Hcv core lipid binding domain monoclonal antibodies

Abstract

The present invention relates to monoclonal antibodies to the HCV core lipid binding domain. The present invention provides monoclonal antibodies for detecting Hepatitis C Virus (HCV) antigens. The antibodies specifically immunoreact with at least one epitope of the lipid binding domain of amino acid residues 134-171 of the HCV core antigen. Further provided are immunoassay methods for detecting HCV infection using the antibodies, kits comprising the antibodies, and compositions.

Description

[0001] Related applications

[0002] This application is a divisional application of international application PCT / US2013 / 077499, filed on December 23, 2013, and entered into China with application number 201380075956.1, entitled "HCV Core Lipid-Binding Domain Monoclonal Antibody". This application is filed as a PCT patent application claiming priority to U.S. Provisional Patent Application No. 61 / 783,529, filed March 14, 2013. The entire text of the aforementioned application is incorporated herein by reference. Technical Field

[0003] This disclosure provides novel monoclonal antibodies against the HCV core protein, as well as methods and compositions for using them to detect HCV infection. Background Technology

[0004] According to WHO statistics, up to 170 million people worldwide are infected with hepatitis C virus (HCV), a viral infection of the liver. 75-85% of those infected with HCV develop chronic infection, and approximately 20% of these cases develop complications of chronic hepatitis C, including cirrhosis or hepatocellular carcinoma 20 years after infection. The currently recommended treatment for HCV infection is a combination of interferon and ribavirin; however, this treatment is not effective in all cases and is indicative of liver transplantation in cases of hepatitis C-related end-stage liver disease. Currently, there is no vaccine available to prevent HCV infection; therefore, all preventative measures must be taken to avoid infection.

[0005] Therefore, patient care and the prevention of hepatitis C virus (HCV) transmission via blood and blood products or through close contact require extreme vigilance using sensitive detection methods. This establishes the need for specific methods for screening and identifying HCV vectors and HCV-contaminated blood or blood products. Serological assays for HCV exposure rely on the detection of HCV present in human plasma or serum. This can be accomplished by detecting the unique structural and non-structural proteins encoded by the virus.

[0006] HCV virus is a (+) sense single-stranded enveloped RNA virus belonging to the genus Hepatitisvirus in the family Flaviviridae. The viral genome is approximately 10 kb in length and encodes a multi-protein precursor of 3011 amino acids. The HCV genome has a single large open reading frame (ORF) encoding a unique multi-protein. This multi-protein is processed by cellular and viral proteases during and after translation into three structural proteins: the core, E1, and E2, and at least six non-structural proteins: NS2, NS3, NS4A, NS4B, NS5A, and NS5B (Choo et al., Science 244:359-362 (1989)).

[0007] Following HCV exposure, the virus enters susceptible hepatocytes and replicates. During the occult period of approximately 10 days, the presence of the virus is not apparent (i.e., viral RNA is undetectable), serum transaminase levels are within normal limits, and there is no evidence of an immune response against HCV (Busch et al., Transfusion 40:143 (2000)). Typically, HCV RNA becomes detectable about 10 days post-exposure, often with a viral load of 100,000–120,000,000 copies of HCV RNA per ml of serum. An increase in ALT levels, indicating liver inflammation, is usually observed several weeks later; antibodies are detected on average about 70 days post-exposure.

[0008] Blood screening for HCV exposure (by detecting HCV antibodies or by detecting virus-specific molecules in serum / plasma, such as HCV RNA or HCV core protein) is an important part of patient care. Blood or blood products derived from individuals identified by these tests as having been exposed to HCV are removed from the blood supply and are not intended for distribution to recipients of blood products (see, for example, U.S. Patent No. 6,172,189). These tests can also be used in clinical settings to diagnose liver disease attributable to HCV infection.

[0009] Serological antibody tests rely on the application of recombinant antigens or synthetic peptides representing selected fragments of viral polyproteins. First-generation anti-HCV screening tests are based on the detection of antibodies against a recombinant protein (HCV genotype 1a) derived from a sequence located in the non-structural NS-4 protein (C100-3) (Choo et al., Science 244:359 (1989); Kuo et al., Science 244:362 (1989)). First-generation assays cannot detect antibodies in approximately 10% of individuals with chronic HCV infection and as many as 10–30% of individuals presenting with acute HCV infection. Second-generation anti-HCV assays now include recombinant proteins derived from three distinct regions of the HCV genome (HCV genotype 1a), including amino acid sequences derived from the core, NS3, and NS4 proteins (Mimms et al., Lancet 336:1590 (1990); Bresters et al., Vox Sang 62:213 (1992)), thus allowing for significant improvements over first-generation assays in identifying HCV infection in blood donors (Aach et al., N Engl J Med 325:1325 (1991); Kleinman et al., Transfusion 32:805 (1992). Second-generation assays detect nearly 100% of chronic HCV cases (Hino K., Intervirology 37:77 (1994)) and almost 100% of acute cases up to 12 weeks post-infection (Alter et al., N Engl J Med 327:1899 (1992); Bresters et al., Vox Sang 62:213 (1992)). The antibody in 62:213 (1992)). The third-generation assay includes a recombinant protein expressing an amino acid sequence derived from the NS5 region, as well as antigens derived from the core, NS3, and NS4. Some studies have indicated a slight increase in sensitivity of the third-generation assay compared to the second-generation assay (Lee et al., Transfusion 35:845 (1995); Courouce et al., Transfusion 34:790-795 (1994)), but this increase is mainly attributed to changes in the NS3 protein rather than the inclusion of NS5 (Courouce et al., Lancet 343:853 (1994)).

[0010] Generally, second- and third-generation HCV antibody tests detect HCV exposure approximately 70 days after exposure. Because HCV establishes a persistent, and in many cases lifelong, infection, HCV antibody testing represents a very effective method for determining HCV exposure. However, antibody tests alone often cannot detect HCV infection in individuals within the first 70 days after exposure.

[0011] It has been proposed that HCV antigen-based assays detect HCV exposure significantly earlier than antibody assays and represent an alternative to nucleic acid assays for detecting HCV exposure in the pre-seroconversion phase. HCV antigen assays are rapid, simple, may not require sample extraction or other pretreatment, and are less prone to operational errors (e.g., contamination) than HCV RNA assays. Therefore, HCV core antigen assays offer a practical alternative to HCV RNA-based assays for screening blood donors or monitoring antiviral therapy.

[0012] Existing HCV antigen assays rely on detecting the presence of HCV core antigen in serum or plasma. The HCV core protein is the structural protein of HCV, comprising the first 191 amino acids of a multiprotein, and forms the inner viral envelope that capsids the genomic RNA. Two different types of serological assays have been developed that allow the detection of HCV core antigen in serum. One assay detects HCV core antigen in subjects before seroconversion and is used for screening blood donors, while the other assay detects core antigen only in hepatitis C patients (regardless of their HCV antibody status) and is used in clinical laboratories to diagnose HCV exposure or monitor antiviral therapy. Currently available core antigen assays all use antibodies targeting the DNA-binding domain located at amino acids 1-125 of the core protein. The core protein also contains a lipid-binding domain located between amino acids 134-171. To date, no antigen derived from this portion of the core protein has been described, and it has been assumed until now that core assays require antibodies targeting the DNA-binding domain.

[0013] Therefore, the ability to accurately detect HCV core antigen-binding proteins would significantly improve the available methods for detecting HCV exposure in patients. Consequently, there is a recognized need for novel antibodies that can be readily used in screening tests. Summary of the Invention

[0014] This invention generally relates to monoclonal antibodies capable of specifically reacting with the lipid-binding domain of an HCV core antigen. More specifically, the HCV core antigen is amino acid residues 134-171 of HCV. In a more specific embodiment, the antibody specifically binds to at least one epitope formed by the amino acid sequence MGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG. In a more specific embodiment, the antibody is capable of reacting with epitopes formed by amino acids 141-161, 134-154, and 151-171 of the HCV core antigen.

[0015] Another aspect of the present invention provides a monoclonal antibody capable of specifically immunizing with a lipid-binding domain of an HCV core antigen, wherein the monoclonal antibody has a subset selected from... Figure 1A The antibody heavy chain variable domains listed in the text and those selected from Figure 1B The variable domains of the light chain of the antibodies are listed in the table.

[0016] It is anticipated that any antibody described herein can be prepared as an immunoassay reagent, and more specifically, such a reagent is preferably labeled with a detectable marker.

[0017] In other embodiments, the immunoassay reagent of the present invention comprises one or more antibodies disclosed herein that are bound to a solid.

[0018] Immunoassay reagents containing the antibodies of the present invention may further contain additional antibodies against HCV antigens. For example, such additional antibodies are additional anti-core antibodies.

[0019] Another aspect of the present invention relates to an immunoassay for detecting HCV in a test sample, said immunoassay comprising:

[0020] (i) Contact a test sample suspected of containing HCV with a first antibody against the HCV core antigen to form a complex between the first antibody and the antigen located within the test sample;

[0021] (ii) Contacting the complex formed in step (i) with the antibody of any one of claims 1-6 to form a complex between the antibody of any one of claims 1-6 and the antigen in the complex formed in step (i), wherein the antibody of any one of claims 1-6 is detectably labeled, and

[0022] (iii) Detect the marker of the complex formed in step (ii).

[0023] In a more specific embodiment, the immunoassay may be further characterized in that the first antibody targets the DNA-binding domain of the HCV core antigen. In a more specific embodiment, the antibody used in step (ii) is labeled with a fluorescent marker. In an exemplary embodiment, the marker is acridine orange.

[0024] In some embodiments, the immunoassay is an immunoassay in which the antibody of step (i) is coated onto a solid phase. In a particular preferred embodiment, the antibody of step (i) comprises an antibody different from the antibody of step (ii). Alternatively, the immunoassay is an immunoassay in which the antibody of step (i) comprises the same antibody as the antibody of step (ii).

[0025] Any immunoassay of the present invention can be used on test samples obtained from patients, and the method further includes diagnosing, predicting, or evaluating the efficacy of a patient's therapeutic / preventive treatment, wherein, if the method further includes evaluating the efficacy of a patient's therapeutic / preventive treatment, the method optionally further includes modifying the patient's therapeutic / preventive treatment as needed to improve efficacy.

[0026] As will be understood by those skilled in the art as described in more detail herein, any immunoassay of the present invention can be readily adapted for use in automated or semi-automated systems. Attached Figure Description

[0027] Figure 1A The heavy chain variable domain of the preferred antibody of the present invention is shown.

[0028] Figure 1B The light chain variable domain of the preferred antibody of the present invention is shown.

[0029] Figure 2 Showing from Figure 1A Two illustrations of the cluster diagram derived from the comparison. Detailed Implementation

[0030] This invention describes the development of monoclonal antibodies against the hepatitis C virus core antigen (specifically, the lipid-binding domain between amino acids 134-171 of the core antigen). The immunogen used is a synthetic peptide, and hybridoma screening utilizes the immunogenic peptide and a set of three overlapping smaller peptides within the 134-171 region. Additionally, a recombinant core antigen representing amino acids 1-169 is used for screening to determine the potency of monoclonal antibodies identified as reacting with the HCV core protein. The antibodies are characterized by their reactivity with the antigen, the immunogenic peptide, and the smaller overlapping peptides containing the immunogen. The binding kinetics of the antibody to the immunogenic peptide are determined by SPR (surface plasmon resonance) using a BIAcore4000 instrument. Immunoreactivity with the recombinant core antigen is determined by standard ELISA.

[0031] Furthermore, to confirm that the monoclonal antibodies of the present invention can be used to analyze the presence of the core antigen, core antigen capture microtiter assays were performed using monoclonal antibodies targeting epitopes within the DNA-binding domains (e.g., amino acids 1-125) of the HCV core as capture reagents and the antibody of the present invention targeting 134-171 as detection reagents. The results of these assays confirmed the practicality of the antibody of the present invention targeting 134-171 for HCV core antigen detection immunoassays. This is the first confirmation of antigen capture assays independently targeting two major domains of the HCV core for capture and detection. Previously reported core antigen detection assays used antibodies that bind to epitopes within the DNA-binding domains (e.g., amino acids 1-125).

[0032] To generate the antibody of the present invention, mice were immunized with a synthetic peptide comprising a concordant sequence of HCV core genotype 1 derived from amino acids 134-171 linked to BSA. More specifically, the immunogen has the following sequence:

[0033]

[0034] Furthermore, the binding of the monoclonal antibody to three specific N-terminal biotinylated epitope regions was also characterized, and further discussed in the examples. Specifically, the three overlapping epitopes are derived from the above regions and have the following sequences:

[0035]

[0036] An immunogen is conjugated to BSA to generate antibodies. In other embodiments, the immunogen is conjugated to TT and fibrils. The TT sequence is frequently used to provide a more robust immune response in mice. The sequence of the TT conjugate is:

[0037]

[0038] The sequence of the filament conjugate is:

[0039]

[0040] B-lymphocytes were fused with myeloma fusion couples to create hybridomas, which were then screened for reactivity to immunogenic peptides, three overlapping peptides within the immunogenic peptide sequence, and the recombinant HCV core antigen. Kinetic profiling using Biacore4000 allowed the identification of antibody clusters (defined by their ability to bind immunogenic peptides) or shorter peptides overlapping the 134-171 region. Combining these results with immunoreactivity to the recombinant antigen, or its absence, determined by ELISA, allowed for further profiling of antibodies into groups with similar characteristics (specificity).

[0041] To briefly summarize the screening results discussed in more detail in the examples, the strongest immune response was observed in mice immunized with the BSA-linked peptide. Furthermore, the responses from these mice were primarily concentrated in the amino acid 141-161 region, although some responses were also observed in the amino acid 134-154 and 151-171 regions. An immune response was observed using the TT-linked HCV peptide; however, this response was not as strong as that using BSA. The response was distributed across all three epitope regions. On the other hand, mice immunized with the amino acid 134-171 peptide linked to the peptide that will form a fibrillary network did not show a significant immune response. Figure 1A and 1B The antibodies of the present invention are described in more detail in the examples.

[0042] The HCV core antigen used in these studies was expressed in Escherichia coli and purified by a two-step method using IMAC and subsequent reversed-phase HPLC based on a previously published method (Boulant et al., J.Virol. (2005), 79(17): 11353-11365).

[0043] In this manner, a large number of monoclonal antibodies specific to the lipid-binding domain of the HCV core have been produced. These monoclonal antibodies are used to develop diagnostic assays for detecting HCV core antigens in the serum and plasma of infected individuals. Prior to this invention, the production of monoclonal antibodies targeting multiple epitopes within the core amino acid region 134-171, which have demonstrated binding activity against the full-length HCV core peptide, had not been reported. The availability of the monoclonal antibodies of this invention allows for the development of immunoassays for the detection of the core antigen, targeting two major domains of the HCV core antigen. Previous core antigen assays described the application of monoclonal antibodies targeting epitopes within amino acids 1-125 (nucleic acid-binding domain). Because the previously described monoclonal antibodies could only target the nucleic acid-binding domain of the HCV core, they were at best inefficient, and often ineffective, in detecting core protein fragments, degradation products, or smaller core proteins derived through internal translation initiation. This invention overcomes these shortcomings of previous assays for the first time by providing a specific monoclonal antibody that can be used as a reagent to more effectively and rapidly detect the HCV core present in test samples.

[0044] More specifically, the antibodies described herein are reagents that can be used to detect HCV core antigens and can be used to advance research on the life cycle of HCV. As noted above, HCV-encoded proteins are expressed in a concerted manner, in which ribosomes bind to the ribosome entry site (IRES) and begin translation, leading to the synthesis of viral polyproteins, which are cleaved to produce the classic HCV protein, p21 core, E1, E2, p7, and non-structural proteins. Without translation initiation in the core gene region, it is impossible to produce viral enzymes, including viral polymerases. Because of this temporary association, it is believed that translation events in this region control the expression of all HCV proteins. Therefore, a complete understanding of the core gene and its gene products is essential for understanding the viral life cycle and can advance our understanding of the mechanisms of viral pathogenicity. In recent years, a new family of conserved viral proteins has been described, which is called microcore (Eng et al., J Virol. 2009 Apr; 83(7): 3104-3114). These proteins encode within the same reading frame as the core gene, but are thought to derive from an internal translation initiation event rather than post-translational processing of the full-length core protein. One of the described microcore proteins is called the "91 microcore," named after the putative start codon within the core. A "134 microcore" is also hypothesized, derived from translation initiation at codon 134, which encodes methionine in many HCV isolates. However, reagents for detecting microcores that are essentially derived from lipid-binding domains are not readily available. Such proteins may play an important role in HCV persistence.

[0045] Since monoclonal antibodies are generated targeting linear synthetic peptides derived from HCV core 134-171, it is unknown whether they will bind to the naturally occurring intact core antigen or the processed form of the HCV core present in infected individuals. However, the prerequisite is that the monoclonal antibodies of the present invention are capable of binding to at least one or more epitopes presented by the linear HCV core region derived from amino acids 134-171, and it is anticipated that such binding is sufficient to provide these monoclonal antibodies that are clearly usable in HCV detection assays. Some of the monoclonal antibodies of the present invention react with the recombinant core antigen, while others do not, suggesting the presence of both linear and conformational epitopes within the core amino acid 134-171 region. Antibodies that recognize linear and conformational epitopes are generally very useful tools for studying viral assembly and the viral life cycle within infected cells.

[0046] Finally, these reagents can also be used in immunoassays in which it is desired to determine the presence of only the lipid-binding domain. Since little is known about the circulating levels of the microcore in infected individuals, they may be present at much higher levels than the core protein containing the region of amino acids 1-125. In providing antibodies to detect HCV core peptides outside the region of amino acids 1-125, the present invention provides an HCV core antigen detection assay with a much greater sensitivity than those currently available.

[0047] definition

[0048] As used herein, the terms "specific binding" or "specifically binding" relating to the interaction of antibodies, proteins, or peptides with a second chemical substance mean that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical substance; for example, an antibody recognizes and binds to a specific protein structure, rather than generally recognizing and binding to proteins. If an antibody is specific to epitope "A," the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody reduces the amount of labeled A that binds to the antibody.

[0049] As used herein, the term "antibody" broadly refers to any immunoglobulin (Ig) molecule comprising four polypeptide chains (two heavy (H) chains and two light (L) chains) or any functional fragment thereof, mutant, variant, or derivative thereof, which retains the essential epitope binding characteristics of an Ig molecule. Such mutant, variant, or derivative antibody forms are known in the art. Non-limiting embodiments thereof are discussed below.

[0050] In a full-length antibody, each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain CL. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0051] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, which can be generated by digestion of an intact antibody with papain. The Fc region can be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally contains two constant domains: a CH2 domain and a CH3 domain, and optionally includes a CH4 domain. Amino acid residue substitutions in the Fc moiety that alter antibody effector functions are known in the art (Winter, et al., U.S. Patents 5,648,260 and 5,624,821). The Fc moiety of an antibody mediates several important effector functions, such as cytokine induction, ADCC, phagocytosis, complement-dependent cytotoxicity (CDC), and the half-life / clearance of the antibody and antigen-antibody complex. In some cases, these effector functions are desirable for therapeutic antibodies, but in others they may be unnecessary or even harmful, depending on the therapeutic purpose. Some human IgG isotypes (particularly IgG1 and IgG3) mediate ADCC and CDC by binding to Fc.γ.R5 and complement Clq, respectively. The neonatal Fc receptor (FcRn) is a key component determining the circulating half-life of an antibody. The substitution of at least one amino acid residue in the constant region of an antibody (e.g., the Fc region) alters the effector function of the antibody. Dimerization of two identical heavy chains of immunoglobulins is mediated by dimerization of the CH3 domain and stabilized by disulfide bonds within the hinge region (Huber et al., Nature; 264: 415-20; Thies et al., 1999 J Mol Biol; 293: 67-79.). Mutations of cysteine ​​residues within the hinge region that prevent heavy chain-heavy chain disulfide bonds destabilize the dimerization of the CH3 domain. Residues responsible for CH3 dimerization have been identified (Dall'Acqua, 1998 Biochemistry 37: 9266-73.). Therefore, monovalent half-Ig molecules may be generated. Interestingly, these monovalent half-Ig molecules of IgG and IgA subclasses have been found in nature (Seligman 1978 Ann Immunol 129:855-70; Biewenga et al. 1983 Clin Exp Immunol 51:395-400). The stoichiometry of the FcRn:Ig Fc region has been determined to be 2:1 (West et al. 2000 Biochemistry 39:9698-708), and the half-Fc is sufficient to mediate FcRn binding (Kim et al. 1994 Eur J Immunol; 24:542-548). Mutations disrupting the dimerization of the CH3 domain may not have a significant adverse effect on FcRn binding, because the residues important for CH3 dimerization are located on the inner interface of the CH3 β-sheet structure, while the region responsible for FcRn binding is located on the outer surface of the CH2-CH3 domain.However, half-Ig molecules may have certain advantages in tissue penetration due to their smaller size compared to conventional antibodies. At least one amino acid residue in the constant region (e.g., the Fc region) of the binding protein of this disclosure can be replaced, thereby disrupting the dimerization of the heavy chain and producing a half-DVD Ig molecule. The anti-inflammatory activity of IgG depends entirely on the sialylation of the N-linked glycan of the IgG Fc fragment. Precise glycan requirements for anti-inflammatory activity have been identified, allowing the production of appropriate IgG1 Fc fragments, resulting in fully recombinant sialylated IgG1 Fc with significantly enhanced titers (Anthony, RM, et al. (2008) Science 320: 373-376).

