Antigen detection

Abstract

This disclosure provides an immunoassay with exceptional sensitivity for detecting a target antigen in a sample. In one disclosure, the capture and detection steps of such an assay utilize recombinant antibodies. The disclosed immunoassay may find specific applications in the detection of HBV antigen.

Description

Technical Field

[0001] The present disclosure provides an improved method for detecting the presence of hepatitis B virus or an antigen derived therefrom in a sample.

Background Art

[0002] Hepatitis B virus (HBV) is one of the most prevalent infectious pathogens worldwide that can cause lifelong chronic infection, liver cirrhosis, liver cancer, liver failure and death (Liang et al., Hepatology, 49(5): S13 - 21 (2009)). Early screening and detection of HBV infection are crucial in the disease management and control of HBV transmission (Franco et al., World J. Heptaol., 4: 74 - 80 (2012)). Detection methods for HBV infection typically involve serological detection of HBV antigens, such as ELISA - based techniques or other immunoassays that detect hepatitis B surface antigen (HBsAg), hepatitis core antigen (HBcAg) and / or hepatitis B surface antibody (Mao et al., Anal. Chim. Acta, 909: 101 - 108 (2016)). Instead, real - time PCR or various sequencing techniques can be employed to detect the presence of HBV DNA, which can be useful, for example, in the management of chronic HBV - infected patients receiving antiviral therapy, and measuring HBV DNA levels at baseline or during treatment can help evaluate the response to treatment (Terrault et al., Hepatology, 63(1): 261 - 282 (2015)). There are a number of commercially available antibodies made against hepatitis B antigens, but there is still a need in the art for an improved method that enables accurate detection of HBV infection and provides the required level of sensitivity, and / or a method that can detect HBV infection quickly and cost - effectively. An object of the present disclosure is, inter alia, to provide an improved method for detecting hepatitis B virus in a sample, including the steps provided herein.

Summary of the Invention

[0003] This disclosure is based on the finding that an immunoassay comprising a target capture step and a target detection step, which uses a recombinant antibody in both the capture and detection steps, represents an improvement over prior art immunoassays that can at best use a recombinant antibody in only one of the capture or detection steps. Please note that the terms “comprise,” “comprising,” and / or “comprises” are used to indicate that aspects and embodiments of the present invention “comprise” a particular singular or plural feature. It should also be understood that these terms may encompass aspects and / or embodiments that “essentially consist of” or “consist of” a particular singular or plural feature. Various aspects and embodiments of this disclosure are applicable to immunoassays for the detection of target antigens in a sample. In such cases, the term “target” may include “single target antigen” or “multiple target antigens.”

[0004] In consideration of the foregoing, 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 recombinant antibody (or "recombinant antibody"). A second aspect of this disclosure provides recombinant antibodies for use in target capture and target detection steps of an immunoassay for detecting a target in a sample. 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 recognize that a vast number of immunoassays exist, and that the disclosures presented herein are applicable to any assay utilizing antibody-based capture and detection systems. As mere examples, the term “immunoassay” may encompass enzyme immunoassays (EIA: e.g., ELISA), radioimmunoassays (RIA), fluoroimmunoassays (FIA); Western blotting (WB); chemiluminescent immunoassays (CIA) and counting immunoassays (CIA); immunohistochemical typing assays (IHC); and immunocytochemical typing assays (ICC). In the context of this disclosure, the term “immunoassay” may not encompass radioimmunoassays (RIA).

[0005] As described, and in one instruction, the target may be an antigen. Further instruction may include antigens known to be expressed by pathogens, such as fungal, bacterial, or viral antigens. The immunoassays of this disclosure may include a target capture step in which the sample is brought into contact with a target capture agent under conditions that allow binding between any target present in the sample and the target capture agent. As described, the target capture agent may include recombinant antibodies. Recombinant antibodies intended for use as target capture agents can bind to targets (e.g., target antigens). Binding between any target and target capture agent present in the sample will result in a target / target capture agent complex. The target capture agent can be immobilized on the substrate. The immunoassays of this disclosure may further include a detection step in which a target / target capture agent complex (formed during the capture step of the immunoassay) is brought into contact with a detection agent.

