RAPID MULTI-LINE HEPATITIS C VIRUS ASSAY FIELD [0001] The present invention relates generally to the field of diagnostic assays. More particularly, the subject invention provides a point of care test (POCT) to detect for recent or past exposure of a subject to Hepatitis C virus (HCV) or an immunologically related virus or to an HCV vaccine. Point of care (POC) diagnostic and sero-evaluation kits for HCV, HCV profiles and HCV-specific antibodies also form part of the present invention.

BACKGROUND

[0002] Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

[0003] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country. [0004] Chronic infection with Hepatitis C virus (HCV) is a major cause of liver-related morbidity and mortality. An estimated 180 million people worldwide, including 120 million in China and 4 million in the US are infected with HCV (WHO-2008 http://www.who.int/vaccine research/diseases/viral cancer/en/indexx2.) In the US, end- stage liver disease due to chronic HCV infection is the most common indication of liver transplantation (http://www.optn.org) and markers for the virus has been found in at least half of all cases of hepatocellular cancer (Snowberger et al, Alimentary Pharmacology & Therapeutics 26(9): 1 187-1 194, 2007; Di Bisceglie et al, Gastroenterology 9: 1 129-1 144, 2003). Data from the 1999-2002 National Health and Nutrition Examination Survey (NHANES, US) have found the prevalence of HCV in US to be 1.6%, equating to an estimated 4.1 million individuals (Armstrong et al, Pediatrics 109(5):839-845, 2002). Of these 80% (3.2 million) are chronically infected with HCV. [0005] Many people infected with HCV do not present with adverse symptoms and have no specific indications on physical examination. Hence, HCV infection frequently remains undiagnosed presenting a barrier to patients receiving appropriate treatment. Even the vast majority of people with liver disease who have advanced to cirrhosis present with no adverse physical symptoms. Therefore, the evaluation and treatment of liver disease, in particular caused by HCV, place a significant burden on laboratory tests to determine the stage of the disease and to evaluate the response to therapy. [0006] The liver is a complex organ and is involved inter alia in biological manufacture and fluid filtering. Blood tests used to evaluate the liver can be divided into those representing liver cell damage, cholestasis and liver function. The serum aminotransaminases, alanine aminotransferase (ALT or SGPT) and aspartate aminotransferase (AST or SGOT) are part of most automated blood chemistry panels. Elevation of these enzymes is caused by damage to the hepatocyte or liver cells. The degree of elevation may be important in acute disease but is less important in chronic disease. The most common causes of elevated aminotransaminases are fatty liver, viral hepatitis, medication induced hepatitis, autoimmune hepatitis and alcoholic liver disease. The tests are a reflection of cell damage and death but are not liver function tests. Therefore, an abnormality in these tests does not mean that the liver is not functioning. In fact, the vast majority of patients with elevated aminotransaminases, regardless of the degree of elevation, have normal liver function.

[0007] Bilirubin is the final breakdown product of heme, the majority of which comes from hemoglobin. Bilirubin can be elevated in many liver-related and non-liver-related conditions and it may be elevated in conditions which lead to liver cell damage and cholestasis. The level of serum bilirubin is not a sensitive indicator of liver function and it may not accurately reflect the degree of liver damage. [0008] The most common laboratory abnormality seen in chronic HCV infection is an elevated ALT although as many as 60% of HCV infected patients have a normal ALT levels. The level of serum ALT elevation does not correlate with histological disease and may be normal in any stage of chronic HCV infection. Therefore, patients with minimal ALT elevations should be evaluated for the presence of chronic hepatitis. In advanced disease cases, an increase in alkaline phosphatase and total bilirubin as well as thrombocytopenia (low platelets) may be seen.

[0009] In a patient with risk factors for HCV or an abnormal ALT, the most practical method of diagnosing HCV infection is by obtaining a second generation enzyme linked immunosorbent assay (EIA) antibody to HCV (anti-HCV). False-positive results occur at a rate of 10-20% and are usually seen in the presence of autoimmune disease, hypergammaglobulinemia and low-risk blood donors. False negative results may occur in immunosuppressed patients, including people infected with the human immunodeficiency virus (HIV). In early infection, anti-HCV testing may be negative, as antibodies may not develop until 4-6 weeks after exposure. Unfortunately, a positive HCV antibody does not distinguish acute from chronic disease or active from past infection nor is it a sign of immunity or protection. Therefore, a positive EIA anti-HCV test is only a marker that HCV may be present and any such test needs to be followed by confirmatory viral load testing. [0010] EIA antibody tests do not distinguish between acute or chronic disease or between past and active infection. Therefore, it adds little to the care of a patient who tests positive for HCV antibody by the EIA method and has known risk factors. The National Institute of Health (NIH) consensus conference on HCV has recommended that it be used as a confirmatory test in patients without known risk factors who test positive for anti-HCV antibodies by EIA to eliminate the possibility of a false positive EIA. The recombinant immunoblot assay (RIBA) is another type of antibody test which is not influenced by the presence of autoimmune disease or hypergammaglobulinemia. In clinical practice, this test provides entire antibody profiles against different HCV antigens which are synthesized during active virus multiplication in liver cells. [0011] Confirmatory tests for the presence of HCV infection are those tests which determine the presence of HCV particles (HCV-RNA) in the blood. A positive HCV-RNA in the serum confirms the diagnosis of active HCV. This type of viral testing may be either qualitative or quantitative. Qualitative testing is more sensitive and specific than quantitative testing and results are reported as either positive or negative. Quantitative testing reports on the actual measured amount of viral particles in the serum and the viral levels are usually expressed as thousands or millions of international units. Therefore, qualitative HCV-RNA testing is used for diagnosis while quantitative testing should be reserved for use during treatment.

[0012] Several other liver tests are frequently performed on in patients with HCV. The serum alpha-fetoprotein is a marker of liver cancer but it may be mildly elevated in patients with chronic HCV infection in the absence of liver cancer. If it is elevated, this test should be closely followed. Autoimmune markers may be present in as many as 25% of patients with HCV without the presence of autoimmune disease. These markers include an anti-nuclear antibody, smooth muscle antibody, anti-mitochondrial antibody or anti-thyroid antibodies. The presence of these antibodies does not appear to influence disease progression. Patients in whom autoimmune disease is suspected should be adequately evaluated before the presence of autoantibodies is attributed to HCV infection.

[0013] The accurate interpretation of laboratory test results is important in the management of HCV infection.

[0014] Transmission of HCV has been strongly associated with intravenous and percutaneous drug and needle use. Reported cases of HCV transmission from intravenous drug use is on the rise in the US. In a study of injection drug users in Baltimore, Maryland from 1988 to 1996, 30.3% of participants developed anti-HCV antibodies with most in the first 2 years of the study (Villano et al, J. Clin. Microbiol. 35:3274-3277, 1997). Among 310 drug users in Antwerp and Limburg in Belgium, 71% and 46% had anti-HCV antibody, respectively (Mathei et al, Epidemiol. Infection 735: 127-136, 2005). The Hepatitis C European Network for C-operative Research (HENCORE) group reported a prevalence of HCV of 80% among intravenous drug users (IVDU) [Touzet / al, Eur J Gastroenterol Hepatol. 72:5667-678, 2000]. In the District Buner study in Pakistan, all 751 anti-HCV patients had a history of injections (Muhammad and Jan, JCPSP 15(1): \ 1 - 14, 2005). 90% of IVDU in Chang Rai, Thailand were positive for HCV (Apichartpiyakul et al, Jpn J Infect Dis. 52: 121-123, 1999) 36.6% of randomly selected IVDU in Sydney, Australia and 74%» of IVDU in Melbourne, Australia were HCV positive (Maher et al, J Gastoenterology Hepatology 19(10) \ \ 14-1 120, 2004; Bradshaw et al, Sex Transm Infect 8I(I):53-5S, 2005). A recent study in London, England took 428 IVDU below the age of 30 and found that 44% had antibodies to HCV compared to 4% with HIV. This came out to an incidence of 41.8 cases per 100 person years of antibody to HCV (Judd et al, BMJ 330:24-25, 2005).

[0015] The importance of intravenous drug use cannot be overemphasized. The prevalence of HCV among people who acquired HIV through intravenous drug use reaches 90% (Sulkowski and Thomas, Ann Intern Med 735: 197-207, 2003). Co-infection of the two viruses can make treatment all the more difficult. Most countries with a young population of HCV infection must deal with intravenous drug use as the leading cause for spread of the virus. Many of these intravenous drug users do not know they are infected. Screening for HCV and treatment of substance abuse are extremely important in this group.