[0052] As used herein, the term "antigen-binding portion" (or simply "antibody portion") of an antibody refers to one or more segments of the antibody that retain the ability to specifically bind to an antigen. It has been demonstrated that the antigen-binding function of an antibody can be performed by segments of a full-length antibody. Such antibody implementations can also be bispecific, dual-specific, or multispecific; specifically binding to two or more different antigens. Examples of binding fragments included in the term "antigen-binding portion" of an antibody include: (i) Fab fragments, i.e., monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab′)2 fragments, i.e., bivalent fragments consisting of two Fab fragments linked by disulfide bonds in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of the antibody; (v) dAb fragments (Ward et al., (1989) Nature 341:544-546, Winter et al., PCT Publication WO 90 / 05144 A1, incorporated herein by reference), which contain a single variable domain; and (vi) separate complementarity-determining regions (CDRs). Furthermore, although the two domains (VL and VH) of the Fv fragment are encoded by separate genes, they can be linked together using recombination methods via synthetic linkers that allow them to be made into a single protein chain, where the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding portion" of antibody. Other forms of single-chain antibodies, such as dimeric antibodies, are also included. A bivalent bispecific antibody is a single polypeptide chain on which the VH and VL domains are expressed, but using a linker that is too short to allow pairing between the two domains on the same chain. This forces the domain to pair with a complementary domain of another chain, resulting in two antigen-binding sites (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, RJ, et al. (1994) Structure 2: 1121-1123). Such antibody-binding moieties are known in the art (see, for example, Kontermann and Dubel, eds., Antibody Engineering (2001) Springer-Verlag. New York. p. 790 (ISBN 3-540-41354-5)).In addition, single-chain antibodies also include “linear antibodies” which contain a pair of tandem Fv segments (VH--CH1-VH--CH1), which together with complementary light chain polypeptides form a pair of antigen-binding regions (Zapata et al., Protein Eng. 8(10): 1057-1062(1995); and U.S. Patent No. 5,641,870).

[0053] The term "multivalent binding protein" is used throughout this specification to refer to a binding protein containing two or more antigen-binding sites. In one aspect, said multivalent binding protein is engineered to have three or more antigen-binding sites and is generally not a naturally occurring antibody. Dual variable domain (DVD) binding proteins contain two or more antigen-binding sites and are tetravalent or multivalent binding proteins. DVDs as described herein can be monospecific, i.e., capable of binding one antigen such as the HCV core protein, or multispecific, i.e., capable of binding two or more antigens. A DVD-binding protein containing two heavy-chain DVD peptides and two light-chain DVD peptides is referred to as DVD-Ig and is described, for example, in U.S. Patent No. 7,612,181, the disclosure of which is incorporated herein by reference in its entirety. Each half of DVD-Ig contains a heavy-chain DVD peptide and a light-chain DVD peptide, as well as two antigen-binding sites. Each binding site contains a heavy-chain variable domain and a light-chain variable domain, wherein a total of six CDRs are involved in antigen binding at each antigen-binding site.

[0054] The “functional antigen-binding site” of a binding protein is a binding site capable of binding to a target antigen. The antigen-binding affinity of an antigen-binding site may not be as strong as that of the parent antibody from which the antigen-binding site originates, but the ability to bind the antigen must be measurable using any of the various methods known for evaluating antibody-antigen binding. Furthermore, the antigen-binding affinity of each antigen-binding site of the multivalent antibodies described herein does not need to be numerically identical.

[0055] "Immunoglobulin constant domain" refers to either the heavy chain or the light chain constant domain. The amino acid sequences of the heavy and light chain constant domains of human IgG are known in the art.

[0056] As used herein, the terms "monoclonal antibody" or "mAb" refer to antibodies derived from a population of substantially homologous antibodies, meaning that the individual antibodies constituting the population are identical except for possibly naturally occurring mutations that may be present in minute quantities. Monoclonal antibodies are highly specific against a single antigen. Furthermore, unlike polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each mAb targets a single determinant on an antigen. The modifier "monoclonal" should not be interpreted as requiring any particular method to produce the antibody.

[0057] The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of this disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced through random or site-specific in vitro mutagenesis or in vivo somatic mutations), such as in CDRs, and particularly in CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which a germline CDR sequence derived from another mammalian species (such as a mouse) has been grafted onto a human frame sequence.

[0058] The term “recombinant human antibody” as used herein is intended to include all human antibodies prepared, expressed, constructed, or isolated via recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (described further in Section II C below), antibodies isolated from recombinant combined human antibody libraries (Hoogenboom HR (1997) TIB Tech. 15: 62-70; Azzazy H., and Highsmith WE (2002) Clin. Biochem. 35: 425-445; Gavilondo JV, and Larrick JW (2002) BioTechniques 29: 128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21: 371-378), and antibodies isolated from transgenic animals (e.g., mice) of human immunoglobulin genes (see Taylor, LD, et al. (1992) Nucl. Acids Res. 20: 6287-6295; Kellermann SA. and Green LL (2002) Current Opinion in Biotechnology 13: 593-597; Little M. et al. (2000) Immunology Today 21: 364-370), or antibodies prepared, expressed, constructed, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. Such recombinant human antibodies can be mutagenized in vitro (or in vivo somatic mutagenized when using transgenic animals with human Ig sequences) so that the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, although derived from and related to human germline VH and VL sequences, cannot be naturally present in the human antibody germline repertoire.

[0059] Affinity-matured antibodies are antibodies that have one or more alterations in one or more of their CDRs, which result in increased affinity for the antigen compared to parent antibodies without those alterations. Exemplary affinity-matured antibodies will have nanomolar or even picomolar affinity for the target antigen. Affinity-matured antibodies are produced using procedures known in the art. Marks et al., Bidl Technology 10:779-783 (1992), describe affinity maturation via VH and VL domain shuffling. The following literature describes the random mutagenesis of CDR and / or framework residues: Barbas et al. Proc Nat. Acad. Sci, USA 91: 3809-3813 (1994); Schier et al. Gene 169: 147-155 (1995); Yelton et al. J. Immunol. 155: 1994-2004 (1995); Jackson et al. J. Immunol. 154(7): 3310-9 (1995); Hawkins et al. J. Mol. Biol. 226: 889-896 (1992), and selective mutagenesis of amino acid residues with enhanced activity at selective mutagenesis sites, contact or high mutation sites as described in U.S. Patent No. 6,914,128 B1.

[0060] The term "chimeric antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies with mouse heavy and light chain variable regions linked to human constant regions.

[0061] The term "CDR-transplantation antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species, but in which one or more CDR regions of VH and / or VL are replaced by CDR sequences from another species, such as antibodies with mouse heavy and light chain variable regions, in which one or more mouse CDRs (e.g., CDR3) have been replaced by human CDR sequences.

[0062] The term "humanized antibody" refers to an antibody that contains heavy and light chain variable region sequences derived from a non-human species (e.g., mouse), but in which at least a portion of the VH and / or VL sequences has been modified to be more "human-like," i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which a human CDR sequence is introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. "Humanized antibody" is also an antibody or its variants, derivatives, analogs, or fragments that immunely and specifically bind to a target antigen and contains a frame (FR) region having substantially the amino acid sequence of a human antibody and a complementarity-determining region (CDR) having substantially the amino acid sequence of a non-human antibody. The term "substantially" as used herein, in the context of a CDR, refers to a CDR whose amino acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the amino acid sequence of a non-human antibody CDR. Humanized antibodies comprise substantially all, and typically two, variable domains (Fab, Fab′, F(ab′)2, FabC, Fv), wherein all or substantially all CDR regions correspond to those CDR regions of non-human immunoglobulins (i.e., donor antibodies), and all or substantially all frame regions are those frame regions of the human immunoglobulin common sequence. In one aspect, humanized antibodies also comprise at least a portion of the immunoglobulin constant region (Fc) (typically the constant region of human immunoglobulins). In some embodiments, humanized antibodies comprise a light chain and at least a variable domain of the heavy chain. The antibody may also comprise CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, humanized antibodies comprise only the humanized light chain. In some embodiments, humanized antibodies comprise only the humanized heavy chain. In certain embodiments, humanized antibodies comprise only the humanized variable domains of the light chain and / or the humanized heavy chain.

[0063] The terms “Kabat numbering,” “Kabat definition,” and “Kabat labeling” are used interchangeably herein. These terms, generally accepted in the art, refer to an amino acid residue numbering system where the amino acid residues are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody or its antigen-binding portion (Kabat et al. (1971) Ann. NY Acad. Sci. 190: 382-391 and Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., USDapartment of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable region is amino acid positions 31-35 for CDR1, 50-65 for CDR2, and 95-102 for CDR3. For the light chain variable region, the hypervariable region is amino acid positions 24-34 for CDR1, 50-56 for CDR2, and 89-97 for CDR3.

[0064] As used herein, the term "CDR" refers to the complementarity-determining region within the variable sequence of the antibody. Three CDRs exist in each variable region of the heavy and light chains, designated CDR1, CDR2, and CDR3 for each variable region. The term "CDR set" as used herein refers to the set of three CDRs present in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently depending on the system. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a definitive residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining three CDRs. These CDRs can be referred to as Kabat CDRs. Chothia and colleagues (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987) and Chothia et al., Nature 342: 877-883 (1989)) found that certain sub-regions within the Kabat CDR, despite their great diversity at the amino acid sequence level, exhibit almost identical peptide backbone conformations. These sub-regions are named L1, L2, and L3 or H1, H2, and H3, where “L” and “H” represent the light chain region and the heavy chain region, respectively. These regions can be referred to as Chothia CDRs, which have similar characteristics to the Kabat CDRs. CDR overlap boundaries. Other boundaries defining CDR overlap with the Kabat CDR have been described by Padlan (FASEB J.9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Other CDR boundary definitions may not strictly follow one of the systems described herein, but will still overlap with the Kabat CDR, although they may be shortened or lengthened given the following predictions or experimental findings: specific residues or groups of residues or even the entire CDR do not significantly affect antigen binding. The methods used herein can utilize CDRs defined according to any of these systems, although some embodiments use Kabat or Chothia-defined CDRs.

[0065] As used herein, the term "frame" or "frame sequence" refers to the sequence remaining after subtracting the CDR from the variable region. Because the precise definition of the CDR sequence can be determined by different systems, the meaning of the frame sequence can be interpreted accordingly. The six CDRs (CDRs -L1, -L2, and -L3 for the light chain, and CDRs -H1, -H2, and -H3 for the heavy chain) also distinguish the frames on the light and heavy chains into four sub-regions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 is located between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying a particular sub-region as FR1, FR2, FR3, or FR4, a frame region, as mentioned by others, represents a combination of FRs within the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR represents one of the four sub-regions, and FR represents two or more of the four sub-regions that constitute a frame region. As used herein, the terms “germ antibody gene” or “gene fragment” refer to an immunoglobulin sequence encoded by a non-lymphoid cell that has not yet undergone a maturation process that results in gene rearrangement and expression mutation of a particular immunoglobulin (see, for example, Shapiro et al., Crit. Rev. Immunol. 22(3): 183-200 (2002); Marhalonis et al., Adv Exp Med. Biol. 484: 13-30 (2001)). One of the advantages provided by different embodiments of this disclosure stems from the understanding that germ antibody genes are more likely than mature antibody genes to retain the basic amino acid sequence structure specific to an individual in the species, and are therefore less likely to be identified as originating from a foreign source when used therapeutically in that species.

[0066] As used herein, the term "humanized antibody" refers to an antibody or its variants, derivatives, analogs, or fragments that immunely bind to a target antigen and comprises a frame (FR) region having substantially the amino acid sequence of a human antibody and a complementarity-determining region (CDR) having substantially the amino acid sequence of a non-human antibody. The term "substantially" as used herein, in the context of a CDR, means a CDR whose amino acid sequence has at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the amino acid sequence of a non-human antibody CDR. Humanized antibodies comprise substantially all at least one and generally two variable domains (Fab, Fab′, F(ab′)2, FabC, Fv), wherein all or substantially all of the CDR regions correspond to those CDR regions of the non-human immunoglobulin (i.e., the donor antibody), and all or substantially all of the frame regions are those frame regions of the common sequence of human immunoglobulins. Preferably, humanized antibodies also comprise at least a portion of the immunoglobulin constant region (Fc) (typically the constant region of human immunoglobulins). In some embodiments, the humanized antibody contains a light chain and at least a variable domain of the heavy chain. The antibody may also include CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only a humanized light chain. In some embodiments, the humanized antibody contains only a humanized heavy chain. In a particular embodiment, the humanized antibody contains only a humanized variable domain of the light chain and / or the humanized heavy chain.

[0067] As used herein, the term "neutralization" means that when a binding protein specifically binds to an antigen, it neutralizes the biological activity of said antigen. In one aspect, a neutralizing binding protein binds to a cytokine and reduces its biological activity by at least about 20%, 40%, 60%, 80%, 85%, or more.

[0068] The term "epitope" includes any polypeptide determinant capable of specifically binding to immunoglobulins or T-cell receptors. In some embodiments, the epitope determinant comprises chemically active surface groupings of molecules (e.g., amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups) and, in some embodiments, may have specific three-dimensional structural features and / or specific charge features. An epitope is an antigenic region that is bound by an antibody. In some embodiments, an antibody is said to specifically bind to an antigen when it recognizes its target antigen in a complex mixture of proteins and / or macromolecules. An antibody is said to "bind to the same epitope" if it cross-competes (one blocks the binding of another or regulates an effect). Furthermore, while structural definitions of epitopes (overlapping, similar, identical) are informative, functional definitions are often more relevant because they encompass both structural (binding) and functional (regulatory, competitive) parameters.

[0069] The term "surface plasmon resonance" as used in this paper refers to an optical phenomenon that allows for the analysis of real-time, biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using... The system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ). For further description, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51: 19-26; Jonsson, U., et al. (1991) Biotechniques 11: 620-627; Johnson, B., et al. (1995) J. Mol. Recognit. 8: 125-131; and Johnson, B., et al. (1991) Anal. Biochem. 198: 268-277.

[0070] As used herein, the term "Kon" is intended to refer to the binding rate constant of a binding protein (e.g., an antibody) to form an antibody / antigen complex, as is known in the art. "Kon" is also referred to as the term "binding rate constant" or "ka," as used interchangeably herein. This value indicates the rate at which an antibody binds to its target antigen or the rate at which a complex forms between an antibody and an antigen.

[0071] As used herein, the term "Koff" is intended to refer to the dissociation rate constant or "dissociation rate constant" of a bound protein (e.g., an antibody) from an antibody / antigen complex, as is known in the art. This value indicates the rate at which an antibody dissociates from its target antigen or an Ab-Ag complex over time into free antibody and antigen.

[0072] As used herein, the term "KD" is intended to represent "equilibrium dissociation constant" and refers to the value obtained at equilibrium in a titration measurement, or the value obtained by dividing the dissociation rate constant (koff) by the binding rate constant (kon). The binding rate constant, dissociation rate constant, and equilibrium dissociation constant are used to represent the binding affinity of an antibody for an antigen. Methods for determining the binding and dissociation rate constants are well known in the art. Using fluorescence-based techniques provides high sensitivity and the ability to examine samples at equilibrium in physiological buffers. Other experimental methods and instruments, such as... (Biomolecular interaction analysis) assays (e.g., instruments available from BIAcore International AB (GE Healthcare, Uppsala, Sweden)). Alternatively, [the following methods can be used]. The Kinetic Exclusion Assay (KEA) is available from Sapidyne Instruments (Boise, Id.).

[0073] "Label" and "detectable label" refer to a portion that is linked to a specific binding partner, such as an antibody or analyte, to make the reaction between members of the specific binding pair (such as an antibody and an analyte) detectable, and such labeled specific binding partners (e.g., antibodies or analytes) are referred to as "detectably labeled." Thus, the term "labeled binding protein" as used herein refers to a protein with an incorporated label that provides for the identification of the binding protein. In one aspect, the label is a detectable marker that can generate a signal detectable by visual or instrumental means, such as the incorporation of a radiolabeled amino acid or the linking of a biotinylated moiety (e.g., streptavidin containing a fluorescent marker or enzyme activity detectable by optical or colorimetric methods) to a polypeptide. Examples of labels used for peptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, or 153Sm); chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanides), enzyme-catalyzed labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent labels; biotinylated groups; predetermined peptide epitopes recognized by secondary reporter molecules (e.g., leucine zipper pairs, binding sites of secondary antibodies, metal-binding domains, epitope tags); and magnetic reagents, such as gadolinium chelates. Representative examples of labels commonly used in immunoassays include light-producing parts, such as acridinium compounds, and fluorescence-producing parts, such as fluorescein. Other labels are described herein. At this point, a part may not be detectably labeled on its own, but may become detectable after reacting with another part. The intended application of “detectably marked” includes the latter type of detectable marking.

[0074] The term "conjugated antibody" refers to a binding protein (such as an antibody) chemically linked to a second chemical component (such as a therapeutic agent or cytotoxic agent). The term "reagent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract prepared from biological material. In one aspect, the therapeutic agent or cytotoxic agent includes, but is not limited to, pertussis toxin, paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, anthraquinone, mitoxantrone, procainox, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogues or homologues. When used in the context of immunoassay, a conjugated antibody can be a detectably labeled antibody used as a detection antibody.

[0075] The terms “isolated polynucleotide” and “isolated nucleotide molecule” used interchangeably herein refer to a polynucleotide (e.g., having a genomic, cDNA, or synthetic origin, or some combination thereof) that does not accompany all or part of a polynucleotide found in nature with the “isolated polynucleotide” or “isolated nucleotide molecule”, or does not occur in nature as part of a larger sequence. An “isolated polynucleotide” or “isolated nucleotide molecule” can be operatively linked to a polynucleotide that is not linked in nature.

[0076] The terms “regulation” and “adjustment” used interchangeably herein refer to a change or alteration in the activity of a target molecule (e.g., the biological activity of a cytokine). Regulation can be an increase or decrease in the magnitude of some activity or function of a target molecule. Exemplary activities and functions of a molecule include, but are not limited to, binding characteristics, enzyme activity, cell receptor activation, and signal transduction. Accordingly, the term “regulator” as used herein is a compound capable of changing or altering the activity or function of a target molecule (e.g., the biological activity of a cytokine). For example, a regulator can cause an increase or decrease in the magnitude of some activity or function of a molecule compared to the magnitude of activity or function observed in the absence of said regulator. In some embodiments, a regulator is an inhibitor that reduces the magnitude of at least one activity or function of a molecule. Exemplary inhibitors include, but are not limited to, proteins, peptides, antibodies, peptide bodies, carbohydrates, or small organic molecules. Peptides are described, for example, in WO01 / 83525.

[0077] "Patient" and "subject" may be used interchangeably herein to refer to animals, such as mammals, including primates (e.g., humans, monkeys, and chimpanzees), non-primates (e.g., cattle, pigs, camels, vicuñas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, whales), birds (e.g., ducks or geese), and sharks. Preferably, a patient or subject is a person, such as a person treating or assessing a disease, disorder, or condition, a person at risk of a disease, disorder, or condition, a person having a disease, disorder, or condition, and / or a person treating a disease, disorder, or condition.

[0078] The term "sample" as used herein is used in its broadest sense. "Biological sample" as used herein includes, but is not limited to, any amount of substance derived from living or formerly living organisms. Such living organisms include, but are not limited to, humans, mice, rats, monkeys, dogs, rabbits, and other animals. Such substances include, but are not limited to: blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0079] "Component" and "at least one component" generally refer to capture antibodies, detection antibodies or conjugated antibodies, controls, calibrators, calibrator series, sensitivity test subject groups, containers, buffers, diluents, salts, enzymes, enzyme cofactors, detection reagents, pretreatment reagents / solutions, substrates (e.g., as solutions), stop solutions, etc., which may be included in a kit for determining test samples (such as patient urine, serum, or plasma samples) according to the methods described herein and other methods known in the art. Therefore, in the context of this disclosure, "at least one component" and "component" can include peptides or other analytes as described above, such as compositions containing analytes (such as peptides) optionally immobilized on a solid support, such as by binding to an anti-analyte (e.g., anti-peptide) antibody. Some components may be in solution or reconstituted by low-pressure lyophilization for assay.

[0080] "Control" refers to a composition known to contain either an analyte ("negative control") or an analyte ("positive control"). A positive control may contain a known concentration of the analyte. "Control," "positive control," and "calibrator" are used interchangeably herein to refer to a composition containing a known concentration of the analyte. A "positive control" can be used to establish assay performance characteristics and is a useful indicator of the integrity of the reagent (e.g., the analyte).

[0081] "Predetermined cutoff value" and "predetermined level" generally refer to an assay cutoff value used to assess diagnostic / prognostic / treatment outcomes by comparing assay results to a predetermined cutoff value / level, wherein the predetermined cutoff value / level has been associated with or correlated with various clinical parameters (e.g., disease severity, progression / non-progression / improvement, etc.). While this disclosure may provide exemplary predetermined levels, it is well known that cutoff values ​​may vary depending on the nature of the immunoassay (e.g., the antibody used, etc.). Furthermore, it is entirely within the ordinary skill of those skilled in the art to modify this disclosure for use with other immunoassays to obtain cutoff values ​​with respect to the immunoassay specificity of those other immunoassays. Although the precise value of the predetermined cutoff value / level may vary between assays, the correlations (if any) described herein should be generally applicable.

[0082] As described herein, “pretreatment reagents” (e.g., lysis, precipitation, and / or solubilizing reagents) used in diagnostic assays are reagents that lyse any cells present in the test sample and / or solubilize any analyte. As further described herein, not all samples require pretreatment. Among other things, solubilizing an analyte (e.g., a target peptide) may cause the release of that analyte from any endogenous binding proteins present in the sample. Pretreatment reagents can be homogeneous (not requiring a separation step) or heterogeneous (requiring a separation step). When using heterogeneous pretreatment reagents, any precipitated analyte-binding proteins are removed from the test sample before proceeding to the next step of the assay.