[0006] As described, the detection agent may contain recombinant antibodies. Recombinant antibodies intended for use as detection agents can bind to a target (e.g., a target antigen). Recombinant antibodies intended for use as detection agents can bind to a target at a site or epitope different from that to which they are bound by a capture agent. The detection agent may be conjugated to a detectable portion, for example, an optically detectable portion. Detection systems for use in 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-stained systems and / or alkaline phosphatase-based systems. In one example, a section system for use in the methods or assays of this disclosure may include a streptavidin polyHRP conjugate (a biotin-binding protein conjugated with a polymer of horseradish peroxidase).

[0007] Considering the foregoing, a third aspect of this disclosure is a method for detecting a target in a sample, for example, a target antigen, The steps include: contacting the sample with the first recombinant antibody under conditions suitable for enabling binding between the first recombinant antibody and any target in the sample; and then The steps include: contacting one of the first recombinant antibody / target complexes (formed by a binding event between the first antibody and any of the targets present in the sample) with the second antibody under conditions suitable for enabling binding between the second antibody and any of the first recombinant antibody / target complexes; and then The steps include detecting a second antibody bound to a first recombinant antibody / target complex, and The present invention provides a method in which the detection of a second antibody bound to a first recombinant antibody / target complex indicates that the sample contains the target. 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 on a substrate. Further teachings indicate that the methods described above (or in fact, 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 subjected to a wash designed to wash away any unbound first antibody. The method may also include a second wash after the step of contacting any first recombinant antibody / target complex with a second recombinant antibody, this additional wash designed to remove any unbound second recombinant antibody.

[0008] (i) Immunoassays utilizing recombinant antibodies (in either capture or detection step) demonstrate improved sensitivity; (ii) Recombinant antibody technology allows for customization of both the capture and detection antibodies to desired specifications; in this way, the highest quality target conjugates can be selected. This results in immunoassays with improved sensitivity and specificity (compared to prior art assays that do not use recombinant antibodies in both the capture and detection steps); (iii) Recombinant antibodies can be easily manipulated and / or converted to take any desired predetermined format, fragment type, or class; Therefore, the use of recombinant antibodies in both the capture and detection steps of the immunoassays described herein is advantageous. In addition, antibodies produced using recombinant technology can be generated rapidly and cost-effectively. Those skilled in the art will recognize that immunoassays for detecting antigens in a sample can be used in the diagnosis of a particular disease or condition. As described, the antigen may include pathogenic antigens (e.g., bacterial, viral, or fungal antigens), and the presence of such antigen in the sample may be associated with a specific disease, infection, or condition caused by or contributed to by a pathogen expressing such antigen. Thus, the finding that a sample contains a particular antigen may indicate that the sample was provided by or obtained from a subject who has been infected with a pathogen expressing such antigen, or who suffers from / is recovering from a disease or condition caused by or contributed to by such pathogen.

[0009] Recombinant antibodies for use as capture or detection agents can be produced using established recombination methods. For example, recombination methods for producing useful antibodies may include the use of molecular techniques for generating recombinant antibody libraries, antibody display techniques, antibody selection protocols, and affinity maturation techniques. Further details regarding recombination techniques used for antibody production can be found in Hoogenboom, (2005); Nature Biotechnology. For example, preferred antibody display technologies may include technologies referred to as phage (antibody) displays, ribosome (antibody) displays, and / or microbial (antibody) cell displays. Techniques for recombinant antibody selection may include the step of exposing antibodies from a display library to an antigen. Such techniques may include, for example, biopanning to select clones (e.g., phage clones) that exhibit desired binding characteristics. The selection procedure may include repeated rounds of selection to derive an antibody exhibiting superior binding properties. Such selection techniques can be used to select antibodies for use in the capture and detection steps of the immunoassays described herein.

[0010] Affinity maturation is a process by which properties, such as antibody potency and antibody affinity, can be improved, developed, and / or manipulated. The process may involve generating a library with diversity concentrated on a small number of residues most likely to be involved in target / antigen interactions. CDRs are constructed, for example, using amplification techniques (e.g., PCR) and libraries that are screened to identify variants with desired characteristics. Other approaches involve the use of "selective pressure" to select antibodies that bind best (to their target) under specific conditions. These approaches can result in antibodies exhibiting substantial acquisition of affinity / potency, etc. Antibodies for use in the immunoassays of this disclosure may include antibodies that can be obtained using recombinant techniques, including the techniques described herein. As a further example, antibodies for use as capture and detection agents in the immunoassays of the present invention can be obtained, for example, as antibody (capture and detection) pairs exhibiting specific and predetermined target binding and / or potency and / or affinity characteristics. In this way, the immunoassays of this disclosure (compared to prior art methods that do not use recombinant capture and detection antibodies produced using the techniques described herein) can exhibit an improved level of sensitivity.