[0016] Transfusion of blood products has also been a leading cause of transmission of HCV; however, due to improved screening, transmission through transfusions has decreased in most developed countries. In Japan, the incidence of post-transfusion non-A non-B hepatitis among those with less than 10 transfusions dropped from 4.9% (1988-Oct '89) to 1.9% (Nov'89-90) after screening with a first-generation anti-HCV test was introduced. In the US, incidence of post-transfusion hepatitis C dropped from 3.84% to 0.57% per patient (0.03% per unit blood) after HCV screening was introduced in 1990. In England, the frequency HCV infected donations dropped from 1 in 520,000 (1993-98) to 1 in 30 million (1999-2001) when donations were tested for HCV-RNA (Soldan et al, Vox Sang. 84 (4 ):214-286, 2003). [0017] However, the incidence of transfusion related HCV is still higher in other areas of the world. In a study of 147 Chilean patients with chronic HCV, the most common risk factor was blood transfusion in 54% versus just 5% with IVDU. A study was done in the largest blood bank in Santa Catarina, Brazil from 1991-2001 showing a significant drop in the risk of acquiring HCV, but the lowest risk of 1 : 13721 was still almost 10 times higher than that of developed countries ( upek, The Brazilian Journal of Infectious Diseases 5(3^:236-240, 2004). Despite better screening for selecting blood donors, there remains a need for a highly sensitive and specific assay for HCV. [0018] The role of sexual activity in the transmission of HCV remains unclear. In the NHANESIII study, number of sexual partners (OR 2.54 for 2-49 partners) and age at first sexual intercourse (OR 2.94) had significant correlation with HCV Ab and this has been confirmed in other studies (Alter et al, N Engl J Med 341:556-562, 1999; Perez et al, Int J Epidemiol 34(3):593,599, 2005). Among 1257 non-IVDU in Baltimore at a STD clinic 9.7% were positive for HCV (Thomas et al, J Infect Dis 169:990-995, 1994). One hypothesis is that many of the HCV patients may have injecting sexual partners. In one study, 15% of non IVDU women with an injecting partner had HCV (Goldberg et al, Br J Obstet Gynaecol 705:365-370, 2001). More recently, a 10-year prospective follow-up study (8060 person-years) showed no evidence of sexual transmission among monogamous couples in Italy (Vandelli et al, Am J Gastroenterol 99(5 : 855-859, 2004). However, in a study among spouses in Egypt, it was estimated that wife to husband transmission was 34% and 10% among women with and without detectable HCV RNA. Husband to wife transmission was estimated at 3%. Overall, 6% were estimated to have contracted HCV from their spouse (Magder et al, International Journal of Epidemiology 34:160-165, 2005). Also recently, there was lack of evidence found for sexual transmission of HCV among men who have sex with men in the prospective ongoing Omega Cohort Study in the US (2653 person-years of follow-up) [Alary et al, American Journal of Public Health 95(3,1:502-505, 2005]. All of this new evidence supports that sexual transmission of HCV is still rare but for some reason is higher among those with high-risk sexual activity. [0019] It has been well documented that dialysis patients have a higher rate of HCV infection. In the 1990's much of the world reported anti-HCV prevalence rates of 10-50% among hemodialysis patients with lower rates in such places as Ireland (1.7%) (55-60). Previously, rates in Europe were as high as 20-30% (Touzet et al, 2000 supra). A more recent report from Saudi Arabia showed a prevalence rate of HCV among hemodialysis patients to be 9.24% compared to 0.30% among blood donors (Qadi et al, Am J Infect Control 32 (8) .493-495, 2004). In a tertiary-care hospital in Mexico City, Mexico, the rate of anti-HCV was 6.7% compared to the roughly 1.2% prevalence in the population of Mexico (Mendez-Sanchez et al, Journal of Clinical Microbiology 42(9):432 \ -4322, 2004). The rate of seroconversion among hemodialysis patients with no other risk factors has been reported 1.38-1.9%/year (Halfon et al, J Viral Hepat. 5: 1 15-121 , 1998; Fabrizi et al, Am J Nephrol 79:383-388, 1999).These studies generally conclude that the transmission of HCV to hemodialysis patients is generally nosocomial with possible risk factors being failure to disinfect devices between patients, sharing of single-use vials for infusions, poor sterile technique, poor cleaning of dialysis machines, and poor distance between chairs (Zampieron et al, EDTNA ERCA J. 30(2) 84-90, 2004).

[0020] The prevalence of HCV has been noted to be higher in other populations as well. Among kidney transplants, the prevalence was reported to be as high as 33.3% in Italy with the frequency higher prior to 1990(50%) than after 1990 (27%) [Angelico et al, Ital J Gastroenterology Hepatology 29:448-455, 1997]. clearly, most of these kidney transplant patients underwent dialysis as well.

[0021] The US Veteran Affairs medical centers have also reported a higher prevalence of HCV than the general population with percentages as high as 35% in the VA Palo Alto system (Cheung, Am J Gastroenterol 95:740-747, 2000). The most recent study among 20 centers reported an estimated prevalence of 5.4% with 78% reporting a risk factor of either transfusion or intravenous drug use. Seropositivity was also associated with tattoo use and incarceration in this study (Zampieron et al, 2004 supra). [0022] There is also an increased prevalence of HCV also among prison inmates. One example is the Riverside county jail system where 25% adults incarcerated carried the virus while only 2% of the juvenile detention population carried HCV (Feldman et al, Journal of Adolescent Health 25:505-508, 2004). The juvenile detention population, therefore, provides a target for teaching and intervention since many of these juveniles acquire the virus early in their adult years.

[0023] HCV is divided among six genotypes with numerous subtypes. These genotypes can differ up to 30% from each other in nucleotide sequence. Depending on the HCV genotype, length of treatment can differ. Genotype lb is less responsive to alpha-interferon therapy compared to genotypes 2 and 3. It is, therefore, important to track the different genotypes of the HCV virus. In the NHANESIII study done in the US, 56.7% were classified as la, 17% as lb, 3.5% as 2a, 1 1.4% as 2b, 7.4% as 3a, 0.9% as 4, 3.2% as type 6 (Alter et al, 1999 supra). 50% of all infections were by genotype 1 with a higher percentage of genotype 3 among the IVDU population in England (Mohsen and Trent, Gut 48:101-m, 2001 ; Thomson and Finch, Clin Microbiol Infect 77:86-94, 2005). Genotype lb was the predominant genotype (46% among blood donors) in Chile and was found in all infected patients with hepatocarcinoma in one study (Munoz et al, Rev Med Chil 726f9 : 1035-1042, 1998). This same genotype was also found in 82% of 147 chronic hepatitis C patients in Chile as well. Genotype lb is also dominant in Japan (Yamada et al, J Gastroenterol Hepatol. 70:538-545, 1995; Ohno et al, J Clin Microbiol. 35:201 -207, 1997). In Beijing, China, of 63 HCV-RNA samples, 52% were genotype 2 and 29% type 3 (Wang et al, J Hepatol 27:634-640, 1994). In Thailand, HCV genotype-3a was the most common genotype at 50-60% with la, lb, and 6 comprising the rest (10-20% each) [Apichartpiyakul et al, 1999 supra . Out of 90 patients in Estonia, 73.3% carried genotype- lb, 20% with genotype-3a, and 6.7% with genotype-2a (Zusinaite et al, World J gastroenterology 77(¾):488-491 , 2005). Genotype-3 is most common on the Indian subcontinent whilst genotype-4 is the most common genotype in Africa and the Middle East (Das et al, Indian J Pathol Microbiol 45:323-328, 2002; Shah et al, J Gastroenterology Hepatology 72:758-761 , 1997; Valliammai et al, J General Virol 76(3):Ί\ \ -Ί\6, 1995; Osoba, Saudi Med J 23:7-12, 2002; Bdour, J Med Microbiol 57:700- 704, 2002). Genotype-5 can be found in South Africa and as mentioned above, genotype 6 can be found in south-east Asia (Smuts and Kannemeyer, J Clin Microbiol 33: 1679- 1681 , 1995; Huy and Abe, Pediatrics International 46:223-230, 2004). [0024] Primary prevention of HCV infection should target reduction of transmission of the virus. Prevention should target those at risk of acquiring the virus and should involve providing education, risk reduction counseling, HCV screening and substance abuse treatment. In the US, the Centers for Disease Control (CDC) suggest screening for the following populations:

· Persons who ever injected illegal drugs, including those who injected once or a few times many years ago.

Persons who received a blood transfusion or organ transplant before July 1992.

Persons who received clotting factor concentrates before 1987.

Persons who were ever on long-term dialysis.

· Children born to HCV-positive women.

Healthcare, emergency medical, and public safety workers after needle sticks, sharps, or mucosal exposures to HCV-positive blood.

• Persons with evidence of chronic liver disease. [0025] Extra attention should be given to populations in specific settings such as correctional institutions, drug treatment programs, programs for high risk youth, HIV counseling and testing sites, and STD clinics. In these settings, physicians should always screen for intravenous drug use. Unlike HIV, HCV is found in high concentrations in filters, spoons, and rinsing liquids that may be used in association with needle drug use. Patients should be counselled on contaminated equipment being a source of infection. Addiction care and counseling should be focused on with possible referrals for psychotherapy and detoxification (Viral Hepatitis C http://www/cdc.gov/neidod/diseases hepatitits/c/plan/Prev Control.htm; Backmund et al, Clin Infect Dis 40(Suppl 5,):S330-335, 2005; Edlin et al, Clin Infect Dis 40(Suppl 5) S276- 285, 2005). [0026] Prevention in healthcare setting should also take place by having better sterilization, safer injections, reducing opportunities for percutaneous exposures to blood. In developing countries, better screening for donors and blood screening should take place to reduce the number of transfusion related transmissions.

[0027] Once a patient is found to have HCV, that patient needs to be counselled to reduce the risk of transmission of the virus to others. The physician should also offer counseling on treatment, reducing alcohol usage and immunization with hepatitis A, hepatitis B, pneumococcal and influenza vaccines. HCV negative persons with ongoing risk factors also require counseling and immunization with hepatitis A and hepatitis B vaccines (Viral Hepatitis C http://\vww/cdc.gov/neidod/diseases hepatitits/c/plan/Prev Control.htm; Backmund et al, 2005 supra; Edlin et al, 2005 supra).

[0028] Better prevention, screening and treatment methods for HCV are needed. There is a particular need to be able to rapidly identify acute or chronic exposure to HCV with a high degree of sensitivity and specificity.

[0029] The present invention provides an effective and sensitive point of care test (POCT) for the detection of exposure of subjects to HCV and to monitor the efficacy of HCV immunity.

