Antigen detection

Abstract

The present disclosure provides an immunoassay method with very high sensitivity for detecting a target antigen in a sample. In one teaching, the capture and detection steps of these assay methods use recombinant antibodies. The disclosed immunoassay methods may be particularly suitable for the detection of HBV antigens.

Description

TECHNICAL FIELD

[0001] The present disclosure provides improved methods of detecting the presence of hepatitis B virus or antigen thereof in a sample. BACKGROUND

[0002] Hepatitis B virus (HBV) is one of the most widespread infectious agents in the world, which can lead to lifelong chronic infection, cirrhosis, liver cancer, liver failure and death (Liang et al., Hepatology, 49(5): S13-21 (2009)). Early screening and detection of HBV infection is critical for disease management and control of HBV transmission (Franco et al., World J. Heptaol., 4: 74-80 (2012)). Detection methods for HBV infection generally involve serological detection of one or more HBV antigens, such as ELISA-based techniques or other immunoassays that detect HBV surface antigen (HBsAg), hepatitis core antigen (HBcAg), and / or hepatitis B surface antibodies (Mao et al., Anal. Chim. Acta, 909: 101-108 (2016)). Alternatively, real-time PCR or various sequencing techniques can be employed to detect the presence of HBV DNA, such as to assist in managing chronically HBV-infected patients receiving antiviral therapy, measuring HBV DNA levels at baseline or during treatment, to assist in assessing treatment response (Terrault et al., Hepatology, 63(1): 261-282 (2015)).

[0003] While there are many antibodies on the market that are directed to one or more hepatitis B antigens, there remains a need in the art for improved methods that can accurately detect HBV infection and provide the required level of sensitivity, and / or methods that can rapidly and cost-effectively detect HBV infection.

[0004] It is an object of the present disclosure to provide improved methods for detecting hepatitis B virus in a sample, which include the steps provided herein. SUMMARY

[0005] The present disclosure is based on the discovery that immunoassays comprising a target capture step and a target detection step, in which both the capture step and the detection step use recombinant antibodies, represent an improvement over prior art immunoassay methods, which at most can use recombinant antibodies in only one of the capture step or the detection step.

[0006] It is noted that the terms "comprise", "comprising", and / or "comprises", when used in this specification and in the following claims, are taken to specify the presence of stated features, integers, steps and / or components, but not to preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof. It is further noted that the claims can be drafted to exclude any optional feature, unless this is explicitly stated in the claims or

[0007] Various aspects and embodiments of the present disclosure are applicable to immunoassays used to detect a target antigen in a sample. Thus, the term "target" can include "a target antigen" or "a plurality of target antigens".

[0008] In view of the above, one aspect of the present disclosure provides an immunoassay for detecting a target in a sample, wherein the immunoassay comprises a target capture step and a target detection step, and both the target capture step and the target detection step comprise a recombinantly produced antibody (or "recombinant antibody").

[0009] A second aspect of the present disclosure provides a recombinant antibody for use in a target capture step and a target detection step of an immunoassay used to detect a target in a sample.

[0010] As used herein, the term "immunoassay" refers to an assay method used to determine whether a sample contains a particular target. Those skilled in the art will appreciate that there are a large number of immunoassays, and the disclosure presented herein is applicable to any assay that utilizes an antibody-based capture and detection system. By way of example only, the term "immunoassay" can include enzyme immunoassays (EIA: e.g. ELISA), radioimmunoassays (RIA), fluorescence immunoassays (FIA); Western Blot (WB); chemiluminescence immunoassays (CIA) and counting immunoassays (CIA); immunohistochemistry type assays (IHC); immunocytochemistry type assays (ICC). In the context of the present disclosure, the term "immunoassay" can exclude radioimmunoassays (RIA).

[0011] As previously mentioned, in one teaching, the target can be an antigen. In another teaching, the target can include an antigen known to be expressed by a pathogen, such as a fungal, bacterial or viral antigen.

[0012] The immunoassay of the present disclosure can comprise a target capture step, wherein the sample is contacted with a target capture agent under conditions that allow binding between any target present in the sample and the target capture agent.

[0013] As previously mentioned, target capture agents may include recombinant antibodies. Recombinant antibodies used as target capture agents can bind to a target (e.g., a target antigen). Binding between any target present in the sample and the target capture agent will produce a target / target capture agent complex.

[0014] The target trapping agent can be immobilized onto the matrix.

[0015] The immunoassay disclosed herein may further include a detection step in which a target / target capture complex (formed during the capture step of the immunoassay) contacts a detection agent.

[0016] As previously mentioned, the detection agent may include recombinant antibodies. Recombinant antibodies used as detection agents can bind to a target (e.g., a target antigen). Recombinant antibodies used as detection agents can bind to the target at a site or epitope different from the binding site of the capture agent.

