the HBV treatment based on the presence or level of HBcAg and P-HBcAg in the at least one sample.

4. A method for assessing and monitoring the stage or staging of chronic hepatitis B (HBV) infection, the method comprising the steps of: 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, wherein the assay comprises 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 received HBV treatment or has not received HBV treatment prior to performing the determining of step a); and c) providing chronic HBV treatment to the subject when the amount of infectious HBV particles in the at least one sample is equal to or above a threshold value.

5. A method for monitoring response to treatment of chronic hepatitis B (HBV) infection in a subject, the method comprising the steps of: 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 receiving chronic HBV treatment, wherein the assay comprises 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; as well as c) determining that the treatment is effective when the amount of infectious HBV particles in the at least one sample is less than a reference level; or d) determining that the treatment is ineffective when the amount of infectious HBV particles in the at least one sample is greater than or equal to a reference level.

6. A method for monitoring response to treatment of chronic hepatitis B (HBV) infection in a subject, the method comprising the steps of: 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 receiving chronic HBV treatment, wherein the assay comprises contacting the at least one sample with an antibody that specifically binds to HBcAg and an antibody that specifically binds to 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; as well as d) determining a response to said chronic HBV treatment based on the amount of infectious and / or non-infectious HBV particles in said at least one sample.

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

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

9. The method of claim 8, wherein: a) determining that the treatment is effective when 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; b) determining that the treatment is effective when 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 wherein 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 when the amount of infectious HBV particles in the second sample has not decreased by at least an absolute amount compared to the amount of infectious HBV particles in the first sample.

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

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

12. The method of claim 11, wherein changing the chronic HBV treatment comprises 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.

13. A method as described in any of the preceding claims, wherein the antibody that specifically binds to HBcAg binds to an epitope comprising at least 3 amino acids of SEQ ID NO: 2 or SEQ ID NO: 25, and / or wherein the antibody that specifically binds to P-HBcAg binds to an epitope comprising at least 3 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. The method of any of the preceding claims, wherein the chronic HBV treatment is an interferon, a nucleoside(acid) analog, a nucleic acid, an immunomodulator, a core protein assembly inhibitor, a capsid assembly regulator (CAM), an HBsAg release inhibitor, an entry inhibitor, an interfering RNA, a DNA or RNA modifier, or a combination thereof.

15. The method of claim 14, wherein: a) the interferon is interferon α-2a or pegylated interferon α-2a; b) the nucleoside (acid) analogue is lamivudine, adefovir, tenofovir, telbivudine or entecavir; c) the nucleic acid is siRNA, antisense oligonucleotide, shRNA or 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 9AC; g) the entry inhibitor is Myrcludex-B; or h) Any combination of a)-g).

16. The method of any one of the preceding claims, wherein performing the assay to measure the level of HBcAg in the at least one sample comprises contacting the at least one sample simultaneously or sequentially in any order with: 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, thereby forming a capture antibody-HBcAg-detection antibody complex; and c) measuring the level of HBcAg in the sample based on the signal generated by the detectable label in the capture antibody-HBcAg-detection antibody complex.

17. The method of any one of the preceding claims, wherein performing the assay to measure the level of P-HBcAg in the sample comprises contacting the sample simultaneously or sequentially in any order with: 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, thereby forming a capture antibody-P-HBcAg-detection antibody complex; and c) measuring 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.

18. Use of reagents 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 staging of hepatitis B (HBV) infection in a subject or monitoring the response to chronic HBV treatment.

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

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