SUMMARY

[0030] The present invention provides a "point of care test" (POCT) for detecting a subject who has had exposure to Hepatitis C virus (HCV) or an immunological relative thereof or who has achieved some level of immunity against an HCV antigen. The method is predicated in part on the detection of binding partners (generally IgG antibodies) which are specific for multiple HCV antigens including structural and non-structural proteins. The POCT comprises contacting a biological sample from the subject with a mixture of HCV specific component; and determining the presence of a complex that forms between a binding partner (e.g. IgG antibodies) in the biological sample and an HCV specific immunogenic component and characterizing the binding partner in the complex with an anti-binding partner antibody conjugated with gold colloid using reverse flow technology.

[0031] In an embodiment, the POCT relies on the identification of IgG antibodies specific for multiple antigens from HCV or its immunological relative including structural and nonstructural HCV proteins in the biological sample. The assay is referred to herein as an HCV multi-line rapid test (HCV-MRT) for detecting, in an embodiment, IgG antibodies specific to more than two HCV antigens. The antigens include structural and nonstructural proteins and nucleocapsid protein (also referred to as capsid protein and core protein [Cp]).

[0032] The POCT is a low cost, rapid and straightforward assay to determine present or prior recent exposure to HCV or an HCV vaccine. In an embodiment, the POCT utilizes a solid support such as a nitrocellulose membrane to hold a mixture of HCV immunogens, which subsequently capture binding partners identified as anti-HCV antibodies from a biological sample such as whole blood, serum, plasma, saliva, urine or other tissue fluid from subjects to be tested for exposure to HCV and/or suspected or at risk of having HCV and finally visualized by anti-antibodies conjugated with gold particle, generally using reverse flow technology. In an embodiment, the antibodies are IgG antibodies. However, the present invention extends to other antibody types and sub-types such as IgM and IgA. The visualization is via multiple lines depending on the location of the immunogens. [0033] Accordingly, the POCT of the present invention shows greater specificity and sensitivity compared to other conventional and most currently used HCV detection assays. The reverse flow rapid platform specifically identifies antibodies (e.g. IgG) produced against HCV immunogens or immunologically cross-reactive proteins after recent or past exposure to HCV or an HCV vaccine. In an embodiment, the POCT identifies current (acute) HCV infection. In another embodiment, the POCT identifies chronic HCV infection. [0034] In another aspect of the present invention, there is provided a solid support for use in a POCT for detecting exposure of a subject to HCV or immunological relative thereof or a vaccine to HCV or its immunological relative, the support comprising HCV-specific immunogenic components immobilized on the support. In use, the solid support is used in a method which comprises contacting a biological sample from the subject with a mixture of HCV-specific immunogenic components or an immunogenic equivalent thereof; determining the presence of a complex that forms between a binding partner present in the biological sample and a HCV- specific immunogenic component; and optionally characterizing the binding partner in the complex to correlate the binding partner to exposure to HCV.

[0035] In an embodiment, the biological sample is applied to a nitrocellulose membrane previously loaded with HCV immunogens which include structural and non-structural proteins. Conveniently, the antigen or immunogenic component is derived from purified HCV recombinant proteins or comprise recombinant HCV components. The complex formed by an immunogenic component of the HCV and the binding partner is then detected using a detection agent that contains a reporter group, wherein the detection agent specifically binds to the component/binding partner complex. Examples of immunogens include non-structural (NS) proteins 3, 4 and 5 and the structural protein nucleocapsid (also referred to as core protein, capsid protein and Cp). [0036] More particularly, the binding partner is IgG induced in a subject infected with or exposed to HCV and the detection agent is an anti-IgG antibody. In a particular embodiment, detection is by an anti-IgG antibody conjugated gold with colloid using reverse flow technology.

[0037] Yet another aspect of the present invention provides a POCT kit in a cassette form for detecting a binding partner in a subject that is specific for HCV or an immunological relative thereof or for detecting HCV exposure, the kit comprising a solid support comprising an HCV-specific immunogenic components or immunological relatives thereof; or a solid support comprising an HCV-specific immunogenic component or immunological relatives thereof attached to a second support; at least one detection agent conjugated to a reporter group for detecting a binding partner in a biological sample that forms a complex with the HCV-specific immunogenic component; and optionally instructions for using the kit to further identify the binding partner of the complex. In an embodiment, the binding partner is IgG. In that aspect, the reporter group generally comprises anti-IgG antibodies conjugated with gold particles and the platform is reverse flow technology. The present invention extends to other antibody types and sub-types such as IgGl , IgG2, IgM and IgA. [0038] The present invention also provides individual components of the POCT kit for use in the method of the present invention. The present invention also serves as a method of assessing the relative risk of one or more subjects being exposed to HCV or an immunological relative thereof or a vaccine for same within a defined location (e.g. geographical area, housing estate, means of transport or center for medical treatment or assessment), comprising: obtaining samples from a representative population within a defined location; and assessing evidence of exposure of individual members of a sample population to HCV or immunological relative thereof by the method comprising the steps of contacting a biological sample from the subject with a mixture of HCV-specific immunogenic components; and determining the presence of a complex that forms between a binding partner in the biological sample and an HCV-specific immunogenic component and wherein the presence of the complex is indicative of exposure of the subject to HCV or equivalent thereof; and assessing the relative risk of exposure within the defined location by characterizing the binding partner in the complex. In an embodiment, the binding partner is IgG.

[0039] Risk analysis may be conducted using software in a computer readable form. Consequently, the present invention further relates to a computer readable program and computer comprising suitable for analyzing exposure of subjects or group of subjects or a risk of exposure of a subject or group of subjects to HCV or immunological relative thereof.

[0040] The present invention is further directed to the use of IgG antibodies specific for HCV or an immunological relative thereof or an HCV^ antigen or an immunogenic fragment thereof in the manufacture of a POCT to detect current or recent prior infection or exposure to HCV or an immunological relative thereof.

Table 1

Abbreviations

Abbreviation Definition

ALT Alanine aminotransaminase (also referred to as SGPT)

AST Aspartate aminotransferase (also referred to as SGOT)

CMV Cytomegalovirus

Cp Core protein from HCV (also referred to as nucleocapsid protein and capsid protein)

EIA Enzyme immunosorbent assay

ELISA Enzyme linked immunosorbent assay

HAV Hepatitis A virus

HBsAg HBV surface antigen

HBV Hepatitis B virus

HCV Hepatitis C virus

HCV-MRT HCV multi-line rapid test

HCV-NAT HCV nucleic acid test

HCV-RNA HCV particles, when present in blood, they are a sign of active HCV infection

HIV Human immunodeficiency virus

HTLV Human T-lymphocyte virus

IVDU Intravenous drug users

NS Non-structural proteins from HCV

PCR Polymerase chain reaction

POC Point of care

POCT Point of care test

RIBA Recombinant immunoblot assay

r-Proteins Recombinant proteins BRIEF DESCRIPTION OF THE FIGURES

[0041] Some figures contain color representations or entities. Color photographs are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

[0042] Figure 1 is a schematic diagram of test (T) lines specific against different HCV proteins at various stages of infection. [0043] Figure 2 is a schematic representation of the HCV multi-line rapid test (HCV- MRT results).

DETAILED DESCRIPTION

[0044] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0045] As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a single protein, as well as two or more proteins; reference to "an HCV" includes an HCV genotype, as well as two or more HCV genotypes; reference to "the invention" includes a single or multiple aspects of an invention; and so forth.

[0046] The present invention provides an assay to detect HCV infection or exposure. The "assay" may also be referred to as a "method" or "protocol" or "test" and is a point of care (POC) test or "POCT". The terms "infection" and "exposure" mean recent or past exposure to HCV and acute and chronic HCV infection.

[0047] Reference to "HCV" or "Hepatitis C virus" includes an immunological relative thereof. The latter term encompasses variants of one of the serotypes or sub-serotypes of HCV. In this context a serotype includes a genotype of HCV. An immunological relative includes another genus or member within the HCV family or a species or genotype of HCV. Reference to "exposure" includes current or recent prior infection as well as an infection which does not lead to a disease condition. In one embodiment, the POCT screens for a binding partner such as IgG in a subject that is specific for HCV or immunological thereof or immunogen within HCV and the test comprises contacting a biological sample from the subject with a mixture of HCV-specific immunogenic components; and determining the presence of a complex that forms between a binding partner in the biological sample and a HCV-specific immunogenic component; and characterizing the binding partner in the complex with an anti-IgG antibody conjugated with gold colloid. The present invention extends to other antibodies specific to HCV such as sub-types of IgG (e.g. IgGl , IgG2) as well as IgM and IgA. In those cases, anti-IgM or anti-IgA or other anti-antibodies conjugated to colloid of gold may be used.

[0048] In a particular embodiment, multiple immunogens from HCV are employed referred to as a multi-lined assay or an HCV-multi-line rapid test or HCV-MRT.

[0049] This method is specific for the identification of HCV-specific binding partner molecules. In a particular embodiment, the binding partner is IgG. This method may be further enhanced by the use of immunogenic components that have been isolated using a HCV-specific IgG. Accordingly, the immunogenic components may be HCV-specific IgG immunogenic components. The use of IgG reactive immunogenic components renders the POCT specific for IgG antibodies in the biological sample which are specific for HCV or its immunological relative. However, other binding partners may also be employed such as IgM or IgA or sub-types thereof or sub-types of IgG.

[0050] Accordingly, one aspect of the present invention provides a method for detecting a binding partner in a subject that is specific for HCV, the method comprising contacting a biological sample from the subject with a mixture of HCV reactive immunogenic components; and determining the presence of a complex that forms between the binding partner in the biological sample and an HCV-specific immunogenic component; and characterizing the binding partner in the complex with an antibody specific for the binding partner conjugated with gold colloid.