[0017] The detection reagent may be conjugated to a detectable portion (e.g., an optically detectable portion). Detection systems used for any of the methods or assays described herein may include, for example, biotin-based systems, HRP-based systems, streptavidin-based systems, silver and / or gold staining-based systems, and / or alkaline phosphatase-based systems. In one example, a section system used for the methods or assays of this disclosure may include a streptavidin poly-HRP conjugate (a biotin-binding protein polymerically conjugated to horseradish peroxidase).

[0018] In view of the above, a third aspect of this disclosure provides a method for detecting a target (e.g., a target antigen) in a sample, the method comprising:

[0019] Under conditions suitable for allowing the first recombinant antibody to bind to any target in the sample, the sample is contacted with the first recombinant antibody; and then...

[0020] Contact any first recombinant antibody / target complex (formed by a binding event between the first antibody and any target present in the sample) with the second antibody under conditions suitable for allowing binding between the second antibody and any first recombinant antibody / target complex; and then

[0021] Detect the second antibody that binds to the first recombinant antibody / target complex;

[0022] The detection of a second antibody that binds to the first recombinant antibody / target complex indicates that the sample contains a target.

[0023] In one teaching of a method for detecting a target (e.g., a target antigen) in a sample, a first recombinant antibody may be immobilized to a matrix.

[0024] In further teaching, the above methods (or any of the immunoassays described herein) may include one or more washing steps. For example, after the step of contacting the sample with a first recombinant antibody, the assay may be washed to remove any unbound first antibody. The method may include a second wash after the step of contacting any first recombinant antibody / target complex with a second recombinant antibody, the additional wash being intended to remove unbound second recombinant antibody.

[0025] As described in this article, the use of recombinant antibodies in both the capture and detection steps of an immunoassay has the following advantages:

[0026] (i) Immunoassays using recombinant antibodies (in any capture and detection step) exhibit improved sensitivity;

[0027] (ii) Recombinant antibody technology allows for the customization of both capture and detection antibodies to the desired specifications; this enables the selection of the best target conjugate. This provides immunoassays with improved sensitivity and specificity (compared to existing techniques that do not use recombinant antibodies in both capture and detection steps); and

[0028] (iii) Recombinant antibodies can be readily manipulated and / or transformed to take any desired predetermined form, fragment type, or class;

[0029] Furthermore, antibodies generated using recombinant technology can be produced rapidly and cost-effectively.

[0030] Those skilled in the art will understand that immunoassays used to detect antigens in a sample can be used for the diagnosis of certain diseases or conditions. As previously described, antigens may include pathogen antigens (e.g., bacterial, viral, or fungal antigens), and the presence of such an antigen in a sample may be associated with a specific disease, infection, or condition caused or facilitated by a pathogen expressing that antigen. Therefore, the discovery of a specific antigen in a sample may indicate that the sample was provided by or obtained from a subject who is infected with a pathogen expressing that antigen, or who has a disease or condition caused or facilitated by / recovered from a disease or condition caused or facilitated by a pathogen expressing that antigen.

[0031] Established recombinant methods can be used to generate recombinant antibodies that can be used as capture or detection agents. For example, recombinant methods for generating useful antibodies may include molecular techniques for generating recombinant antibody libraries, antibody display techniques, antibody selection schemes, and affinity maturation techniques. More detailed information on recombinant technologies for antibody generation can be found in Hoogenboom (2005); Nature Biotechnology.

[0032] For example, suitable antibody display technologies may include those called phage (antibody) display, ribosome (antibody) display, and / or microbial (antibody) cell display.

[0033] Techniques for selecting recombinant antibodies may include exposing antibodies in a display library to an antigen. For example, such techniques may include biopanning to select clones that exhibit the desired binding characteristics (e.g., phage clones).

[0034] The selection process may include several rounds of repeated selection to obtain antibodies exhibiting optimal binding properties. Such selection techniques can be used to select antibodies for use in the capture and detection steps of the immunoassay described herein.

[0035] Affinity maturation is a process that can improve, develop, and / or manipulate properties such as antibody potency and antibody affinity. These processes can include generating libraries with concentrated diversity on a small number of residues most likely to participate in target / antigen interactions. For example, constructing a CDR using amplification techniques (such as PCR) and screening libraries to identify variants with the desired characteristics. Other methods include using “selection pressure” to select antibodies that bind best to their target under specific conditions. These methods can enable antibodies to exhibit substantial improvements in affinity / potency, etc.

[0036] Antibodies used in the immunoassays of this disclosure may include antibodies obtained using recombinant techniques, including those described herein. As a further example, antibodies used as capture and detection agents in the immunoassays of this invention may be obtained as antibody (capture and detection) pairs exhibiting, for example, specific and predetermined target binding and / or potency and / or affinity properties. Thus, the immunoassays of this disclosure (where the antibodies are recombinant and generated using the techniques described herein, compared to prior art methods that do not use capture and detection antibodies) can exhibit a higher level of sensitivity.

