Abstract:
The present invention describes peptides and recombinant proteins containing Hepatitis C virus core protein sequence in which one or more of the amino acids have been modified or deleted to remove the ability of these proteins to bind to specific anti-HCV murine monoclonal antibodies. The deletions and modifications are designed as to maintain the ability of this protein to be used in immunoassays used for the detection of anti-HCV antibodies in individuals infected with HCV.

Description:
BACKGROUND OF THE INVENTION 
     An estimated 170 million people worldwide have been infected by hepatitis C virus (HCV). In the next few years, the number of U.S. deaths for HCV-caused liver disease and cancer may overtake deaths caused by Acquired Immune Deficiency Syndrome (AIDS). 
     The transmission of HCV seems to require blood-to-blood contact. Carrying a single strand of ribonucleic acid (RNA), HCV contains just one gene, coding for a polyprotein that is subsequently cleaved into at least 10 functional proteins. Clearly, the ability to test the blood supply for HCV is of great importance. A sensitive assay that can detect infection at an early stage would be helpful. 
     HCV detection assays typically detect antibodies against HCV virus. These antibodies are detected in immunoassays using recombinant proteins and peptides containing HCV protein sequences. Most commercial anti-HCV assays use proteins from the following regions, core protein, NS3, NS4, and NS5 protein sequences. 
     Anti-HCV core antibodies are one of the most prevalent anti-HCV antibodies detected in chronic HCV infected individuals. HCV core protein contains multiple epitopes. Using synthetic peptides in the HCV core region, it has been shown that most of these epitopes are in the amino terminal end of this protein. For example, using overlapping peptides, each approximately 15 amino acids in length, an ELISA was developed to screen for anti-HCV antibodies in chronic HCV infected individuals. Table 1 below shows the peptide sequences. As shown in Table 2 below, HCV infected individuals have antibodies to two or more of these core peptides. Thus, it has been shown that the complete core peptide is not required to detect anti-core antibodies. 
     
       
         
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                   
                   
                 HCV 
                   
               
               
                 Peptide 
                 Amino Acid 
                 Polyprotein 
               
               
                 ID # 
                 Sequence 
                 AA Location 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 0 
                 MSTNPKPQKKNKRNT 
                  1-15 
                 SEQ ID NO.:1 
                   
               
               
                   
               
               
                 1 
                 KNKRNTNRRPQDVKF 
                 10-24 
                 SEQ ID NO.:2 
               
               
                   
               
               
                 2 
                 QDVKFPGGGQIVGGV 
                 20-34 
                 SEQ ID NO.:3 
               
               
                   
               
               
                 3 
                 QIVGGVYLLPRRGPR 
                 29-43 
                 SEQ ID NO.:4 
               
               
                   
               
               
                 4 
                 RRGPRLGVPATRKTS 
                 39-53 
                 SEQ ID NO.:5 
               
               
                   
               
               
                 5 
                 ATRKTSERSQPRGRR 
                 48-62 
                 SEQ ID NO.:6 
               
               
                   
               
               
                 6 
                 PRGRRQPIPKARRPE 
                 58-72 
                 SEQ ID NO.:7 
               
               
                   
               
               
                 7 
                 KARRPEGRTWAQPGY 
                 67-81 
                 SEQ ID NO.:8 
               
               
                   
               
               
                 8 
                 AQPGYPWPLYGNEGC 
                 77-91 
                 SEQ ID NO.:9 
               
               
                   
               
               
                 9 
                 YGNEGCGWAGWLLSP 
                  86-100 
                 SEQ ID NO.:10 
               
               
                   
               
               
                 10 
                 WLLSPRGSRPSWGPT 
                  96-110 
                 SEQ ID NO.:11 
               
               
                   
               
               
                 11 
                 SWGPTDPRRRSRLNG 
                 106-120 
                 SEQ ID NO.:12 
               
               
                   
               
               