[0051] In another embodiment, the present invention provides a method for detecting IgG in a subject that is specific for HCV thereof, the method comprising contacting a biological sample from the subject with a mixture of HCV reactive immunogenic components; and determining the presence of a complex that forms between a binding partner in the biological sample and an HCV-specific immunogenic component; and characterizing the binding partner in the complex with an anti-IgG antibody conjugated with gold colloid.

[0052] In a further aspect, the present invention provides a POCT using reverse flow technique for detecting exposure of a subject to a HCV or immunological relative thereof, the method comprising contacting a biological sample from the subject with a mixture of HCV-specific immunogenic components; determining the presence of a complex that forms between a binding partner in the biological sample and an HCV- specific immunogenic component; characterizing the binding partner in the complex; and correlating the binding partner to exposure to the HCV.

[0053] In a particular embodiment, the binding partner is IgG. Reference to "IgG" includes its various sub-types such as IgGl , IgG2, etc.

[0054] Hence, in a particular aspect, the present invention provides a POCT reverse flow rapid test for HCV reactive IgG which, in an embodiment, uses saliva, whole blood, serum, plasma, or other body fluid including urine, semen, tissue fluid or sputum as the biological sample. The technique shows a high level of performance in terms of detecting anti-HCV IgG antibodies compared to the detection of HCV-specific antibodies using ELISA techniques. The POCT of the present invention is compatible to diagnostics at the primary health care level where molecular and ELISA-based HCV diagnostic assays are not feasible. [0055J The POCT is rapid (approximately 10-20 minutes, average time 15 minutes), low cost and straightforward without needing laboratory facilities and special technical expertise to determine present or prior recent exposure to HCV. In accordance with the present invention, the subjects including animals such as mammals and in particular humans are screened for the presence of binding partners and in particular IgG to HCV or an immunological relative thereof using reverse flow technology. The binding partners are generally subject-derived binding partners such as, but not limited to, immunointeractive molecules. The most useful immuno-interactive molecules are antibodies particularly IgG. The identification of such binding partners is then used as evidence of present or prior recent exposure of the subject to HCV or an immunological relative thereof.

[0056J The present invention specifically utilizes reverse flow technique, where HCV antigen is sprayed or loaded onto the nitrocellulose or other suitable solid support for capturing of HCV reactive IgG from the biological samples of the subject exposed to HCV infection. The resultant complex is later detected by anti-IgG antibody conjugated with gold particle and the technique can be useful at any level of health care system which is called point of care (POC) testing.

[0057] Accordingly, the present invention shows greater sensitivity and specificity compared to other conventional and most currently used antibody and antigen capture IgG (GAC and GAC-ELISAs) by providing a platform that can identify antibody produced against HCV or an immunological relative thereof at an early stage of the infection by reverse flow technique.

[0058] Therefore, the present invention provides a specific, rapid and economical POCT using HCV antigen, such as structural and non-structural immunogenic protein components of the virus, which permit the specific detection of HCV binding partners such as IgG that may be present in the test samples (e.g. in whole blood, serum, plasma, saliva, urine or tissue fluid). The test is conducted at room temperature (RT). It is a convenient technique for specific antibody detection. The POCT utilizes reverse flow platform in a cassette format and anti-IgG antibodies conjugated with gold for the detection of anti-HCV IgG from saliva, whole blood, serum, plasma, urine or tissue fluid. Furthermore, the POCT is used for testing a single sample with high sensitivity.

[0059] As indicated above, the term "HCV" or "Hepatitis C virus" as used herein refers to all HCV serotypes and genotypes associated acute or chronic HCV infection or symptoms of HCV infection. It also applies to asymptomatic subjects. The present invention is applicable to detecting HCV infection or exposure in any subjects including human, non- human animals and laboratory animals. Human subjects, however, are the most useful subjects in accordance with the present invention. Notwithstanding, the present invention includes any subject that can respond to an infection or immunization by the HCV or an immunological relative thereof. [0060] However, the term "infection" includes exposure to HCV but without necessarily causing disease symptoms. The exposure may also be in the form of an experimental vaccine. [0061] The term "HCV" is used herein the claims and description refers to all HCV genotypes (HCV genotype-1 , genotype-2, genotype-3, genotype-4, genotype-5, genotype-6 including their sub-genotypes such as HCV genotype- l a and HCV genotype- lb) associated with a HCV infection. Preferably, the present invention is applicable to detecting HCV infection or exposure in any subjects including human. Human subjects, however, are preferred in accordance with the present invention. However, the invention includes any subject that can respond to an infection or immunization by the HCV or an equivalent thereof.

[0062] The Hepatitis C virus (HCV) is a small (50 nm in size), enveloped, single-stranded, positive sense RNA virus. It is the only known member of the Hepacivirus genus in the family Flaviviridae. There are six major genotypes of the HCV, which are indicated numerically (e.g., genotype-1 , genotype-2, etc.).

[0063] The HCV is transmitted by blood-to-blood contact. In developed countries, it is estimated that 90% of persons with chronic HCV infection were infected through transfusion of unscreened blood or blood products or via injecting drug use or, by inhalational drug use. Inhalational drug usage (snorting) has evolved into one of the most common means of infection in the US. In developing countries, the primary sources of HCV infection are unsterilized injection equipment and infusion of inadequately screened blood and blood products.

[0064] The diagnosis of "hepatitis C" is rarely made during the acute phase of the disease because the majority of people infected experience no symptoms during this phase of the disease. Those who do experience acute phase symptoms are rarely ill enough to seek medical attention. The diagnosis of chronic phase HCV infection is also challenging due to the absence or lack of specificity of symptoms until advanced liver disease develops, which may not occur until decades into the disease.

[0065] Chronic HCV infection may be suspected on the basis of the medical history (particularly if there is any history of IV drug abuse or inhaled substance usage such as cocaine), a history of piercings or tattoos, unexplained symptoms, or abnormal liver enzymes or liver function tests found during routine blood testing. Occasionally, HCV infection is diagnosed as a result of targeted screening such as blood donation (blood donors are screened for numerous blood-borne diseases including hepatitis C) or contact tracing.

[0066] HCV testing begins with serological blood tests used to detect antibodies to HCV. Anti-HCV antibodies can be detected in 80% of patients within 15 weeks after exposure, in >90% within 5 months after exposure, and in >97% by 6 months after exposure. [0067] Overall, HCV antibody tests have a strong positive predictive value for exposure to the HCV, but may miss patients who have not yet developed antibodies (seroconversion), or have an insufficient level of antibodies to detect. In some situations, subjects infected with HCV do not develop antibodies to the virus and, therefore, never test positive using HCV antibody screening. Because of this possibility, RNA testing (see nucleic acid testing methods below) should be considered when antibody testing is negative but suspicion of hepatitis C is high (e.g. because of elevated transaminases in someone with risk factors for hepatitis C).

[0068] Anti-HCV antibodies indicate exposure to the virus, but cannot determine if ongoing infection is present. All persons with positive anti-HCV antibody tests must undergo additional testing for the presence of HCV virus itself to determine whether current infection is present. The presence of the virus is tested for using molecular nucleic acid testing methods such as polymerase chain reaction (PCR), transcription mediated amplification (TMA), or branched DNA (b-DNA). All HCV nucleic acid molecular tests have the capacity to detect not only whether the virus is present, but also to measure the amount of virus present in the blood (the HCV viral load). The HCV viral load is an important factor in determining the probability of response to interferon-based therapy, but does not indicate disease severity nor the likelihood of disease progression.

[0069] In people with confirmed HCV infection, genotype testing is generally recommended. HCV genotype testing is used to determine the required length and potential response to interferon-based therapy.

[0070] Reference to "HCV" include immunological relatives, also referred to as an equivalent.

[0071] The term "equivalent" as used herein and applied to the HCV is intended to include similar molecules that can elicit the same or similar response that the HCV or a structural or non-structural protein of the HCV could elicit. For instance, various antigens expressed by the HCV at various stages of infection or various virus particles or fragments may cause similar effects that the whole virus causes. The response may be an immunological response (non-clinical response) or it may be an infectious response (clinical response) or due to vaccination.

[0072] HCV antigens used in this development are recombinant proteins and are constructed as a fusion protein containing {structural-C protein (8 to 56) and non-structural NS3 (1 192-1457), NS4 (1961 -1947) and NS5 (2212-2313)} . This fusion protein is expressed in E. coli and is subsequently stabilized in 5M urea, and purified in the presence of 8M urea in 0.05 moles/L ΤΕ buffer and then purified sequentially by Q-Sepharose FF, and Sephadex G-50 to homogeneity, the purity is above 95% by SD-PAGE. The final solvent is 0.02moles/L Tris buffer, pH 8.0 containing 0.02 SDS and lyophilized. The purified protein is refolded by serial dilution in carbonate buffer (pH 9.6) at 4°C prior to application.

[0073] The present invention is applicable to detecting exposure to the HCV or an immunological relative thereof. Exposure may be present or prior exposure to HCV or an immunological relative thereof. Generally, the exposure is sufficient to elicit an immune reaction or response in the body so as to induce a binding partner in response to the HCV or immunological thereof. Once the subject is exposed, the method of the present invention may be applied at any stage of exposure as described above. Generally, the method is used to detect exposure where there are no signs and symptoms that are obvious of a HCV infection. The POCT herein detects exposure of the subject at a phase of HCV infection at an early acute phase or late convalescence or chronic stage of exposure to HCV or equivalent thereof for the primary infection or vaccination. The exposure may not always manifest in a HCV infection or notable signs or symptoms but it will cause a response so as to induce a binding partner (e.g. IgG). The response is particularly an immunological response.