[0037] In view of the above, this disclosure provides a method for selecting capture antibodies and detection antibodies for use in an immunoassay to detect a target, the method comprising preparing recombinant capture antibodies and recombinant detection antibodies, and selecting a capture / detection recombinant antibody pair that exhibits the desired level of target detection sensitivity and / or selectivity in the immunoassay.

[0038] As an example, this disclosure may relate to an immunoassay for detecting hepatitis B antigen in a sample. It is noteworthy that the various definitions provided for an immunoassay for detecting hepatitis B antigen may also apply to general disclosures of immunoassays in which both the capture and detection steps utilize recombinant antibodies (e.g., sample source and subject type).

[0039] In view of the above, an immunoassay for detecting hepatitis B antigen in a sample may include an antigen capture step and an antigen detection step, both of which involve recombinant antibodies—in other words, recombinant antibodies used as capture agents (reagents for capturing and / or immobilizing targets from a sample) and recombinant antibodies used to detect said targets.

[0040] For example, an immunoassay for detecting hepatitis B antigen in a sample may include a capture step and a detection step. In the capture step, a recombinant antibody is used as a capture agent (to capture any hepatitis B antigen in the sample), and in the detection step, any captured targets are labeled for detection using the recombinant antibody. As described above, the recombinant antibody used as a capture agent can be immobilized to a matrix. The recombinant antibody used as a detection agent can be conjugated to a detectable moiety.

[0041] This disclosure is also based on an unexpected discovery that certain combinations of capture and detection (recombinant) antibodies against hepatitis B antigen exhibit anomalous sensitivity. Without wishing to be bound by theory, this suggests that the improved method described herein is a result of the high binding capacity of the antibody combinations.

[0042] In one aspect, a method is provided for detecting hepatitis B virus (HBV) antigens (e.g., HBV target antigens) in a sample, the method comprising contacting the sample with a first (capture) antibody and a second (detection) antibody, wherein both the first and second antibodies bind to the HBV antigen.

[0043] In one teaching, the primary and secondary antibodies are recombinant antibodies or fragments thereof.

[0044] In one teaching, the contact step includes contacting the first and second antibodies with the sample under conditions that allow the first and second antibodies to bind to any HBV target antigens present in the sample.

[0045] The contact step can generate an antigen-antibody complex, wherein the antigen-antibody complex includes a first antibody and a second antibody that bind to the HBV antigen.

[0046] The method may further include a detection step. This detection step can be used to detect the presence of the antigen-antibody complex described herein. The detection of the antigen-antibody complex indicates that the sample contains HBV antigen.

[0047] In view of the above, one embodiment of the method includes: contacting the sample with the first antibody and the second antibody under conditions that allow the first antibody and the second antibody to bind with any HBV antigen present in the sample, and detecting the resulting antigen-antibody complex, wherein the detection of the antigen-antibody complex indicates that the sample contains HBV antigen.

[0048] In one example, the antibody (e.g., the first antibody) may be immobilized on or to the matrix.

[0049] The matrix may include glass, polystyrene, or any other material suitable for antibody immobilization. The matrix may be suitable for imaging, microscopic observation, and / or optical procedures / measurements and / or cell culture. In some examples, the matrix may contain a coating, for example, to promote the binding and / or immobilization of antibodies (e.g., primary antibodies) to the matrix, and / or to inhibit the immobilization or binding of other antibodies, proteins (peptides), molecules, and / or reagents. These other antibodies, proteins (peptides), molecules, and / or reagents may be present in the sample.

[0050] One teaching suggests that once an antibody (such as a primary antibody) is immobilized to the surface of a matrix, the matrix can be blocked to mask other binding sites on the matrix. Blocking may also help reduce nonspecific binding events that could lead to false positive results. For example, the "blocking" procedure can use gelatin, BSA, skim milk, or some other blocking agents.

[0051] Therefore, in one embodiment, this disclosure provides a method for detecting hepatitis B virus (HBV) antigen in a sample, the method comprising contacting the sample with a first antibody and a second antibody, wherein both the first antibody and the second antibody bind to the HBV antigen, and the first antibody is immobilized to a matrix.

[0052] Those skilled in the art will understand that by immobilizing, for example, a first antibody, onto the matrix, any antigen-antibody complexes formed during the use of the method (i.e., through binding events between the first and second antibodies and any target HBV antigens present in the sample) will also be immobilized on the matrix.

[0053] As previously described, the detection steps may include detecting antigen-antibody complexes, wherein successful detection of antigen-antibody complexes indicates that the sample contains the target HBV antigen (and may be obtained from or provided by a subject with HBV infection). The detection steps are not necessarily limited to a specific detection method, and antigen-antibody complexes of the type described herein can be detected using any suitable technique known in the art. In one embodiment, the first antibody and / or the second antibody (preferably the second antibody) may contain a detectable portion. For example, the first antibody and / or the second antibody (preferably the second antibody) may bind to or conjugate to the detectable portion.