                 12 
                 SRLNGKVIDTLTCGF 
                 116-130 
                 SEQ ID NO.:13 
               
               
                   
               
               
                 13 
                 LTCGFADLMGYIPLV 
                 126-140 
                 SEQ ID NO.:14 
               
               
                   
               
               
                 14 
                 YIPLVGAPLGGAARA 
                 136-150 
                 SEQ ID NO.:15 
               
               
                   
               
               
                 15 
                 GAARALAHGVRVLED 
                 146-160 
                 SEQ ID NO.:16 
               
               
                   
               
               
                 16 
                 RVLEDGVNYATGNLP 
                 156-170 
                 SEQ ID NO.:17 
               
               
                   
               
               
                 17 
                 TGNLPGCSFSIFLLA 
                 166-180 
                 SEQ ID NO.:18 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Peptide 
                 0 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
                 8 
                 9 
                 10 
                 11 
                 12 
                 13 
                 14 
                 15 
                 16 
                 17 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Neg 
                 0.044 
                 0.004 
                 0.021 
                 0.075 
                 0.059 
                 0.069 
                 0.076 
                 0.103 
                 0.020 
                 0.034 
                 0.002 
                 0.088 
                 0.051 
                 0.028 
                 0.023 
                 0.034 
                 0.041 
                 0.042 
               
               
                 Neg 
                 0.045 
                 0.065 
                 0.017 
                 0.081 
                 0.051 
                 0.081 
                 0.103 
                 0.111 
                 0.033 
                 0.035 
                 0.002 
                 0.085 
                 0.038 
                 0.030 
                 0.023 
                 0.030 
                 0.038 
                 0.052 
               
               
                 Neg 
                 0.050 
                 0.003 
                 0.014 
                 0.075 
                 0.087 
                 0.079 
                 0.094 
                 0.102 
                 0.025 
                 0.034 
                 0.002 
                 0.071 
                 0.038 
                 0.033 
                 0.021 
                 0.031 
                 0.039 
                 0.047 
               
               
                 Neg 
                 0.014 
                 0.043 
                 0.134 
                 0.023 
                 0.034 
                 0.039 
                 0.034 
                 0.039 
                 0.012 
                 0.018 
                 −0.001 
                 0.025 
                 0.009 
                 0.009 
                 0.008 
                 0.013 
                 0.010 
                 0.020 
               
               
                 Neg 
                 0.015 
                 0.039 
                 0.119 
                 0.021 
                 0.033 
                 0.030 
                 0.030 
                 0.034 
                 0.013 
                 0.019 
                 0.000 
                 0.026 
                 0.011 
                 0.010 
                 0.005 
                 0.012 
                 0.010 
                 0.016 
               
               
                 Neg 
                 0.013 
                 0.036 
                 0.126 
                 0.027 
                 0.053 
                 0.032 
                 0.037 
                 0.042 
                 0.013 
                 0.016 
                 0.001 
                 0.037 
                 0.013 
                 0.009 
                 0.009 
                 0.012 
                 0.009 
                 0.017 
               
               
                 Neg 
                 0.016 
                 0.004 
                 0.104 
                 0.020 
                 0.039 
                 0.034 
                 0.003 
                 0.041 
                 0.019 
                 0.019 
                 0.000 
                 0.041 
                 0.011 
                 0.017 
                 0.006 
                 0.010 
                 0.008 
                 0.021 
               
               
                  9 
                 0.256 
                 2.500 
                 2.500 
                 0.425 
                 1.443 
                 0.016 
                 0.218 
                 0.248 
                 0.062 
                 0.015 
                 0.025 
                 0.049 
                 0.030 
                 0.013 
                 0.012 
                 0.021 
                 0.012 
                 0.028 
               