[0083] In the context of the immunoassays and kits described herein, "quality control reagents" include, but are not limited to, calibrators, controls, and sensitivity test subjects. "Calibrators" or "standards" (e.g., one or more, such as multiple) are typically used to establish calibration (standard) curves for interpolating the concentration of an analyte (such as an antibody or analyte). Alternatively, a single calibrator close to a predetermined positive / negative cutoff value may be used. Multiple calibrators (i.e., more than one calibrator or different amounts of calibrators) may be used in combination to constitute a "sensitivity test subject set."

[0084] "Risk" refers to the likelihood or probability of a particular event occurring now or at some point in the future. "Risk grading" refers to an array of known clinical risk factors that allows physicians to classify patients as low, medium, high, or highest risk of developing a particular disease, disorder, or condition.

[0085] The terms "specific" and "specific" in the context of an interaction between members of a specific binding pair (e.g., an antigen (or a fragment thereof) and an antibody (or an antigen-reactive fragment thereof)) indicate the selective reactivity of the interaction. The phrase "specific binding" and similar phrases indicate the ability of an antibody (or an antigen-reactive fragment thereof) to specifically bind to an analyte (or a fragment thereof) and not specifically bind to other entities.

[0086] A "specific binding pair" is a member of a specific binding pair. A specific binding pair comprises two distinct molecules that bind specifically to each other through chemical or physical means. Therefore, in addition to the antigen-antibody specific binding pair in common immunoassays, other specific binding pairs can include biotin and avidin (or streptavidin), carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzyme inhibitors and enzymes, etc. Additionally, specific binding pairs can include analog members that are the initial specific binding member, such as analyte analogs. Immunoreactive specific binding members include antigens, antigen fragments, and antibodies, including monoclonal and polyclonal antibodies and their complexes, fragments, and variants (including fragments of variants), whether isolated or recombinant.

[0087] Monoclonal antibodies

[0088] Figure 1A and 1B Sequences of various antibodies have been shown, said antibodies having been determined to be specific for the HCV core antigen, and more specifically, specific for the lipid-binding domain of the HCV core antigen. These monoclonal antibodies have been found to specifically immunoreact with the lipid-binding domain of the HCV core antigen. More specifically, the antibodies of the present invention have been found to specifically bind to at least one epitope formed by the amino acid sequence MGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG. More specifically, said monoclonal antibodies are at least immunoreacting with epitopes formed by amino acids 141-161, 134-154, and 151-171 of the HCV core antigen. In view of the disclosure of these monoclonal antibodies, the present invention anticipates their use in specific immunoassays to facilitate the rapid and efficient detection of the presence of HCV in test samples by determining the presence of the HCV core antigen in such test samples.

[0089] Anti-HCV core-binding proteins can be used in immunoassays for the diagnosis or prediction of hepatitis C virus infection in mammals. These anti-HCV core-binding proteins comprise monoclonal antibodies and any derivatives thereof (e.g., fragments or variants), the derivatives comprising the heavy and light chain CDRs of the monoclonal antibodies described herein (see [link to documentation]). Figure 1A and1B The prerequisite is that such derivatives retain the property of specifically binding to the lipid-binding domain of the HCV core protein. As used throughout this disclosure, "mammal" includes humans and non-human primates, as well as other animals. It should be understood that the target analyte in the immunoassay and related methods is the lipid domain of the HCV core protein, and therefore the target analyte is the HCV core protein present in the sample, for example, after HCV infection. Furthermore, it should be understood that the immunoassay can detect two or more target analytes, provided that at least one analyte is the HCV core protein, and the second or additional target analyte can be another core protein analyte (e.g., the DNA-binding domain of the HCV core protein), or it can be an analyte that is not an HCV core protein.

[0090] By immunizing mice with a synthetic peptide, the nucleotide (DNA) sequence encoding the heavy and light chain variable domains of an anti-HCV core monoclonal antibody and the inferred protein sequence are obtained, said synthetic peptide comprising the HCV core genotype 1 common sequence derived from amino acids 134-171 and the tetanus toxoid (TT) peptide sequence. In some embodiments, the amino acid sequence 134-171 is conjugated to BSA. However, in other embodiments, the synthetic peptide is also conjugated to the TT sequence, as this is often used to provide a more robust immune response in mice by methods known to those skilled in the art, such as those detailed below and in, for example, the following literature: Goding, JW 1983. Monoclonal Antibodies: Principles and Practice, Pladermic Press, Inc., NY, NY, pp. 56-97. Briefly, for the production of human-human hybridomas, human lymphocyte donors are selected. HCV-infected donors (wherein the infection has been confirmed, for example, by the presence of antiviral antibodies in the blood or by viral culture) are known to serve as suitable lymphocyte donors. Lymphocytes can be isolated from peripheral blood samples, or spleen cells can be used if splenectomy is to be performed on the donor. Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes, or human fusion couples can be used to produce human-human hybridomas. Primary in vitro immunization using peptides can also be used to prepare human monoclonal antibodies. Antibodies secreted by immortalized cells are screened to identify clones that secrete antibodies with the desired specificity. For monoclonal anti-HCV core antibodies, the antibody must bind to the HCV core protein, and more specifically, the lipid-binding domain of the HCV core protein. Cells that produce antibodies with the desired specificity are selected. Other methods for obtaining monoclonal antibodies can be used, as known in the art. The following examples describe how anti-HCV core monoclonal antibodies are obtained and characterized after isolating mRNA from hybridoma cells grown in cell cultures. The inferred amino acid sequences of the heavy and light chain variable regions of the anti-HCV core monoclonal antibody of this invention are listed in... Figure 1A and Figure 1B middle.

[0091] The inferred amino acid sequences of the heavy and light chain domains are assigned SEQ ID NO, and the corresponding cDNA sequences encoding them are shown in the sequence listing in Appendix A.

[0092] The cDNA sequences set forth in the sequence listing represent exemplary embodiments of the disclosed cDNA. Variations are anticipated in the cDNA sequences shown therein. Such variations include those that will produce a specific nucleic acid sequence capable of directing the production of an analogue of the corresponding protein shown in the sequence listing. It should be understood that, due to the degeneracy of the genetic code, many nucleotide substitutions may be made, which will produce DNA sequences that still direct the production of the corresponding protein or its analogues. This disclosure includes all such variant DNA sequences that are functionally equivalent to any sequence described herein.

[0093] Any binding proteins described in this article (such as...) Figure 1A and 1BThe variants of the monoclonal antibodies of the present invention illustrated herein refer to proteins (or polypeptides) that differ from a given protein (e.g., an anti-HCV core monoclonal antibody) in their amino acid sequence by the addition (e.g., insertion), deletion, or conserved substitution of amino acids, but which retain the biological activity of the given protein. Conserved substitution of amino acids, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., degree and distribution of hydrophilicity and charged regions), is generally recognized in the art as involving minor variations. As understood in the art, these minor variations can be identified in part by taking into account the hydrophilicity index of the amino acid (see, for example, Kyte et al., J. Mol. Biol. 157: 105-132 (1982)). The hydrophilicity index of an amino acid is based on consideration of its hydrophobicity and charge. It is known in the art that amino acids with similar hydrophilicity indices can be substituted and still retain protein function. In one aspect, amino acids with a hydrophilicity index of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that will result in the retention of biological function in the protein. In the context of peptides, consideration of the hydrophilicity of amino acids allows for the calculation of the maximum local average hydrophilicity of the peptide, a useful metric reportedly well correlated with antigenicity and immunogenicity (see, for example, U.S. Patent No. 4,554,101, which is incorporated herein by reference). As understood in the art, substitution of amino acids with similar hydrophilicity values ​​can produce peptides that retain biological activity (e.g., immunogenicity). In one aspect, substitutions are made with amino acids having hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with this observation, biologically compatible amino acid substitutions are understood to depend on the relative similarity of the amino acids (especially the side chains of those amino acids), as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties. The term "variant" can also be used to describe peptides or fragments thereof that have undergone differential processing (such as by proteolysis, phosphorylation, or other post-translational modifications) but still retain their biological activity or antigenic reactivity (e.g., the ability to bind IL-18). The term “variant” is intended to be used in this article to include fragments of a variant, unless the context otherwise contradicts it.

[0094] The antibodies of the present invention, or antigen-binding fragments of those antibodies (e.g., fragments containing the heavy and light chain CDRs of the antibodies of the present invention), can also be produced through genetic engineering. For example, techniques for expressing heavy and light chain genes in *E. coli* are the subject of the following literature: PCT patent applications WO 901443, WO 901443, and WO 9014424, and Huse et al., 1989 *Science* 246: 1275 1281. This disclosure also includes recombinant vectors comprising isolated nucleic acid molecules as described herein, and host cells comprising such recombinant vectors. A vector is a nucleic acid molecule capable of transporting another nucleic acid to which it is linked; it may be a construct. A vector may include any preferred or desired operational element. Preferred vectors are those whose restriction sites have been described and which contain operational elements required for transcription of the nucleic acid sequence. Such operational elements include, for example, at least one suitable promoter, at least one operator gene, at least one leader sequence, at least one terminator codon, and any other DNA sequence necessary or preferred for proper transcription and subsequent translation of the nucleic acid sequence. Such vectors contain at least one origin of replication recognized by the host organism, at least one selection marker, and at least one promoter sequence capable of initiating transcription of a nucleic acid sequence. The vector can be a plasmid in which an additional DNA segment can be ligated. The vector can be a viral vector in which an additional DNA segment can be ligated into a viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episodic mammalian vectors). Other vectors (e.g., non-episodic mammalian vectors) can integrate into the host cell's genome after introduction and thereby replicate along with the host genome. Furthermore, some vectors can direct the expression of the gene to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors used in recombinant DNA technology are typically in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vector. However, this disclosure is intended to include other forms of expression vectors, such as equivalent viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0095] Operablely linked sequences are in a relationship that allows them to function in their intended manner. Control sequences operablely linked to coding sequences are connected in such a way that the expression of the coding sequence is achieved under conditions compatible with the control sequence. Operablely linked sequences include expression control sequences adjacent to the target gene and expression control sequences that function trans- or at a distance from the target gene. Expression control sequences are polynucleotide sequences that are essential for the expression and processing of the coding sequence to which they are linked. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals such as splicing and polyadenylation signals; sequences stabilizing cytoplasmic mRNA; sequences enhancing translation efficiency (i.e., Kozak concordant sequences); sequences enhancing protein stability; and sequences enhancing protein secretion when needed. The nature of the control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include promoters, ribosome binding sites, and transcription termination sequences; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. Control sequences include components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0096] Host cells can be transformed using vectors that introduce exogenous DNA into the host cells so that the cells become recombinantly producing the antibodies of the present invention. Transformation can be performed under natural or artificial conditions using a variety of methods well known in the art. Transformation can rely on any known method of inserting a foreign nucleic acid sequence into prokaryotic or eukaryotic host cells. The method is selected based on the host cells to be transformed and may include, but is not limited to, viral infection, electroporation, lipid transfection, and particle bombardment. Transformed cells include stably transformed cells (where the inserted DNA is capable of replicating as an autonomously replicating plasmid or as part of the host chromosome) and cells that transiently express the inserted DNA or RNA for a limited time.

[0097] Suitable host organisms include, for example, eukaryotic cell systems, such as, but not limited to, cell lines such as HeLa, MRC-5, or CV-1. The host organism, such as host cells, is cultured under conditions suitable for amplifying the vector and expressing the protein, as is well known in the art. The expressed recombinant protein can be detected by any of a number of methods also well known in the art.

[0098] Although the HCV detection aspect of this invention only requires that the antibodies be monoclonal antibodies that specifically recognize the HCV core antigen, some embodiments may require the production of humanized forms of the antibodies of this invention. “Humanized” antibodies and their production are well known to those skilled in the art. Morrison S., 1985 Science 229:1202 and Oi et al., 1986 BioTechniques 4:214 provide general reviews of “humanized” antibodies. Suitable “humanized” antibodies can be alternatively produced by CDR or CEA substitution (Jones et al., 1986 Nature 321:552; Verhoeyan et al., 1988 Science 239:1534; Biedler et al., 1988 J. Immunol. 141:4053, the entire disclosure of which is incorporated herein by reference).

[0099] In other embodiments, the monoclonal antibody of the present invention can serve as a useful starting material for the production of engineered and derivatized binding proteins, including dual variable-domain immunoglobulin (DVD-Ig) binding proteins comprising one or more anti-HCV monoclonal antibodies as described herein. For example, DVD-Ig with a unique binding affinity for the HCV core protein can be produced as described, for example, in U.S. Patent No. 7,612,181 (the entire disclosure of which is hereby incorporated by reference). The DVD-Ig binding protein is capable of binding to one or more targets. Preferably, the binding protein comprises a polypeptide chain containing VD1-(X1)n-VD2-C--(X2)n, wherein VD1 is a first variable domain, VD2 is a second variable domain, C is a constant domain, X1 represents an amino acid or polypeptide, X2 represents an Fc region, and n is 0 or 1. The binding protein can be prepared using various techniques.

[0100] In an exemplary technique, DVD-Ig can be formed using four polypeptide chains, which form four functional antigen-binding sites. Thus, for example, DVD-Ig is capable of binding the HCV core protein. The binding protein may be able to modulate the biological function of the HCV core protein or neutralize the HCV core protein. An exemplary such binding protein has at least one heavy chain variable domain and at least a corresponding light chain variable domain, the heavy chain variable domain containing an amino acid sequence having at least 90% identity with one of the antibodies of the present invention, and the light chain variable domain containing an amino acid sequence having at least 90% identity with the sequence of the light chain variable domain.

[0101] The variable domains of DVD-binding proteins can be derived from parental antibodies, including polyclonal and monoclonal antibodies capable of binding to the target antigen. Monoclonal antibodies that specifically bind to the HCV core protein described herein are suitable parental antibodies. Typically, antibodies for DVD-binding proteins can be naturally occurring or prepared using recombinant technologies.

[0102] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma, recombinant, and phage display techniques, or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma techniques, including those described herein for the preparation of anti-HCV core protein monoclonal antibodies and those known in the art and taught in, for example, the following: Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988); Hammerling et al., see: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981) (the references are incorporated herein by reference in their entirety). The term “monoclonal antibody” as used herein is not limited to antibodies produced by hybridoma techniques. The term “monoclonal antibody” means an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone) and not a method for producing it. As discussed in Example 1 below, hybridomas are selected, cloned, and further screened for desired characteristics, including robust hybridoma growth, high antibody production, and desired antibody signatures. Hybridomas can be cultured and propagated in vivo in syngeneic animals, in animals lacking an immune system (e.g., nude mice), or in in vitro cell cultures. Methods for selecting, cloning, and propagating hybridomas are well known to those skilled in the art. In a preferred embodiment, the hybridoma is a mouse hybridoma. In another preferred embodiment, the hybridoma is produced in a non-human, non-mouse species (such as rats, sheep, pigs, goats, cattle, or horses). In yet another embodiment, the hybridoma is a human hybridoma in which a human non-secreting myeloma is fused with human cells expressing antibodies capable of binding specific antigens.

[0103] As described in the following literature, recombinant monoclonal antibodies are also prepared from single isolated lymphocytes using a method known in the art as the Selected Lymphocyte Antibody Method (SLAM): U.S. Patent No. 5,627,052, PCT Publication WO92 / 02551, and Babcock, JS et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848. In this method, single cells secreting the target antibody are identified, for example, lymphocytes derived from an immunized animal, and heavy and light chain variable region cDNAs are rescued from said cells by reverse transcriptase-PCR. These variable regions can then be expressed in mammalian host cells (such as COS or CHO cells) in the context of appropriate immunoglobulin constant regions (e.g., human constant regions). Host cells transfected with amplified immunoglobulin sequences (derived from selected lymphocytes in vivo) can then undergo further in vitro analysis and selection, for example by panning transfected cells to isolate cells expressing antibodies against the target antigen. Immunoglobulin sequences can be further amplified in vitro, such as by in vitro affinity maturation methods, as described in PCT Publications WO 97 / 29131 and PCT Publications WO 00 / 56772.

[0104] Monoclonal antibodies can also be produced by immunizing non-human animals containing some or all of the human immunoglobulin loci with a target antigen. In a preferred embodiment, the non-human animal is... Transgenic mice are engineered mouse strains that contain large fragments of human immunoglobulin gene loci and exhibit a deficiency in mouse antibody production. See, for example, Green et al., Nature Genetics 7:13-21 (1994) and U.S. Patents 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598, and 6,130,364. See also WO 91 / 10741 published on July 25, 1991; WO 94 / 02602 published on February 3, 1994; WO 96 / 34096 and WO 96 / 33735 published on October 31, 1996; WO 98 / 16654 published on April 23, 1998; WO 98 / 24893 published on June 11, 1998; WO 98 / 50433 published on November 12, 1998; WO 99 / 45031 published on September 10, 1999; WO 99 / 53049 published on October 21, 1999; WO 00 09560 published on February 24, 2000; and WO 00 / 037504 published on June 29, 2000. Transgenic mice produce a full suite of adult-like human antibodies, including antigen-specific human Mabs. This is achieved by introducing megabase-sized, germline YAC fragments from the human heavy chain locus and the x light chain locus. The transgenic mice contain approximately 80% of the complete human antibody panel. See Mendez et al., Nature Genetics 15:146-156 (1997), Green and Jakobovits J. Exp. Med. 188:483-495 (1998), the contents of which are hereby incorporated by reference.

[0105] In vitro methods can also be used to prepare parental antibodies, in which antibody libraries are screened to identify antibodies with the desired binding specificity. Such methods for screening recombinant antibody libraries are well known in the art and include those described in, for example, the following publications: Ladner et al., U.S. Patent No. 5,223,409; Kang et al., PCT Publication No. WO 92 / 18619; Dower et al., PCT Publication No. WO 91 / 17271; Winter et al., PCT Publication No. WO 92 / 20791; Markland et al., PCT Publication No. WO 92 / 15679; Breitling et al., PCT Publication No. WO 93 / 01288; McCafferty et al., PCT Publication No. WO 92 / 01047; Garrard et al., PCT Publication No. WO 92 / 09690; Fuchs et al. (1991) Bio / Technology 9: 1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3: 81-85; Huse et al. (1989) Science 246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffiths et al. (1993) EMBO J.12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio / Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, U.S. Patent Application Publication 20030186374, and PCT Publication WO97 / 29131, the contents of each of which are incorporated herein by reference.

[0106] Parental antibodies can also be prepared using a variety of phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of a phage particle carrying a polynucleotide sequence encoding them. Specifically, such phages can be used to display antigen-binding domains expressed from a full suite of antibodies or a combined antibody library (e.g., human or mouse). Phages expressing antigen-binding domains that bind to target antigens can be selected or identified using antigen selection, for example, using labeled antigens, or antigens that bind to or are captured by solid surfaces or beads. The phages used in these methods are typically filamentous phages, including those expressing fd and M13 binding domains from phages having Fab, Fv, or disulfide-stabilized Fv antibody domains recombinantly fused to phage gene III or gene VIII proteins. Examples of phage display methods that can be used to prepare antibodies as described herein include those disclosed in the following literature: Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application number PCT / GB91 / 01134; PCT publications WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO WO 93 / 11236, WO 95 / 15982, WO 95 / 20401 and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108; each of these is incorporated herein by reference in its entirety.

[0107] As described in the references above, after phage selection, antibody coding regions derived from phages can be isolated and used to prepare intact antibodies, including human antibodies or any other desired antigen-binding fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, for example, as described in detail below. For example, techniques for the recombinant production of Fab, Fab′, and F(ab′)2 fragments can also be employed using methods known in the art, such as those disclosed in the following publications: PCT Publication WO 92 / 22324; Mullinax et al., BioTechniques 12(6): 864-869 (1992); and Sawai et al., AJRI 34: 26-34 (1995); and Better et al., Science 240: 1041-1043 (1988) (the entire references are incorporated by reference). Examples of techniques that can be used to produce single-chain Fv and antibodies include those described in the following literature: U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988).

[0108] As an alternative to screening recombinant antibody libraries via phage display, other methods known in the art for screening large combinatorial libraries can be applied to identify parental antibodies. One class of alternative expression systems involves expressing a recombinant antibody library as an RNA-protein fusion, as described by Szostak and Roberts in PCT Publication No. WO 98 / 31700 and in Roberts, RW and Szostak, JW (1997) Proc. Natl. Acad. Sci. USA 94: 12297-12302. In this system, a covalent fusion is established between mRNA and a peptide or protein, the mRNA encoding the peptide or protein through in vitro translation of a synthetic mRNA carrying puromycin (a peptide receptor antibiotic) at its 3′ end. Thus, based on the properties of the encoded peptide or protein (e.g., antibody or a portion thereof), such as the binding of the antibody or a portion thereof to a bispecific antigen, specific mRNAs can be enriched from complex mixtures of mRNAs (e.g., combinatorial libraries). The nucleic acid sequences encoding antibodies or portions thereof recovered from such libraries can be expressed via recombination as described above (e.g., in mammalian host cells), and can further undergo affinity maturation via additional screening cycles of mRNA-peptide fusions (in which mutations have been introduced into the originally selected sequences) or via other methods of in vitro affinity maturation of recombinant antibodies as described above.