[0011] In consideration of the foregoing, the present disclosure provides a method for selecting capture and detection antibodies for use in an immunoassay for the detection of a target, comprising the steps of preparing recombinant capture antibodies and recombinant detection antibodies, and selecting a capture / detection recombinant antibody pair that exhibits a desired level of target detection sensitivity and / or selectivity in an immunoassay. For example, this disclosure may also relate to an immunoassay for the detection of hepatitis B antigens in a sample. Note that the various definitions provided for immunoassays for the detection of hepatitis antigens may also apply to general disclosures of immunoassays using recombinant antibodies for both the capture and detection steps (e.g., source of sample and type of target). Considering the foregoing, an immunoassay for detecting hepatitis B antigen in a sample may include the use of antigen capture and detection steps, both of which include recombinant antibodies—in other words, recombinant antibodies for use as a capture agent (a drug for capturing and / or immobilizing a target from a sample) and recombinant antibodies for use in the detection of the target. For example, an immunoassay for detecting hepatitis B antigens in a sample may include a capture step in which a recombinant antibody is used as a capture agent (for capturing any hepatitis B antigens in the sample), and a detection step in which any captured target is labeled for detection using the recombinant antibody. As described, the recombinant antibody for use as a capture agent may be immobilized on a substrate. The recombinant antibody for use as a detection agent may be conjugated to a detectable portion.

[0012] This disclosure is further based on the unexpected finding that specific combinations of capture and detection (recombinant) antibodies against hepatitis B antigens exhibit exceptional sensitivity. While we do not wish to be constrained by theory, it is suggested that the improved methods described herein are a result of the high binding capacity of the antibody combinations. In one embodiment, a method is provided for detecting hepatitis B virus (HBV) antigen (e.g., HBV target antigen) in a sample, comprising the step of contacting the sample with first (capture) and second (detection) antibodies, wherein both the first and second antibodies bind to the HBV antigen. In one instruction, the first and second antibodies are recombinant antibodies or fragments thereof. One instruction involves contacting the first and second antibodies with the sample under conditions that allow binding between the first and second antibodies and any of the HBV target antigens present in the sample. The contact step may also produce antigen-antibody complexes, which contain first and second antibodies bound to the HBV antigen.

[0013] The method may further include a detection step. Using the detection step, the presence of an antigen-antibody complex can be detected as described herein. Detection of an antigen-antibody complex indicates that the sample contains HBV antigen. In consideration of the foregoing, one embodiment of the described method comprises the steps of contacting a sample with the first and second antibodies under conditions that allow binding between the first and second antibodies and any HBV antigen present in the sample, and detecting the resulting antigen-antibody complex, the detection of the antigen-antibody complex indicating that the sample contained HBV antigen. In one example, an antibody, such as a first antibody, can be immobilized on or attached to the substrate surface. The substrate may contain glass, polystyrene, or any other material suitable for antibody immobilization. The substrate may be suitable for use in imaging, microscopy and / or optical procedures / measurements and / or cell culture. In some examples, the substrate may include a coating to facilitate the binding and / or immobilization of an antibody (e.g., the first antibody) to the substrate, and / or to inhibit the immobilization or binding of other antibodies, proteins (peptides), molecules and / or reagents, etc. Such other antibodies, proteins (peptides), molecules and / or reagents may be present in the sample.