[0054] The detectable portion can directly or indirectly generate a detectable signal, such as an optically detectable signal. The detectable portion can be used for fluorescence detection, absorbance detection, enzyme-mediated detection, colorimetric and / or chemiluminescent detection. For example, the detectable portion can be radioactive opaque or radioactive isotopes, such as 3H, 14C, 32P, 35S, 123I, 125I, 131I; biotin; fluorescent (fluorophore) or chemiluminescent (chromophore) compounds, such as fluorescein isothiocyanate, rhodamine, or luciferin; enzymes, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase; imaging agents; or metal ions.

[0055] In view of the above, one teaching of the method described herein utilizes a fixed (first) antibody conjugated to the detectable portion and another (second) antibody (which is not fixed).

[0056] The antibodies used in the methods of this disclosure can be used in any order. For example, antibodies can be added to the sample simultaneously. Alternatively, the sample can be contacted with one of the antibodies before contacting the other antibody. In one teaching, a second antibody can be provided with the sample, and / or the second antibody can be provided after the first antibody has been contacted with the sample. Thus, the methods of this disclosure can include multiple contact steps, such as in a contact step, contacting the sample with a first (optionally immobilized) antibody and incubating the sample / first antibody mixture under conditions that allow the first antibody to bind to any target HBV antigen. After an appropriate incubation period, the sample / first antibody mixture can be subjected to a washing step. When the first antibody has been immobilized to the matrix, washing does not remove the immobilized first antibody; in fact, washing at this point will remove the sample, leaving the immobilized first antibody, which (depending on the antigen content of the sample) may or may not bind to the target antigen. In other words, washing leaves behind a mixture of immobilized first antibody and / or immobilized first antibody / target antigen complexes. The amount of immobilized first antibody / target antigen complexes depends on the content of the target HBV antigen in the sample.

[0057] The method may further include a subsequent contact step, wherein the sample is contacted with a washed and immobilized first antibody and / or any immobilized first antibody / target antigen complex. This step may be performed under conditions that allow the second antibody to bind to any target antigen present in the (immobilized) first antibody / target antigen complex. After an appropriate period of time, the system (or assay) may be washed. Washing at this stage will remove unbound second antigen, leaving only the second antibody bound to the target antigen, which is bound to the first (and immobilized) antibody. The method may then be performed in the detection step to detect the presence of the first antibody / target antigen / second antibody complex.

[0058] Therefore, the methods of this disclosure may include one or more washing steps. For example, the assay may be washed after a contact step or after each of multiple contact steps to remove antibodies that have not bound to their target. One or more washing steps will reduce the occurrence of false positive results.

[0059] This invention is based on the identification of anti-hepatitis B antigen antibody combinations that can be used to enhance the detection of HBV antigens in samples. Importantly, the methods described herein can detect hepatitis B antigens in samples obtained from patients with early infection within required regulatory limits. The improved methods and / or assays of this disclosure can be used to aid in the diagnosis of hepatitis B in subjects—whereby the detection of HBV target antigens in a sample using the methods described herein may indicate that the subject providing the sample or the subject obtaining the sample may be suffering from or recovering from HBV infection.

[0060] Samples processed using the methods of this disclosure can be provided or obtained from any suitable subject, including, for example, healthy subjects without obvious signs or symptoms of hepatitis B virus infection. Samples can also be provided or obtained from subjects who have or are suspected of having hepatitis B virus infection.

[0061] The term "sample" can include any type of sample believed to be capable of carrying the HBV antigen. For example, a sample can include bodily fluids from a subject, such as serum, blood (including whole blood or fragments or portions thereof, such as serum or plasma), saliva, or urine. Alternatively or additionally, a sample can include one or more cells or tissues, such as a tissue biopsy. In one teaching, a sample can include a liver biopsy.

[0062] The term “subject” may cover any human or animal subject, including (as previously stated) healthy subjects or subjects who are considered susceptible to or vulnerable to hepatitis B virus infection, subjects who are known to have been infected with or have hepatitis B virus infection, and / or subjects who are recovering from hepatitis B virus infection.

[0063] According to the method of this disclosure, a sample (of any type) is contacted with a first antibody and a second antibody. The first and second antibodies provided herein specifically bind to HBV antigens. The first and second antibodies may bind to the same HBV antigen or different HBV antigens in the sample. The first and second antibodies may bind to the same epitope of a specific HBV antigen or different epitopes on the same HBV antigen.

[0064] As used herein, the term "antigen" refers to a substance that can elicit an immune response and promote antibody production in a subject's body. As used herein, "epitope," also known as "antigenic determinant," refers to the portion of an antigen that elicits an immune response; it is the antigenic portion that antibodies specifically bind to. For use in the methods of this disclosure, an antigen or any fragment thereof may contain one or more epitopes that are recognized and specifically bound by the antibodies of this disclosure.