[0109] In another approach, parental antibodies can also be prepared using yeast display methods known in the art. In yeast display methods, antibody domains are attached to the yeast cell wall using genetic methods and displayed on the surface of the yeast. Specifically, such yeast can be used to display antigen-binding domains expressed from a full suite of antibodies or a combined antibody library (e.g., human or mouse). Examples of yeast display methods that can be used to prepare parental antibodies include those disclosed in Wittrup et al., U.S. Patent No. 6,699,658 (incorporated herein by reference).

[0110] The monoclonal antibodies described herein can be further modified to prepare CDR-grafted and humanized parental antibodies. The CDR-grafted parental antibody comprises heavy and light chain variable region sequences derived from a human antibody, wherein one or more CDR regions of VH and / or VL are replaced by CDR sequences of a mouse antibody capable of binding the target antigen. Frame sequences derived from any human antibody can serve as templates for CDR grafting. However, straight-chain substitutions on such frames often result in some loss of binding affinity to the antigen. The higher the homology between the human antibody and the original mouse antibody, the less likely combining mouse CDRs with human frames will cause CDR deformation that could reduce affinity. Therefore, it is preferred that the selected human variable frame used to replace the mouse variable frame away from the CDR has at least 65% sequence identity with the mouse antibody variable region frame. More preferably, the human and mouse variable regions away from the CDR have at least 70% sequence identity. Even more preferably, the human and mouse variable regions away from the CDR have at least 75% sequence identity. Most preferably, the human and mouse variable regions away from the CDR have at least 80% sequence identity. Methods for producing such antibodies are known in the art (see EP 239,400; PCT Publication WO 91 / 09967; U.S. Patent Nos. 5,225,539, 5,530,101 and 5,585,089), as well as surface cladding or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4 / 5): 489-498 (1991); Studnicka et al., Protein Engineering 7(6): 805-814 (1994); Roguska et al., PNAS 91: 969-973 (1994)), and chain truncation (U.S. Patent No. 5,565,352).

[0111] Humanized antibodies are antibody molecules derived from non-human species antibodies that bind to desired antigens, and have one or more complementarity-determining regions (CDRs) derived from non-human species and a framework region derived from human immunoglobulin molecules. Known human Ig sequences are publicly available at, for example, www.ncbi.nlm.nih.gov / entrez- / query.fcgi; www.atcc.org / phage / hdb.html; www.sciquest.com / ; www.abcam.com / ; www.antibodyresource.com / onlinecomp.html; www.public.iastate.edu / .about.pedro / research_tools.html; www.mgen.uni-heidelberg.de / SD / IT / IT.html; www.whfreeman.com / immunology / CH-05 / kuby05.html; www.library.thinkquest.org / 12429 / Immune / Antibody.html; www.hhmi.org / grants / lectures / 1996 / vlab / ; www.path.cam.ac.uk / .about.mrc7 / m-ikeimages.html; www.antibodyresource .com / ;mcb.harvard.edu / BioLinks / Immunology.html.www.immunologylink.com / ;pathbox.wustl.edu / .about.hcenter / index.-html ;www.biotech.ufl.edu / .about.hcl / ;www.pebio.com / pa / 340913 / 340913.html-;www.nal.usda.gov / awic / pubs / antibody / ;www.m.ehi me-u.acjp / .about.yasuhito- / Elisa.html; www.biodesign.com / table.asp; www.icnet.uk / axp / facs / davies / lin-ks.html; www.biote ch.ufl.edu / .about.fccl / protocol.html; www.isac-net.org / sites_geo.html; aximtl.imt.uni-marburg.de / .about.rek / AEP-Start.html; baserv.uci.kun.nl / .aboutjraats / linksl.html; www.recab.uni-hd.de / immuno.bme.nwu.edu / ; www.mrc-cpe.cam.ac.uk / imt-doc / pu-blic / INTRO.html; www.ibt.unam.mx / virV_-mice .html;imgt.cnusc.fr:8104 / ;www.biochem.ucl.ac.uk / .about.martin / abs / index.html;antibody.bath.ac.uk / ;abgen.cvm.tamu.edu / lab / wwwabgen.html;www.unizh.ch / .about.honegger / AHOseminar / Slide01.html; www.cryst.bbk.ac.uk / .about.ubcg07s / ; www.nimr.mrc.ac.uk / CC / ccaewg / ccaewg.htm; www.path.cam.ac.uk / .about.mrc7 / humanisation / TAHHP.html; www.ibt.unam.mx / vir / structure / stataim.html; www.biosci.missouri.edu / smithgp / index.html; www.cryst.bioc.cam.ac.uk / .abo-ut.fmolina / Webpages / Pept / spottech.html; www.jerini.de / fr roducts.htm; www.patents.ibm.con / ibm.html. Kabat et al., Sequences of Proteins of Immunological Interest, USDept. Health (1983), each fully cited and incorporated herein by reference. As is known in the art, such introduced sequences can be used to reduce immunogenicity, or to reduce, enhance, or modify binding, affinity, binding rate, dissociation rate, specificity, half-life, or any other suitable characteristic.

[0112] Framework residues in the human framework region can be replaced with corresponding residues from CDR donor antibodies to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well-known in the art, for example, by modeling the interaction between the CDR and framework residues (to identify framework residues important for antigen binding) and sequence comparison (to identify rare framework residues at specific locations) (see, e.g., Queen et al., U.S. Patent No. 5,585,089; Riechmann et al., Nature 332:323 (1988), both incorporated herein by reference in their entirety). Three-dimensional immunoglobulin models are generally available and are well-known to those skilled in the art. Computer programs are available to display and visualize possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these displays allows analysis of the possible roles of residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues affecting the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected from shared and introduced sequences and combined to achieve desired antibody characteristics, such as increased affinity for target antigens. Generally speaking, CDR residues are directly and most significantly involved in affecting antigen binding.Antibodies can be humanized using a variety of techniques known in the art, such as, but not limited to, those described in the following literature: Jones et al., Nature 321:522 (1986); Verhoeyen et al., Science 239:1534 (1988); Sims et al., J. Immunol. 151:2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993); Padlan, Molecular Immunology 28(4 / 5):489-498 (1991); Studnicka et al., Protein Engineering 7(6): 805-814 (1994); Roguska et al., PNAS 91: 969-973 (1994); PCT Publication WO91 / 09967, PCT / : US98 / 16280, US96 / 18978, US91 / 09630, US91 / 05939, US94 / 01234, GB89 / 01334, GB91 / 01134, GB92 / 01755; WO90 / 14443, WO90 / 14424, WO90 / 14430, EP 229246, EP592,106; EP 519,596, EP 239,400, U.S. Patent Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539, and 4,816,567, each of which is incorporated herein by reference in its entirety.

[0113] Parental monoclonal antibodies can be selected from a variety of monoclonal antibodies that can bind to specific targets such as those well known in the art (including HCV protein, or other than HCV protein).

[0114] Parental monoclonal antibodies can also be selected from a variety of approved therapeutic antibodies that are in clinical trials or under development for clinical application, especially those that may be suitable for treating symptoms of HCV infection or for treating conditions or diseases coexisting with HCV infection (such as cancer, particularly hepatocellular carcinoma).

[0115] As indicated throughout this invention, it may be desirable to label the antibodies of this invention. Labeled antibodies (or binding proteins derived from one of the antibodies of this invention) comprise derivatized antibodies or antibodies linked to another functional molecule (e.g., another peptide or protein). For example, monoclonal antibodies can be derivatized by functionally linking (through chemical coupling, gene fusion, non-covalent binding, or other means) them to one or more other molecular entities (such as another antibody (e.g., a bispecific antibody or dimer), a detectable reagent, a cytotoxic agent, a pharmaceutical reagent, and / or a protein or peptide that can mediate the binding protein's binding to another molecule (such as a streptavidin core region or a polyhistidine tag).

[0116] Useful detectable reagents that can be used to derivatize monoclonal antibodies include fluorescent compounds. Exemplary fluorescent detectable reagents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, etc. The antibodies can also be derivatized with detectable enzymes such as alkaline phosphatase, horseradish peroxidase, glucose oxidase, etc. When derivatized with a detectable enzyme, detection is achieved by adding another reagent to the enzyme to generate a detectable reaction product. For example, in the presence of the detectable reagent horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine results in a detectable colored reaction product. The monoclonal antibodies of the present invention can also be derivatized with biotin and detected by indirect measurement of binding to avidin or streptavidin, or vice versa.

[0117] While the compositions of the present invention have demonstrated use in diagnostic applications for determining the presence of HCV core antigen in test samples, it is anticipated that the compositions of the present invention may also be used for diagnostic or therapeutic purposes for in vivo administration to mammals. Thus, in some embodiments, the present invention provides pharmaceutical and diagnostic compositions comprising one or more anti-HCV core binding proteins disclosed herein as active ingredients. Pharmaceutical or diagnostic compositions may comprise any monoclonal antibody described herein or any combination thereof and pharmaceutically acceptable carriers, diluents, and / or excipients. Typically, pharmaceutical and diagnostic compositions are prepared by combining the active ingredient with a carrier, diluent, and / or excipient.

[0118] Compositions containing binding proteins as described herein are used, but not limited to, for the diagnosis, detection, or monitoring of disorders, but may also be used for the prevention, treatment, control, or improvement of a disorder or one or more symptoms thereof and / or for use in research. In one specific embodiment, the composition comprises one or more monoclonal antibodies of the present invention or binding proteins derived from one or more monoclonal antibodies of the present invention. In another embodiment, the composition comprises one or more monoclonal antibodies as described herein or binding proteins derived therefrom, and one or more diagnostic, preventative, or therapeutic agents other than monoclonal antibodies or binding proteins derived therefrom as described herein.

[0119] Immunoassay

[0120] Immunoassays according to this disclosure include techniques generally known in the art, such as radioimmunoassay, Western blotting, immunofluorescence assay, enzyme immunoassay, chemiluminescence assay, immunohistochemistry assay, immunoprecipitation, etc. Standard techniques for ELISA known in the art are well-known and described in, for example, Methods in Immunodiagnosis, 2nd edition, Rose and Bigazzi, eds., John Wiley and Sons, 1980 and Campbell et al., Methods of Immunology, WABenjamin, Inc., 1964, both of which are incorporated herein by reference. Immunoassays can be direct, indirect, competitive, or non-competitive immunoassays as described in the art (Oellerich, M. 1984. J. Clin. Chem. Clin. BioChem 22: 895 904). Suitable biological samples for such assays include, but are not limited to, blood, plasma, serum, liver, saliva, lymphocytes, or other mononuclear cells.

[0121] In a preferred embodiment, the antibodies described herein are used in immunoassays specific for HCV detection. Examples include, but are not limited to, sandwich immunoassays, radioisotope assays (radioimmunoassays (RIA)) and enzyme assays (enzyme immunoassays (EIA) or enzyme-linked immunosorbent assays (ELISA) (e.g., Quantikine ELISA assay, R&D Systems, Minneapolis, Minn.), competitive inhibition immunoassays (e.g., forward and reverse), fluorescence polarization immunoassays (FPIA), enzyme multiple immunoassay techniques (EMIT), bioluminescent resonance energy transfer (BRET), and homogeneous chemiluminescence assays. In SELDI-based immunoassays, a capture reagent that specifically binds to a target analyte, such as the HCV core (or fragment thereof), is attached to the surface of a mass spectrometry probe (such as a pre-activated protein chip array). The analyte (or fragment thereof) is then specifically captured on the biochip, and the captured analyte (or fragment thereof) is detected by mass spectrometry. Alternatively, the analyte (or a fragment thereof) can be eluted from the capture reagent and detected by conventional MALDI (matrix-assisted laser desorption / ionization) or by SELDI. Chemiluminescent microparticle immunoassays, especially those employing... The chemiluminescent microparticle immunoassay of the automated analyzer (Abbott Laboratories, Abbott Park, Ill.) is an example of a preferred immunoassay.

[0122] Immunoassays for determining the presence or amount of human hepatitis C virus (HCV) in a sample may include, for example, combining an HCV core protein-binding protein with the sample for a time sufficient for the binding protein to bind to any HCV that may be present in the sample, and determining the presence or amount of HCV in the sample based on the specific binding of the binding protein to the HCV core protein. This disclosure also includes immunoassay devices for detecting the presence or absence of human HCV in a sample, wherein said devices comprise any antibody described herein immobilized on a solid support. Anti-HCV core antibodies and any analogues thereof can be prepared in kit form, either alone or in combination with other reagents (such as a secondary antibody) for use in immunoassays.

[0123] Methods well-known in the art for collecting, manipulating, and processing urine, blood, serum, plasma, and other bodily fluids are used in the practice of this disclosure, for example, when the anti-HCV core antibody of the present invention is used as an immunodiagnostic reagent and / or in an analyte immunoassay kit. The test sample may contain portions other than the HCV core antigen, including, for example, antibodies, antigens, haptens, hormones, drugs, enzymes, receptors, proteins, peptides, polypeptides, oligonucleotides, and / or polynucleotides. For example, the sample may be a whole blood sample obtained from a subject. Prior to the immunoassay as described herein, it may be necessary or desirable to treat the test sample, particularly whole blood, for example, with a pretreatment reagent. Even where pretreatment is not necessary (e.g., for most urine samples), pretreatment may be optionally performed (e.g., as part of a protocol on a commercial platform).

[0124] The pretreatment reagent can be any reagent suitable for use with the immunoassays and kits of this disclosure. The pretreatment optionally comprises: (a) one or more solvents (e.g., methanol and ethylene glycol) and optionally a salt, (b) one or more solvents and a salt, and optionally a detergent, (c) a detergent, or (d) a detergent and a salt. Pretreatment reagents are known in the art, and such pretreatments can be used as previously described, for example, for use in Abbott TDx, and Determination on an analyzer (Abbott Laboratories, Abbott Park, Ill.), as described in the literature (see, for example, Yatscoff et al., Abbott TDx Monoclonal Antibody Assay Evaluated for Measuring Cyclosporine in Whole Blood, Clin. Chem. 36: 1969-1973 (1990), and Wallemaq et al., Evaluation of the New AxSYM Cyclosporine Assay Comparison with TDx Monoclonal Whole Blood and EMIT Cyclosporine Assays, Clin. Chem. 45: 432-435 (1999)), and / or as commercially available. Alternatively, preprocessing can be performed as described in the following documents: U.S. Patent No. 5,135,875, European Patent Publication No. 0 471 293, U.S. Provisional Patent Application No. 60 / 878,017, filed December 29, 2006, and U.S. Patent Application Publication No. 2008 / 0020401 (in whole, by reference, its teachings on preprocessing are incorporated herein by reference).

[0125] The application of pretreatment reagents makes the assay more sensitive by disrupting pre-formed / pre-existing immune complexes or viral particles in the test sample. In such pretreated test samples, the anti-HCV core antibody in the sample is separated from the antigen, and then the remaining antigen in the sample is tested for the presence of the HCV core antigen using the monoclonal antibody of the present invention. Thus, an antibody capture step is performed on the HCV core antigen in the test sample to capture any HCV antigen present in the test sample.

[0126] In some other embodiments, such a separation step is not required for the pretreatment application. The entire mixture of the test sample and the pretreatment reagent is contacted with an antibody specific to the targeted antigen (in this case, the HCV core antigen, or more specifically, the lipid-binding domain of the HCV core antigen). The pretreatment reagent used for such an assay is typically diluted in the pretreated test sample mixture before or during capture with the first antibody (which is used to capture the HCV antigen). Despite such dilution, a certain amount of the pretreatment reagent may still be present in the test sample mixture during the capture process. The capture reagent can be the antibody of the present invention; alternatively, it can be another anti-HCV core antigen antibody, or it can actually be an antibody against non-core protein antigens of HCV (e.g., antibodies against the HCV envelope protein, E1 or E2, or other parts).

[0127] In one assay, after obtaining the test sample from the subject, a first mixture is prepared. The mixture contains the test sample to assess the presence of a given antigen (e.g., in this case, the presence of the HCV core antigen) and a first specific binding partner (typically an antibody recognizing an HCV epitope), wherein the first specific binding partner and any HCV antigen contained in the test sample form a first antibody-antigen complex. The order in which the test sample and the first specific binding partner are added to form the mixture is not critical. The first specific binding partner may be immobilized on a solid phase, but in alternative embodiments, the first specific binding partner may be in a solution phase. The solid phase used in the immunoassay (for the first specific binding partner, and optionally, the second specific binding partner) may be any solid phase known in the art, such as, but not limited to, magnetic particles, beads, test tubes, microplates, cuvettes, membranes, scaffold molecules, thin films, filter paper, disks, and chips.

[0128] The method is suitable for use in systems utilizing particulate technology, including automated and semi-automated systems, wherein the solid phase contains particulates. Such systems include those described in pending U.S. Patent Application Serials 425,651 and 425,643 (corresponding to published EPO applications EP 0 425 633 and EP 0 424 634, respectively, which are incorporated herein by reference).

[0129] After forming a mixture containing a first specific binding partner-analyte complex, any unbound analyte is removed from the complex using any technique known in the art. For example, unbound analyte can be removed by washing. However, ideally, the first specific binding partner is present in an amount exceeding that of any analyte present in the test sample in order to optimize the maximum binding of the first specific binding partner to the analyte present in the test sample.

[0130] After removing unbound analytes, a second specific binding coupler is added to the mixture to form a first specific binding coupler-analyte-second specific binding coupler complex. The second specific binding coupler is preferably an anti-analyte antibody that binds to an epitope on the analyte, the epitope being different from the epitope on the analyte bound by the first specific binding coupler. Simply as an example, assuming the assay is for detecting HCV core antigen, a first "capture" antibody specific to the DNA-binding domain of the HCV core antigen is used (or alternatively, the first antibody is an anti-HCV core antibody specific to the lipid-binding domain of the HCV core antigen, such as the antibody described herein). Once the first capture antibody captures the HCV core protein from the sample, the second anti-core antigen antibody binds to the lipid-binding domain of the HCV core antigen (wherein the first antibody binds to the DNA-binding domain, or alternatively, where the first antibody is specific to the lipid-binding domain of the HCV core antigen, and the second antibody may be specific to the DNA-binding domain of the HCV core antigen). Preferably, in such an embodiment, the second specific binding partner is labeled with or contains a detectable marker as described above to facilitate the detection of the [capture antibody-antigen-second antibody] complex.

[0131] Any suitable detectable marker known in the art can be used. For example, the detectable marker could be a radioactive marker (such as...). 3 H, 125 I, 35 S, 14 C 32 P and 33P), enzyme labels (such as horseradish peroxidase, alkaline peroxidase, glucose-6-phosphate dehydrogenase, etc.), chemiluminescent labels (such as acridinium ester, thioester or sulfonamide; luminol, isoluminol, phenanthrene dimethyl ester, etc.), fluorescent labels (such as fluorescein (e.g., 5-fluorescein, 6-carboxyfluorescein, 3′6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachlorofluorescein, 6-tetrachlorofluorescein, isothiocyanate fluorescein, etc.), rhodamine, phycobiliprotein, R-phycoerythrin, quantum dots (e.g., zinc sulfide capped cadmium selenide), thermometric labels or immunopolymerase chain reaction labels. For an introduction to the markers, labeling methods, and labeling detection, see: Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, NY (1997), and Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996), a combined handbook and catalog published by Molecular Probes, Inc., Eugene, Oreg. Fluorescent markers can be used in FPIA (see, for example, U.S. Patent Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093, and 5,352,803, which are hereby incorporated in their entirety by reference). Acridinium compounds can be used as detectable labels in homogeneous chemiluminescence assays (see, for example, Adamczyk et al., Bioorg. Med. Chem. Lett. 16: 1324-1328 (2006); Adamczyk et al., Bioorg. Med. Chem. Lett. 4: 2313-2317 (2004); Adamczyk et al., Bioorg. Med. Chem. Lett. 14: 3917-3921 (2004); and Adamczyk et al., Org. Lett. 5: 3779-3782 (2003)).

[0132] A preferred acridinene compound is acridinene-9-carboxamide. Methods for preparing acridinene-9-carboxamide are described in Mattingly, J. Biolumin. Chemilumin. 6: 107-114 (1991); Adamczyk et al., J. Org. Chem. 63: 5636-5639 (1998); Adamczyk et al., Tetrahedron 55: 10899-10914 (1999); Adamczyk et al., Org. Lett. 1: 779-781 (1999); Adamczyk et al., Bioconjugate Chem. 11: 714-724 (2000); Mattingly et al., In Luminescence Biotechnology: Instruments and Applications; edited by Dyke, KV; CRC Press: Boca Raton, pp. 77–105 (2002); Adamczyk et al., Org. Lett. 5: 3779–3782 (2003); and U.S. Patent Nos. 5,468,646, 5,543,524 and 5,783,699 (each of which is incorporated herein by reference in its entirety for its teachings on the same subject).