(00741 The subject may have been exposed to HCV but need not show visual symptoms of the infection. The present method detects exposure that may lead to infection or may indicate prior exposure with no symptoms manifested.

(00751 An "immune response" or "immunological response" is understood to be a selective response mounted by the immune system of vertebrates in which specific antibodies or fragments of antibodies and/or cytotoxic cells are produced against invading pathogens and antigens which are recognized as foreign in the body.

[0076J The binding partner is any molecule or cell that is produced against the foreign HCV or immunological relative thereof. The binding partner is generally an antibody or immunologically active fragment thereof, or a cytotoxic cell. The binding partner includes an immuno-interactive molecule that can interact with HCV antigen or equivalent and is most particularly an IgG molecule. Other binding partners include IgM and IgA and subtypes thereof as well as sub-types of IgG (e.g. IgGl , IgG2).

[0077] As indicated herein, the binding partner is generally an immuno-interactive molecule, which refers to any molecule comprising an antigen binding portion or a derivative thereof. Particularly, the immuno-interactive molecule is an antibody against any portion of HCV proteins or epitopes thereon produced during a humoral response in the subject infected or exposed to HCV.

[0078] As indicated herein, a particular binding partner is an antibody produced in the subject to an HCV or related virus components. However, a binding partner of the targeted antibody may also be used. An example of such a binding partner is an anti-idiotypic antibody or an antibody specific for and discriminatory of a subject antibody specific for an HCV or related virus components.

[0079J As used herein, an "anti-idiotypic antibody" is an antibody which binds to the specific antigen binding site of another antibody generated in response to exposure to a component derived from an HCV or immunological relative thereof.

[0080] As used herein, the terms "antibody" or "antibodies" include the entire antibody and antibody fragments containing functional portions thereof. The term "antibody" includes any monospecific or bispecific compound comprised of a sufficient portion of the light chain variable region and/or the heavy chain variable region to effect binding to the epitope to which the whole antibody has binding specificity. The fragments can include the variable region of at least one heavy or light chain immunoglobulin polypeptide, and include, but are not limited to, Fab fragments, F(ab') 2 fragments, and Fc fragments.

[0081) In a particular embodiment, the binding partner is an antibody and more particularly is an HCV -specific IgG molecule or an HCV antigen-specific IgG molecule.

[0082] The method of the present invention detects exposure to the HCV or immunological relative thereof via the use of a biological sample obtained from a subject having been potentially exposed to the virus. The biological sample may be any sample from the body that may contain a binding partner. Such biological samples may be selected from the group including blood, saliva, cord fluid, B cells, T-cells, plasma, serum, urine and amniotic fluid. Conveniently, the biological sample is serum or plasma. Most conveniently, the biological sample is serum or saliva. [0083] It is also useful that the biological sample be obtained from subjects suspected of exposure to HCV. A biological sample may also be modified prior to use, such as by centrifugation, dilution, and the like. Accordingly, a biological sample may refer to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof.

[0084] It should be noted that a biological sample might also be devoid of a binding partner that can interact with HCV or an immunological relative thereof. This occurs when the subject has not been exposed to HCV or an immunological relative thereof. Hence "determining the presence of a complex that forms between a binding partner in the biological sample and an HCV-specific immunogenic component" may yield a zero result, as a complex cannot form in the absence of binding partners. A control may be performed with an HCV-specific immunological agent such as a monoclonal antibody designed to compete with binding partners in the biological sample.

[0085] Reference to a biological sample being placed in contact with a component, such as an immunogenic component or its immunological relative thereof should be understood as a reference to any method to facilitating the interaction of one or more immuno-interactive molecules of the biological sample with a component of HCV antigen derived from infection of cells with HCV or an immunological relative thereof. The interaction should be such that coupling or binding or otherwise association between the immuno-interactive molecule and a specific immunogenic component of HCV antigen derived from cells infected with the HCV or an immunological relative thereof can occur. [0086] The biological sample is contacted with a mixture of HCV specific immunogenic components, particularly derived from cells infected with HCV or an immunological relative thereof. The HCV provides viral immunogenic components that may be provided by the HCV at any stage of its development. In the early convalescent stages of HCV infection, antibody, particularly IgG developed or derived from previous HCV infection is one of the indications of either primary or secondary HCV infection and this may be detected by the formation of a complex between it and an HCV-specific immunogenic component, particularly an immunological component of the virus.

[0087] HCV infected vero cells may be cultured for any period, generally for a period that allows the HCV to establish and infect the cell. More particularly, the cells are cultured until a cytopathic effect is apparent in the cell culture thereby indicating active infection of the virus in the cells.

[0088] At this point, HCV is collected from the infected cells either by lysed infected cells or from the supernatant of the infected cells by any other method available. Later the viral antigen is purified and inactivated using standard protocol and concentrated at lmg/ml.

[0089] Conveniently, the HCV antigen is a structural or non-structural protein.

[0090] The HCV specific immunogenic component is generally from lysate and culture supernatant. The biological sample is contacted so that a complex may form between the viral immunogenic components of the lysate and the binding partner contained within the biological sample. Particularly, immunogens of the HCV particles, including but not limited to those of the structural and non-structural proteins captured by anti-HCV IgG, will form complexes with binding partners. Preferably, the specific binding partners are antibodies or fragments thereof derived from the biological sample. These will only be present when the subject has been exposed/infected or immunized to the HCV.

[0091] A complex forms between an antibody, particularly an IgG reactive with HCV or immunological relative thereof.

[0092] The methods and kits of the present invention seek to detect components and binding partners which form complexes and are indicative of a HCV infection. These components and binding partners are generated in the course of an HCV infection/immunization.

[0093] The complex may comprise one or more binding partners bound to one or more components derived from HCV or an equivalent thereof. However, not all will be HCV- specific IgG. Other immunological molecules such as IgM and IgA may also bind.

[0094] A second binding molecule is also loaded on the nitrocellulose membrane which binds all immunoglobulins (IgG, IgA, and IgM) called Protein-L or Protein-A and this protein is used as a control line to indicate the validity of the assay.

[0095] The biological sample is left in contact with the component derived from HCV or an equivalent thereof for a period of time sufficient and conditions, which allow the stable formation of the complex.

[0096] The HCV specific immunogenic components and the biological sample are contacted so that a complex may form between the components and a binding partner present within the biological sample. Conveniently, immunogens of the HCV particles, including but not limited to those of the structural and non- structural proteins particularly captured by anti-HCV virus IgG having a binding site specific to HCV antigen, will form complexes with either binding partners or a competing HCV-specific immunological agent such as a specific IgG. [0097] Particularly, the specific binding partners are antibodies or fragments thereof present in the biological sample. These will only be present when the subject has been exposed/immunized to the HCV.

[0098] Particularly, the complex forms between an antibody, particularly an IgG specific for the member of the HCV antigen or equivalent thereof and an anti-HCV IgG captured HCV viral component. This is then indicative of HCV-specific IgG in the sample and hence recent or prior exposure.

[0099] In one embodiment, the present invention relies upon the detection of HCV specific binding partners particularly IgG present in the biological sample that are specific for a component of the HCV antigen present in cell lysate derived from a cell infected with the HCV or an immunological relative thereof which has been captured using anti-HCV IgG. The complex may comprise one or more binding partners bound to one or more components derived from HCV or an equivalent thereof. However, it is the identification of an IgG bound to the complex that is indicative of prior exposure in this present invention.

[0100] A non-immunological complex is formed with biological samples (salvia, serum) along the line loaded with protein-L (or Protein-A) containing all three types of immunoglobulins (IgG, IgA and IgM). This complex does not indicate the exposure of HCV to the subject but does provide the validity of the assay.

[0101] Another aspect of the present invention provides a solid support for use in a method for detecting exposure of a subject to a HCV or equivalent thereof, the method comprising contacting a biological sample from the subject with a mixture of HCV specific immunogenic components or an immunological relative thereof; determining the presence of a complex that forms between a binding partner present in the biological sample and a HCV specific immunogenic component; and optionally characterizing the binding partner in the complex to correlate the binding partner to exposure to the HCV; the support comprising HCV specific immunogenic components immobilized on the support.

[0102] The solid support may be any material known to those of ordinary skill in the art to which a binding partner or HCV specific immunogenic component may be attached. For example, the solid support may be a test well in a microtitre plate or a nitrocellulose or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass, fibreglass, latex or a plastic material such as polystyrene or polyvinylchloride.

[0103] The binding partner or the HCV specific immunogenic component may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature. In the context of the present invention, the term "immobilization" refers adsorption of antigen on the nitrocellulose membrane following spraying by BioDot machine. Immobilization by adsorption of antigen is onto the membrane (prima-85). In such cases, adsorption may be achieved by spraying of antigen at 7" from the bottom of membrane using BioDot machine in a suitable buffer. Two more lines are sprayed on the same membrane at 14" and 12" using crystal violet and Protein-L. Following spraying membrane is allowed dry in a dryer and finally is blocked by blocking buffer.

[0104] The HCV specific immunogenic component derived from an HCV or an equivalent thereof loaded/sprayed onto the membrane is allowed to contact with biological samples which is passed over the membrane once added onto the sample pad, which allow the stable formation of a complex. Once the biological sample meets the indicator line (crystal violet) a detection system which is anti-IgG antibodies conjugated with gold particle is allowed to flow over the membrane from other side of the cassette which then detect the specific immunocomplex through the specific binding with IgG molecule. [0105] Detecting the complex between the components derived from HCV or an equivalent thereof and a subject-derived binding partner such as immunoreactive molecules, may be based on any convenient method, which will be known to those of the skill of the art. [0106] It is contemplated that procedures useful for detecting components and binding partners which form complexes and are indicative of an HCV infection in a biological sample include, but are not limited to, immunological assays, such as immunoblotting, immunocytochemistry, immunohistochemistry or antibody-affinity chromatography, Western blot analysis, lateral flow or variations or combinations of these or other techniques such as are known in the art.