[0065] As used herein, the term "antibody" (including "first" and "second" antibodies) refers not only to recombinant (first and second) antibodies, but also to any (recombinant) immunoglobulin or intact molecule, and its fragment that binds to a target HBV antigen and / or a specific epitope of the target HBV antigen. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies, Fab fragments, Fab' fragments, F(ab)' fragments, and / or the F(v) portion of the entire antibody.

[0066] Furthermore, the term "antibody fragment" as used herein refers to an incomplete or separated portion of the complete antibody sequence that retains the antigen-binding function of the parent antibody. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. All antibody fragments disclosed herein are included within the scope of this invention, provided they retain the affinity required for a full-length antibody. Specifically, antibody fragments may be at least one amino acid shorter than a full-length antibody.

[0067] As used herein, the terms “specific binding” or “specifically binding” refer to the interaction between an antibody (such as the first and second antibodies described herein) and the target HBV antigen.

[0068] In this disclosure, the term "HBV antigen" may cover hepatitis B surface antigen (HBsAg).

[0069] Therefore, the first and second antibodies used in the methods or assays of this disclosure can bind to (or have affinity for) hepatitis B surface antigen (HBsAg). In other words, each of the first and second antibodies can bind to an epitope present on HBsAg.

[0070] In one example, the first and second antibodies of this disclosure are typically monoclonal antibodies.

[0071] In one example, the antibody used in the methods of this disclosure (e.g., a first antibody or capture antibody) can specifically bind to an antigen comprising the following sequence (SEQ ID NO:1) or a fragment or variant thereof (which also binds the antibody and / or contains a related epitope), substantially consisting of the following sequence (SEQ ID NO:1) or a fragment or variant thereof (which also binds the antibody and / or contains a related epitope) or consisting of the following sequence (SEQ ID NO:1) or a fragment or variant thereof (which also binds the antibody and / or contains a related epitope):

[0072] MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGS TTTSTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYI

[0073] (SEQ ID NO: 1)

[0074] As described above in this disclosure, SEQ ID NO:1 may contain at least one, for example, two epitopes that are recognized and / or bound by the antibodies of this disclosure. Therefore, antigens detectable using the methods of this disclosure may contain any one or more of the following:

[0075] (i) SEQ ID NO: 1; and / or

[0076] (ii) The antibody-binding fragment of SEQ ID NO:1; and / or

[0077] (iii) The first antibody-binding fragment of SEQ ID NO:1; and / or

[0078] (iv) The second antibody-binding fragment of SEQ ID NO:1; and / or

[0079] (v) a tabletop defined by SEQ ID NO:1; and / or

[0080] (vi) The first antibody-binding epitope defined by SEQ ID NO:1; and / or

[0081] (vii) The second antibody-binding fragment of SEQ ID NO:1.

[0082] In one teaching, the HBsAg antigen detected by the method of this disclosure may comprise a sequence showing a certain degree of sequence identity or homology with SEQ ID NO:1. For example, the HBsAg antigen to be detected by the method of this disclosure may comprise a sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity / homology with the sequence of SEQ ID NO:1.

[0083] In one teaching, the HBsAg antigen to be detected by the method of this disclosure may comprise a sequence comprising any suitable portion or fragment of the complete 226 amino acids of SEQ ID NO: 1. The detectable portion or fragment of SEQ ID NO: 1 should always bind to or be bound by the antibody described herein and may comprise “n” amino acids from the complete SEQ ID NO: 1 sequence. The term “n” may represent any number from about 5 (continuous) amino acids to about 225 (continuous) amino acids of SEQ ID NO: 1. The detectable portion or fragment of SEQ ID NO: 1 may comprise amino acids from SEQ ID NO: 1. 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 221, 222, 223, or 224 (continuous) amino acids. The detectable portion or fragment may contain a first antibody-binding epitope and / or a second antibody-binding epitope.

[0084] As used herein, the term "variant" refers to a detectable antigen / amino acid sequence altered by one or more amino acids. Such variants may have "conserved" variations, where the substituted amino acids have similar structural or chemical properties (e.g., leucine replaced by isoleucine). Additionally or alternatively, detectable variants may have "non-conserved" variations (e.g., glycine replaced by tryptophan). Similar minor variations may also include amino acid deletions or insertions, or both. Those skilled in the art will recognize various methods by which amino acid sequences (e.g., substitutions, insertions, deletions) can be altered without eliminating biological activity (in this case, the ability to bind to the antibody described herein), for example, by using bioinformatics software available in the art (e.g., PROVEAN, DNASTAR). For example, a detectable antigen variant, such as the HBsAg antigen described herein, may have 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with a reference protein or peptide sequence (e.g., SEQ ID NO:1). Therefore, in all cases, variants and detectable forms of SEQ ID NO:1, or any detectable fragment or portion thereof, should bind to or be bound to any antibody described herein.