[0133] Another preferred acridinene compound is an aryl acridinene-9-carboxylic acid ester. An example of an aryl acridinene-9-carboxylic acid ester of Formula II is 10-methyl-9-(phenoxycarbonyl)acidinene fluorosulfonate (available from Cayman Chemical, Ann Arbor, Mich.). Methods for preparing aryl acridinene-9-carboxylic acid esters are described in McCapra et al., Photochem. Photobiol. 4: 1111-21 (1965); Razavi et al., Luminescence 15: 245-249 (2000); Razavi et al., Luminescence 15: 239-244 (2000); and U.S. Patent No. 5,241,070 (each of which is incorporated herein by reference in its entirety for the purposes of its teachings on the same subject matter). Such acridine-9-carboxylic acid aryl esters are effective chemiluminescent indicators of the intensity and / or speed of the signal generated by hydrogen peroxide produced during the oxidation of an analyte by at least one oxidase. The chemiluminescent emission process of acridine-9-carboxylic acid aryl esters is rapid (i.e., less than 1 second), while the chemiluminescent emission of acridine-9-carboxamide is prolonged to more than 2 seconds. However, acridine-9-carboxylic acid aryl esters lose their chemiluminescent properties in the presence of proteins. Therefore, their application requires the absence of proteins during signal generation and detection. Methods for separating or removing proteins from samples are well known to those skilled in the art and include, but are not limited to, ultrafiltration, extraction, precipitation, dialysis, chromatography, and / or digestion (see, for example, Wells, High Throughput Bioanalytical Sample Preparation. Methods and Automation Strategies, Elsevier (2003)). The amount of protein removed or separated from the test sample may be about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. Further details regarding acridine-9-carboxylic acid aryl esters and their uses are set forth in U.S. Patent Application Serial No. 11 / 697,835, filed April 9, 2007, and published October 9, 2008, as U.S. Patent Application Publication No. 2008 / 0248493. Acridine-9-carboxylic acid aryl esters can be dissolved in any suitable solvent, such as degassed anhydrous N,N-dimethylformamide (DMF) or an aqueous solution of sodium cholate.

[0134] Chemiluminescence assays can be performed according to the method described in Adamczyk et al., Anal. Chim. Acta 579(1): 61-67 (2006). Although any suitable assay method can be used, a microplate chemiluminometer (Mithras LB-940, Berthold Technologies USA, LLC, Oak Ridge, Tenn.) enables rapid assays of multiple small volumes of sample. Using a 96-well black polystyrene microplate (Costar #3792), the chemiluminometer can be equipped with multiple reagent syringes. Each sample can be added to a separate well, followed by other reagents, as determined by the type of assay employed, added simultaneously or sequentially. Ideally, the formation of false bases in neutral or alkaline solutions of acridine aryl esters, such as by acidification, is avoided. The chemiluminescent response is then recorded well by well. At this point, the time to record the chemiluminescent response depends in part on the delay between the reagents used and the addition of the specific acridine ester.

[0135] The order in which the test sample and specific binding partners are added to form the chemiluminescent assay mixture is not critical. If the first specific binding partner is detectably labeled with a chemiluminescent reagent (such as an acridine compound), a detectably labeled first specific binding partner-analyte complex is formed. Alternatively, if a second specific binding partner is used and is detectably labeled with a chemiluminescent reagent (such as an acridine compound), a detectably labeled first specific binding partner-analyte-second specific binding partner complex is formed. Any unbound specific binding partners, whether labeled or unlabeled, can be removed from the mixture using any technique known in the art, such as washing.

[0136] Hydrogen peroxide can be generated in situ in the mixture before, during, or after the addition of the aforementioned acridine compounds, or hydrogen peroxide can be supplied to the mixture (e.g., the source of hydrogen peroxide is one or more buffer solutions or other solutions known to contain hydrogen peroxide). Hydrogen peroxide can be generated in situ in many ways (such as those that will be apparent to those skilled in the art).

[0137] After at least one alkaline solution is added to the sample simultaneously or subsequently, a detectable signal indicating the presence of the analyte, i.e., a chemiluminescent signal, is generated. The alkaline solution contains at least one base and has a pH greater than or equal to 10, preferably greater than or equal to 12. Examples of alkaline solutions include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide, sodium carbonate, sodium bicarbonate, calcium hydroxide, calcium carbonate, and calcium bicarbonate. The amount of alkaline solution added to the sample depends on the concentration of the alkaline solution. Based on the concentration of the alkaline solution used, those skilled in the art can readily determine the amount of alkaline solution added to the sample.

[0138] The generated chemiluminescent signal can be detected using conventional techniques known to those skilled in the art. The amount of analyte in the sample can be quantified based on the intensity of the generated signal. Specifically, the amount of analyte in the sample is proportional to the intensity of the generated signal. The amount of analyte present can be quantified by comparing the amount of generated light with a standard curve of the analyte or by comparing it with a reference standard. A standard curve can be generated using a series of dilutions or solutions of known concentrations of the analyte by mass spectrometry, gravimetric analysis, and other techniques known in the art. Although the above description emphasizes the use of acridinium compounds as chemiluminescent reagents, those skilled in the art can readily modify this description to use other chemiluminescent reagents.

[0139] Analytical immunoassays can typically be performed using any form known in the art (e.g., but not limited to, sandwich assays). Specifically, in one immunoassay format, at least two antibodies are used to capture and quantify an analyte (such as a human analyte) or fragment thereof in a sample. More specifically, preferably, at least two antibodies bind to different epitopes on the analyte (or fragment thereof) to form an immune complex, referred to as a “sandwich.” Typically, in an immunoassay, one or more antibodies can be used to capture an analyte (or fragment thereof) in a test sample (these antibodies are often referred to as “capture” antibodies), and one or more antibodies can be used to bind a detectable (i.e., quantifiable) marker to the sandwich (these antibodies are often referred to as “detection antibodies” or “conjugates”). Therefore, in the context of sandwich immunoassay formats, the anti-HCV core antibody of the present invention can be used as a capture antibody, a detection antibody, or both. For example, an anti-HCV core antibody having a domain that can bind to a first epitope on an analyte (e.g., a lipid-binding domain of an HCV core antigen) can be used as a capture antibody, and / or another anti-HCV core antibody having a domain that can bind to a second epitope (e.g., a DNA-binding domain of an HCV core antigen) can be used as a detection antibody, or vice versa. Alternatively, an antibody having a first domain that can bind to an epitope on an HCV core antigen and a second antibody that binds to epitopes on different HCV antigens can be used as a capture antibody and / or a detection antibody to detect and optionally quantify two or more analytes.

[0140] Generally, a sample tested against (e.g., suspected of containing) an analyte can be contacted simultaneously or sequentially with at least one capture antibody (or multiple capture antibodies) and at least one detection antibody (which may be a second or third detection antibody or even sequentially numbered antibodies, such as in cases where the capture antibody and / or detection antibody comprises multiple antibodies). For example, the test sample can be contacted first with at least one capture antibody and then (sequentially) with at least one detection antibody. Alternatively, the test sample can be contacted first with at least one detection antibody and then (sequentially) with at least one capture antibody. In another alternative, the test sample can be contacted simultaneously with both the capture antibody and the detection antibody.

[0141] In a sandwich assay, the sample suspected of containing the analyte (or a fragment thereof) is first contacted with at least one first capture antibody under conditions that allow for the formation of a first antibody / analyte complex. If more than one capture antibody is used, a first capture antibody / analyte complex comprising two or more capture antibodies is formed. In a sandwich assay, an antibody is used in a molar excess of the maximum amount of the analyte (or a fragment thereof) expected in the test sample, i.e., preferably at least one capture antibody. For example, about 5 μg to about 1 mg of antibody / mL buffer (e.g., microparticle coating buffer) can be used.

[0142] Competitive inhibition immunoassays (often used to measure small analytes because binding requires only one antibody) include sequential and classical forms. In a sequential competitive inhibition immunoassay, a capture antibody against the target analyte is coated onto the wells of a microtiter plate or other solid support. When a sample containing the target analyte is added to the well, the target analyte binds to the capture antibody. After washing, a known amount of labeled analyte (e.g., with biotin or horseradish peroxidase (HRP)) is added to the well. The enzyme-labeled substrate is necessary to generate a signal. An example of a suitable substrate for HRP is 3,3′,5,5′-tetramethylbenzidine (TMB). After washing, the signal generated by the labeled analyte is measured, and it is inversely proportional to the amount of analyte in the sample. In a classical competitive inhibition immunoassay, an antibody against the target analyte is coated onto a solid support (e.g., the wells of a microtiter plate). However, unlike a sequential competitive inhibition immunoassay, both the sample and the labeled analyte are added to the well simultaneously. Any analyte in the sample competes with the labeled analyte for binding to the capture antibody. After washing, the signal generated by the labeled analyte is measured, and it is inversely proportional to the amount of analyte in the sample.

[0143] Optionally, before contacting the test sample with at least one capture antibody (e.g., a first capture antibody), the at least one capture antibody may bind to a solid support, which facilitates the separation of the first antibody / analyte (or fragment thereof) complex from the test sample. The matrix to which the capture antibody binds may be any suitable solid support or solid phase that facilitates the separation of the capture antibody-analyte complex from the sample.

[0144] Examples of solid phases or supports are well known to those skilled in the art and include wells in plates (such as microwell titration plates), test tubes, porous gels (e.g., silica gel, agarose, dextran, or gelatin), polymer films (e.g., polyacrylamide), beads (e.g., polystyrene beads or magnetic beads), strips of filter paper / membranes (e.g., nitrocellulose or nylon), and microparticles (e.g., latex particles, magnetizable microparticles (e.g., microparticles with an iron oxide or chromium oxide core and a homopolymer or hybrid coating and a radius of about 1-10 micrometers)). The matrix may contain suitable porous materials with appropriate surface affinity for binding antigens and sufficient porosity to allow detection antibodies to reach them. Microporous materials are generally preferred, although hydrated gel-like materials may be used. Such porous matrices are preferably in the form of sheets having a thickness of about 0.01 to about 0.5 mm, preferably about 0.1 mm. Although pore sizes may vary considerably, preferably pore sizes are about 0.025 to about 15 micrometers, more preferably about 0.15 to about 15 micrometers. The surface of such a matrix can be activated by a chemical process that causes covalent bonding between the antibody and the matrix. The antigen or antibody binds irreversibly to the matrix, typically through adsorption via hydrophobic forces; alternatively, chemical coupling agents or other methods can be used to covalently bind the antibody to the matrix, provided that such binding does not interfere with the antibody's ability to bind analytes. Alternatively, the antibody can bind to microparticles previously treated with streptavidin (e.g., [missing information]). Beads, Invitrogen, Carlsbad, Calif.) or biotin (e.g., using Power-Bind) TM-SA-MP streptavidin-coated microparticles (Seradyn, Indianapolis, Ind.) or anti-species-specific monoclonal antibody coating. If necessary, the matrix can be derivatized to allow reactivity with different functional groups on the antibody. Such derivatization requires the use of certain coupling agents, examples of which include, but are not limited to, maleic anhydride, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. If desired, one or more capture reagents (such as antibodies (or fragments thereof), each specific to the analyte) can be attached to a solid phase at different physical or accessible locations (e.g., in a biochip configuration (see, for example, U.S. Patent No. 6,225,047; International Patent Application Publication No. WO 99 / 51773; U.S. Patent No. 6,329,209; International Patent Application Publication No. WO 00 / 56934, and U.S. Patent No. 5,242,828). If the capture reagent is attached to a mass spectrometry probe serving as a solid support, the amount of analyte bound to the probe can be detected by laser desorption / ionization mass spectrometry. Alternatively, a single column can be filled with different beads derivatized with one or more capture reagents, thereby capturing the analyte in a single location (see antibody-derived bead-based techniques, such as Luminex's (Austin, Tex.) xMAP technique).

[0145] After contacting the test sample, which is to be measured against an analyte (or a fragment thereof), with at least one capture antibody (e.g., a first capture antibody), the mixture is incubated to allow the formation of a first antibody (or multiple antibodies)-analyte (or a fragment thereof) complex. Incubation can be performed at a pH of about 4.5 to about 10.0 and at a temperature of about 2°C to about 45°C for a period of at least about one (1) minute to about eighteen (18) hours, preferably about one minute to about 24 minutes, and most preferably about four minutes to about 18 minutes. The immunoassay described herein can be performed in one step (meaning the test sample, at least one capture antibody, and at least one detection antibody are added sequentially or simultaneously to the reaction vessel) or in more than one step (such as two steps, three steps, etc.).

[0146] After forming one or more capture antibody / analyte complexes, the complexes are then contacted with at least one detection antibody under conditions that allow for the formation of one or more capture antibody / analyte / second detection antibody complexes. Although referred to as "second" antibody (e.g., second detection antibody) for clarity, in practice, when multiple antibodies are used for capture and / or detection, the at least one detection antibody can be a second, third, fourth, etc., antibody used in immunoassays. If the capture antibody / analyte complex is contacted with more than one detection antibody, then one or more capture antibody / analyte (or fragment thereof) / (multiple) detection antibody complexes are formed. Similar to the capture antibody (e.g., the first capture antibody), when at least one (e.g., the second and any subsequent) detection antibody is contacted with the capture antibody / analyte (or fragment thereof) complex, it needs to be incubated for a period of time under conditions similar to those described above to form one or more capture antibody / analyte / (second or more) detection antibody complexes. Preferably, at least one detection antibody contains a detectable marker. The detectable marker can be bound to at least one detection antibody (e.g., a second detection antibody) before, simultaneously with, or after the formation of (a first or more) capture antibody / analyte / (a second or more) detection antibody complex. Any detectable marker known in the art can be used (see the discussion above).

[0147] The detectable marker can be bound to the antibody directly or via a conjugation agent. An example of a usable conjugation agent is EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride), commercially available from Sigma-Aldrich, St. Louis, Mo. Other usable conjugations are known in the art. Methods for binding the detectable marker to the antibody are known in the art. Additionally, many detectable markers can be purchased or synthesized that already contain end groups that promote conjugation of the detectable marker to the antibody, such as CPSP-acridone ester (i.e., 9-[N-toluenesulfonyl-N-(3-carboxypropyl)]-10-(3-sulfopropyl)acridone carboxamide) or SPSP-acridone ester (i.e., N10-(3-sulfopropyl)-N-(3-sulfopropyl)-acridone-9-carboxamide).

[0148] The (first or more) capture antibody / analyte / (second or more) detection antibody complex can, but does not necessarily, separate from the rest of the test sample before quantification of the label. For example, if at least one capture antibody (e.g., a first capture antibody) is bound to a solid support (such as a well or bead), separation can be achieved by removing the fluid (of the test sample) from contact with the solid support. Alternatively, if at least a first capture antibody is bound to a solid support, it can be simultaneously contacted with the sample containing the analyte and at least one second detection antibody to form a first(multiple) antibody / analyte / second(multiple) antibody complex, after which the fluid (test sample) is removed from contact with the solid support. If at least one first capture antibody is not bound to a solid support, then the (first or more) capture antibody / analyte / (second or more) detection antibody complex does not need to be removed from the test sample to quantify the amount of label.

[0149] After forming a labeled capture antibody / analyte / detection antibody complex (e.g., a first capture antibody / analyte / second detection antibody complex), the amount of the label in the complex is quantified using techniques known in the art. For example, if an enzyme label is used, the labeled complex is reacted with a substrate of the label, which produces a quantifiable reaction such as color development. If the label is a radiolabel, the label is quantified using an appropriate method (such as a scintillation counter). If the label is a fluorescent label, it is quantified by stimulating the label with light of one color (called the “excitation wavelength”) and detecting another color emitted by the label in response to the stimulation (called the “emission wavelength”). If the label is a chemiluminescent label, it is quantified by visual inspection or by detecting the emitted light using a photometer, X-ray film, high-speed photographic film, CCD camera, etc. Once the amount of the label in the complex has been quantified, the concentration of the analyte or its fragment in the test sample is determined by an appropriate method (such as by using a standard curve generated by a series of dilutions of the analyte or its fragment with known concentrations). In addition to using a series of dilutions of the analyte or its fragments, standard curves can be generated by specific gravity determination, by mass spectrometry, and by other techniques known in the art.

[0150] In Adoption In the chemiluminescent particle determination of the analyzer, the pH of the conjugate diluent should be about 6.0 ± 0.2, the particle coating buffer should be maintained at about room temperature (i.e., from about 17 to about 27°C), the pH of the particle coating buffer should be about 6.5 ± 0.2, and the pH of the particle diluent should be about 7.8 ± 0.2. The solid content is preferably less than about 0.2%, such as less than about 0.15%, less than about 0.14%, less than about 0.13%, less than about 0.12%, or less than about 0.11%, such as about 0.10%.

[0151] FPIA is based on the principle of competitive binding immunoassay. When excited by linearly polarized light, a fluorescently labeled compound emits fluorescence with a polarization inversely proportional to its rotation rate. When a fluorescently labeled tracer-antibody complex is excited by linearly polarized light, the emitted light remains highly polarized because the rotation of the fluorophore between the absorption and emission times is restricted. When a "free" tracer compound (i.e., a compound without bound antibodies) is excited by linearly polarized light, its rotation is much faster than that of the corresponding tracer-antibody conjugate produced in a competitive binding immunoassay. FPIA is advantageous over RIA because it does not involve radioactive materials requiring special handling or disposal. Furthermore, FPIA is a homogeneous assay that can be performed easily and rapidly.

[0152] In view of the above, a method is provided for determining the presence, amount, or concentration of HCV core (or fragment thereof) in a test sample. The method includes determining the HCV core antigen (or fragment thereof) in the test sample by: (i) employing at least one of (i') an antibody, an antibody fragment capable of binding to an analyte, an antibody variant capable of binding to an analyte, an antibody variant fragment capable of binding to an analyte, or a DVD-Ig (or fragment thereof, variant, or variant fragment thereof) capable of binding to the HCV core antigen; and (ii') at least one detectable marker; and (ii) comparing the signal generated by the detectable marker, which serves as a direct or indirect indicator of the presence, amount, or concentration of the HCV core antigen (or fragment thereof) in the test sample, with a signal generated as a direct or indirect indicator of the presence, amount, or concentration of the HCV core antigen (or fragment thereof) in a control or calibrator. The calibrator is optionally part of a series of calibrators, wherein each calibrator differs from the others in the concentration of the analyte.

[0153] The method may include (i) contacting a test sample with at least one first specific binding coupler of an HCV core (or a fragment thereof), the first specific binding coupler being selected from antibodies of the present invention, fragments of such antibodies capable of binding HCV core antigens, variants of antibodies capable of binding HCV core antigens, fragments of antibody variants capable of binding HCV core antigens, or DVD-Ig (or a fragment thereof, variant, or variant fragment thereof) capable of binding HCV core antigens, thereby forming a first specific binding coupler / HCV core antigen (or a fragment thereof) complex, and (ii) contacting the first specific binding coupler / HCV core antigen (or a fragment thereof) complex with at least one second specific binding coupler of an HCV core antigen (or a fragment thereof), the second specific binding coupler being selected from detectably labeled (ii) The presence, amount, or concentration of HCV core antigen in the test sample is determined by detecting or measuring the signal generated by the detectable marker in the first specific binding partner / HCV core antigen (or fragment thereof) / second specific binding partner complex formed in (ii). This is achieved by using an anti-HCV core antibody, a fragment of an anti-HCV core antibody detectably labeled to bind HCV core antigen, a variant of an anti-HCV core antibody detectably labeled to bind HCV core antigen, a variant fragment of an anti-HCV core antibody detectably labeled to bind HCV core antigen, and a DVD-Ig (or a fragment thereof, or a variant fragment thereof).

[0154] Alternatively, the method may include contacting the test sample with at least one first specific binding partner of an HCV core (or a fragment thereof), the first specific binding partner being selected from antibodies, antibody fragments capable of binding to an HCV core, antibody variants capable of binding to an HCV core, fragments of antibody variants capable of binding to an HCV core, and DVD-Ig (or fragments, variants, or variant fragments thereof), and simultaneously or sequentially, in any of these orders, contacting the test sample with at least one second specific binding partner, the second specific binding partner being competitive with the HCV core (or a fragment thereof) for binding to at least one first specific binding partner and being selected from detectably labeled HCV cores, detectably labeled fragments of HCV cores capable of binding to first specific binding partners, detectably labeled variants of HCV cores capable of binding to first specific binding partners, and detectably labeled fragments of variants of HCV cores capable of binding to first specific binding partners. Any HCV core (or fragment thereof) present in the test sample and at least one second specific binding partner compete with each other to form a first specific binding partner / HCV core (or fragment thereof) complex and a first specific binding partner / second specific binding partner complex, respectively. The method further includes determining the presence, amount, or concentration of HCV cores in the test sample by detecting or measuring a signal generated by a detectable marker in the first specific binding partner / second specific binding partner complex formed in (ii), wherein the signal generated by the detectable marker in the first specific binding partner / second specific binding partner complex is inversely proportional to the amount or concentration of HCV cores in the test sample.

[0155] The above methods may further include diagnosing, predicting, or evaluating the efficacy of therapeutic / preventive treatments for patients receiving test samples therefrom. If the methods further include evaluating the efficacy of therapeutic / preventive treatments for patients receiving test samples therefrom, the methods may optionally further include modifying the patient's therapeutic / preventive treatment as needed to improve efficacy. The methods may be suitable for use in automated or semi-automated systems.

[0156] Regarding the assay method (and the kits used therein), commercially available anti-HCV core antibodies or methods for producing anti-HCV core antibodies as described in the literature may be employed. Commercial suppliers of various antibodies include, but are not limited to, Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.), GenWay Biotech, Inc. (San Diego, Calif.), and R&D Systems (RDS; Minneapolis, Minn.).

[0157] Typically, a predetermined level can be used as a benchmark against which to evaluate results obtained after measuring the HCV core or fragments of a test sample, for example, to detect a disease or risk of disease. Typically, in making such a comparison, the predetermined level is obtained by running a specific assay a sufficient number of times under appropriate conditions to establish an association or correlation between the presence, amount, or concentration of the HCV core and a specific stage or endpoint of a disease, disorder, or condition, or with a specific clinical marker. The predetermined level is typically obtained using assays from a reference subject (or subject population). The measured HCV core may include its fragments, its degradation products, and / or its enzymatic cleavage products.

