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Matched Legal Cases: ['Application No. 60', 'Application No. 60', 'Application No. 04720548', 'Application No. 04720548', 'Application No. 04720548', 'Application No. 200480013005', 'Application No. 04720548', 'Application No. 04720548', 'Application No. 200480013005', 'Application No. 200480013005']

Patent US8183348 - Non-invasive marker for liver function and disease - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsA monoclonal antibody or fragment thereof, capable of specifically binding to at least one epitope of sH2a and/or being elicited by at least one epitope, and assays, kits, and methods of use thereof diagnosing liver disease or condition, detecting liver function and assessing the efficacy of therapy...http://www.google.com/patents/US8183348?utm_source=gb-gplus-sharePatent US8183348 - Non-invasive marker for liver function and diseaseAdvanced Patent SearchPublication numberUS8183348 B2Publication typeGrantApplication numberUS 12/758,848Publication dateMay 22, 2012Filing dateApr 13, 2010Priority dateMar 13, 2003Also published asUS7741065, US20060286615, US20100190185Publication number12758848, 758848, US 8183348 B2, US 8183348B2, US-B2-8183348, US8183348 B2, US8183348B2InventorsGerardo Z. Lederkremer, Maria KondratyevOriginal AssigneeRamot At Tel-Aviv University Ltd.Export CitationBiBTeX, EndNote, RefManPatent Citations (16), Non-Patent Citations (43), Classifications (11) External Links: USPTO, USPTO Assignment, EspacenetNon-invasive marker for liver function and diseaseUS 8183348 B2Abstract A monoclonal antibody or fragment thereof, capable of specifically binding to at least one epitope of sH2a and/or being elicited by at least one epitope, and assays, kits, and methods of use thereof diagnosing liver disease or condition, detecting liver function and assessing the efficacy of therapy to a liver disease.
1. A monoclonal antibody capable of specifically binding to at least one epitope of sH2a, wherein said antibody is described by deposit number 04030801, deposited at the European Collection of Cell Cultures.
2. A hybridoma cell line capable of producing the monoclonal antibody of claim 1.
3. A kit for detecting abnormal liver function in a subject, the kit comprising the monoclonal antibody of claim 1 which specifically binds to at least one epitope of sH2a and instructions for detecting sH2a in a sample of a subject and interpreting results of the detecting.
4. The kit of claim 3, wherein said antibody further comprises a label.
5. The kit of claim 3, further comprising one or more of a washing buffer, a blocking buffer and a sample dilution buffer.
6. The kit of claim 3, further comprising a solid phase for immobilizing said antibody.
7. The kit of claim 3, further comprising a control protein.
8. The kit of claim 3, further comprising an indicator for indicating the level of sH2a in said sample.
9. The kit of claim 3, wherein said sample comprises a serum sample, a plasma sample, a urine sample, a whole blood sample or a blood fraction sample.
10. The kit of claim 3, for performing at least one of an ELISA, a competitive ELISA, a flow through assay or an immunoblot.
11. The kit of claim 3, wherein the subject is a human subject.
12. The kit of claim 3, wherein said abnormal liver function comprises a liver disease.
13. The kit of claim 12, wherein said liver disease is selected from a group consisting of hepatocellular carcinoma, liver cirrhosis, liver fibrosis, hepatitis, Wilson's disease, HHC and α-1-AntiTrypsin deficiency.
RELATED APPLICATIONS This Application is a Divisional of U.S. patent application Ser. No. 11/224,148, filed on Sep. 13, 2005, which is a Continuation-In-Part (CIP) of PCT Patent Application No. PCT/IL2004/000244, filed on Mar. 14, 2004, which claims priority from U.S. Provisional Application No. 60/453,944, filed on Mar. 13, 2003. U.S. patent application Ser. No. 11/224,148 also claims priority from U.S. Provisional Application No. 60/691,241, filed on Jun. 17, 2005. The contents of the above Applications are incorporated herein by reference.