[0085] In a preferred embodiment, the antibody (e.g., the first antibody) used (as a capture agent) in the disclosed method may comprise the following heavy chain CDR sequence:

[0086] CDRH1: SYGVH (SEQ ID NO: 2)

[0087] CDRH2: VIWAGGITNYNSALMS (SEQ ID NO: 3)

[0088] CDRH3: GGGVYYGINYAMDY (SEQ ID NO: 4)

[0089] The antibody (e.g., the first antibody) used in the methods of this disclosure may comprise or further comprise the following light chain CDR sequences:

[0090] CDRL1: RASESVEYYGTSLMQ (SEQ ID NO: 5)

[0091] CDRL2: AASNVEP (SEQ ID NO: 6)

[0092] CDRL3: QQSRNVPWT (SEQ ID NO: 7)

[0093] The first antibody used in the methods of this disclosure (as a capture agent) may contain the following heavy chain (HC) sequence:

[0094] LHQSGAGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWAGGITNYNSALMSRLSIRKDNFKSQVFLKMNSLQNDDTAMYYCARGGGVYYGINYAMDYWGQGTTVTV (SEQ ID NO: 8);

[0095] and / or the following light chain (LC) sequences:

[0096] ELTQTPVSLAVSLGQRATISCRASESVEYYGTSLMQWYQQKPGQPPKLLIYAASNVEPGVPARFSGSGSGTDFSLNIQPVEEDDIAMYFCQQSRNVPWTFGGGTKLE (SEQ ID NO: 9).

[0097] Exemplary heavy and light chain sequences of the first (or capture) antibody used in the methods of this disclosure may be included in the sequences deposited in GenBank with accession numbers AF110502 (heavy chain sequence) and AF110503 (light chain sequence). Further exemplary heavy and light chain sequences of the first (or capture) antibody used in the methods of this disclosure are disclosed in “Intracellular Expression of a Cloned Antibody Fragment Interferes with Hepatitis B Virus Surface Antigen Secretion”: Jasper zu Putlitz, Arne Skerra, Jack R. Wands: Vol. 255, No. 3, February 24, 1999, pp. 785-791, the contents of which are incorporated herein by reference.

[0098] The first (or capture) antibody used in the methods of this disclosure may comprise CDR, light chain, and / or heavy chain sequences that have a degree of identity with any of the related sequences in SEQ ID NOS: 2-9. The term "degree of identity" may cover sequences that have at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the related sequences.

[0099] For example, the antibody used (as a capture agent) in the methods of this disclosure, such as the first antibody, may contain an anti-HBsAg monoclonal antibody called “5C3” (product catalog number Ab00769-2.0; Absolute antibody).

[0100] As previously stated, while the methods of this disclosure may utilize the complete or full form of 5C3, the methods may also use or utilize its antigen (i.e., HBsAg) binding fragment.

[0101] In a preferred embodiment, the antibody (e.g., a second antibody) used in the method (detection step) of this disclosure may comprise the following heavy chain CDR sequence:

[0102] CDRH1: GFAFSDYSIN (SEQ ID NO: 10)

[0103] CDRH2: IISYDGRITYYRDSVKG (SEQ ID NO: 11)

[0104] CDRH3: QYYDFWSGSSVGRNYDGMDV (SEQ ID NO: 12)

[0105] Antibodies (e.g., second antibodies) used in the methods of this disclosure may contain or further contain the following light chain CDR sequences:

[0106] CDRL1: RSSQSLLHRSGNNYLD (SEQ ID NO: 13)

[0107] CDRL2: VGSNRAS (SEQ ID NO: 14)

[0108] CDRL3: MQALQTPRT (SEQ ID NO: 15)

[0109] The second antibody used in the methods of this disclosure (as a detection agent) may contain the following heavy chain (HC) sequence:

[0110] QVQLVESGGGVVRPGRSLRLSCAASGFAFSDYSINWVRQAPGKGLEWVAIISYDGRITYYRDSVKGRFTISRDDSKNTLYLQMNSLRTEDTAVYYCARQYYDFWSGSSVGRNYDGMDVWGLGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 16);

[0111] and / or the following light chain (LC) sequence:

[0112] DIVMTQSPLSLSVTPGEPASISCRSSQSLLHRSGNNYLDWYLQKPGHSPQLLIYVGSNRASGVPDRFSGSGSGTEYTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 17).

[0113] The second (or detection) antibody used in the methods of this disclosure may comprise CDR, light chain, and / or heavy chain sequences that have a degree of identity with any of the relevant sequences in SEQ ID NOS: 10-17. The term "degree of identity" may cover sequences that have at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the relevant sequences. The second (or detection) antibody used in the methods of this disclosure may comprise an anti-HBsAg monoclonal antibody called Livivirumab (catalog number PX-TA1189; ProteoGenix). As previously stated, while the methods of this disclosure may utilize the whole or complete form of Livivirumab, the methods may also use or utilize its antigen (i.e., HBsAg) binding fragment.