               
                 11 
                 0.413 
                 2.500 
                 2.500 
                 0.917 
                 0.229 
                 0.647 
                 0.114 
                 0.262 
                 0.166 
                 0.159 
                 0.002 
                 0.055 
                 0.022 
                 0.027 
                 0.021 
                 0.042 
                 0.247 
                 0.088 
               
               
                 15 
                 0.044 
                 0.661 
                 2.500 
                 0.067 
                 0.577 
                 0.057 
                 0.110 
                 0.072 
                 0.061 
                 0.032 
                 −0.003 
                 0.045 
                 0.018 
                 0.031 
                 0.020 
                 0.039 
                 0.035 
                 0.080 
               
               
                 18 
                 1.643 
                 0.887 
                 2.500 
                 0.641 
                 0.100 
                 0.052 
                 0.032 
                 0.576 
                 0.037 
                 0.022 
                 −0.001 
                 0.040 
                 0.006 
                 0.010 
                 0.009 
                 0.025 
                 0.036 
                 0.039 
               
               
                 20 
                 0.021 
                 0.111 
                 2.500 
                 2.500 
                 0.482 
                 0.032 
                 0.029 
                 0.045 
                 0.028 
                 0.015 
                 0.011 
                 0.035 
                 0.009 
                 0.014 
                 0.009 
                 0.017 
                 0.019 
                 0.059 
               
               
                 21 
                 2.500 
                 2.500 
                 2.500 
                 2.262 
                 2.500 
                 0.516 
                 2.259 
                 0.080 
                 0.378 
                 0.018 
                 0.022 
                 0.064 
                 0.013 
                 0.024 
                 0.017 
                 0.024 
                 0.135 
                 0.035 
               
               
                 27 
                 0.032 
                 2.500 
                 2.500 
                 0.089 
                 2.500 
                 1.652 
                 1.349 
                 0.043 
                 0.039 
                 0.034 
                 0.033 
                 0.026 
                 0.012 
                 0.018 
                 0.013 
                 0.028 
                 0.287 
                 0.008 
               
               
                 30 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
                 0.183 
                 0.044 
                 0.075 
                 0.993 
                 0.078 
                 0.035 
                 0.028 
                 0.190 
                 0.023 
                 0.025 
                 0.022 
                 0.028 
                 0.042 
                 0.043 
               
               
                 35 
                 0.022 
                 0.278 
                 0.912 
                 0.031 
                 0.036 
                 0.116 
                 0.079 
                 0.049 
                 0.062 
                 0.010 
                 0.005 
                 0.026 
                 0.005 
                 0.006 
                 0.009 
                 0.020 
                 0.007 
                 0.020 
               
               
                 36 
                 0.059 
                 0.129 
                 2.500 
                 0.090 
                 0.131 
                 0.060 
                 0.093 
                 0.148 
                 0.090 
                 0.037 
                 0.003 
                 0.050 
                 0.018 
                 0.060 
                 0.077 
                 0.062 
                 0.129 
                 0.079 
               
               
                 37 
                 2.500 
                 2.500 
                 2.500 
                 0.219 
                 0.130 
                 0.090 
                 0.169 
                 0.223 
                 0.147 
                 0.040 
                 0.018 
                 0.065 
                 0.016 
                 0.051 
                 0.051 
                 0.025 
                 0.080 
                 0.110 
               
               
                 39 
                 0.165 
                 2.500 
                 2.500 
                 2.500 
                 0.124 
                 1.217 
                 2.351 
                 2.500 
                 0.397 
                 0.056 
                 0.015 
                 1.319 
                 0.017 
                 0.050 
                 0.056 
                 0.041 
                 0.200 
                 0.190 
               
               
                 40 
                 0.058 
                 2.500 
                 2.500 
                 2.500 
                 0.098 
                 0.066 
                 0.060 
                 0.755 
                 0.093 
                 0.024 
                 0.000 
                 0.059 
                 0.013 
                 0.028 
                 0.017 
                 0.032 
                 0.068 
                 0.101 
               