[0107] In general, components and binding partners, which form complexes and are indicative of an HCV infection, may be detected in a biological sample obtained from a subject by any means available to the skilled addressee. In an embodiment, the method of detection employs a further detection agent such as specific anti-body conjugated with enzyme, gold particle/colloid which permits detection of the complexes with the binding partners.

[0108J In an embodiment, the methods as herein described involve the use of an HCV antigen onto nitrocellulose membrane to which a binding partner of a biological sample may absorb/bind. The complex formed by a component of the HCV antigen and the binding partner may then be detected using a detection agent that contains a reporter group and specifically binds to the component/binding partner complex. Such detection agent may comprise, for example, an anti-antibody or other agent that specifically binds to the binding partner, such as an anti-immunoglobulin (i.e. antibody), protein G, protein A, protein-L or a lectin.

[0109] In an embodiment, the detection reagent is an antibody or secondary antibody or an antigen-binding fragment thereof, capable of binding to the binding partner of the biological sample. Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art (See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988).

[0110] The secondary antibody which may be conjugated to a label, can be added to the complex to facilitate detection. A range of labels providing a detectable signal may be employed. The label may be selected from a group including chromogen, an enzyme, a catalyst, a fluorophore, gold colloid and a direct visual label. In the case of a direct visual label, use may be made of a colloidal metallic or non-metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, or a latex particle. A large number of enzymes suitable for use as labels are disclosed in United States Patent Nos. 4,366,241 ; 4,843,000 and 4,849,338. Suitable enzyme labels in the present invention include alkaline phosphates, horseradish peroxidase, preferably horseradish peroxidase. The enzyme label may be used alone or in combination with a second enzyme, which is in solution. In the present invention a secondary antibody attached with horseradish peroxidase, which then reacts with its substrate DAB and produces a visually detectable color change, preferably achieves the detection of the complex. [0111] Generally, the antibody is an anti-IgG antibody conjugated with gold colloid and therefore detects IgG binding partners that have bound to the HCV specific immunogenic components.

[0112] This assay may be performed by first contacting a binding partner of a biological sample that has been immobilized on a solid support, such as a nitorcellulose membrane, with the HCV-specific immunogenic components as herein described, such that a component is allowed to bind to the immobilized binding partner such as an antibody. Alternatively, the HCV-specific immunogenic components may be bound to the solid support such that binding partners are allowed to bind to the immobilized component and then detection reagent is added which may be specific reporter group. Unbound sample is then removed from the immobilized complex through reverse flow technique. [0113] More specifically, once the binding partner or an HCV-specific immunogenic component is immobilized on the support as described above, the remaining binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin or milk casein with either Triton X 100 or Tween 20 (Trademark) [Sigma Chemical Co., St. Louis, Mo.]. The component or binding partner is used undiluted form during contact with HCV immunogenic component. In general, an appropriate contact time is 5-10 minutes that is sufficient to allow a an HCV specific immunological component presence in the biological samples to bind to the immobilized binding partner, or vice versa. Preferably, the contact time is sufficient to achieve a level of binding to the target epitope on the attached HCV specific immunogenic component that is at least about 95% of that achieved at equilibrium between the bound and unbound binding partner or HCV-specific immunogenic component. Those of ordinary skill in the art will recognize that the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. [0114] The detection agent is generally an anti-lgG antibody. Preferred reporter groups include those groups recited herein. The detection agent is then released from the gold-pad over the membrane where immunocomplex is formed with HCV antigen and anti-HCV IgG from biological samples. An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time. Unbound detection agent is then removed and bound detection agent is detected using the reporter group. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups, chromogenic enzymes and fluorescent groups. Chromogenic enzymes include, but are not limited to, peroxidase and alkaline phosphatase. Fluorescent groups include, but are not limited to, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC) 1 rhodamine, Texas Red, Gold colloid, and phycoerythrin. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). [0115] As used herein, "binding" refers to a non-covalent association between two separate molecules such that a complex is formed. The ability to bind may be evaluated by, for example, determining a binding constant for the formation of the complex. The binding constant is the value obtained when the concentration of the complex is divided by the product of the component concentrations. In general, two compounds are said to "bind," in the context of the present invention, when the binding constant for complex formation exceeds about 10 ³ L/mol. The binding constant may be determined using methods well known in the art.

[0116] Membranes contemplated by the method and kits of the present invention include any membrane to which either the binding partner or components derived from the flavivirus or equivalent thereof can bind. Examples of membranes include without being limited to, nitrocellulose membranes, polytetrafluorethylene membrane filters, cellulose acetate membrane filters and cellulose nitrate membrane filters with filter paper carriers. Most preferably, the membrane is a nitrocellulose membrane. [0117] Alternatively, the diagnostic methods of the present invention may adopt an automated analytic method using a biological microchip. For instance, a diagnostic kit can be structured to perform immuno-blotting using a glass slide coated with the component of the cell lysate. This diagnostic kit may comprise a biological microchip onto the surface of which an HCV specific immunogenic component is immobilized, an appropriate buffer, a standardized sample comprising a detectable level of binding agent, and a secondary detection reagent, as herein described.

[0118] The method and kits of the present invention can detect specific exposure of human or animals to HCV or any specific member or serotype or sub-serotype of the family or equivalent thereof either during acute infection or in convalescent phase. As used therein "acute infection" refers to the period of time when a virus has infected a host and is actively replicating and/or causing symptoms associated with infection like fever, rush, joint pain and or abdominal pain. The "convalescent phase" refers to the stage of HCV infection cycle when HCV is no longer multiplying or remains in the host blood and has developed binding partners such as, but not limited to antibodies. Using the method and kit of the present invention, exposure can be detected at any time after generation of a binding partner in the infected patient or patient derived it from his/her previous infection infections. The method and assay can also be used to detect chronic infection such as in patients with chronic liver disease caused by HCV.

[0119] In another aspect of the present invention there is provided a POCT kit for detecting IgG in a subject that is specific for an HCV or equivalent thereof or for detecting HCV exposure comprising: a solid membrane support including a HCV specific immunogenic component or equivalent thereof; or a solid support including a HCV specific immunogenic component or equivalent thereof attached to a second support; at least one detection agent conjugated to a reporter group for detecting a binding partner in a biological sample that forms a complex with the HCV specific immunogenic component; and optionally instructions for using the kit to further identify the binding partner of the complex. The kit may also be in the form of a cassette for detecting IgG specific for an HCV component. [0120] Optionally, the kit will also include additional parts such as plastic dropper, chase buffers, cassette and instructions as are necessary for conducting the method. [0121] Accordingly the present invention provides a kit for detecting exposure of a subject to HCV or any member of the family or serotype or sub-serotype or genotype or sub- genotype or an equivalent thereof. The kit may be any convenient form of POC testing which allows for a binding partner in a biological sample to interact with an anti-HCV IgG captured HCV viral component. The result is an indication, by the presence of HCV specific binding partners such as IgG in the biological sample, of prior exposure to HCV. Preferably, the kit comprises a solid support such as described herein adapted to receive or comprise anti-HCV IgG captured components of HCV or an equivalent thereof. The kit may also comprise reagents, reporter molecules capable of providing detectable signals and optionally instructions for use.

[0122] The kit may be a modular kit comprising one or more members wherein at least one member is a solid support comprising loaded/sprayed HCV component of virus particle or equivalent or cell lysate comprising an immunogenic component derived from an HCV or equivalent thereof.

[0123] The present invention also provides individual components of the kit for use in the method of the present invention. The invention provides solid supports that include anti- HCV IgG reactive components of the HCV for use in the detection of exposure to the HCV. In one embodiment, a nitrocellulose membrane is used to attach viral antigen, either for use as an immobilized anti-HCV IgG reactive HCV components or as a line blot. Preferably, the membranes include components selected from the group of HCV antigens, structural and non-structural proteins, viral particles and fragments thereof, glycoproteins, lipids and carbohydrates derived from the flavivirus or any mixture thereof. [0124] The solid support may also be a microtiter plate, glass slide or biological microchip wherein the components of the cell lysate are immobilized. These solid supports can then be subjected to the biological sample to detect flavivirus exposure. Preferably, nitrocellulose membrane is used to attach HCV antigen by spraying onto the membrane from HCV infected cells. [0125] In yet another aspect, the present invention also provides a method of assessing the relative risk of one or more subjects being exposed to HCV or an equivalent thereof within a defined location (e.g. geographical area, housing estate, means of transport or center for medical treatment or assessment), comprising: obtaining samples from a representative population within a defined location; and assessing evidence of exposure of individual members of a sample population to an HCV or equivalent thereof by the method comprising the steps of contacting a biological sample from the subject with a mixture of HCV specific immunogenic components; and determining the presence of a complex that forms between a binding partner in the biological sample and an HCV specific immunogenic component and wherein the presence of the complex is indicative of exposure of the subject to an HCV or equivalent thereof; and assessing the relative risk of exposure within the defined location by characterizing the binding partner in the complex.

[0126] The method or technique of the present invention allows for the epidemiological study or sero-surveillance of outbreaks of infection caused by HCV or any member of the family or serotype or genotype or sub-serotype or sub-genotype or equivalent thereof. Such studies provide valuable information, which advance multiple facets of research in the area of HCV disease. For example, epidemiological studies aid in the identification of the index of an infection. Such information enables the identification of a defined location from which the source of virus responsible for a viral outbreak originated.