[0114] It should also be noted that in the methods disclosed herein, the first antibody and the second antibody described above are interchangeable—an antibody referred to as the first antibody (e.g., 5C3) is used after an antibody referred to herein as the second antibody (e.g., rivirumab).

[0115] The antibodies disclosed herein typically exhibit high affinity for target epitopes. More specifically, the antibodies disclosed herein typically exhibit high affinity for epitopes of HBV antigens (e.g., HBsAg). For example, the KD value of the antibody may be 10. -9 Up to 10 -13 Within the range.

[0116] In view of the above, a method for detecting HBsAg in a sample is provided, the method comprising contacting the sample with 5C3 and rivirumab, wherein both 5C3 and rivirumab can bind to HBsAg. The contact step may include first contacting the sample with 5C3 under conditions allowing binding between any HBsAg present in the sample and the 5C3 antibody, thereby generating a 5C3 / HBsAg complex. The contact step may further include contacting any 5C3 / HBsAg complex with rivirumab under conditions allowing binding between any HBsAg component of the 5C3 / HBsAg complex and rivirumab, thereby forming a 5C3 / HBsAg / rivirumab complex. The method may further include a step of detecting the 5C3 / HBsAg / rivirumab complex, wherein the detection of the 5C3 / HBsAg / rivirumab complex indicates that the sample was obtained or provided from or to a subject who has had HBV infection or has recovered from HBV infection.

[0117] In addition to the steps described above, the methods of this disclosure may optionally include one or more washing steps. In one example, the washing step may be performed after the first antibody has contacted the sample but before the second antibody has contacted the sample. Alternatively, the washing step may be performed after the second antibody has contacted the sample. As an example, the methods of the present invention may include a washing step designed, for example, to remove unbound 53C and / or ribavirin antibody. This helps to reduce the occurrence of false positive results.

[0118] In one example, the method may optionally include one or more control assays. For example, a negative control assay may include contacting a first and a second antibody with a sample known to be free of the relevant target antigen. In this case, no antigen-antibody complex will form, and the detection step (followed by an appropriate washing step) will produce a negative result. A positive control may include using a sample known to contain or have been added to the relevant target antigen. In this case, an antigen-antibody complex will form, and the detection step should produce a positive result.

[0119] Those skilled in the art will understand that, in addition to the first and second antibodies of this disclosure, the method may also include one or more additional antibodies to further improve the sensitivity and / or specificity of the assay. By way of example only, the one or more additional antibodies may specifically bind to another different hepatitis B antigen, such as hepatitis B core antigen (HBcAg) and / or hepatitis B e antigen (HBeAg).

[0120] It should be noted that in any disclosed method, the method is not necessarily limited to the examples provided herein and can be performed in any suitable form, including in a single container (such as an Eppendorf tube or other test tube) or in multiple forms, such as a multiwell plate. Furthermore, cartridge forms, such as those based on lateral flow or microfluidics, are also conceivable. Those skilled in the art will understand that, depending on the detection method used and / or the sample provided (e.g., bodily fluids or tissue), the form of the method may need to be adapted using various methods known in the art.

[0121] In other teachings, this disclosure also relates to a kit for use in the methods described herein. The kit may include, substantially consist of, or comprise of:

[0122] First antibody and second antibody, wherein the first antibody and / or second antibody are selected from:

[0123] Rivirumab (Catalogue No. PX-TA1189; ProteoGenix)

[0124] and

[0125] Anti-HBsAg [5C3] (Product Catalog No.: Ab00769-2.0; Absolute antibody)

[0126] And optionally, a first or second antibody immobilized on the matrix,

[0127] Furthermore, optionally, a first antibody and / or a second antibody labeled with the detection portion are used.

[0128] This disclosure also provides the use of ribavirin (catalog number PX-TA1189; ProteoGenix) and / or 5C3 (catalog number Ab00769-2.0; Absolute antibody) in methods or assays for detecting HBsAg in samples. Attached Figure Description

[0129] The invention will now be described in detail with reference to the following accompanying drawings, which show:

[0130] Figure 1 An immunoassay (ELISA) used to detect HBsAg, comparing various combinations of capture and detection antibodies.

[0131] Figure 2 An immunoassay (ELISA) used to detect HBsAg, comparing various combinations of capture and detection antibodies.

[0132] Figure 3 The MosaiQ immunoassay is used to detect HBsAg and compares various combinations of capture and detection antibodies.

[0133] Figure 4 The MosaiQ immunoassay is used to detect HBsAg and compares various combinations of capture and detection antibodies.

