Method for determining deletions in hbv pre-s2 region

Abstract

A method of detecting pre-S 2 deletion mutant LHBS is disclosed herein. The method comprises incubating a biological sample with a first antibody to captured HBS proteins; detecting the LHBS and WT LHBS bound to the immobilized first antibody, respectively; and calculating the amount of the pre-S 2 deletion mutant LHBS protein by subtracting the amount of the WT LHBS protein from that of the LHBS protein. Advantageously, by the method described herein, the amount of the pre-S 2 deletion mutant LHBS, a potential high-risk marker for HCC incidence in chronic HBV carriers and recurrence in HCC patients after hepatectomy surgery, in a biological sample may be easily calculated without mutual influence between the WT and pre-S mutant LHBS while reducing the labor-intensive process for cloning each gene product before analysis.

What is claimed is: 
     
         1 . A method of detecting a pre-S 2  deletion mutant large hepatitis B virus surface protein in a biological sample, comprising:
 incubating a biological sample with a first antibody immobilized on a substrate to bind a hepatitis B virus surface protein to the first antibody, wherein the first antibody specifically binds to amino acid residues 1-226 of hepatitis B virus surface protein;   detecting a large hepatitis B virus surface protein and a wild type large hepatitis B virus surface protein bound to the immobilized first antibody by incubating the bound hepatitis B virus surface protein with a second antibody specifically binding to amino acid residues 1-119 of large hepatitis B virus surface protein and a third antibody specifically binding to amino acid residues 125-142 of large hepatitis B virus surface protein, respectively; and   calculating the amount of the pre-S 2  deletion mutant large hepatitis B virus surface protein by subtracting the amount of the wild type large hepatitis B virus surface protein from that of the large hepatitis B virus surface protein.   
     
     
         2 . The method of  claim 1 , wherein the first antibody is a hepatitis B virus surface protein-specific antibody produced by a hybridoma cell line deposited on Oct. 22, 2014 under accession number BCRC960491 at the Food Industry Research and Development Institute, 331 Shih-Pin Road, Hsinchu 300, Taiwan, and also on Oct. 31, 2014 under accession number DSM ACC3252 at Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, 38124 Braunschweig, Germany, or progeny thereof, the second antibody is a large hepatitis B virus surface protein-specific antibody produced by a hybridoma cell line deposited on Sep. 1, 2014 under accession number BCRC960490 at the Food Industry Research and Development Institute, 331 Shih-Pin Road, Hsinchu 300, Taiwan, and also on Oct. 31, 2014 under accession number DSM ACC3253 at Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, 38124 Braunschweig, Germany, or progeny thereof, and the third antibody is a wild type large hepatitis B virus surface protein-specific antibody produced by a hybridoma cell line deposited on Jul. 16, 2014 under accession number BCRC960488 at the Food Industry Research and Development Institute, 331 Shih-Pin Road, Hsinchu 300, Taiwan, and also on Oct. 31, 2014 under accession number DSM ACC3251 at Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, 38124 Braunschweig, Germany, or progeny thereof. 
     
     
         3 . The method of  claim 1 , wherein the biological sample is isolated from a human. 
     
     
         4 . The method of  claim 3 , wherein the human is a HBV carrier or a liver cancer patient. 
     
     
         5 . The method of  claim 4 , wherein the HBV carrier is HBeAg-negative, with HBV viral load higher than 10 4  copies/ml of the biological sample, or the combination thereof. 
     
     
         6 . The method of  claim 5 , further comprising a step of assessing a risk of developing hepatocellular carcinoma or cirrhosis of the HBV carrier. 
     
     
         7 . The method of  claim 4 , wherein the HBV carrier has undergone an antiviral therapy using nucleoside analogs or interferon drugs. 
     
     
         8 . The method of  claim 7 , further comprising a step of assessing a risk of developing hepatocellular carcinoma or cirrhosis after the antiviral therapy. 
     
     
         9 . The method of  claim 4 , wherein the liver cancer patient is a hepatocellular carcinoma patient undergone a hepatectomy surgery. 
     
     
         10 . The method of  claim 9 , further comprising a step of assessing risk of cancer recurrence of the hepatocellular carcinoma patient at 1 to 12 month(s) after the surgery. 
     
     
         11 . The method of  claim 3 , wherein the biological sample is whole blood, plasma or serum. 
     
     
         12 . The method of  claim 1 , further comprising a step of isolating hepatitis B virus surface protein from the biological sample.