[0014] In one instruction, once an antibody (e.g., the first antibody) is immobilized on the surface of the substrate, the substrate can be blocked to mask further binding sites on the substrate surface. Blocking can also help reduce nonspecific binding events that may contribute to false positive results. The "blocking" procedure can use, for example, gelatin, BSA, skim milk, or several other blocking agents. Accordingly, in one embodiment, the present disclosure provides a method for detecting hepatitis B virus (HBV) antigen in a sample, comprising the step of contacting the sample with first and second antibodies, wherein both the first and second antibodies bind to the HBV antigen and the first antibody is immobilized on a substrate. One skilled in the art would recognize that, for example, by immobilizing the first antibody on a substrate, any antigen-antibody complexes formed when the method is used (i.e., by binding events between the first and second antibodies and any target HBV antigen present in the sample) would also become immobilized on the substrate. As described, the detecting step may include detecting the antigen-antibody complex, and success in detecting the antigen-antibody complex indicates that the sample contains the target HBV antigen (and may have been obtained from or provided by a subject suffering from HBV infection). The detecting step need not be limited to a particular 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 and / or second antibody (preferably the second antibody) may include a detectable moiety. For example, the first and / or second antibody (preferably the second antibody) may be bound or conjugated to a detectable moiety.

[0015] The detectable moiety can produce a detectable signal, either directly or indirectly, such as an optically detectable signal. The detectable moiety can be suitable for fluorescence detection, absorbance detection, enzyme-mediated detection, chromogenic and / or chemiluminescent detection. By way of example, the detectable moiety can be a radio-opaque or radioisotope, such as 3H, 14C, 32P, 35S, 123I, 125I, 131I; biotin; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, β-galactosidase or horseradish peroxidase; a contrast agent; or a metal ion. In view of the above, one teaching of the methods described herein utilizes an immobilized (first) antibody and a separate (second) antibody (not immobilized) conjugated to a detectable moiety.

[0016] The antibodies used in the methods of the present disclosure can be used in any order. For example, the antibodies can be added to the sample simultaneously. Alternatively, the sample can be contacted with one of the antibodies and then with the other antibody. In one teaching, the second antibody can be provided together with the sample and / or the second antibody can be provided after contacting the first antibody with the sample. Thus, the methods of the present disclosure can include multiple contacting steps, for example, the sample is contacted with a first (optionally immobilized) antibody and the sample / first antibody mix is incubated under conditions that allow binding between the first antibody and any target HBV antigen. After a suitable period of incubation, the sample / first antibody mix can be subjected to a washing step. If the first antibody is immobilized on a substrate, the washing will not remove the immobilized first antibody; indeed, washing at this point will remove the sample while leaving the immobilized first antibody that is either bound or not bound (depending on the antigen content of the sample) to the target antigen. In other words, the washing will leave the immobilized first antibody and / or a mix of the immobilized first antibody / target antigen complex. The number of immobilized first antibody / target antigen complexes will depend on the target HBV antigen content of the sample.

[0017] The method can further include a subsequent contacting step where the sample is contacted with the washed and immobilized first antibody and / or any immobilized first antibody / target antigen complex. This step can be carried out under conditions that allow binding between the second antibody and any target antigen present in the (immobilized) first antibody / target antigen complex. After a suitable period, the system (or assay) can be washed. Washing at this stage will remove the unbound second antibody while leaving only the second antibody bound to the target antigen that is bound to the first (and immobilized) antibody. Next, the method can be subjected to the detection steps described to detect the presence of the first antibody / target antigen / second antibody complex. Accordingly, the method of this disclosure may include one or more washing steps. For example, the assay may be washed after or after each contact step to remove antibodies that are not bound to its target. One or more washing steps would reduce the occurrence of false positive results. The present invention is based on the identification of combinations of anti-hepatitis B antigen antibodies that can be used to enhance the detection of HBV antigen in a sample. Importantly, the methods described herein can detect hepatitis B antigen in samples obtained from early-infected patients, within the required regulatory limits. The improved methods and / or assays of this disclosure can facilitate the diagnosis of hepatitis B in a subject (detection of HBV target antigen in a sample using the methods described herein) and can indicate that the subject providing the sample or the subject from which the sample was obtained may have or be recovering from an HBV infection.

[0018] The samples to be subjected to the methods of this disclosure may be provided or obtained from any preferred subject, including, for example, a healthy subject with no clear signs or symptoms of hepatitis B virus infection. The samples may be provided or obtained from a subject that has or is suspected of having hepatitis B virus infection. The term "sample" can encompass any sample type that is thought to be capable of containing the HBV antigen. For example, a sample may include, for instance, bodily fluids derived from the subject, such as serum, blood (whole blood or fragments or fractions thereof, such as serum or plasma), saliva, or urine. Alternatively, or in addition to these, a sample may include cells (or a number of cells) or tissue, such as a tissue biopsy. In one teaching, a sample may include a liver biopsy. The term "subject" can encompass any human or animal subject, including (as described) a healthy subject, a subject considered to be predisposed to or susceptible to HBV infection, a subject known to have had HBV infection or currently has HBV infection, and / or a subject recovering from HBV infection.