               
                 42 
                 0.022 
                 0.043 
                 2.500 
                 0.031 
                 0.023 
                 0.150 
                 0.018 
                 0.036 
                 0.023 
                 0.011 
                 −0.001 
                 0.032 
                 0.005 
                 0.014 
                 0.006 
                 0.010 
                 0.012 
                 0.031 
               
               
                 43 
                 0.158 
                 0.612 
                 0.368 
                 0.033 
                 0.065 
                 0.103 
                 0.069 
                 0.075 
                 0.053 
                 0.051 
                 0.001 
                 0.055 
                 0.025 
                 0.022 
                 0.015 
                 0.023 
                 0.029 
                 0.035 
               
               
                 44 
                 0.042 
                 2.240 
                 2.500 
                 0.511 
                 0.070 
                 0.060 
                 0.086 
                 0.301 
                 0.108 
                 0.066 
                 −0.002 
                 0.143 
                 0.028 
                 0.041 
                 0.028 
                 0.047 
                 0.061 
                 0.076 
               
               
                 45 
                 1.002 
                 2.500 
                 2.500 
                 0.174 
                 0.713 
                 0.055 
                 0.129 
                 1.722 
                 1.896 
                 0.029 
                 −0.003 
                 0.069 
                 0.038 
                 0.038 
                 0.055 
                 0.022 
                 0.136 
                 0.039 
               
               
                 46 
                 0.016 
                 0.031 
                 2.500 
                 0.022 
                 0.021 
                 0.021 
                 0.038 
                 0.035 
                 0.031 
                 0.027 
                 −0.002 
                 0.027 
                 0.016 
                 0.015 
                 0.013 
                 0.026 
                 0.019 
                 0.037 
               
               
                 47 
                 2.484 
                 2.500 
                 2.500 
                 0.449 
                 0.750 
                 0.593 
                 0.102 
                 2.006 
                 0.331 
                 0.017 
                 −0.004 
                 0.034 
                 0.012 
                 0.014 
                 0.007 
                 0.026 
                 0.014 
                 0.032 
               
               
                 48 
                 0.427 
                 0.201 
                 2.500 
                 0.364 
                 0.356 
                 0.038 
                 0.049 
                 0.070 
                 0.027 
                 0.029 
                 −0.001 
                 0.040 
                 0.018 
                 0.011 
                 0.013 
                 0.025 
                 0.014 
                 0.095 
               
               
                 49 
                 0.019 
                 0.483 
                 2.500 
                 0.044 
                 0.045 
                 0.047 
                 0.050 
                 0.246 
                 0.039 
                 0.025 
                 −0.005 
                 0.033 
                 0.019 
                 0.014 
                 0.013 
                 0.030 
                 0.016 
                 0.085 
               
               
                 50 
                 0.668 
                 2.500 
                 2.500 
                 0.568 
                 1.233 
                 0.277 
                 0.154 
                 2.500 
                 0.882 
                 0.024 
                 −0.001 
                 0.035 
                 0.014 
                 0.012 
                 0.007 
                 0.017 
                 0.044 
                 0.055 
               
               
                 52 
                 2.500 
                 2.500 
                 2.500 
                 0.948 
                 1.116 
                 1.619 
                 0.026 
                 0.267 
                 0.112 
                 0.019 
                 0.026 
                 0.042 
                 0.012 
                 0.007 
                 0.009 
                 0.019 
                 0.008 
                 0.044 
               
               
                 53 
                 0.823 
                 2.500 
                 2.500 
                 0.428 
                 2.500 
                 0.780 
                 0.086 
                 2.500 
                 1.566 
                 0.025 
                 0.001 
                 0.050 
                 0.014 
                 0.020 
                 0.011 
                 0.022 
                 0.019 
                 0.028 
               
               
                 54 
                 1.577 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
                 0.020 
                 0.032 
                 0.053 
                 0.021 
                 0.012 
                 0.012 
                 0.020 
                 0.017 
                 0.038 
               