[01271 Additionally the technique/method of the present invention permits for the rapid identification or isolation of subjects who are infected with an HCV or equivalent thereof without major laboratory equipment or even in field conditions. Such information aids in identifying subjects, who require medical treatment as well as defining locations that require further investigation or disease control approaches such as identification of breeding places and its control. Further, the technique of the present invention allows for the monitoring of an infected patient to determine the presence of anti-HCV specific IgG. Alleviation of IgG titre or its presence in an early phase of infection may be the indication of secondary infection and thus, help the monitoring of the subsequent phases of HCV infection such as chronic liver disease.

[0128] Further, the technique of the present invention provides a means for identifying subjects who are infected with any specific serotype or genotype of HCV involved, allowing for the rapid detection, risk of further infection, pointing to the location of an infection and disease control strategy.

[0129] The present invention is further directed to the use of IgG antibodies specific for HCV or an immunological relative thereof or an HCV antigen or an immunogenic component thereof in the manufacture of a POCT to detect current or recent prior infection or exposure to HCV or an immunological relative thereof.

[0130] Risk analysis may be conducted using software in a computer readable form. Consequently, the present invention further relates to a computer readable program and computer comprising suitable for analyzing exposure of subjects or group of subjects or a risk of exposure of subject or group of subjects to an HCV or equivalent thereof.

[0131] The present invention contemplates, therefore, the use of a knowledge base of training data comprising the presence or level of anti-HCV antibodies to one or more HCV components from a subject with a known HCV acute or chronic infection state to generate an algorithm which, upon input of a second knowledge base of data comprising levels of the same antibodies from a patient with an unknown state of a HCV exposure condition, provides an index of probability that predicts whether the subject has recently or at some point been exposed to or infected with HCV.

[0132] The term "training data" includes knowledge of levels or presence of HCV-specific antibodies relative to a control. A "control" includes a comparison to levels of biomarkers in a subject devoid of HCV exposure or cured of the HCV infection or may be a statistically determined level based on trials.

[0133] The "training data" also include the concentration of HCV-specific antibodies. The data may comprise information on an increase or decrease in the level of HCV-specific antibodies.

[0134] The present invention further provides, therefore, an algorithm-based screening assay to screen samples from patients for HCV infection or recent or prior exposure. Generally, input data are collected based on the presence of IgG antibodies to an HCV specific immunogen and subjected to an algorithm to assess signal to cut off ratios to determine if these are high or low relative to a predetermined value which then become output data. Computer software and hardware for assessing input and output data are encompassed by the present invention. Antibodies include IgG as well as other types such as IgM or IgA.

[0135] The assay of the present invention permits integration into existing or newly developed diagnostic architecture or platform systems. For example, the present invention contemplates a method of allowing a user to determine the status of a subject with respect to HCV infection or HCV-associated condition, the method including:

(a) receiving data in the form of the presence or absence of HCV specific antibodies; from the user via a communications network;

(b) processing the subject data via an algorithm which provides a disease index value;

(c) determining the status of the subject in accordance with the results of the disease index value in comparison with predetermined values; and

(d) transferring an indication of the status of the subject to the user via the communications network. [0136] Conveniently, the method generally further includes:

(a) having the user determine the data using a remote end station; and (b) transferring the data from the end station to the base station via the communications network.

[0137 J The base station can include first and second processing systems, in which case the method can include:

(a) transferring the data to the first processing system;

(b) transferring the data to the second processing system; and

(c) causing the first processing system to perform the algorithmic function to generate the disease index value.

[01381 The method may also include:

(a) transferring the results of the algorithmic function to the first processing system; and

(b) causing the first processing system to determine the status of the subject.

[0139] In this case, the method also includes at least one of:

(a) transferring the data between the communications network and the first processing system through a first firewall; and

(b) transferring the data between the first and the second processing systems through a second firewall.

[0140] The second processing system may be coupled to a database adapted to store predetermined data and/or the algorithm, the method include:

(a) querying the database to obtain at least selected predetermined data or access to the algorithm from the database; and

(b) comparing the selected predetermined data to the subject data or generating a predicted probability index.

[0141] The second processing system can be coupled to a database, the method including storing the data in the database. [0142] The method can also include having the user determine the data using a secure array, the secure array of elements capable of determining the presence of HCV specific antibody and having a number of features each located at respective position(s) on the respective code. In this case, the method typically includes causing the base station to:

(a) determine the code from the data;

(b) determine a layout indicating the position of each feature on the array; and

(c) determine the parameter values in accordance with the determined layout, and the data.

(0143] The method can also include causing the base station to:

(a) determine payment information, the payment information representing the provision of payment by the user; and

(b) perform the comparison in response to the determination of the payment information.

[0144] The present invention also provides a base station for determining the status of a subject with respect to HCV infection or HCV associated condition, the base station including:

(a) a store method;

(b) a processing system, the processing system being adapted to:

(i) receive subject data from the user via a communications network, the data including the presence or absence of HCV specific antibodies;

(ii) performing an algorithmic function including comparing the data to predetermined data;

(iii) determining the status of the subject in accordance with the results of the algorithmic function including the comparison; and

(c) output an indication of the status of the subject to the user via the communications network.

[0145] The processing system can be adapted to receive data from a remote end station adapted to determine the data. [0146] The processing system may include:

(a) a first processing system adapted to:

(i) receive the data; and

(ii) determine the status of the subject in accordance with the results of the algorithmic function including comparing the data; and

(b) a second processing system adapted to:

(i) receive the data from the processing system;

(ii) perform the algorithmic function including the comparison; and

(iii) transfer the results to the first processing system.

[0147] The base station typically includes:

(a) a first firewall for coupling the first processing system to the communications network; and

(b) a second firewall for coupling the first and the second processing systems.

[0148] The processing system can be coupled to a database, the processing system being adapted to store the data in the database. [0149] Reference to an "algorithm" or "algorithmic functions" as outlined above includes the performance of a univariate or multivariate analysis function such as determining signal to cut off ratios. A range of different architectures and platforms may be implemented in addition to those described above. It will be appreciated that any form of architecture suitable for implementing the present invention may be used. However, one beneficial technique is the use of distributed architectures.

[0150] It will also be appreciated that in one example, the end stations can be hand-held devices, such as PDAs, mobile phones, or the like, which are capable of transferring the subject data to the base station via a communications network such as the Internet, and receiving the reports. [0151] In the above aspects, the term "data" means the levels or concentrations or presence or absence of the HCV-specific antibodies. The "communications network" includes the internet. When a server is used, it is generally a client server or more particularly a simple object application protocol (SOAP).

[0152] The present invention is now described with reference to the following non-limiting Examples. It should be understood, however, that the following description is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

EXAMPLE 1

HCV antigen

[0153] HCV antigen: the HCV recombinant proteins, core, NS3, NS4 and NS5 (Biolink, USA) were used to develop an HCV-MRT using reverse flow technology. HCV recombinant antigens were expressed in E. coli system and purified standard protein purification protocol.

[0154] Protein-L: Protein-L (Pierce Biotechnology, USA) is an immunoglobulin-binding recombinant protein that was originally derived from the bacterium Peptostreptococcus magnus. Protein-L has the ability to bind with Alpha light chain of all subclasses of immunoglobulin G (IgG) without interfering with antigen-antibody reactions and was used to bind IgG molecule from the biological samples used in this development. [0155] Protein-A: (Pierce Biotechnology, USA) is an immunoglobulin-binding recombinant protein that was originally derived from the bacterium Staphylococcus aureus. Protein-A binds with high affinity to IgG 1 and IgG2 and has moderate affinity to IgM, IgA and IgE. [0156] Anti-human IgG-Gold conjugate (40nm): Goat anti-human IgG antibodies conjugated with gold colloid (Code-HIAIO, Millenia Diagnostics, Inc, USA) were used as secondary antibodies at a dilution of 7 OD (520 ran).

[0157] Serum samples: A total 400 serum samples positive by HCV PCR and ELISA were used as HCV positive samples. 1000 serum samples collected from non-HCV infected and 1000 healthy donors were used as negative controls. Another 150 samples obtained from non-HCV illness including hepatitis B, hepatitis E and Dengue virus was used as cross-reactive sera. [0158] Serological assays: HCV IgG ELISA, a commercial kit (Abbott, USA) was used for the measurement of anti-HCV specific IgM antibodies in the samples. The test was performed according to the procedures described by the manufacturer. [01591 HCV Nucleic Acid Test (HCV-NAT): HCV virus quantification was carried out using real-time in vitro quantification of HCV RNA from clinical samples via amplification of 5' untranslated region. The reliability of the test was compared with WHO calibrated synthetic virus control and conducted according to the method described by RoboGene (registered trade mark) Hepatitis C virus Quantification kit (AJ Roboscreen GmbH, D-04-129 Leipzig, Delitzscher Str. 135, Germany).

[0160] HCV-MRT (multi-line rapid test): An HCV rapid diagnostic kit available from MP Biomedicals in the form of cassette was used as a comparator in the validation and evaluation of the HCV-MRT of the present invention.

[0161] HCV Immunoblots: RIBA (Chiron) and MPD Bolt 2.4 (MP Biomedicals) were used for the detection of HCV specific IgM and IgG from the serum samples. The test was carried out according to the procedures described by the manufacturer. [0162] HBV and HCV diagnostics: HBsAg rapid antigen detection kit (MP Biomedicals) and Biokit HBsAg ELISA were used to screen samples for the presence of HBV infection. HCV antibody (IgG) rapid test (MP Biomedicals) was also used to screen sera samples. Positive serum against HCV was detected and confirmed by the PCR technique. [0163] Optimization of HCV Antigens: The optimization of HCV antigen (MP Biomedicals) is carried out lx PBS according to Table 2. A minimum volume of 500μ1 was made for each concentration before starting the optimization. Table 2

Optimization of HCV antigens

[0164] HCV recombinant proteins (r-proteins) were sprayed at positions set for HCV- MRT striping on 3 pieces of membrane (15 cm each). Protein-A was used as as control line and crystal violet as indicator line. Following spraying, the membrane is dried, blocked using blocking buffers and dried again and then laminated, cut and assembled for test.