[0019] According to the method of this disclosure, a sample (of any type) is brought into contact with first and second antibodies. The first and second antibodies provided herein specifically bind to HBV antigens. The first and second antibodies can bind to the same antigen or different HBV antigens in the sample. The first and second antibodies can bind to the same epitope of a particular HBV antigen or to different epitopes on the same HBV antigen. As used herein, the term “antigen” refers to a substance that can induce an immune response in a subject and promote the production of antibodies. “Epitope,” also known as an “antigenic determinant,” as used herein refers to the portion of an antigen that induces an immune response and is the portion of an antigen to which an antibody specifically binds. For use in the methods of this disclosure, an antigen or any antigenic fragment thereof may comprise one or more epitopes that are recognized and specifically bound by the antibodies of this disclosure.

[0020] 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 that binds to the target HBV antigen and / or its specific epitope, as well as fragments thereof. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, humanized, single-chain, Fab, Fab', F(ab)' fragments and / or F(v) portions of the entire antibody. Furthermore, as used herein, the term “antibody fragment” refers to an incomplete or isolated portion of the full sequence of an antibody that retains the antigen-binding function of the parent antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabody; linear antibodies; single-chain antibody molecules; and polyspecific antibodies formed from antibody fragments. Antibody fragments disclosed herein are encompassed by the present invention insofar as they retain the desired affinity of the full-length antibody. In particular, antibody fragments may be at least one amino acid shorter than the full-length antibody. As used herein, the terms “specific binding” or “specifically binding” refer to the interaction between an antibody (e.g., the first and second antibodies described herein) and the target HBV antigen.

[0021] In this disclosure, the term "HBV antigen" may include hepatitis B surface antigen (HBsAg). Therefore, the first and second antibodies for use 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 in HBsAg. For example, the first and second antibodies of this disclosure are typically monoclonal antibodies. In one example, an antibody for use in the methods of this disclosure (e.g., a first or capture antibody) may contain, be essentially, or specifically bind to an antigen comprising the following sequence (SEQ ID NO: 1) or a fragment or variant thereof (this fragment or variant also includes an antibody-binding and / or related epitope): MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGS TTTSTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYI (Sequence ID 1)

[0022] As previously described in this disclosure, Sequence ID No. 1 may include at least one, e.g., two epitopes that are recognized and / or bound by the antibody of this disclosure. Antigens detectable using the method of this disclosure include one or more of the following: (i) Sequence ID 1; and / or (ii) Antibody-binding fragment of SEQ ID NO: 1; and / or (iii) the first antibody-binding fragment of SEQ ID NO: 1; and / or (iv) The second antibody-binding fragment of Sequence ID No. 1; and / or (v) Epitope defined by Sequence ID 1; and / or (vi) A first antibody-binding epitope defined by SEQ ID NO: 1; and / or (vii) Second antibody-binding fragment of Sequence ID No. 1 It may include. In one teaching, the HBsAg antigen to be detected by the method of the present disclosure may include a sequence that exhibits a certain level of sequence identity or homology with SEQ ID NO: 1. For example, the HBsAg antigen to be detected by the method of the present disclosure may include a sequence that has 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.

[0023] In one teaching, the HBsAg antigen to be detected by the method of the present disclosure may include a sequence comprising any preferred portion or fragment of the full 226 amino acids of SEQ ID NO: 1. The detectable portion or fragment of SEQ ID NO: 1 is always bound to or to be bound by the antibody described herein and may include "n" amino acids derived from the full SEQ ID NO: 1 sequence. The term "n" can represent any number of amino acids from approximately 5 (consecutive) amino acids of SEQ ID NO: 1 to approximately 225 (consecutive) amino acids of SEQ ID NO: 1. The detectable portion or fragment of SEQ ID NO: 1 may contain 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 221, 222, 223, or 224 (consecutive) amino acids derived from SEQ ID NO: 1. The detectable portion or fragment may contain a first antibody-binding epitope and / or a second antibody-binding epitope.