[0158] Specifically, regarding predetermined levels, such as those used for monitoring HCV disease progression and / or treatment, the amount or concentration of the analyte or a fragment thereof can be “unchanged,” “favorable” (or “favorable change”), or “unfavorable” (or “unfavorable change”). “Increased” or “elevated” means that the amount or concentration in the test sample is higher than a typical or normal level or range (e.g., the predetermined level), or higher than another reference level or range (e.g., a previous or baseline sample). The term “decreased” or “reduced” means that the amount or concentration in the test sample is lower than a typical or normal level or range (e.g., the predetermined level) or lower than another reference level or range (e.g., a previous or baseline sample). The term “changed” means that the amount or concentration in the sample has changed (increased or decreased) compared to a typical or normal level or range (e.g., the predetermined level) or compared to another reference level or range (e.g., a previous or baseline sample).

[0159] The typical or normal level or range of HCV core antigen is determined according to standard practice. Because HCV core levels are very low in some cases, any net change that cannot be explained by experimental error or sample bias compared to the typical or normal level or range or reference level or range can be considered a so-called altered level or change. Therefore, the level measured in a particular sample will be compared with the level or range of levels determined in similar samples obtained from so-called normal subjects. In this context, a “normal subject” is, for example, an individual without a detectable disease, and “normal” (sometimes called a “control”) patients or groups are, for example, patients or groups that do not exhibit a detectable disease. Furthermore, given that HCV core is not typically found at high levels in most populations, a “normal subject” can be considered an individual without a significantly detectable increase or elevation in the amount or concentration of HCV core, and a “normal” (sometimes called a “control”) patient or group is a patient or group that does not exhibit a significantly detectable increase or elevation in the amount or concentration of HCV core. An “apparently normal subject” is a subject in which HCV core has not yet been evaluated or is currently being evaluated. The level of HCV core is referred to as "elevated" when it is normally undetectable (e.g., normal levels are zero, or in the range of about 25% to about 75% of the normal population) but is detected in a test sample, and when HCV core is present in a test sample at a level higher than normal. Therefore, among other things, this disclosure provides a method for screening subjects who have a specific disease, disorder, or condition, or who are at risk of having a specific disease, disorder, or condition. The assay method may also involve measuring other markers, etc.

[0160] Therefore, the methods described herein can also be used to determine whether a subject has HCV disease, disorder, or condition, or is at risk of developing HCV disease, disorder, or condition. Specifically, such a method may include the following steps:

[0161] (a) Determine the concentration or amount of HCV core (or fragments thereof) in a test sample obtained from the subject (e.g., using the methods described herein or methods known in the art); and

[0162] (b) The concentration or amount of the HCV core (or a fragment thereof) determined in step (a) is compared with a predetermined level, wherein if the concentration or amount of the HCV core determined in step (a) is favorable relative to the predetermined level, then the subject is determined not to have the given disease, disorder, or condition, or is not at risk of the given disease, disorder, or condition. However, if the concentration or amount of the HCV core determined in step (a) is unfavorable relative to the predetermined level, then the subject is determined to have the given disease, disorder, or condition, or is at risk of the given disease, disorder, or condition.

[0163] In addition, this article provides a method for monitoring disease progression in subjects. Preferably, the method includes the following steps:

[0164] (a) Determine the concentration or amount of HCV core in the test sample obtained from the subject;

[0165] (b) Determine the concentration or amount of HCV core in a later test sample obtained from the subject; and

[0166] (c) Compare the concentration or amount of HCV core determined in step (b) with the concentration or amount of HCV core determined in step (a). If the concentration or amount determined in step (b) is unchanged or unfavorable compared to the concentration or amount of HCV core determined in step (a), then the subject's disease is determined to have persisted, progressed, or worsened. If, by comparison, the concentration or amount of HCV core determined in step (b) is favorable compared to the concentration or amount of HCV core determined in step (a), then the subject's disease is determined to have stopped, regressed, or improved.

[0167] Optionally, the method further includes comparing the concentration or amount of HCV core as determined in step (b) with, for example, a predetermined level. Further, optionally, if the comparison indicates an unfavorable change in the concentration or amount of HCV core as determined in step (b), for example, relative to a predetermined level, then the method includes treating the subject with one or more pharmaceutical compositions for a period of time.

[0168] In other embodiments, any of the assays described herein for monitoring the presence or level of HCV core antigen can be advantageously combined with other assays that also determine HCV infection. For example, any HCV core assay method of the present invention may further include determining the level of another HCV antigen or HCV antibody against antigens other than the core protein, including, but not limited to, determining the presence of HCV core, E1, E2, NS2, NS3, NS4a, NS4b, and NS5.

[0169] Additionally, the method can be used to monitor the treatment of subjects receiving one or more pharmaceutical compositions. Specifically, such a method involves providing a first test sample from the subject before administering one or more pharmaceutical compositions to the subject. The concentration or amount of HCV core in the first test sample from the subject is then determined (e.g., using methods described herein or known in the art). After determining the concentration or amount of HCV core, optionally the concentration or amount of HCV core is then compared to a predetermined level. If the concentration or amount of HCV core determined in the first test sample is below the predetermined level, then the subject is not treated with one or more pharmaceutical compositions. However, if the concentration or amount of HCV core determined in the first test sample is above the predetermined level, then the subject is treated with one or more pharmaceutical compositions for a period of time. The duration of treatment with one or more pharmaceutical compositions can be determined by those skilled in the art (e.g., the duration can be from about seven (7) days to about two years, preferably from about fourteen (14) days to about one (1) year).

[0170] During treatment with one or more drug compositions, a second test sample and subsequent test samples are subsequently obtained from the subject. The number of test samples and the timing of obtaining said test samples from the subject are not critical. For example, a second test sample may be obtained seven (7) days after the first administration of one or more drug compositions to the subject, a third test sample may be obtained two (2) weeks after the first administration of one or more drug compositions to the subject, a fourth test sample may be obtained three (3) weeks after the first administration of one or more drug compositions to the subject, a fifth test sample may be obtained four (4) weeks after the first administration of one or more drug compositions to the subject, and so on.

[0171] After obtaining each second or subsequent test sample from the subject, the concentration or amount of HCV core in the second or subsequent test sample is determined (e.g., using methods described herein or known in the art). The concentration or amount of HCV core determined in each of the second and subsequent test samples is then compared with the concentration or amount of HCV core determined in the first test sample (e.g., a test sample initially optionally compared to a predetermined level). If the concentration or amount of HCV core determined in step (c) is advantageous when compared to the concentration or amount of HCV core determined in step (a), then it is determined that the subject's disease has stopped, subsided, or improved, and the subject should continue to receive one or more pharmaceutical compositions from step (b). However, if the concentration or amount determined in step (c) is unchanged or adverse compared to the concentration or amount of HCV core determined in step (a), then it is determined that the subject's disease has persisted, progressed, or worsened, and the subject should be treated with a higher concentration of one or more drug compositions administered to the subject in step (b), or with one or more drug compositions different from those administered to the subject in step (b). Specifically, the subject may be treated with one or more drug compositions different from those previously received by the subject to reduce or lower the subject's HCV core level.

[0172] Typically, for assays that may be replicated (e.g., monitoring disease progression and / or response to treatment), a second or subsequent test sample is obtained sometime after the first test sample has been obtained from the subject. Specifically, the second test sample obtained from the subject can be obtained minutes, hours, days, weeks, or years after the first test sample has been obtained from the subject. For example, it can be obtained approximately 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, or 17 hours after the first test sample has been obtained from the subject. Approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, approximately 6 weeks, approximately 7 weeks, approximately 8 weeks, approximately 9 weeks, approximately 10 weeks, approximately 11 weeks, approximately 12 weeks, approximately 13 weeks, approximately 14 weeks, approximately 15 weeks, approximately 16 weeks, approximately 17 weeks, approximately 18 weeks, approximately 19 weeks, approximately 20 weeks, approximately 21 weeks, approximately 22 weeks, approximately 23 weeks, approximately 24 weeks, approximately 25 weeks, approximately 26 weeks, approximately 27 weeks, approximately 28 weeks, approximately 29 weeks, approximately 30 weeks, approximately 31 weeks, approximately 32 weeks, approximately 33 weeks, approximately 34 weeks, approximately 35 weeks, approximately 36 weeks, approximately 37 weeks, approximately 38 weeks, approximately 39 weeks, approximately 40 weeks, approximately 41 weeks, approximately 42 weeks, approximately 43 weeks, approximately 44 weeks, approximately 45 weeks, approximately 46 weeks, approximately 47 weeks, approximately 4 Second trial samples were obtained from subjects at time intervals of 8 weeks, approximately 49 weeks, approximately 50 weeks, approximately 51 weeks, approximately 52 weeks, approximately 1.5 years, approximately 2 years, approximately 2.5 years, approximately 3.0 years, approximately 3.5 years, approximately 4.0 years, approximately 4.5 years, approximately 5.0 years, approximately 5.5 years, approximately 6.0 years, approximately 6.5 years, approximately 7.0 years, approximately 7.5 years, approximately 8.0 years, approximately 8.5 years, approximately 9.0 years, approximately 9.5 years, or approximately 10.0 years.

[0173] When used to monitor disease progression, the above measurements can be used to monitor the disease progression in subjects suffering from acute illness. Acute illness (also known as critical care illness) refers to an acute, life-threatening illness or other critical medical condition involving, for example, the cardiovascular or excretory systems. Typically, critical care illness refers to those conditions requiring acute medical intervention in a hospital setting (including, but not limited to, the emergency room, intensive care unit, trauma center, or other emergency care settings) or administered by nurses or other location-based healthcare personnel. For critical care conditions, repeated monitoring is typically performed over a short timeframe, i.e., minutes, hours, or days (e.g., about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days), and the initial measurement is also typically performed over a short timeframe of the onset of the disease or condition (e.g., about minutes, hours, or days).

[0174] The assay can also be used to monitor disease progression in subjects suffering from chronic or non-acute conditions. Non-critical care or non-acute conditions refer to conditions other than acute, life-threatening illnesses or other critical medical conditions involving, for example, the cardiovascular and / or excretory systems. Typically, non-acute conditions include those that are long-term or of a long duration. For non-acute conditions, repeated monitoring is usually performed over longer timeframes, such as hours, days, weeks, months, or years (e.g., approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours). Approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, approximately 6 weeks, approximately 7 weeks, approximately 8 weeks, approximately 9 weeks, approximately 10 weeks, approximately 11 weeks, approximately 12 weeks, approximately 13 weeks, approximately 14 weeks, approximately 15 weeks, approximately 16 weeks, approximately 17 weeks, approximately 18 weeks, approximately 19 weeks, approximately 20 weeks, approximately 21 weeks, approximately 22 weeks. Approximately 23 weeks, approximately 24 weeks, approximately 25 weeks, approximately 26 weeks, approximately 27 weeks, approximately 28 weeks, approximately 29 weeks, approximately 30 weeks, approximately 31 weeks, approximately 32 weeks, approximately 33 weeks, approximately 34 weeks, approximately 35 weeks, approximately 36 weeks, approximately 37 weeks, approximately 38 weeks, approximately 39 weeks, approximately 40 weeks, approximately 41 weeks, approximately 42 weeks, approximately 43 weeks, approximately 44 weeks, approximately 45 weeks, approximately 46 weeks, approximately 47 weeks, approximately 48 weeks, approximately 49 weeks, approximately 50 weeks, approximately 51 weeks, approximately 52 weeks, approximately 1 The duration of the disease or symptom is approximately 2.5 years, 2.5 years, 3.0 years, 3.5 years, 4.0 years, 4.5 years, 5.0 years, 5.5 years, 6.0 years, 6.5 years, 7.0 years, 7.5 years, 8.0 years, 8.5 years, 9.0 years, 9.5 years, or 10.0 years), and the initial determination is also usually performed over a longer timeframe of the disease or symptom onset (e.g., approximately hours, days, months, or years).

[0175] Furthermore, the above-described measurements can be performed using a first test sample obtained from the subject, wherein the first test sample is derived from a source such as urine, serum, or plasma. Optionally, the above-described measurements can then be repeated using a second test sample obtained from the subject, wherein the second test sample is derived from another source. For example, if the first test sample is derived from urine, then the second test sample can be derived from serum or plasma. The results of the measurements obtained using the first and second test samples can be compared. This comparison can be used to assess the state of the subject's disease or condition.

[0176] Furthermore, this disclosure also relates to methods for determining whether a subject susceptible to or suffering from a given disease, disorder, or condition will benefit from treatment. Specifically, this disclosure relates to HCV core companion diagnostic methods and products. Therefore, the methods for "monitoring treatment of a subject's disease" as described herein may further preferably include selecting or identifying candidates for therapy.

[0177] Therefore, in certain embodiments, the disclosure also provides a method for determining whether a subject with a given disease, disorder, or condition, or at risk of a given disease, disorder, or condition, is a candidate for the therapy. Typically, a subject is one who has experienced some symptom of a given disease, disorder, or condition, or who has actually been diagnosed with or at risk of a given disease, disorder, or condition, and / or exhibits an unfavorable concentration or amount of HCV core or fragments as described herein.

[0178] The method optionally includes the assays described herein, wherein the HCV core is assessed before and after treatment with one or more pharmaceutical compositions (e.g., particularly with drugs involved in the mechanism of action of HCV core), with immunosuppressive therapy, or by immunoadsorption therapy, or wherein the HCV core is assessed after such treatment and the concentration or amount of the HCV core is compared to a predetermined level. Adverse concentrations or amounts of the HCV core observed after treatment confirm that the subject will not benefit from further or continued treatment, while favorable concentrations or amounts of the HCV core observed after treatment confirm that the subject will benefit from further or continued treatment. This confirmation contributes to the administration of clinical studies and the provision of improved patient care.

[0179] The assay method described herein can also be used to identify compounds that improve a given disease, disorder, or condition. For example, cells expressing an HCV core can be exposed to a candidate compound. The assay method described herein can be used to compare the expression level of the HCV core in cells exposed to the compound with the expression level in control cells.

[0180] In another detection method, each binding protein as described herein can be used to detect HCV antigens in fixed tissue sections and to detect fixed cells by immunohistochemical analysis.

[0181] In addition, these binding proteins can bind to a matrix similar to CNBr-activated Sepharose and can be used for affinity purification of specific HCV proteins from cell cultures or biological tissues such as blood and liver.

[0182] The monoclonal antibodies described herein can also be used to prepare chimeric antibodies for therapeutic or other similar uses. Additionally, as discussed throughout this document, the antibodies can also be used to produce DVD-Ig molecules.

[0183] Monoclonal antibodies or fragments thereof can be provided individually for the detection of HCV core antigen. It is anticipated that combinations of monoclonal antibodies (and fragments thereof) provided herein can also be used together as components in a mixture or "mixture" of at least one anti-HCV core antibody as described herein and antibodies targeting other HCV regions (each with different binding specificities). Thus, such a mixture may include monoclonal antibodies targeting the HCV core protein as described herein and other monoclonal antibodies targeting other antigenic determinants of the HCV genome. Examples of other monoclonal antibodies useful for these anticipated mixtures include those targeting HCV C-100, HCV 33C, HCV core, HCV NS5, and / or the putative HCV ENV, disclosed in, for example, U.S. Serial No. 07 / 610,175 entitled "MONOCLONAL ANTIBODIES TO HEPATITIS C VIRUS AND METHOD FORUSING SAME", U.S. Serial No. 07 / 610,175 entitled "MONOCLONAL ANTIBODIES TO HCV 33CPROTEINS AND METHODS FOR USING SAME", U.S. Serial No. 07 / 648,475 entitled "MONOCLONAL ANTIBODIES TO HCV CORE PROTEINS AND METHODSFOR USING SAME", and others. SAME is commonly owned in U.S. Serial No. 07 / 648,473 and in a co-filed patent application entitled MONCLONAL ANTIBODIES TO HCV NS5PROTEIN AND METHODS FOR USING SAME, U.S. Serial No. 07 / 748,563, which are incorporated herein by reference. This mixture of monoclonal antibodies as described herein can be used in the assay form detailed herein, replacing the monoclonal antibody against the HCV core, and thus enabling the detection of both the HCV core and other HCV antigens.

[0184] The polyclonal antibody or fragment thereof used in the assay should specifically bind to the HCV core or other HCV proteins used in the assay, such as HCV C-100 protein, HCV 33C protein, HCV ENV, HCV E2 / NS1, or HCV NS5 protein. The polyclonal antibody used is preferably of mammalian origin; human, goat, rabbit, or sheep anti-HCV polyclonal antibodies can be used. Most preferably, the polyclonal antibody is a rabbit polyclonal anti-HCV antibody. The polyclonal antibody used in the assay can be used alone or as a mixture of polyclonal antibodies. Because the mixtures used in the assay contain monoclonal or polyclonal antibodies with different HCV specificities, they can be used for the diagnosis, evaluation, and prognosis of HCV infection, as well as for studying HCV protein differentiation and specificity.

[0185] As noted elsewhere throughout this document, the test samples that can be tested using the methods described herein include human and animal bodily fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, biological fluids such as cell culture supernatants, fixed tissue samples, and fixed cell (ceil) samples.

[0186] Indicator reagents contain a signal-generating compound (label) capable of producing a measurable signal that can be detected by an external device conjugated (linked) to a specific binding member of the HCV core. As used herein, "specific binding member" refers to a member of a specific binding pair. That is, two distinct molecules, one of which specifically binds to the second molecule by a chemical or physical means. Besides being an antibody member of a specific binding pair of the HCV core, an indicator reagent can also be a member of any specific binding pair, including hapten-anti-hapten systems such as biotin or anti-biotin, avidin or biotin, carbohydrates or lectins, complementary nucleotide sequences, effector or receptor molecules, enzyme cofactors and enzymes, enzyme inhibitors or enzymes, etc. Immunoreactive specific binding members can be antibodies, antigens, or antibody / antigen complexes capable of binding to the HCV core (e.g., in sandwich assays), capture reagents (e.g., in competitive assays), or auxiliary specific binding members (e.g., in indirect assays).

[0187] Various anticipated signal-generating compounds (labels) include chromogens, catalysts such as enzymes, luminescent compounds such as luciferin and rhodamine, chemiluminescent compounds such as acridine, phenanthrene, and dioxane compounds, radioactive elements, and direct visual markers. Examples of enzymes include alkaline phosphatase, horseradish peroxidase, and β-galactosidase. The choice of a specific label is not critical, but it will be able to generate a signal either on its own or in combination with one or more other substances.

[0188] The application of scanning probe microscopy (SPM) for immunoassays is also a technique readily adaptable to monoclonal antibodies as described herein. In SPM, particularly in atomic force microscopy, a capture phase (e.g., at least one of the monoclonal antibodies described herein) is attached to a solid phase, and antigen / antibody complexes that may be present on the surface of the solid phase are detected using a scanning probe microscope. The application of scanning tunneling microscopy eliminates the need for markers that are typically required in many immunoassay systems to detect antigen / antibody complexes. Such a system is described in pending U.S. Patent Application Serial No. 662,147, which is commonly owned and incorporated herein by reference.

[0189] The use of SPM to monitor specific binding responses can be performed in many ways. In one embodiment, a member of the specific binding partner (HCV core-specific material, which is a monoclonal antibody as described herein) is attached to a surface suitable for scanning. The attachment of the HCV core-specific material can be performed by adsorption onto a test strip comprising a solid phase of a plastic or metal surface, as known to those skilled in the art. Alternatively, the specific binding partner (HCV core-specific material) can be covalently linked to a test strip comprising a derivatized solid phase of plastic, metal, silicon, or glass. Covalent linking methods are known to those skilled in the art and include various methods for irreversibly linking the specific binding partner to the test strip. If the test strip is silicon or glass, the surface must be activated before linking the specific binding partner. Activated silane compounds such as triethoxyaminopropylsilane (available from Sigma Chemical Co., St. Louis, Mo.), triethoxyvinylsilane (Aldrich Chemical Co., Milwaukee, Wis.), and (3-mercaptopropyl)trimethoxysilane (Sigma Chemical Co., St. Louis, Mo.) can be used to introduce reactive groups such as amino-, vinyl-, and mercapto groups, respectively. Such activated surfaces can be used for direct attachment of binding partners (in the case of amino or thiol groups), or the activated surfaces can be further reacted with connectors such as glutaraldehyde, bis(succinimide) octanoate, SPPD (succinimide 3-[2-pyridyldithio]propionate), SMCC (succinimide 4-[N-maleimidemethyl]cyclohexane-1-carboxylic acid), SIAB (succinimide 4-iodoacetyl]aminobenzoate), and SMPB (succinimide 4-[1-maleimidephenyl]butyrate). Vinyl groups can be oxidized to provide covalent bonding. It can also be used as an anchor for the polymerization of various polymers, such as polyacrylic acid, providing multiple attachment sites for specific binding partners. Amino surfaces can be reacted with oxidized dextran of different molecular weights to provide hydrophilic connectors of varying sizes and capabilities. Examples of oxidizable dextran include dextran T-40 (molecular weight 40,000 Daltons), dextran T-110 (molecular weight 110,000 Daltons), dextran T-500 (molecular weight 500,000 Daltons), dextran T-2M (molecular weight 2,000,000 Daltons) (all available from Pharmacia, Piscataway, NJ), or sucrose (molecular weight 70,000 Daltons) (available from Sigma Chemical Co., St. Louis, Mo.).Furthermore, using the techniques and chemical reagents described in pending U.S. Patent Application Serial No. 150,278 (filed January 29, 1988) and Serial No. 375,029 (filed July 7, 1989) (each of which is jointly owned and incorporated herein by reference), specific binding partners can be immobilized on the surface of a test piece using polyelectrolyte interactions. A preferred attachment method is covalent attachment. After the attachment of the specific binding member, the surface can be further treated with materials such as serum, protein, or other blocking agents to minimize nonspecific binding. The surface can also be scanned at the manufacturing or usage site to verify its suitability for the assay purpose. It is anticipated that the scanning process will not alter the specific binding properties of the test piece.

