Invented by Robert Ziemann, April Ahlberg, David Hawksworth, Bryan Tieman, A. Scott Muerhoff, Christopher Marohnic, Kathy Otis, Abbott Laboratories

The market for detection methods employing HCV core lipid and DNA binding domain monoclonal antibodies has been witnessing significant growth in recent years. Hepatitis C virus (HCV) is a major global health concern, affecting millions of people worldwide. Early and accurate detection of HCV infection is crucial for effective treatment and prevention of further transmission. Monoclonal antibodies (mAbs) have emerged as powerful tools in the field of diagnostics, offering high specificity and sensitivity in detecting HCV antigens. Among the various HCV antigens, the core lipid and DNA binding domain have gained attention due to their crucial role in viral replication and pathogenesis. Antibodies targeting these domains can aid in the detection of HCV infection at an early stage. The market for detection methods employing HCV core lipid and DNA binding domain monoclonal antibodies is driven by several factors. Firstly, the increasing prevalence of HCV infection globally has created a demand for accurate and efficient diagnostic tools. According to the World Health Organization (WHO), an estimated 71 million people have chronic HCV infection, and around 399,000 people die each year from HCV-related liver diseases. This alarming statistic highlights the urgent need for effective detection methods. Secondly, advancements in biotechnology and antibody engineering have led to the development of highly specific and sensitive monoclonal antibodies targeting the core lipid and DNA binding domain of HCV. These antibodies can be used in various diagnostic platforms, including enzyme-linked immunosorbent assays (ELISAs), lateral flow assays, and immunofluorescence assays, among others. The versatility of these detection methods makes them suitable for use in different healthcare settings, from laboratories to point-of-care facilities. Furthermore, the growing focus on personalized medicine and precision diagnostics has also contributed to the market growth. Monoclonal antibodies targeting the HCV core lipid and DNA binding domain can aid in the identification of specific viral strains and genotypes, allowing for tailored treatment regimens. This personalized approach improves patient outcomes and reduces the risk of drug resistance. In terms of geographical distribution, the market for detection methods employing HCV core lipid and DNA binding domain monoclonal antibodies is witnessing significant growth in regions with a high burden of HCV infection, such as Asia, Africa, and the Middle East. These regions have a large population at risk of HCV infection, and the demand for accurate diagnostic tools is on the rise. However, challenges such as high costs associated with monoclonal antibody production and limited access to advanced diagnostic technologies in resource-limited settings hinder the market growth to some extent. Efforts are being made to address these challenges through collaborations between research institutions, pharmaceutical companies, and global health organizations. In conclusion, the market for detection methods employing HCV core lipid and DNA binding domain monoclonal antibodies is poised for significant growth in the coming years. The increasing prevalence of HCV infection, advancements in antibody engineering, and the focus on personalized medicine are driving the demand for accurate and efficient diagnostic tools. With ongoing research and development, these detection methods have the potential to revolutionize the diagnosis and management of HCV infection, ultimately improving patient outcomes and reducing the global burden of this disease.

The Abbott Laboratories invention works as follows

The present disclosure describes detection methods using monoclonal HCV core lipid-binding domain and DNA-binding domain antibodies. The monoclonal lipid-binding domain antibody can recognize an epitope between amino acids 141 and 161 in HCV core protein.

Background for Detection Methods Employing HCV Core Lipid and DNA Binding Domain Monoclonal Antibodies

According to WHO statistics up to 170 million people are infected with hepatitis C (HCV), an infection of the liver. Infected individuals with HCV can progress to chronic infection. About 20% of those infected develop chronic hepatitis C complications, such as cirrhosis or hepatocellular cancer, after 20 years. The combination of interferon-ribavirin drugs is the current treatment recommended for HCV infections. However, the treatment does not work in all cases. Liver transplantation may be indicated in hepatitis-C-related end-stage disease. There is currently no vaccine to prevent HCV, so all precautions must be taken to avoid infection.