[0024] As used herein, the term “variant” refers to a detectable antigen / amino acid sequence in which one or more amino acids have been altered. A variant may have a “conservative” change (e.g., substitution of leucine with isoleucine) in which the substituted amino acid has similar structural or chemical properties. In addition or instead, a detectable variant may have a “non-conservative” change (e.g., substitution of glycine with 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 an amino acid sequence can be altered (e.g., substitution, insertion, deletion) without loss of biological activity (in this case, the ability to bind to the antibodies described herein), such as by using bioinformatics software available in the art (e.g., PROVEAN, DNASTAR). For example, the antigens described herein, such as detectable variants of the HBsAg antigen, may have sequence identity of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% with respect to a reference protein or peptide sequence (e.g., SEQ ID NO: 1). In all cases, variants and detectable forms of SEQ ID NO: 1 or any of its detectable fragments or portions are to be conjugated to or by any of the antibodies described herein.

[0025] In a preferred embodiment, the antibody, for example, the first antibody for use (as a scavenger) in the method of this disclosure, has the following heavy chain CDR sequence: CDRH1:SYGVH (Sequence ID 2) CDRH2:VIWAGGITNYNSALMS (Sequence ID 3) CDRH3:GGGVYYGINYAMDY (Sequence ID 4) It may include. An antibody, for example, a first antibody for use in the method of this disclosure, has the following light chain CDR sequence: CDRL1:RASESVEYYGTSLMQ (Sequence ID 5) CDRL2:AASNVEP (Sequence ID 6) CDRL3:QQSRNVPWT(Sequence ID 7) It may include or further include.

[0026] The first antibody for use (as a scavenger) in the method of this disclosure is the following heavy chain (HC) sequence: LHQSGAGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWAGGITNYNSALMSRLSIRKDNFKSQVFLKMNSLQNDDTAMYYCARGGGVYYGINYAMDYWGQGTTVTV(Sequence No. 8); and / or the following light chain (LC) sequence: ELTQTPVSLAVSLGQRATISCRASESVEYYGTSLMQWYQQKPGQPPKLLIYAASNVEPGVPARFSGSGSGTDFSLNIQPVEEDDIAMYFCQQSRNVPWTFGGGTKLE (Sequence ID 9) It may include. Exemplary heavy and light chain sequences of the first (or capture) antibody for use in the methods of this disclosure may include sequences deposited in GenBank under accession numbers AF110502 (heavy chain sequence) and AF110503 (light chain sequence). Further exemplary heavy and light chain sequences of the first (or capture) antibody for use in the methods of this disclosure are published in “Intracellular Expression of a Cloned Antibody Fragment Interferes with Hepatitis B Virus Surface Antigen Secretion”: Jasper zu Putlitz, Arne Skerra, Jack R. Wands: Volume 255, Issue 3, 24 February 1999, Pages 785-791” – the contents of which are incorporated herein by reference.

[0027] The first (or capture) antibody for use in the methods of the present disclosure may include a CDR, light chain, and / or heavy chain sequence that indicates a degree of identity to any of the relevant sequences of SEQ ID NOs. 2-9. The term “degree of identity” can encompass sequences that are at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the relevant sequence. The antibody, for example, the first antibody for use (as a scavenger) in the method of this disclosure may include an anti-HBsAg monoclonal antibody known as "5C3" (catalog number Ab00769-2.0; Absolute antibody). As described, the method of this disclosure may utilize the full or complete form of 5C3, but the method may also use or utilize its antigen (i.e., HBsAg)-binding fragment.

[0028] In a preferred embodiment, the antibody, for example, the second antibody for use in the detection step of the method of this disclosure, is the following heavy chain CDR sequence: CDRH1:GFAFSDYSIN (Sequence ID 10) CDRH2:IISYDGRITYYRDSVKG (Sequence ID 11) CDRH3:QYYDFWSGSSVGRNYDGMDV (Sequence ID 12) It may include. An antibody, for example, a second antibody for use in the method of this disclosure, has the following light chain CDR sequence: CDRL1:RSSQSLLHRSGNNYLD(Sequence ID 13) CDRL2:VGSNRAS (Sequence ID 14) CDRL3:MQALQTPRT(Sequence ID 15) It may include or further include.