[0165] Optimization of Protein-A and Protein-L: Proteins-A and -L (Pierce Biotechnology, USA) were diluted in 1 x PBS according to Table 3 mentioned below. A minimum volume of 500μ1 was made for each concentration for the process of optimisation.

Table 3

Optimization of Protein-A and Protein-L

[0166] Following assembly of the cassette, the cassette was used for the optimization of antigen and Protein-A sprayed/load onto the membrane-using panel of strong, weak and negative anti-HCV IgG positive serum samples. For Protein- A/-L, the C-Iine with 2.0 to 3.0 intensity with serum was considered as optimum level of reaction. For the optimization of viral protein the T-line with strong positive serum 3.0, weak positive 1.5 while for a negative result, no line or line with +/— intensity were considered as optimum level of reactions.

[0167] Following optimization of antigen and Protein-L, 1500 cassettes were prepared to assess sensitivity, specificity, positive predictive value and negative predictive value.

Assay procedure

Serum / Plasma/Saliva samples [0168] Step 1- Added 25μ1 of whole blood sample to the samples square well followed by 1 drop of chase buffer to the same well.

[0169] Step 2 - Sample absorbed by sample pad and then move over the membrane and once reached the blue control line (A), three (3) drops Chase Buffer was added to the conjugate pad.

[0170] Step 3 -Pulled the "plastic tab (labeled with HCV-MRT) until resistance is felt and added another drop of chase buffer onto the sample pad square and started timing. Read the result after 15 minutes.

[0171] Figure 1 shows the HCV-MRT device which contains three lines: one visible blue line known as indicator line while the remaining five lines are visible following the test with a positive sample. The details description and interpretations are given below. Interpretation of HCV-MRT

[0172] The result of HCV-MRT was interpreted using an interpretation card which has a range of color intensities varied from 0.25 to 3.0. For test lines (Cp [core protein], NS3, NS4 and NS5) any intensities between 0.5 to 3.0 is considered as an HCV-MRT positive sample while 0.0 to 0.25 is considered as negative. If only the core protein (Cp) line along with C-line is visible and all non-structural (NS) proteins specific bands are negative, the patient may be considered as recovered, whereas a Cp line with any of the non-structural proteins (NS3, NS4, NS5) is considered as chronic case. Only NS3 line along with C- line indicates that the patent is in a very early stage of HCV infection. A control line (C-Line) intensity of between 2.5 to 3.0 is considered to validate the test. See Figure 2.

[0173] HCV IgG ELISA: Commercially available kits were used as comparative kits to assess the performance of the HCV-MRT of the present invention.

[0174] Comparative analytical sensitivity of HCV-MRT: The analytical sensitivity of HCV-MRT was studied by using strong, medium and weak HCV IgG positive serum samples. Serum samples were diluted at 1 :5 to 1 : 160 in whole blood, serum or plasma from a healthy person negative to HCV antibodies (IgG, IgM and IgA).Twenty-microliter (25μ1) of each diluted sample was then added onto the cassette sample pad and test was performed as described above.

EXAMPLE 2

Comparative analysis of HCV-MRT against HCV ELISAs

[0175] The level of sensitivity of HCV-MRT carried out using HCV positive sera (PCR) showed that HCV-MRT exhibited 100% sensitivity and 99.90% specificity respectively (see Table 4 which shows the overall diagnostic performances of HCV-MRT).

Table 4

Diagnostic performance of HCV-MRT using 2x2 table

HCV PCR

POS NEG Total

HCV-MRT POS 326 1 327

NEG 0 999 999

Total 326 1000 1326

[0176] The overall sensitivity and specificity of the HCV-MRT of the present invention exceeded expectations and was shown to be a rapid screening and confirmatory test at all levels of the health care system. EXAMPLE 3

HCV-MRT performance [0177] The performance of the HCV-MRT of the present invention was further evaluated using a commercially available screening ELISA (Abbott). Results showed 100% sensitivity (see Table 5 which shows the comparative performance of HCV-MRT against PCR and HCV ELISA). Table 5

HCV-MRT Abbott ELISA (n=100)

Positive Negative Total

Positive 100 0 100

Negative 0 0 0

Total 100 0 100

EXAMPLE 4

HCV-MRT compared with commercially available HCV-RT

[0178J The performance of the HCV-MRT of the present invention was compared with commercially available HCV-RT and is presented in Table 6 (comparative performance of HCV-MRT with HCV Immunoblots (RIBA and MPD HCV Blot 2.4)).

Table 6

Comparative performance of HCV MRT and HCV Blot 3.0 (2x 2 tables)

[0179] The overall diagnostic results of HCV-MRT of the present invention and HCV-RT showed that HCV-MRT was as sensitive as the #CV Blot approach.

EXAMPLE 5

HCV-MRT compared with Western Blot

[0180] The diagnostic performances of the HCV-MRT of the present invention was also compared with Western Blot (RIBA, Chiron) and is presented in Table 7 (specificity of HCV IgA POCT against non-HCV viral hepatitis and other febrile illness).

Table 7

Comparative performance of HCV-MRT against HCV blot (RIBA, Chiron) using 2x2 table

HCV -Blot (RIBA

POS NEG Total

HCV-MRT POS 50 0 50

NEG 0 91 91

Total 50 91 141

[0181] In 2x2 table analysis of HCV-MRT and HCV Blot (RiBA) showed 100% agreement in diagnostic performance which indicates the consistency of the performance of the HCV-MRT of the present invention. EXAMPLE 6

Specificity of HCV-MRT using sera collected from non-HCV febrile illness (n-760) including HBV, HA V and HEV infections.

[0182] The cross-reactivity of HCV-MRT was carried against non-HCV viral hepatitis infections (HAV, HBV and HCV) along with other five febrile illnesses and showed very low level of reactivity. HBV positive sera were confirmed as HBV using both HBsAg rapid test (MP Biomedicals) as well as antigen ELISA (Biokit Ag ELISA) while HCV positive samples were positive to PCR technique (Table 8).

Table 8

Performance with Cross-reactive serum samples

Serum group and patient status No. of positive & Performance %

From other hepatitis patients:

HEV antibody positive 0/100

HBV antibody positive 0/250

HAV antibody positive 0/40

HIV antibody positive 0/100

HTLV antibody positive 0/40

Dengue antibody positive 0/100

CMV positive 0/10

Other conditions

Pregnant women serum samples 0/100

Rheumatoid Arthritis 0/20

High Bilirubin 1/10

Total 1/760

Total Specificity 99.87%

(95% CL: 99.27% to 99.98%)

[0183] HCV-MRT showed very high level of specificity against other viral hepatitis infected patient's serum samples as well as other disease conditions. Test also showed 100% specificity against non-specific molecules produced during pregnancy. EXAMPLE 7

HCV-RTand MPD HCV-Blot 3.0

[0184] The comparative performances of the HCV-RT of the present invention and and MPD HCV-Blot 3.0 against antibody molecule directed to structural and non-structural proteins was carried out and is presented in Table 9 (n=326 PCR positive samples).

Table 9

Structural and non-structural proteins wise reactivity of HCV-MRT and MPD HCV Blot

3.0 assay

HCV Core NS3 NS4 NS5 All

Tests

HCV-Blot 297 256 138 206 46

(90.82%) (78.28%) (42.20%) (62.99%) (14.06%)

HCV- 285 297 248 219 147

MRT (87.15%) (90.82%) (75.84%) (66.97%) (44.95%)

[0185] In terms of HCV protein specific reactivity, HCV-MRT showed better performance than HCV Blot 3.0. Only antibody against core protein showed more reactive with HCV Blot 3.0 (90.82%) compared to HCV-MRT (87.15%). In HCV-RT platform 44.95% samples showed antibodies against all proteins compared to 14.06% with HCV Blot 3.0, although the incubation times of HCV Blot 3.0 was 2.5 hours compared to only 15 minutes by HCV-MRT. These results clearly showed higher level assay performance of HCV-MRT compared to HCV Blot 3.0.

EXAMPLE 8

Analytical sensitivity of HCV-MRT

[0186] Analytical sensitivity of HCV-MRT was conducted using HCV positive samples and is presented in Table 10.

Table 10

Analytical sensitivity

EXAMPLE 9

Detection of HCV

[0187] The HCV-MRT of the present invention was used to study the suitability of this platform to establish how early HCV cases can be detected using sero-converted samples. The results are presented in Table 1 1.

Table 11

Detection of HCV cases

[01881 It should be noted that Ortho-ELIA 3.0->1.0 is reactive and Ortho-RIBA is positive with one of the antigen lines. This is considered as ^determinate. In contrast, HCV-MRT is considered as positive when one line is >1 and two lines >0.3. [0189] HCV-MRT detected HCV clinical sample as positive on day 13 of first bleeding while Ortho-EIA 3.0 on day 19 and Ortho RIBA 2.0 showed indeterminate on day 27.

EXAMPLE 10

Detection of all HCV Genotypes by HCV-MRT

[0190] The performance of HCV-MRT was also evaluated to check the ability to recognize all 6 genotypes of HCV virus and is presented in Table 12.

Table 12

Performance of HCV-MRT in detecting all HCV Genotypes

The above study clearly indicated that all 6 HCV genotypes could be detected using HCV- MRT platform. [0191] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

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