Generally, blood tests for liver function are based on the level of several markers such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamic transpeptidase (GGT), bilirubin, albumin and prothrombin time (PT) in the serum. However, while the markers used in such �liver function tests� are capable of assessing hepatocyte integrity, which might be indicative to liver damage, most of them, except albumin and prothrombin, are not indicative of the synthesis function of the liver. Albumin, which is produced in the liver and circulates in the blood, is affected only when a liver disease is at a severe stage. On the other hand non-hepatic diseases such as nephrotic syndromes can affect albumin levels. Similarly, prothrombin, which is used to evaluate blood clotting disorders, is insensitive to mild liver disease and can be also affected by non-hepatic conditions such as dietary deficiencies or the use of anti-coagulants Likewise, abnormal levels of bilirubin can result from hemolysis, ineffective erythropoiesis and other non-hepatic syndromes. In addition, as ALT, AST, ALP and GGT are also produced in organs other than the liver, their blood levels can be elevated in a wide range of non-hepatic diseases. Biochemical screening of healthy, asymptomatic people has revealed that up to 6% of the population exhibit abnormal levels of liver enzymes. However, the prevalence of liver disease in the general population is significantly lower (about 1%) (Gopal and Rosen, 2000). Even though the current serum biochemical test pattern may suggest a specific diagnosis, confirmation usually requires further investigation using imaging studies and, possibly, liver biopsy. Even mild liver test abnormalities may be an early clue to the presence of potentially significant liver disease (Hay, J. E., et al., 1989). For instance, patients with chronic hepatitis C virus (HCV) infection are often asymptomatic unless they have advanced liver disease. They usually have mild elevation of the serum ALT level, and about one third have persistently normal liver enzyme levels. Accordingly, as mentioned above, lack of sensitivity and specificity limit the use of liver function tests. For example, in some clinical conditions (e.g., cirrhosis), patients may have serum aminotransferase levels in the normal to near-normal range. In addition, several nonhepatic factors (Moseley, R. H. 1996) can affect the results of tests that measure specific hepatic function, such as serum albumin, total bilirubin, and prothrombin time (PT).
According to features of the present invention, the at least one epitope comprises SEQ ID NO:1. According to still further features of the present invention, the monoclonal antibody or fragment thereof is described by deposit number, Cell Name�B9; Provisional Accession Number�04030801, which was done on Mar. 8, 2004 at the European Collection of Cell Cultures, Center for Applied Microbiology and Research, Porton Down, Salisbury, Wilshire, SP4 0JG, United Kingdom. According to further features of the present invention, the monoclonal antibody or fragment thereof comprises a chimeric antibody, a humanized antibody, a Fab fragment, a single chain antibody, an immobilized antibody or a labeled antibody.
A deposit of hybridoma cells of the present invention is maintained by ECACC (European Collection of Cell Cultures) (Center for Applied Microbiology and Research, Porton Down, Salisbury, Wilshire, SP4 0JG, United Kingdom) since Mar. 8, 2004 under the following depository number: B9 04030801.
1.2 ml cell supernatants from 90 mm petri-dishes of 3T3 (lanes 1-2) or HepG2 (lanes 4-5), or 0.3 ml of normal human sera from 3 donors (S1, lanes 5-6; S2, lane 7; S3, lanes 8-9) were immunoprecipitated with anti-H2 carboxy-terminal antibodies and subjected to 12% SDS-PAGE. The proteins were then transferred to a nitrocellulose membrane and the blot was reacted with the anti-H2 carboxy-terminal antibody, followed by goat anti-rabbit peroxidase. The detection of the bands was performed using the TMB membrane peroxidase substrate (3,3′,5,5′-tetramethylbenzidine). Samples in lanes 2, 4, 6, 7 and 9 were treated with N-glycanase after immunoprecipitation. All the methods were performed substantially as described in the materials and methods section above.