[0029] The second antibody for use (as a detection agent) in the method of this disclosure is the following heavy chain (HC) sequence: QVQLVESGGGVVRPGRSLRLSCAASGFAFSDYSINWVRQAPGKGLEWVAIISYDGRITYYRDSVKGRFTISRDDSKNTLYLQMNSLRTEDTAVYYCARQYYDFWSGSSVGRNYDGMD VWGLGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 16); and / or the following light chain (LC) sequence: DIVMTQSPLSLSVTPGEPASISCRSSQSLLHRSGNNYLDWYLQKPGHSPQLLIYVGSNRASGVPDRFSGSGSGTEYTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 17) It may include.

[0030] The second (or detecting) antibody for use in the methods of this disclosure may include a CDR, light chain, and / or heavy chain sequence indicating a degree of identity to any of the relevant sequences of SEQ ID NOs. 10-17. The term “degree of identity” can encompass sequences that are at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the relevant sequence. The second (or detecting) antibody for use in the methods of this disclosure may include an anti-HBsAg monoclonal antibody known as livivirumab (catalog number PX-TA1189; ProteoGenix). As described, the method of this disclosure may utilize the full or complete form of rivivirumab, but the method may also use or utilize its antigen (i.e., HBsAg) binding fragment. In the method of this disclosure, the first and second antibodies described above can be interchanged; note that the antibody referred to as the first antibody (e.g., 5C3) is used after the antibody referred to herein as the second antibody (e.g., rivivirumab).

[0031] The antibodies disclosed herein typically exhibit high affinity for target epitopes. More specifically, the antibodies disclosed herein typically exhibit high affinity for HBV antigen, for example, the epitope of HBsAg. For example, the antibody has a high affinity for 10 -9 ~10 -13 K within the range D It can show a value. In consideration of the foregoing, a method is provided for detecting HBsAg in a sample, comprising the step of contacting the sample with 5C3 and rivivirumab, wherein both 5C3 and rivivirumab bind to HBsAg. The contacting step may first include contacting the sample with 5C3 under conditions that allow binding between any HBsAg and 5C3 antibodies present in the sample to obtain a 5C3 / HBsAg complex. The contacting step may further include contacting any 5C3 / HBsAg complex with rivivirumab under conditions that allow binding between the HBsAg component of any 5C3 / HBsAg complex and rivivirumab to form a 5C3 / HBsAg / rivivirumab complex. The method may further include the step of detecting the 5C3 / HBsAg / ribivirumab complex, the detection of the 5C3 / HBsAg / ribivirumab complex indicating that the sample was obtained from or provided by a subject who has or is recovering from an HBV infection.

[0032] In addition to the steps of the method provided above, the method of the present disclosure may optionally further include one or more washing steps. For example, the washing step may be performed after contact with the sample with the first antibody but before contact with the second antibody. In addition or instead, the washing step may be performed after contact with the sample with the second antibody. For example, the method of the present invention may include a washing step designed to remove, for example, unbound 53C and / or rivivirumab antibodies. This operation helps reduce the occurrence of false positive results. In one example, the method may optionally further include one or more control assays. For example, a negative control assay may include the step of contacting the first and second antibodies with a sample known not to contain the relevant target antigen. In such a scenario, no antigen-antibody complex would form, and the detection step (subjecting to an appropriate washing step) would yield a negative result. A positive control may include the use of a sample known to contain the relevant target antigen or a sample spiked with it. In this scenario, an antigen-antibody complex would form, and the detection step should yield a positive result. Those skilled in the art will recognize that, in addition to the first and second antibodies of this disclosure, the method may further include one or more additional antibodies to further enhance the sensitivity and / or specificity of the assay. As merely one example, one or more additional antibodies may specifically bind to another, different hepatitis B antigen, e.g., hepatitis B core antigen (HBcAg) and / or hepatitis B e antigen (HBeAg).

[0033] It should be noted that in any of the disclosed methods, the methods are not limited to the examples provided herein and may be performed in any preferred format, for example, in a single receptacle, e.g., in an Eppendorf or other tube, or in multiple formats, e.g., in a multiwell plate. Furthermore, cartridge formats, e.g., lateral flow or microfluidic-based cartridges, may be considered. Skilled readers will recognize that, depending on the detection method used and / or the sample provided (e.g., body fluid or tissue), the format of the method may need to be adapted using various methods known in the art. Further teachings further cover the kit for use in the methods described herein. The kit is The first and / or second antibody Rivivirumab (Catalog number PX-TA1189; ProteoGenix) and Anti-HBsAg [5C3] (Catalog No. Ab00769-2.0; Absolute antibody) A first antibody and a second antibody are selected from, Optionally, a first antibody or a second antibody immobilized on the surface of the substrate, Furthermore, optionally, a first antibody and / or a second antibody labeled at the detection portion. It may include, essentially be, or become.