[0190] While this disclosure expresses a preference for solid-phase assays, it is anticipated that monoclonal antibodies, as described herein, can be used in non-solid-phase assay systems. These assay systems are known to those skilled in the art and are considered to be within the scope of this disclosure.

[0191] It is anticipated that reagents for assays can be provided in the form of kits containing one or more containers (such as tubular vials or bottles), each containing individual reagents used in the assay, such as monoclonal antibodies or mixtures of monoclonal antibodies, detection reagents, and washing reagents.

[0192] The antibodies can also be used to enhance the immune response. They can be administered in amounts similar to those used in other therapeutic applications. For example, normal immunoglobulins are administered at 0.02–0.1 ml / lb body weight during the early incubation period of other viral diseases such as rabies, measles, and hepatitis B to interfere with viral entry into cells. Therefore, antibodies that react with the HCV core protein can be passively administered, alone or in combination with another antiviral agent, to an HCV-infected host to enhance the immune response and / or the effectiveness of antiviral drugs.

[0193] When used as a method to induce anti-HCV antibodies in animals, the antibodies are administered in the same manner as for vaccination purposes, i.e., intramuscular, intraperitoneal, subcutaneous, etc., in a physiologically appropriate diluent, with or without adjuvant, to achieve an effective concentration. One or more booster injections may be required.

[0194] Reagent test kit

[0195] This document also anticipates kits for determining the presence, amount, or concentration of HCV core protein (or a fragment thereof) in a test sample. Such kits comprise at least one component for determining the HCV core protein (or a fragment thereof) in a test sample and instructions for determining the HCV core (or a fragment thereof) in the test sample. The at least one component for determining the HCV core (or a fragment thereof) in a test sample may comprise a composition containing, or capable of being immobilized on, a monoclonal antibody against HCV core protein or an anti-HCV core protein DVD-Ig (or a fragment thereof, variant, or variant fragment thereof).

[0196] The kit may contain at least one component for determining the HCV core protein of a test sample by immunoassay (e.g., chemiluminescent microparticle immunoassay) and instructions for determining the HCV core of the test sample by immunoassay (e.g., chemiluminescent microparticle immunoassay). For example, the kit may contain at least one specific binding partner of the HCV core, such as a monoclonal / polyclonal antibody against the anti-HCV core (or a fragment thereof that can bind to the HCV core, a variant thereof that can bind to the HCV core, or a variant fragment thereof that can bind to the HCV core) or anti-HCV core DVD-Ig (or a fragment thereof, a variant thereof, or a variant fragment thereof), either of which may be detectably labeled. Alternatively or additionally, the kit may contain a detectably labeled HCV core (or a fragment of a monoclonal / polyclonal antibody or anti-HCV core DVD-Ig (or a fragment, variant, or variant fragment thereof) that can bind to an anti-HCV core, competing with any HCV core in the test sample for binding to either a monoclonal / polyclonal antibody or anti-HCV core DVD-Ig (or a fragment, variant, or variant fragment thereof), either of which may be immobilized on a solid support. The kit may also contain calibrators or controls, such as isolated or purified HCV cores. The kit may contain at least one container for performing the assay (e.g., test tube, microtiter plate, or strip, which may have been coated with, for example, a first specific binding coupler) and / or buffer (such as assay buffer or wash buffer, either of which may be provided as a concentrated solution), substrate solution or stop solution for detecting a marker (e.g., an enzyme marker). Preferably, the kit contains all the components necessary to perform the assay, i.e., reagents, standards, buffers, diluents, etc. Instructions may be in paper or computer-readable form, such as disk, CD, DVD, etc.

[0197] Any antibody (such as anti-HCV core antibody or anti-HCV core DVD-Ig) or tracer may be incorporated into detectable markers as described herein, such as fluorophores, radioactive moieties, enzymes, biotin / avidin markers, chromophores, chemiluminescent markers, etc., or the kit may include reagents for performing detectable labeling. Antibodies, calibrators, and / or controls may be provided in individual containers or pre-dispensed into appropriate assay formats, such as pre-dispensed into microtiter plates.

[0198] Optionally, the kit includes quality control components (e.g., a sensitivity test group, calibrators, and positive controls). The preparation of quality control reagents is well known in the art and is described in inserts of various immunodiagnostic products. The sensitivity test group members are optionally used to establish assay performance characteristics and are further optionally useful indicators of the integrity of the immunoassay kit reagents and the standardization of the assay.

[0199] The kit may optionally include other reagents required for diagnostic assays or to facilitate quality control evaluation, such as buffers, salts, enzymes, enzyme cofactors, enzyme substrates, and assay reagents. Other components (such as buffers and solutions) (e.g., pretreatment reagents) for separating and / or processing test samples may also be included in the kit. The kit may additionally include one or more other controls. One or more components of the kit may be lyophilized under low pressure; in this case, the kit may further contain reagents suitable for reconstructing the lyophilized components.

[0200] The various components of the kit may optionally be provided in suitable containers (e.g., microplates) as needed. The kit may further include containers for containing or storing samples (e.g., containers or tubes for urine samples). Where appropriate, the kit may also optionally contain reaction vessels, mixing containers, and other components to facilitate the preparation of reagents or test samples. The kit may also include one or more instruments for assisting in obtaining test samples, such as syringes, pipettes, forceps, measuring spoons, etc.

[0201] If the detectable marker is at least one acridine compound, then the kit may contain at least one acridine-9-carboxamide, at least one aryl acridine-9-carboxylic acid ester, or any combination thereof. If the detectable marker is at least one acridine compound, then the kit may also contain a source of hydrogen peroxide, such as a buffer, solution, and / or at least one alkaline solution. If desired, the kit may contain a solid phase, such as magnetic particles, beads, test tubes, microporous burettes, cuvettes, membranes, scaffold molecules, thin films, filter paper, disks, or chips.

[0202] The kit (or its components), and the method for determining the presence, amount, or concentration of HCV cores in a test sample by means of assays (such as immunoassays) as described herein, can be modified for use in a variety of automated and semi-automated systems (including those in which the solid phase contains microparticles), as described, for example, in U.S. Patent Nos. 5,089,424 and 5,006,309, and as, for example, as Commercially sold by Abbott Laboratories (Abbott Park, Ill.).

[0203] Some differences between automated or semi-automated systems and non-automated systems (e.g., ELISA) include the matrix to which the first specific binding partner (e.g., a monoclonal / polyclonal antibody against the anti-HCV core (or a fragment thereof, its variant, or a variant fragment thereof) or anti-HCV core DVD-Ig (or a fragment thereof, its variant, or a variant fragment thereof) attaches (either way can affect sandwich formation and HCV core reactivity), and the duration and timing of capture, detection, and / or any optional washing steps. While non-automated forms (such as ELISA) may require relatively long incubation times with the sample and capture reagent (e.g., approximately 2 hours), automated or semi-automated forms (e.g., ELISA) may require longer incubation times with the sample and capture reagent (e.g., approximately 2 hours). Abbott Laboratories may have relatively short incubation times (e.g., for...). Approximately 18 minutes). Similarly, although non-automated forms (such as ELISA) may require relatively long incubation times (e.g., approximately 2 hours) for detecting antibodies (such as conjugate reagents), automated or semi-automated forms (e.g., ... ) may have a relatively short incubation time (e.g., for (Approximately 4 minutes).

[0204] Other platforms available from Abbott Laboratories include, but are not limited to, those mentioned above. (See, for example, U.S. Patent No. 5,294,404, which is hereby incorporated in its entirety by reference.) EIA (beads) and Quantum TM II. and other platforms. Additionally, the assays, kits, and kit components can be used in other forms, for example, on electrochemical or other portable or care-site assay systems. This disclosure is applicable, for example, to commercial Abbott Point of Care systems performing sandwich immunoassays. Abbott Laboratories’ electrochemical immunoassay system. Immunosensors in single-use test devices and methods of their preparation and operation are described, for example, in the following documents: U.S. Patent No. 5,063,081, U.S. Patent Application Publication No. 2003 / 0170881, U.S. Patent Application Publication No. 2004 / 0018577, U.S. Patent Application Publication No. 2005 / 0054078, and U.S. Patent Application Publication No. 2006 / 0160164, all of which are incorporated herein by reference in their entirety for teachings relating to the same content.

[0205] Specifically, regarding HCV core assays... For system adaptation, the following configuration is preferred. A microfabricated silicon chip is fabricated using a pair of gold galvanometer working electrodes and a silver-silver chloride reference electrode. On one of the working electrodes, polystyrene beads (0.2 mm in diameter) with an immobilized anti-HCV core monoclonal / polyclonal antibody (or a fragment thereof, its variant, or a variant fragment thereof) or anti-HCV core DVD-Ig (or a fragment thereof, its variant, or a variant fragment thereof) are attached to a patterned polyvinyl alcohol polymer coating on the electrode. The chip is assembled into an I-type flow control technology suitable for immunoassays. The cartridge contains a layer on a portion of the wall of the sample retention chamber containing a specific binding partner for the HCV core (such as a monoclonal / polyclonal antibody against the HCV core (or a fragment thereof, a variant thereof, or a variant fragment thereof that can bind to the HCV core) or an anti-HCV core DVD-Ig (or a fragment thereof, a variant thereof, or a variant fragment thereof that can bind to the HCV core, either of which can be detectably labeled). The fluid pouch of the cartridge contains an aqueous reagent comprising p-aminophenol phosphate.

[0206] During operation, a sample suspected of containing HCV cores was added to the retention chamber of the test tube, and the tube was inserted into I- In the reader, after the specific binding coupler of the HCV core has dissolved in the sample, a pump element within the cartridge forces the sample into a conduit containing the chip. Here, it is agitated to promote sandwich formation. In the penultimate step of the assay, fluid is expelled from the bag and into the conduit to wash the sample out of the chip and into the waste chamber. In the final step of the assay, an alkaline phosphatase label reacts with p-aminophenol phosphate to cleave the phosphate groups and allow the released p-aminophenol to be electrochemically oxidized at the working electrode. Based on the measured current, the reader is able to calculate the amount of HCV core in the sample using an embedded algorithm and a factory-defined calibration curve.

[0207] It goes without saying that the methods and kits described herein necessarily include other reagents and methods for performing immunoassays. For example, this includes various buffers, such as those known in the art and / or readily prepared or optimized for, for example, washing, use as conjugate diluents, particulate diluents, and / or calibrator diluents. An exemplary conjugate diluent is one used in certain kits (Abbott Laboratories, Abbott Park, Ill.) and contains 2-(N-morpholino)ethanesulfonic acid (MES), salts, protein blocking agents, antimicrobial agents, and detergents. Conjugate diluent. An exemplary calibrator diluent is used in certain kits (Abbott Laboratories, Abbott Park, Ill.). Human calibrator diluents contain a buffer solution containing MES, other salts, protein blocking agents, and antimicrobial agents. Additionally, as described in U.S. Patent Application No. 61 / 142,048, filed December 31, 2008, improved signal generation can be achieved, for example, in the form of an I-Stat tube, by using a nucleic acid sequence linked to a signal antibody as a signal amplifier.

[0208] Those skilled in the art will readily recognize that other suitable modifications and improvements to the methods of the invention described herein are apparent and can be made using suitable equivalents without departing from the scope of the invention or the embodiments disclosed herein. The invention has now been described in detail and will be more clearly understood by referring to the following examples, which are included for illustrative purposes only and are not intended to be limiting of the invention.

[0209] Example

[0210] Example 1 .

[0211] Animal immunization.

[0212] Female CAF1 / J and RBF / DnJ mice (obtained from The Jackson Laboratory, Bar Harbor, Maine) were immunized at weeks 0, 4, and 10 with 50 μg of HCV core peptides corresponding to amino acids (all numbered according to HCV-1) 134-171, covalently linked to BSA (ALRZ-8 immunogen).

[0213] HCV core peptide-BSA was prepared by AnaSpec, Inc. (Fremont, CA). The immunogenic peptide was emulsified in complete or incomplete Adjulite Freund's adjuvant (Pacific Immunology, Ramona, CA). Complete Freund's adjuvant was used for the primary immunization, and incomplete Freund's adjuvant was used for the second and third immunizations. Each inoculum was prepared as follows: HCV peptide-BSA was first diluted to an appropriate concentration in sterile saline (0.9% sodium chloride), an equal volume of adjuvant was added, and then the mixture was passed back and forth between two syringes via a 3-way plunger until a thick, stable emulsion was formed. Serum samples were collected 10–14 days after the third immunization. On days 4 and 3 prior to B cell harvest, RBF / DnJ mice #306 and 315 were administered 50 μg of peptide-BSA diluted in sterile saline. The inoculum was delivered into the body cavity near the spleen.

[0214] ALRZ-8 Immunogen

[0215]

[0216] Example 2.

[0217] Screening for antigenic immunoreactivity in mouse serum.

[0218] First, the reactivity of mouse serum samples collected 7–10 days after final immunization to each of the three synthetic (Anaspec, Inc.) carboxyl-terminated biotinylated HCV core peptides was tested in a 96-well microtitrative enzyme immunoassay (EIA). The peptides used for screening were derived from the immunogen sequences described in Example 1 and had the following names and sequences: Peptide 1 (all numbered according to HCV-1), amino acids 134–151: MGYIPLVGAPLGGAARALAHG; Peptide 2, amino acids 141–161: GAPLGGAARALAHGVRVLEDG; Peptide 3, amino acids 151–171: LAHGVRVLEDGVNYATGNLPG. Assay plates (NUNC Corporation, Naperville, IL) were coated with 100 μL / well of sheep anti-mouse IgG Fc-specific antibody (Jackson Immuno Research, West Grove, PA) diluted to 2 μg / mL in phosphate-buffered saline (PBS). Incubate the plates at 37°C for approximately 2 hours, then at approximately 21°C for approximately 2 hours. Remove the capture antibody and add 200 μL / well of blocking solution (3% w / v [weight / volume] bovine serum albumin (BSA) and 0.5% (v / v) [volume / volume] polysorbate-20 diluted in PBS). Incubate the plates for approximately 30 minutes, then wash with distilled water. Next, add a series of dilutions of mouse serum or positive control (in the blocking solution) to the assay plates (100 μL / well), incubate for 2–20 hours, then wash with dH2O. Then, add 100 μL / well of normal serum solution (NSS; blocking solution containing 2% (v / v) normal mouse serum) for additional blocking. This solution helps prevent nonspecificity in the assay wells. Binding. Incubate the plates for approximately 30 minutes, then wash with dH2O. Subsequently, add 100 μL / well of a 224 nM solution of each peptide to the assay wells for a brief incubation, followed by washing the plates with dH2O (to test the reactivity of serum samples to each peptide (not a mixture of all three)). Next, add 100 μL / well of horseradish peroxidase-labeled streptavidin (Jackson ImmunoResearch) diluted to 200 ng / mL in blocking solution, incubate for approximately 30 minutes, and then wash the plates; o-phenylenediamine substrate (OPD) is used as a chromophore to generate the signal, and the reaction is quenched using 1N sulfuric acid. Readout the signal at a wavelength of 492 nm.

[0219] Example 3.

[0220] Screening for relative affinity of mouse serum.

[0221] Relative affinity assays were performed on each serum sample-peptide combination for which strong signals were observed in previous assays. To determine the relative affinity of each serum sample for each core peptide, the reactivity of the sample to each biotin-labeled peptide at limited concentrations was tested. The assay format was the same as described above, except that instead of preparing serial dilutions of mouse serum test samples, each sample was prepared at a single dilution in a blocking solution. Furthermore, various peptides were tested at different concentrations, starting with a 500 nM solution in the blocking solution, followed by 10 log 2 dilutions, also in the blocking solution. Binding curves were prepared and used to determine the relative affinity of each serum sample. Based on these results, RBF / DnJ mice #306 and 315 were selected for B-cell fusion.

[0222] Example 4.

[0223] Mouse spleen cell fusion.

[0224] On the day of fusion, mice were euthanized, and their spleen cells were harvested and placed in Iscoves Modified Dulbeccos Medium (IMDM) supplemented with Pen Strep (Invitrogen Corporation). Cell fusion was performed as described by Kohler and Milstein (Nature 1975; 256: 495-7). Each mouse spleen was placed in a culture dish containing IMDM. Using a syringe containing IMDM and a cell scraper, spleen cells were perfused from each spleen. All spleen cells obtained from mice #306 and 315 were isolated and collected in 50 ml centrifuge tubes, and then counted using a hemocytometer with trypan blue dye exclusion to determine viability. A total of approximately 8.0 x 10⁸ cells were recovered from these spleens. 8 The cells had an 89% viability. Based on their physical appearance under a microscope, the estimated number is approximately 7.6 x 10⁻⁶. 6 10 cells / ml is defined as B-cells. Approximately 5 mL of this cell suspension was used for the first fusion experiment (fusion 208A), and the remaining cells were treated using Magnetic Activated Cell Sorting (MACS) and the Pan B-cell Isolation Kit (Miltenyi Biotech) to enrich the B-cell population and remove other cell types. Following the manufacturer's instructions, a total of approximately 6.7 x 10 cells / ml was collected. 8One cell was incubated with a mixture of Pan B-cell biotin-labeled antibodies, followed by incubation with anti-biotin microbeads. The cell suspension / microbead mixture was washed by centrifugation and passed through a Miltenyi Biotech LS column containing a magnetic field. B-cells flowed freely through the column, while other cell types were retained. The column was washed three times with PBS containing 2% FBS to remove all B-cells. The B-cell suspension was centrifuged, and the precipitate was resuspended in IMDM and then counted using a hemocytometer. Approximately 1.4 x 10⁻⁶ cells were recovered from the enrichment process. 8 7.0 x 10⁻⁶ B cells. Approximately 7.0 x 10⁻⁶ cells will be obtained from this suspension. 7 One B-cell was used for the second fusion experiment (fusion 208B), and the remaining B-cells were cryopreserved for future use.

[0225] Non-enriched spleen cells obtained from the spleen (for fusion of 208A ~3.8x10) 7 (7.0 x 10⁻⁶ B-cells) and enriched B-cell aggregates (for fusion of 208B cells) 7B-cells were washed by centrifugation in separate tubes, and the cell pellet was resuspended in IMDM. These spleen cells were mixed with an equal number of NS / O myeloma cells and centrifuged into the pellet. Fusion was achieved by exposing the spleen cells and NS / O cells to 50% polyethylene glycol (PEG) in HSFM (USC Type Culture Collection - molecular weight 1300-1600). 1 mL of PEG solution was added to each cell pellet over 30 seconds, followed by incubation for another minute. The PEG and cell pellet were diluted by slowly adding 30 mL of IMDM over 30 seconds. The fused cells were then removed from the suspension by centrifugation and decanting the supernatant. Cell pellets from each fusion (208A and 208B) were resuspended in ~250 mL of IMDM supplemented with ~10% FBS (Hyclone Laboratories), HAT (hypoxanthine, aminopterin, thymidine) (Sigma Laboratories), HT supplement (Invitrogen Corporation), BM Condimed H1 (Roche Applied Science), cholesterol, and L-glutamine (Invitrogen Corporation) for hybridoma selection. Fusion cells were seeded into T162 culture flasks containing HAT medium and cultured in batches (in bulk) at 37°C and 5% CO2 for approximately 48 hours. After 48 hours of HAT selection, the batch cultures were centrifuged, and the pellets were resuspended in semi-solid tissue culture medium. The semi-solid tissue culture medium consisted of a 50% mixture of 2X IMDM (Invitrogen) and Clone Matrix (Molecular Devices) supplemented with 10% FBS, HT supplement, Penn / Strep, L-glutamine, and anti-mouse IgG-FITC Clone Detect (Molecular Devices). The semi-solid culture plates were incubated for 7–10 days, followed by colony selection on ClonepixFL (Molecular Devices). Colonies grown on the semi-solid medium were considered clones because their individual cells were not yet allowed to move and mix with other cells during growth; however, all target cell lines would be subcloned at a later date to ensure colony-forming ability. An immunoprecipitation reaction occurred between the antibodies produced by the colonies and the fluorescent goat anti-mouse IgG Fc-FITC. The brighter the observed fluorescence signal, the more antibodies produced. Colony fluorescence was analyzed on ClonepixFL, and colonies with the brightest fluorescence signal were selected for automated transfer to 96-well tissue culture plates containing IMDM supplemented with 10% FBS, HT supplement, cholesterol, L-glutamine, and Pen Strep.Allow 96-well tissue culture plates to grow at 37°C for 3-7 days, then screen the supernatant based on antibody production.

[0226] Example 5.

[0227] Hybridoma screening and selection using peptides.

[0228] Anti-HCV antibodies in cell supernatant samples were analyzed by EIA. Sheep anti-mouse IgG Fc (Jackson Immunoresearch) was coated at 1 μg / mL onto 96-well microtiter EIA plates. After the capture reagent was coated onto the solid phase, the remaining solution was removed, and the plates were blocked with 3% BSA in PBS. The wells were washed with distilled water, and cell supernatant was added to the blocked plates, allowing incubation at room temperature for at least 1 hour. Anti-mouse IgG Fc captured anti-HCV mouse antibodies from the supernatant. After incubation, the plates were washed with distilled water. 3% normal mouse serum in BSA blocking solution was added to all wells, and the plates were incubated at room temperature for 30 minutes to block any unbound sheep anti-mouse IgG Fc capture sites coated on the plates. The wells were washed with distilled water, and a mixture of biotinylated HCV peptides (i.e., amino acids 134-154, 141-161, and 151-171 corresponding to HCV-1) was added at 100 ng / mL each, and incubated at room temperature for 30 minutes. Following this incubation, the biotinylated antigen was washed from the plate with distilled water, and streptavidin-HRPO (Jackson Immunoresearch) diluted to approximately 200 ng / mL was added to the plate and incubated for another 30 minutes. The plate was washed with distilled water, and an o-phenylenediamine substrate was used as a chromophore to generate a signal. The plate was readout at 492 nm, and the results were analyzed. A well was considered positive if it had an EIA signal at least 3 times larger than the background. Positive wells were amplified into 24-well plates in IMDM supplemented with 10% FBS, HT supplement, cholesterol, and L-glutamine.