               
                 55 
                 0.017 
                 0.083 
                 2.500 
                 0.047 
                 0.018 
                 0.023 
                 0.071 
                 0.042 
                 0.019 
                 0.017 
                 −0.002 
                 0.026 
                 0.015 
                 0.015 
                 0.012 
                 0.026 
                 0.014 
                 0.030 
               
               
                 58 
                 2.500 
                 2.500 
                 2.500 
                 2.283 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
                 0.810 
                 0.037 
                 0.073 
                 0.067 
                 0.018 
                 0.019 
                 0.021 
                 0.025 
                 0.159 
                 0.059 
               
               
                 59 
                 0.015 
                 1.805 
                 2.500 
                 0.023 
                 2.500 
                 0.019 
                 0.180 
                 0.038 
                 0.070 
                 0.039 
                 0.027 
                 0.017 
                 0.012 
                 0.015 
                 0.018 
                 0.015 
                 0.023 
                 0.049 
               
               
                 60 
                 0.053 
                 2.500 
                 2.500 
                 0.255 
                 2.500 
                 0.102 
                 1.266 
                 0.666 
                 2.500 
                 0.034 
                 −0.003 
                 0.153 
                 0.026 
                 0.019 
                 0.022 
                 0.039 
                 0.097 
                 0.063 
               
               
                 62 
                 0.018 
                 0.053 
                 2.500 
                 1.178 
                 0.025 
                 0.031 
                 0.041 
                 0.060 
                 0.028 
                 0.017 
                 −0.002 
                 0.039 
                 0.013 
                 0.012 
                 0.010 
                 0.017 
                 0.010 
                 0.048 
               
               
                 63 
                 0.009 
                 0.016 
                 0.027 
                 0.012 
                 0.017 
                 0.013 
                 1.225 
                 0.028 
                 0.012 
                 0.017 
                 −0.004 
                 0.026 
                 0.007 
                 0.007 
                 0.006 
                 0.010 
                 0.057 
                 0.020 
               
               
                 65 
                 1.440 
                 0.041 
                 0.026 
                 0.040 
                 0.032 
                 0.056 
                 0.052 
                 0.062 
                 0.020 
                 0.040 
                 0.023 
                 0.051 
                 0.018 
                 0.023 
                 0.023 
                 0.019 
                 0.020 
                 0.035 
               
               
                 66 
                 2.060 
                 2.500 
                 2.500 
                 1.030 
                 2.500 
                 0.508 
                 0.144 
                 2.500 
                 2.500 
                 0.013 
                 0.009 
                 0.060 
                 0.007 
                 0.009 
                 0.009 
                 0.014 
                 0.025 
                 0.018 
               
               
                 67 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
                 1.338 
                 0.406 
                 2.500 
                 0.135 
                 0.031 
                 0.003 
                 0.091 
                 0.021 
                 0.027 
                 0.028 
                 0.032 
                 0.042 
                 0.077 
               
               
                 68 
                 2.500 
                 2.500 
                 2.500 
                 1.105 
                 0.415 
                 0.358 
                 1.025 
                 2.500 
                 0.263 
                 0.028 
                 0.006 
                 0.039 
                 0.023 
                 0.025 
                 0.022 
                 0.024 
                 0.034 
                 0.037 
               
               
                 70 
                 0.745 
                 2.500 
                 2.500 
                 0.342 
                 2.500 
                 0.200 
                 0.049 
                 0.075 
                 1.730 
                 0.030 
                 0.009 
                 0.119 
                 0.015 
                 0.025 
                 0.022 
                 0.027 
                 0.056 
                 0.050 
               
               
                 73 
                 0.031 
                 2.392 
                 2.500 
                 0.049 
                 0.629 
                 0.935 
                 0.596 
                 1.119 
                 0.081 
                 0.040 
                 0.009 
                 0.052 
                 0.028 
                 0.036 
                 0.031 
                 0.038 
                 0.043 
                 0.059 
               