[0034] This disclosure further provides the use of rivivirumab (catalog no. PX-TA1189; ProteoGenix) and / or 5C3 (catalog no. Ab00769-2.0; Absolute antibody) in a method or assay for the detection of HBsAg in a sample. Next, the present invention will be described in detail with reference to the following drawings, which illustrate the following points. [Brief explanation of the drawing]

[0035] [Figure 1] An immunoassay (ELISA) for the detection of HBsAg, comparing various combinations of capture and detection antibodies. [Figure 2] An immunoassay (ELISA) for the detection of HBsAg, comparing various combinations of capture and detection antibodies. [Figure 3] An immunoassay (MosaiQ) for HBsAg detection comparing various combinations of capture and detection antibodies. [Figure 4] An immunoassay (MosaiQ) for HBsAg detection comparing various combinations of capture and detection antibodies. [Figure 5] Analysis demonstrating the sensitivity of the assay using different combinations of capture and detection agents. (A) Capture: Absolute 5C3; Detection: Proteogenix rivivirumab; Conjugate SDT streptavidin polyHRP 80; Cycle time: 48 seconds. (B) Capture: Absolute 5C3; Detection: Meridian B65811; Conjugate SDT streptavidin polyHRP 80; Cycle time: 48 seconds. [Modes for carrying out the invention]

[0036] result [Table 1] [Table 2]

[0037] Table 3

[0038] Table 4

[0039] Table 5

[0040] Table 6

Claims

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

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

3. The method according to claim 1 or 2, wherein the first and second antibodies are antibodies for capturing and detecting HBV antigen, respectively.

4. The method according to any one of claims 1 to 3, wherein both the first and second antibodies are recombinant antibodies.

5. The method according to any one of claims 1 to 4, wherein the contact step includes contacting the first and second antibodies with a sample under conditions that enable binding between the first and second antibodies and any of the HBV antigens present in the sample.

6. The method according to any one of claims 1 to 5, wherein binding between the first and second antibodies and the HBV antigen present in the sample forms an antibody-antigen complex, and the complex comprises the first and second antibodies bound to the HBV antigen.

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

8. The method according to claim 7, wherein the second antibody binds to a complex containing the first antibody bound to the HBV antigen.

9. The method according to any one of claims 1 to 8, wherein the first antibody is immobilized on a substrate.

10. The method according to any one of claims 1 to 9, wherein the second antibody includes a detectable portion.

11. The method according to any one of claims 1 to 10, wherein the HBV antigen is hepatitis B surface antigen (HBsAg).

12. The method according to any one of claims 1 to 11, wherein the first antibody is an anti-HBsAg monoclonal antibody 5C3 (catalog number Ab00769-2.0; absolute antibody) or an HBsAg-binding fragment thereof.

13. The method according to any one of claims 1 to 12, wherein the second antibody is the anti-HBsAG monoclonal antibody rivivirumab (catalog number PX-TA1189; ProteoGenix) or an HBsAg-binding fragment thereof.

14. A method for detecting HBsAg in a sample, A step of contacting a sample with monoclonal antibody 5C3 under conditions suitable for enabling binding between monoclonal antibody 5C3 and any HBsAg in the sample, wherein the binding event between monoclonal antibody 5C3 and any HBsAg in the sample results in the formation of a 5C3 / HBsAg complex. The steps include contacting one of the 5C3 / HBsAg complexes with the monoclonal antibody rivivirumab under conditions suitable for enabling binding between the monoclonal antibody rivivirumab and one of the 5C3 / HBsAg complexes, and then The step of detecting any rivivirumab bound to the 5C3 / HBsAg complex and A method comprising detecting rivivirumab bound to a 5C3 / HBsAg complex, wherein the detection indicates that a sample contains the target.

15. 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 an antibody produced by recombinant.

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

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

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

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

20. The method according to claim 19, wherein the first recombinant antibody is immobilized on a substrate.

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

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