[0229] After 5–14 days of growth, the 24-well cultures were evaluated by EIA in the same manner as described above, but with contrast to each biotinylated HCV core peptide (individually) and BSA titration of the supernatant sample to identify clones that might nonspecifically bind peptides or blocking proteins. 24-well cultures that produced a signal at least 5 times greater than the average BSA background value of 0.08 OD units with at least one screening peptide were considered positive and selected for further evaluation. Values ​​are listed in Table 1.

[0230] Table 1

[0231]

[0232]

[0233]

[0234]

[0235] Example 6.

[0236] Cloning and expression of recombinant HCV core 1-169.

[0237] The nucleotide sequence encoding amino acids 1-169 of HCV-1 was codon-optimized for expression in *E. coli* and cloned into a modified pET32a vector, in which the sequence encoding the thioredoxin fusion protein was eliminated and replaced with methionine (M). Additionally, a C-terminal hexahistine tag was included immediately after codon 169 of the HCV core to facilitate purification via immobilized metal affinity chromatography (IMAC). *E. coli* BL21(DE3) cells were transformed with the purified plasmid DNA, and clones carrying plasmid pET-HCV core 1-169 were identified. The protein expressed from these clones was named HCV core 1-169.

[0238] Protein expression was achieved by culturing pET-HCV core 1-169-transformed *E. coli* BL21(DE3) cells in terrific broth (TB) medium. Cells were cultured in a fermenter to an OD600 nm of 10, then induced with 1 mM IPTG and incubated at 37°C for approximately 3 hours until an OD600 nm of approximately 20 was obtained. Cells were harvested by centrifugation and lysed by sonication in a 25 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl, 1 mM DTT, 5 mM MgCl2, lysozyme, and benzonase. The lysates were clarified by centrifugation, and the insoluble fractions were dissolved in a 25 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl, 6 M urea, 1.0% n-dodecyl-β-D-maltose glycoside, 1 mM DTT, and 5 mM MgCl2. The dissolved lysate was clarified again by centrifugation, and the soluble fraction was loaded onto a HisTrap Fast Flow column (GE Healthcare). The column was then washed with 25 column volumes of 25 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl, 1 mM DTT, 5 mM MgCl2, 6 M urea, 0.1% n-dodecyl-β-D-maltose glycoside, and 10 mM imidazole. Elution was performed using the same buffer and a linear gradient (0–500 mM) of imidazole. The eluted fractions containing the desired target protein (as determined by SDS-PAGE) were combined and dialyzed in 25 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl, 1 mM DTT, 5 mM MgCl2, with and without 6 M urea and 0.1% n-dodecyl-β-D-maltose glycoside.

[0239] HCV core nucleotide sequence 1-169

[0240]

[0241] HCV core amino acid sequence 1-169

[0242]

[0243] Example 7.

[0244] Hybridoma screening using core antigens.

[0245] The ability of 24-well cultures to bind directly coated HCV core 1-169 onto microtiter plates was then evaluated by EIA (as described in Example 6) (solid phase assay). HCV core 1-169 was coated at 1 μg / mL onto 96-well microtiter EIA plates. After the capture reagent was coated onto the solid phase, the solution was removed, and the plates were blocked with a solution of 3% BSA in PBS. The wells were washed with distilled water, and a 5-fold serial dilution of cell culture supernatant was added, and the plates were incubated at room temperature for at least 1 hour. The plates were washed with distilled water, and HRP-labeled goat anti-mouse IgG FC antibody diluted at approximately 200 ng / mL in BSA blocking solution was added to the plates, allowing incubation at room temperature for 30 minutes. The plates were washed with distilled water, and an o-phenylenediamine substrate was used as a chromophore to generate a signal. The plates were readout at 492 nm, and the results were analyzed.

[0246] Antibodies with a BSA background reactivity greater than or equal to the Core 1-169 reactivity value are considered negative and are not used to calculate the average BSA background value. For remaining antibodies to be considered Core 1-169 positive, they must have an EIA signal of at least 0.50 OD units, or an EIA signal at least 5 times greater than the average BSA background signal of 0.10 OD units. Values ​​are listed in Table 2.

[0247] Table 2

[0248]

[0249]

[0250]

[0251]

[0252] Example 8.

[0253] Hybridoma screening via core antigen capture assay.

[0254] Cell lines identified as positive in a 24-well stage by peptide-based EIA (Example 5) or HCV core 1-169 solid-phase immunoassay (Example 7) were expanded for cryopreservation, followed by preparation of high-density depleted cell supernatants. The high-density depleted supernatants obtained from fusion 208A and 208B cell lines were tested to assess the ability of their detection monoclonal antibody (14-153-229, U.S. Patent 7,858,752) to capture HCV core 1-169 from solution. This monoclonal antibody targets an epitope within the nucleic acid-binding domain (e.g., amino acids 1-125) of the HCV core, also referred to as domain 1. Anti-domain 1 monoclonal antibody was coated at 1 μg / mL onto the solid phase of a 96-well microtiter EIA plate. After the capture reagent was coated onto the solid phase, it was removed, and plates were blocked for 30 minutes at room temperature using 5X PBS buffer containing 2% fish gelatin, 0.5% Tween 20, and 0.1% n-dodecyl-N,N-dimethylamine-N-oxide (Affymetrix). The plates were washed with distilled water, and a 50 ng / ml solution of core 1-169 antigen diluted in fish gelatin / detergent solution was added to all wells, and the plates were incubated at room temperature for at least 30 minutes. The wells were washed with distilled water, and cell supernatant was titrated onto the blocked plates, and the plates were incubated at room temperature for at least 30 minutes. The plates were washed with distilled water, and HRP-labeled goat anti-mouse IgG FC antibody diluted to approximately 200 ng / ml in BSA blocking solution was added to the plates, and the plates were incubated at room temperature for 30 minutes. The plates were washed with distilled water, and an o-phenylenediamine substrate was used as a chromophore to generate a signal. The plates were read out at 492 nm, and the results were analyzed. An antibody is considered positive for Core 1-169 if it has an EIA signal of at least 0.50 OD units, or an EIA signal at least 5 times greater than the average BSA background signal of 0.10 OD units. Values ​​are listed in Table 3.

[0255] Table 3

[0256]

[0257]

[0258]

[0259] Example 9.

[0260] Determination of the binding kinetics of the anti-HCV core antibody.

[0261] The affinity / kinetics of the anti-HCV core peptide 134-171 monoclonal antibody was determined using a Biacore 4000 instrument (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). First, after pretreating the CM5 Series S biosensor chip (GE Healthcare) with a double-injection solution of 100 mM HCl, 50 mM NaOH, and 0.1% SDS, a rabbit anti-mouse IgG capture biosensor was prepared by amine-conjugating a rabbit anti-mouse IgG antibody (GE Healthcare, Piscataway, NJ) to spots 1, 2, 4, and 5 in all four flow cells of the biosensor chip via the EDC / NHS / ethanolamine chemical reagents provided in the amine conjugation kit (GE Healthcare). The clarified hybridoma supernatant depleted of anti-HCV core antibody and HCV core peptide were diluted in a filtration buffer consisting of 10x HBS-EP+ buffer (GE Healthcare; hereinafter referred to as “running buffer”) diluted 10-fold in distilled water, supplemented with 0.1% BSA and 0.1% CM-glucan, and filtered through a 0.2 μm filter. Each HCV core antibody supernatant was diluted 1:1 with the running buffer and filtered again through a 0.2 μm filter. A 53-amino acid custom peptide (ALRZ-9 peptide, Anaspec, Fremont, CA) containing HCV core residues 134-171 and a carboxyl-terminal tetanus toxoid (TT) immunogenic T-cell epitope peptide (Eur. J. Immunol. (1989), 19: 2237-2242) with its terminal amino and carboxyl groups acetylated and amidated, respectively. The low-pressure lyophilized HCV core 134-171-tetanus toxoid synthetic peptide immunogen was diluted in distilled water to a stock concentration of 0.7 or 1 mg / mL, and further diluted in running buffer to concentrations of 0.457–3,000 nM or 0.412–2,700 nM, both using a 3-fold dilution series. All antigen solutions were filtered through a 0.2 μm filter before use.

[0262] The HCV core 134-171-TT peptide was administered as follows: 10 μL of HCV core antibody was individually injected at 10 μL / min onto spots 1 and 5 in all four flow cells. After all spots contained the captured antibody, the flow rate was increased to 30 μL / min, and the biosensors were equilibrated at this new flow rate for 2 min; then, 90 μL of HCV core peptide was injected for 3 min, followed by 4 min of run buffer. All biosensor surfaces were regenerated by a single injection of 30 μL of 10 mM glycine (pH 1.7) (GE Healthcare) at a flow rate of 10 μL / min. All concentrations were tested in duplicate. Binding kinetics (binding and dissociation) were monitored via sensor maps during antigen injection and subsequent run buffer administration. The sensor maps were double-referenced and fitted to a 1:1 binding model using Biacore 4000 evaluation software (GE Healthcare Bio-Sciences AB) to determine binding and dissociation rates, as well as total K. D Kinetic and affinity values ​​are listed in Table 4. If a value is not present, then the binding kinetics cannot be determined, or the antibody will not react with the HCV core 134-171-TT peptide in this assay.

[0263] ALRZ-9 peptide

[0264]

[0265] Table 4

[0266]

[0267]

[0268]

[0269] Example 10

[0270] BIAcore antibody binding pair analysis using nucleic acid binding domain mAbs

[0271] Using a Biacore 4000 instrument (GE Healthcare Bio-Sciences AB), the ability of anti-HCV core peptide 134-171 monoclonal antibody to form antibody-antibody binding pairs with anti-HCV core C11-3, C11-7, C11-9, and C11-14 (US Patent 6,727,092; Morota et al., J. Virol. Meth., 2009, 157: 8-14) antibodies and recombinant HCV core 1-169 antigen was determined. First, after pretreating the CM5 Series S biosensor chip (GE Healthcare) with a double injection of 100 mM HCl, 50 mM NaOH and 0.1% SDS, the rabbit anti-mouse IgG Capture Biosensor was prepared by amine-conjugating rabbit anti-mouse IgG antibody (GE Healthcare, Piscataway, NJ) to spots 1, 2, 4 and 5 in all four flow cells of the biosensor chip via the EDC / NHS / ethanolamine chemical reagents provided in the amine conjugation kit (GE Healthcare).

[0272] The clarified hybridoma supernatant depleted of anti-HCV core (peptide amino acid 134-171) antibody, recombinant HCV core 1-169 antigen, three different purified mouse monoclonal IgGs (representing isotypes IgG1, IgG2a, and IgG2b that do not react with the HCV core used as a blocking agent), and anti-HCV core C11-3, C11-7, C11-9, and C11-14 mAb were diluted into a filtered running buffer (hereinafter “running buffer”) consisting of 10x PBS buffer (GE Healthcare) diluted 5-fold in distilled water, supplemented with 3 mM EDTA, 0.1% BSA, 0.1% CM-dextran, 0.1% n-dodecyl-N,N-dimethylamine-N-oxide, an additional 500 mM NaCl, and filtered through a 0.2 μm filter. Each HCV core antibody supernatant was diluted 1:1. Recombinant HCV core 1-169 antigen was diluted to 500 nM according to the calculated dimer molecular weight (39,453 Da). Purified monoclonal antibodies against HCV core C11-3, C11-7, C11-9, and C11-14 were individually diluted to 20 μg / mL. Three mouse IgG blocking reagents were diluted in running buffer to a library, with each isotype having a concentration of at least 100 μg / mL. All dilutions were filtered through a 0.2 μm filter before use.

[0273] The HCV core antibody-antigen-antibody sandwich procedure is as follows: 20 μL of HCV core C11 antibody is injected at a rate of 10 μL / min into spots 1 and 5 of all four flow cells: C11-3 in flow cell 1, C11-7 in flow cell 2, C11-9 in flow cell 3, and C11-14 in flow cell 4. The flow rate is increased to 30 μL / min, and the remaining available anti-mouse IgG binding sites on the biosensor are blocked by injecting 60 μL into spots 1 and 2 of all flow cells, followed by another 60 μL into spots 4 and 5 of all flow cells. 60 μL of HCV core 1-169 antigen is injected into spot 1 of all flow cells, followed by another 60 μL into spot 5 of all flow cells. 60 μL of supernatant diluted with an HCV core antibody was injected into spots 1 and 2 of all flow cells, and another diluted supernatant was injected into spots 4 and 5 of all flow cells. The flow rate was reduced to 10 μL / min, and all biosensor surfaces were regenerated with a single 30 μL injection of 10 mM glycine (pH 1.7) (GE Healthcare).

[0274] The Biacore 4000 epitope mapping software module (GE Healthcare Bio-Sciences AB) was used to assess the binding response of each C11 antibody, antigen, and HCV core antibody supernatant after each injection, and to calculate expected response reference values ​​using the molecular weights of the dimeric antigen and antibody (150,000 Da). Expected percentage binding values ​​were determined for each sample relative to the reference values. Any expected percentage binding value greater than 5.0 was considered positive for antibody sandwich formation with recombinant HCV core 1-169 antigen. The expected percentage values ​​are listed in Table 5.

[0275] Table 5

[0276]

[0277]

[0278]

[0279]

[0280] Example 11.

[0281] Immunoglobulin purification and labeling.

[0282] Anti-HCV core hybridomas were propagated in serum-free hybridoma medium (Invitrogen Corporation) supplemented with L-glutamine and 10% ultra-low IgG FBS, and grown at approximately 0.5 x 10⁻⁶ E⁻¹. 5 Seeds were placed into roller flasks at 1 cell / mL. The culture was incubated at 37°C for 10–14 days, or until the final target culture was obtained, while rotating at approximately 1 rpm. The final roller flask supernatant was harvested and clarified using a 0.45 μm filter. The clarified supernatant was diluted with an equal volume of 1.5 M glycine, 3 M NaCl buffer (pH 8.9) and loaded onto a pre-equilibrated 5 mL Protein A column using an AKTA automated purification system (Amersham / Pharmacia / GE). The column was then washed with approximately 5 column volumes of binding buffer, and at a stable baseline, the mAb was eluted with 0.1 M sodium citrate buffer (pH 2.8). The IgG was then transferred to a desalting column and exchanged for PBS (pH 7.2–7.4), followed by further dialyzing in PBS (pH 7.2–7.4) using a 10,000 molecular weight cutoff dialysis membrane (Pierce Chemical). The selected antibody was biotinylated with a 20-fold molar excess of sulfonyl-NHS-LC-biotin (Pierce) and incubated at room temperature for 30 minutes. Unbound biotin was removed by dialyzing in PBS (pH 7.2–7.4). All biotinylated monoclonal antibodies were tested by EIA to confirm successful labeling.

[0283] Example 12.

[0284] HCV core antigen capture assay.

[0285] The ability of purified anti-HCV core 134-171 monoclonal antibodies to form binding pairs with themselves and two other domain 1 monoclonal antibodies was evaluated in EIA form using the HCV core 1-169 recombinant antigen. Anti-HCV domain 1 monoclonal antibodies C11-7 and C11-9 and anti-HCV core 134-171 monoclonal antibody were coated onto microtiter plates at approximately 1000 ng / mL and allowed to incubate overnight at 2–8 °C. After the capture reagent was coated onto the solid phase, the plates were blocked with 5X PBS buffer containing 2% fish gelatin, 0.5% Tween 20, and 0.1% n-dodecyl-N,N-dimethylamine-N-oxide. The wells were washed with distilled water, and purified core 1-169 antigen (in serial dilutions of 50–0.78 ng / mL in fish gelatin blocks) was added to the blocked plates, which were then allowed to incubate at room temperature for approximately 30 minutes. Wash the wells with distilled water and add biotin-labeled anti-HCV core monoclonal antibody to the plates at a concentration of 100-5000 ng / mL, then incubate at room temperature for 30 minutes. Wash the plates with distilled water and add streptavidin-HRPO diluted to approximately 200 ng / mL, allowing incubation at room temperature for 30 minutes. Wash the plates with distilled water and use o-phenylenediamine substrate as a chromophore to generate a signal, and measure the optical density at 492 nm.

[0286] Table 6 summarizes the assay signal (OD492 nm) for each antibody pair combination using 25 ng / ml core 1-169 antigen, indicating whether each binding pair was able to form a sandwich. An OD492 value at least 3 times larger than that produced by the negative control (NC) monoclonal antibody used as a capture or conjugation reagent was considered positive for core antigen detection.

[0287] Table 6

[0288]

[0289] Example 13.

[0290] The sequence of variable structural domains of the anti-core 134-171.

[0291] A subset of anti-HCV core 134-171 hybridomas was selected to determine the nucleotide and inferred amino acid sequences of the variable heavy chain (VH) and variable light chain (VL). Total RNA was extracted from hybridoma cells using Trizol (Invitrogen) or Tri-Reagent (Sigma) according to the manufacturer's recommendations. Heavy and light chain cDNAs were prepared from the extracted total RNA using Superscript III (Life Technologies) and oligodT primers according to a standard protocol. The variable heavy and light chain cDNA sequences were amplified using the 5′RACE (rapid amplification of cDNA ends) protocol using dC anchoring primers (5'-AAGCAGTGGTATCAACGCAGAGTACCCCCCCCCCCCCCCCCC-3') and universal primers specific to the constant regions of the mouse heavy or light chain (Novogen). The amplicon was cloned into a commercially available vector (pCR2.1-TOPO cloning kit, Invitrogen) according to the manufacturer's instructions and transformed into TOP10 E. coli. At least eight colonies were selected for PCR amplification of the cloned variable domain sequences using M13 forward and reverse primers. Amplicon treatment with ExoSap (Affymetrix) was performed before sequencing using the M13 forward primer and the BigDye Terminator v3.1 Cyclic Sequencing Kit (Applied Biosystems, Foster City, CA). Sequences were obtained using an ABI 3130x1 automated sequencer and assembled and analyzed using Vector NTI software (Invitrogen).

[0292] The inferred amino acid sequences were aligned using ClustalW (Higgins et al., Nucleic Acids Res. 22: 4673-4680, 1994), implemented in the MEGA5 software package (Tamura et al., Molecular Biology and Evolution 28: 2731-2739, 2011). Groups or clusters of related heavy chain amino acid sequences derived from alignments and phylogenetic tree construction (using the Neighbor Joining method, with complete deletion of sequence gaps during alignment) were determined using MEGA5 software. The reliability of the tree topology, and consequently the reliability of the clusters or groups within it, was examined using a guide experiment derived from 1000 copies. As a general rule, if the guide value for a given internal branch is 95% or higher, then the topology at that branch is considered "correct" (Nei and Kumar, Molecular Evolution and Phylogenetics, 2000; Oxford University Press, New York). Analysis of the heavy chain variable domain sequences from 52 anti-HCV core 134-171 monoclonal antibodies revealed the presence of four master groups with guide values ​​>95%. Antibodies containing three of these groups exhibited specificity for binding to one of each peptide used for screening: group B containing peptide 1 (134-154), group A containing peptide 2 (141-161), and group C containing peptide 3 (151-171). Figure 2 Two representations of tree topology are provided.

[0293] Description of the sequence in Appendix A

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Claims

1. A monoclonal antibody that specifically immunizes against the lipid-binding domain of an HCV core antigen, wherein the monoclonal antibody has a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain having the amino acid sequence of SEQ ID NO: 6 and the light chain variable domain having the amino acid sequence of SEQ ID NO:

306.

2. An immunoassay reagent comprising the monoclonal antibody of claim 1, wherein the antibody is labeled with a detectable marker.

3. An immunoassay reagent comprising the monoclonal antibody of claim 1, wherein the antibody is bound to a solid phase.

4. The immunoassay reagent according to claim 2, wherein the immunoassay reagent further comprises an additional antibody against the HCV antigen.

5. The immunoassay reagent according to claim 4, wherein the additional antibody is an additional anti-core antigen antibody.

6. Use of the antibody according to claim 1 in the preparation of an immunoassay apparatus for detecting HCV in a test sample, wherein the immunoassay apparatus comprises: (i) A first antibody against the HCV core antigen, which is used to contact a test sample suspected of containing HCV to form a complex between the first antibody and the antigen located within the test sample; and (ii) The antibody according to claim 1, which is used to contact the complex formed in (i) to form a complex between the antibody and the antigen in the complex formed in (i), wherein the antibody is detectably labeled.

7. The use according to claim 6, wherein the first antibody targets the DNA-binding domain of the HCV core antigen.

8. The use according to claim 6, wherein the antibody in (ii) is labeled with a fluorescent marker.

9. The use according to claim 8, wherein the marker is acridine mononitrate.

10. The use according to claim 6, wherein the antibody of (i) is bound to the solid phase.

11. The use according to claim 6, wherein the antibody in (i) is different from the antibody in (ii).

12. The use according to claim 6, wherein the use is suitable for use in automated or semi-automated systems.

13. A kit comprising the immunoassay reagent of claim 2 and instructions for use of the immunoassay reagent in an immunoassay for detecting HCV in a test sample.

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