               
                 75 
                 0.037 
                 2.500 
                 2.500 
                 1.435 
                 1.342 
                 0.107 
                 0.287 
                 0.094 
                 0.080 
                 0.049 
                 0.012 
                 0.031 
                 0.013 
                 0.030 
                 0.017 
                 0.021 
                 0.058 
                 0.050 
               
               
                 78 
                 2.500 
                 2.500 
                 2.500 
                 0.269 
                 2.500 
                 0.298 
                 0.051 
                 2.500 
                 0.040 
                 0.057 
                 0.006 
                 0.084 
                 0.013 
                 0.022 
                 0.014 
                 0.021 
                 0.022 
                 0.030 
               
               
                 80 
                 0.280 
                 2.500 
                 2.500 
                 0.674 
                 0.318 
                 0.972 
                 0.048 
                 2.118 
                 0.040 
                 0.021 
                 0.022 
                 0.024 
                 0.015 
                 0.010 
                 0.008 
                 0.014 
                 0.066 
                 0.031 
               
               
                 84 
                 0.012 
                 0.075 
                 2.500 
                 0.025 
                 0.190 
                 0.145 
                 0.106 
                 0.099 
                 0.019 
                 0.028 
                 0.050 
                 0.018 
                 0.009 
                 0.010 
                 0.009 
                 0.013 
                 0.009 
                 0.026 
               
               
                 90 
                 0.019 
                 0.306 
                 2.500 
                 0.039 
                 2.500 
                 0.689 
                 0.033 
                 0.301 
                 0.195 
                 0.021 
                 0.005 
                 0.033 
                 0.011 
                 0.017 
                 0.010 
                 0.013 
                 0.014 
                 0.018 
               
               
                   
               
             
          
         
       
     
     A recent report indicates that the HCV core protein can be detected in HCV infected individuals before the appearance of anti-HCV antibodies. (S. Lee et al., Vox Sanguinis, 2001; 80: 19-23). Therefore, we suggest that a more efficient way of early detection of HCV infection would be a combination assay able to detect HCV core protein and anti-HCV antibodies including anti-core antibodies. 
     SUMMARY OF THE INVENTION 
     The present invention describes peptides and recombinant proteins containing Hepatitis C virus core protein sequence in which one or more of the amino acids have been modified or deleted to remove the ability of these proteins to bind to specific anti-HCV murine monoclonal antibodies. These modifications were made in the in the underlined regions shown in Table 3 below. The deletions and modifications are designed as to maintain the ability of this protein to be used in immunoassays used for the detection of anti-HCV antibodies in individuals infected with HCV. These modified core proteins can be used in a combination assay for the simultaneous detection of HCV core protein and anti-HCV antibodies. The combination assay will be able to detect HCV assay earlier than the currently used antibody assays. 
     DETAILED DESCRIPTION OF THE INVENTION 
     One object of the invention is to develop peptide sequences that can detect anti-HCV antibodies in the presence of anti-core monoclonal antibodies to detect HCV core antigen. One use therefore of these modified core antigens will be to use them in an anti-HCV/HCV core assay, or “combination assay”. 
     For this purpose, two or more monoclonal antibodies from Table 4 can be co-coated with one or more modified HCV core proteins on a solid phase thereby enabling the solid phase to capture anti-HCV core antibodies and core antigen. The modification is accomplished by removing the epitope for the antibody being used for detection or capture of HCV core. The removal of epitopes can be achieved by means known in the art such as deleting parts of the core sequence or altering amino acids in the epitope sequence. This can be achieved, for example, by synthesizing these peptides by chemical synthesis using commercially available peptide synthesizers or by modifying recombinant clones expressing HCV core protein. The recombinant sequences can be modified by primer dependent single or multiple site mutagenesis or primer dependent deletions. (B. Tao and K. C. Lee, PCR Technology Current Innovations,1994 by CRC Press, Inc., Chapter 10, Mutagenesis by PCR). 
     Another object of the invention is to identify immunodominant regions of HCV core protein. 
     Another object of the present invention is to determine the pattern of reactivity to core peptides among HCV infected individuals presenting anti-core antibodies. 
     The peptides of the invention were generated by maintaining the highly reactive portions of the core protein and making modifications to the remaining parts of the sequence such that the peptide would not be detected by an antibody used to detect core protein in an assay. 
     In a preferred embodiment, the peptide would be modified by either substitution of amino acids or deletion of amino acids in the regions underlined in Table 3. The remainder of the sequence shown in Table 3 should not be altered. 
     In another preferred embodiment the peptides would be used in a combination assay. That is one that is capable of detecting both HCV antigens and antibodies simultaneously. 
     