In order to be able to measure the absolute concentration of sH2a in the sera, a recombinant version of sH2a with a 6� His-tag in its carboxy-terminus was produced. The construct is expressed in E. coli, and purified on a Ni2+-NTA-sepharose column. After elution and confirmation of the purity of the recombinant protein in SDS-PAGE it can be used as a standard in the competitive ELISA assay.
For this assay, proteins from the obtained sample immunoprecipitated with an anti-H2a carboxyterminal antibody, are separated in SDS polyacrylamide gel electrophoresis and transferred to a nitrocellulose filter. For this purpose, 0.3 ml of serum sample are immunoprecipitated for 16 hours at 4� C. with 20 μl of protein A-sepharose (Repligen, Cambridge, Mass.) that were crosslinked to polyclonal anti-H2a carboxy-terminal antibodies with dimethyl pimelimidate (Pierce). Immunoprecipitates are washed three times with PBS and then boiled in 10 μl of 0.5% SDS in 50 mM sodium citrate pH 6.0. Then, 10 μl of a solution containing 200 mM sodium phosphate pH 8.0, 40 mM EDTA pH 8.0, 3% N-octyl-glucoside is added, together with 40 mU of N-glycosidase F (Boehringer), and incubations are carried out for 16 hours at 37� C. Twenty μl of 2� sample buffer (125 mM Tris-HCl pH 6.8, 4% SDS, 20% Glycerol, 7% β-Mercaptoethanol and Bromphenol blue) are added and the samples boiled before loading for SDS-PAGE.SDS polyacrylamide gels are prepared as follows:
Protein transfer is performed substantially as described above. The gel is located, together with the adjacent nitrocellulose, between Whatmann 3 MM filter paper, conductive, 1 cm-thick foamed material and two carbon plates which conduct the current by way of platinum electrodes. The filter paper, the foamed material and the nitrocellulose are soaked thoroughly with blotting buffer (192 mM glycine, 25 mM tris base, 20% methanol, pH 8.5). The transfer is performed at 2 mA/cm2 for 2 hours. Free binding sites on the nitrocellulose are saturated, at room temperature for 1 hour, with 5% milk in TBS. The blot strips are incubated with an antibody or fragment thereof according to the present invention (dilution, 1:500 in 0.5% milk in TBS (154 mM NaCl and 10 mM Tris-HCl, pH 7.5)) at 4� C. overnight.
Quantitation of the serum levels of sH2a in HCV patients with or without liver fibrosis�Samples of sera from HCV patients with or without liver fibrosis were analyzed by an ELISA assay, essentially as described under Materials and Experimental Methods hereinabove. The patient's sera was compared to normal human sera at various time-points to reveal ALT and sH2a level during therapy for about a year. FIGS. 6 a-b show comparative analyses of the percent of sH2a in serum compared to normal and the normalized ALT level in serum of a patient (patient No. 1) with mild liver fibrosis. The first sample was taken prior to treatment and then the patient was treated with interpheron alpha and ribavirin. Thus, prior to treatment, the ALT level of patient No. 1 was abnormal and, upon treatment, was reduced to the normal range (i.e., less than 1) (FIG. 6 a). On the other hand, sH2a levels displayed an almost minor image, starting with low abnormal levels (i.e., less than 85% of normal) and, upon treatment, increasing to normal levels (FIG. 6 b). Interestingly, there was a delay of about 10 weeks between the time point in which ALT levels reached normal levels to the time point in which sH2a levels were back to normal. This is probably due to the fact that ALT levels reflect on liver damage while sH2a levels reflect on liver function (sH2a). Thus, in order to recover to a normal function following liver damage (as reflected by ALT levels) an additional time is required (as reflected by sH2a levels).
2. Hayasaka, A. and H. Saisho, Serum markers as tools to monitor liver fibrosis. Digestion, 1998. 59(4): p. 381-4.
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