Therefore, the patient’s care as well as prevention of Hepatitis-C Virus (HCV), transmission by blood or blood products, or close personal contact, requires extreme vigilance and sensitive detection assays. It is therefore necessary to develop specific screening methods and identify carriers of HCV or HCV-contaminated products. The detection of HCV in blood plasma or serum is required to determine HCV exposure. This can be achieved by detection of structural and nonstructural proteins encoded from the virus.

The HCV virus belongs to the Hepacivirus family and is a (+-) sense enveloped single-stranded RNA virus. The viral genome measures approximately 10 kb and contains a polyprotein precursor of 3011 amino acids. The HCV genome contains a single large open reading frame (ORF), which codes for a polyprotein. This polyprotein undergoes co-and post-translational processing by cellular proteases and viral proteases to form three structural proteins – core, E1 & E2 – and at least six other non-structural proteins – NS2, NS3, NS4A NS4B NS5A NS5B. (Choo et al., Science 244: 359-362 (1989)).

Following HCV infection, the virus enters into a susceptible liver cell and begins viral replication. During the eclipse phase, which lasts approximately 10 days (i.e. viral RNA is not detectable), serum transaminase values are normal, and there is no evidence of an HCV immune response (Busch, et. al.,Transfusion 40:143, 2000). Usually, 10 days after exposure, HCV-RNA can be detected. The viral load is usually between 100,000 and 120,000,000 copies of HCV-RNA per ml serum. ALT levels are usually elevated several weeks after the initial exposure, indicating liver inflammation. Antibodies can be detected on average 70 days later.

The screening of serum/plasma for HCV exposure, whether by detection of antibodies against HCV or viral-specific molecules, such as HCV RNA and HCV core protein, is an important part of patient treatment. These tests identify individuals who have been exposed to the HCV virus. Blood or blood products from these individuals are then removed from the blood supply, and not used to distribute blood products to recipients (see e.g. U.S. Pat. No. 6,172,189). These tests can also be used in the clinic to diagnose liver diseases caused by HCV infection.

The use of synthetic peptides or recombinant proteins, which represent selected fragments of viral polyproteins are used in serologic antibody tests. The first generation anti HCV screening tests relied on the detection of antibodies against a recombinant (HCV genotype La) originating from nonstructural NS-4 proteins (C100-3). (Choo, Science 244:359 (1989); Kuo, Science 244:362 (1989)). Assays of the first generation failed to detect antibodies for approximately 10% of people with chronic HCV infections and 10-30% in those who had acute HCV infections. Second generation anti-HCV tests incorporate recombinant protein from three different regions on the HCV genome, including amino acid sequences of the core, the NS3, and the NS4 proteins (Mimms, Lancet, 336:1590, 1990; Bresters, Vox Sang, 62:213, 1992)). This allows a marked improvement in the first generation test in identifying HCV positive blood donors (Aach, N Engl. J. Med. 325:1325, 1991; Kleinman, Kleinman, Kleinman, Kleinman, Kleinman, Kleinman, Kleinman, Kleinman, Kleinman, Kleinman, Kleinman Second-generation tests detect antibodies in almost 100% of chronic cases of HCV (Hino, Intervirology, 37:77, 1994) and nearly 100% of acute cases within 12 weeks of infection (Alter, et. al. N Engl. J Med, 327:1899, 1992; Bresters, et. al. Vox Sang, 62:213, 1992) Third generation tests include a recombinant expressing amino acids from the NS5 area, as well antigens from core, NS3 or NS4. There have been some studies that show a slight increase in sensitivity when comparing third generation tests with second generation tests. (Lee, et. al. Transfusion 35:845(1995); Courouce, et. al. This improvement can be attributed to the changes made in the NS3 proteins rather than the addition of NS5 (Transfusion 34:790-795, 1994 )).