       
         
               
               
             
               
               
             
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 HCV core protein sequence: 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 SEQ ID NO.:19  
                   
               
             
          
           
               
                   MSTNPKPQRK TKRNTNRRPQDVKFPGGGQ IVGGVYLL PRRGP RLGVPATR   
                   
               
               
                   
               
               
                 KTSERSQPRGRRQPIPKARRPEGRSW AQPGYPWPLYGNEGCGWAGWLLSP   
               
               
                   
               
               
                 
                   RGSRPSWGPTDPRRRSRNLGKVIDTLTCGF 
                 
               
               
                   
               
             
          
         
       
     
     The effectiveness and advantages of the invention are further illustrated by the following examples. 
    
    
     EXAMPLE 1 
     Synthetic Peptides 
     Synthetic peptides covering the entire sequence of HCV core protein sequence were used. Eighteen overlapping peptides shown in Table 1 were chemically synthesized by solid phase peptide synthesis using a peptide synthesizer. All of the peptides were synthesized with a C-terminal cysteine amide residue. Peptides were cleaved form the resin and purified by reverse phase liquid chromatography. Purity of each of these peptides, based on reverse phase HPLC analysis was greater that ninety-five (95%). The sequence of each peptide was confirmed by amino acid analysis of the acid hydrolysate of the peptide. The structural identities of the peptides were confirmed mass spectrometry. All of the peptides had the molecular weight expected. 
     EXAMPLE 2 
     Synthetic peptides were coated onto Immulon 2 microwells (made by Dyanatech) in 50 mM borate buffer at a concentration of 1 ug/ml. To each microwell, 200 ul of peptide solution was added and the microwells were incubated at 25 degrees Celsius for 16-20 hours. The microwells were aspirated and washed once with phosphate buffer saline (PBS) containing TWEEN 20 to remove any unbound peptide. The microwells were then post coated with 300 ul of PBS containing one percent (1%) bovine serum albumin (BSA) and three percent (3%) sucrose to block all of the available protein binding sites. After 2-4 hours, the plates were aspirated, turbo dried and stored in sealed pouches at 2-8 degrees Celsius. 
     EXAMPLE 3 
     Synthetic Peptide ELISA 
     The sample, 10 ul in 200 ul of diluent, suspected of being infected with HCV was added into the peptide coated microwell. After incubation for approximately one hour the microwells were washed. To the washed microwells anti-human IgG were labeled with horse radish peroxidase was added. After an incubation for thirty minutes the microwells were washed and a solution of ORTHO phenylenedaamine buffer and hydrogen peroxide added to each well. After approximately 30 minutes sulfuric acid were added to each well to stop the reaction. An orange or yellow color indicated the presence of anti-HCV antibodies in the sample. 
     Table 4 
     Monoclonal Antibodies 
     The table below identifies 15 antibodies. The antibodies were screened at every stage of antibody development with microtitrewell plates coated with immunogen. The immunogen used to immunize the mice that produced each strain of monoclonal antibody is identified as one of the following: peptide ODS 243, a large peptide further defined in Example 1; “FLC” means full length core antigen, defined in Example 1; or KLH conjugated core peptide #8, a short peptide, further defined herein. The specificity of each to a numbered peptide is shown and the amino acid sequences of each numbered peptide is identified herein. Furthermore, the epitope to which the antibody specifically binds is included in the last column, defined by the amino acids that encode for the epitope. 
     