[0134] Figure 5: Analysis of the sensitivity of detection methods using different combinations of capture and detection agents. (A) Capture: Absolute 5C3; Detection: proteogenix ribavirin; conjugate SDT streptavidin-polyhortradiction peroxidase 80; Cycle time: 48 seconds. (B) Capture: Absolute 5C3; Detection: Meridian B65811; conjugate SDT streptavidin-polyhortradiction peroxidase 80; Cycle time: 48 seconds. Detailed Implementation

[0135] result

[0136] Table 1 (see also) Figure 1 )

[0137]

[0138] Table 2 (see Table 2) Figure 2 )

[0139]

[0140] Table 3

[0141]

[0142] Table 4 (see Table 4) Figure 3 )

[0143]

[0144] Table 5

[0145]

[0146] Table 6 (see Table 6) Figure 4 )

[0147]

Claims

1. A method for detecting hepatitis B virus (HBV) antigen in a sample, the method comprising contacting the sample with a first antibody and a second antibody, the first antibody comprising a sequence provided by any one of SEQ ID NOs 2-9; and the second antibody comprising a sequence provided by any one of SEQ ID NOs 10-17.

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

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

4. The method according to any one of the preceding claims, wherein, Both the first antibody and the second antibody are recombinant antibodies.

5. The method of any of the preceding claims, wherein the contacting step comprises contacting the first antibody and the second antibody with the sample under conditions that allow the first antibody and the second antibody to bind to any HBV antigen present in the sample.

6. The method of any of the preceding claims, wherein, The first antibody and the second antibody bind to the HBV antigen present in the sample to generate an antibody-antigen complex, the complex comprising the first antibody and the second antibody that bind to the HBV antigen.

7. The method according to any one of claims 1-5, wherein, The binding of the first antibody with the HBV antigen present in the sample generates a first antibody-antigen complex, the complex comprising the first antibody that binds to the HBV antigen.

8. The method of claim 7, wherein, The second antibody binds to a complex comprising the first antibody that binds to the HBV antigen.

9. The method according to any of the preceding claims, wherein, The first antibody was immobilized into the matrix.

10. The method of any one of the preceding claims, wherein, The second antibody contains a detectable portion.

11. The method of any of the preceding claims, wherein the HBV antigen is hepatitis B surface antigen (HBsAg).

12. The method of any of the preceding claims, wherein, The first antibody is anti-HBsAg monoclonal antibody 5C3 (product catalog number Ab00769-2.0; Absolute antibody) or its HBsAg binding fragment.

13. The method of any one of the preceding claims, wherein, The second antibody is the anti-HBsAg monoclonal antibody rivirumab (product catalog number PX-TA1189; ProteoGenix) or its HBsAg binding fragment.

14. A method for detecting HBsAg in a sample, the method comprising: The sample is contacted with monoclonal antibody 5C3 under conditions suitable for allowing binding between monoclonal antibody 5C3 and any HBsAg in the sample, wherein a binding event between monoclonal antibody 5C3 and any HBsAg in the sample results in the formation of a 5C3 / HBsAg complex. Any 5C3 / HBsAg complex is contacted with the monoclonal antibody rivirumab under conditions suitable for allowing binding between the monoclonal antibody rivirumab and any 5C3 / HBsAg complex; and then Detect any rivirumab that binds to the 5C3 / HBsAg complex; The detection of ribavirin, which binds to the 5C3 / HBsAg complex, indicates that the sample contains a target.

15. An immunoassay for detecting a target in a sample, wherein the immunoassay includes a target capture step and a target detection step, wherein both the target capture step and the target detection step include a recombinant antibody.

16. Recombinant antibodies used in target capture and target detection steps of immunoassays for detecting targets in samples.

17. The immunoassay of claim 15 or the use of claim 16, wherein, The immunoassay is used to determine whether a sample contains a specific target or antigen.

18. The immunoassay or use according to claims 15-17, wherein the recombinant antibody for the target capture step is immobilized to a matrix and / or the recombinant antibody for the detection step is conjugated to a detectable portion.

19. A method for detecting a target, such as a target antigen, in a sample, said method comprising: The sample is brought into contact with the first recombinant antibody under conditions suitable for allowing the first recombinant antibody to bind to any target in the sample; and then Contact any first recombinant antibody / target complex (formed by a binding event between the first antibody and any target present in the sample) with the second antibody under conditions suitable for allowing binding between the second antibody and any first recombinant antibody / target complex; and then Detect the second antibody that binds to the first recombinant antibody / target complex; The detection of a second antibody that binds to the first recombinant antibody / target complex indicates that the sample contains the target.

20. The method of claim 19, wherein the first recombinant antibody is immobilized to the matrix.

21. A method for selecting capture antibodies and detection antibodies in an immunoassay for detecting a target, the method comprising preparing recombinant capture antibodies and recombinant detection antibodies, and selecting a capture / detection recombinant antibody pair that exhibits desired or predetermined levels of target detection sensitivity and / or selectivity in the immunoassay.

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