In general the second and third-generation HCV antibodies tests detect exposure about 70 days following exposure. The detection of HCV antibodies is a highly effective method to determine exposure to HCV, since HCV can be a persistent infection that lasts for many years. However, antibody tests alone are often ineffective at detecting HCV-infected individuals within the first 70 days of exposure.

It has been suggested by some that HCV antigen testing detects HCV exposure much earlier than antibody tests and is a viable alternative to nucleic acids testing to detect exposure to the virus during the pre-seroconversion phase. HCV antigen tests are simple and rapid. They do not require any sample preparation or pretreatment. And they are less prone to errors in handling (e.g. contamination) than HCV RNA testing. HCV core antigens are a viable alternative to HCV-RNA tests for screening blood donors and monitoring antiviral treatment.

Existing HCV antigen test rely on detection of the HCV core antibody in serum or plasma. HCV core is the structural protein that comprises the first 191 amino acid of the polyprotein. It forms the viral coat, which encapsulates the genomic RNA. Two serological assays were developed to detect HCV core antibodies in serum. The first format is used to detect HCV core antibodies in blood donors prior to seroconversion. The second format is used to detect core antigens exclusively in hepatitis patients regardless of HCV antibody status and is used in clinical laboratories for diagnosis or monitoring antiviral treatment. All of the current core antigen detection tests use antibodies that target the DNA-binding domain of HCV, which is found at amino acids 1 – 125. The core protein contains a lipid-binding domain between amino acids 134-171. There have not been any antigens identified from this section of the core protein. Until now, it was assumed that detection of core proteins required antibodies to the DNA-binding domain.

Binding proteins that detect core HCV antigens will improve detection methods for HCV in patients. There is an acknowledged need for new anti-bodies that can be used in screening tests.

The present disclosure describes detection methods using monoclonal HCV core lipid-binding domain and DNA-binding domain antibodies. The monoclonal lipid-binding domain antibody can recognize an epitope between amino acids 141 and 161 in HCV core protein.

In certain embodiments of the invention, a monoclonal antigen is provided that is immunoreactive to the lipid-binding domain of the HCV core antibody. The HCV core is more specifically amino acid residues from HCV 134-171. In more particular embodiments, the antibody specifically binds at least one epitope formed by amino acid sequence MGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG (SEQ ID NO:578). In more specific versions, the antibody reacts with an epitope formed from amino acids 141-161, 34-154, and 151-171 HCV core antigen.

Another aspect” of the invention is a monoclonal antigen that is immunoreactive to the lipid-binding domain of the HCV core antibody, and wherein the monoclonal has a variable heavy chain domain chosen from the group of antibodies shown in FIG. “Another aspect of the invention provides a monoclonal antibody that is specifically immunoreactive with the lipid binding domain of HCV core antigen, wherein said monoclonal antibody has a heavy chain variable region selected from the group comprising the antibodies listed on FIG. 1B.

It is envisaged that any antibodies described in this document may be prepared as immunoassays reagents. More specifically, these reagents are preferably labeled with an detectable label.

In other embodiments, the immunoassay reagents disclosed herein include one or more antibodies bound to a liquid phase.

The immunoassay reagents containing the antibodies of the present invention may also contain an additional antigen against HCV. This additional antibody could be an anti-core antibody, for example.

Another aspect of the invention relates to an immunoassay that detects HCV in a sample of test material, the immunoassay consisting of:

(i), contacting a sample of suspected HCV with an antibody directed against the core antigen of HCV to form a compound between said antibody and antigen within said sample;

(ii), contacting the complex formed by step (i), with an antigen and an antibody against a core-lipid binding region, to form a compound between said antigen and said antibody towards a core-lipid binding site in the complex

(iii), detecting the label formed by the complex in step (ii)

In more particular embodiments, the test may be further characterized by the fact that the antibody directed at the DNA-binding domain of the HCV core antigen. In some embodiments, the antibody used in step (ii), is labeled using a fluorescent dye. The label in exemplary embodiments is acridinium.

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