       
         
               
               
               
               
               
               
             
           
               
                   
               
               
                 ATCC 
                 AG-FUSION #, 
                   
                   
                   
                   
               
               
                 # 
                 clone 
                 IMMUNOGEN 
                 ISOTYPE 
                 SPECIFICITY 
                 AA 
               
               
                   
               
             
             
               
                 PTA- 
                 ODS243, 7B4F11 
                 Peptide 
                 IgG 2b 
                 HCV core 
                 77-91 
               
               
                 3811 
                   
                 ODS 243 
                   
                 peptide #8 
               
               
                 PTA- 
                 ODS243, 1E3D12 
                 Peptide 
                 IgG2a 
                 HCV core 
                  86-100 
               
               
                 3803 
                   
                 ODS 243 
                   
                 peptide #9 
               
               
                 PTA- 
                 ODS243, 7C12C4 
                 Peptide 
                 IgG2b 
                 HCV core 
                 77-91 
               
               
                 3802 
                   
                 ODS 243 
                   
                 peptide #8 
               
               
                 PTA- 
                 core#3, 
                 FLC 
                 IgG1 
                 HCV core 
                 106-120 
               
               
                 3813 
                 2A11C6 
                   
                   
                 peptide# 11 
               
               
                 PTA- 
                 Core#12, 
                 FLC 
                 IgG1 
                 HCV core 
                 29-43 
               
               
                 3809 
                 1B7A1 
                   
                   
                 peptide# 3 
               
               
                 PTA- 
                 CORE#13, 
                 FLC 
                 IgG1 
                 HCV core 
                 39-53 
               
               
                 3805 
                 5A12G12 
                   
                   
                 peptide# 4 
               
               
                 PTA- 
                 Core#13, 
                 FLC 
                 IgG1 
                 HCV core 
                 48-62 
               
               
                 3812 
                 4H7E7 
                   
                   
                 peptide# 5 
               
               
                 PTA- 
                 Core#13, 12F4A 
                 FLC 
                 IgG1 
                 HCV core 
                 58-72 
               
               
                 3806 
                 11 
                   
                   
                 peptide# 6 
               
               
                 PTA- 
                 Core#13, 
                 FLC 
                 IgG1 
                 HCV core 
                 67-81 
               
               
                 3804 
                 14D12A12 
                   
                   
                 peptide# 7 
               
               
                 PTA- 
                 c22-8#4, 
                 KLH 
                 IgG1 
                 HCV core 
                 77-91 
               
               
                 3807 
                 6D8E8 
                 Conjugated 
                   
                 peptide# 8 
               
               
                   
                   
                 core 
               
               
                   
                   
                 peptide 
               
               
                   
                   
                 #8 
               
               
                 PTA- 
                 Core#12, 
                 FLC 
                 IgG2b 
                 HCV core 
                  96-110 
               
               
                 3800 
                 4G10G6 
                   
                   
                 peptide# 10 
               
               
                 PTA- 
                 Core#13, 
                 FLC 
                 IgG2a 
                 HCV core 
                 106-120 
               
               
                 3801 
                 6E7E1 
                   
                   
                 peptide# 11 
               
               
                 PTA- 
                 Core#13, 
                 FLC 
                 IgG2b 
                 HCV core 
                 106-120 
               
               
                 3810 
                 11D12A6 
                   
                   
                 peptide# 11 
               
               
                 PTA- 
                 Core#13, 
                 FLC 
                 IgG3 
                 HCV core 
                 106-120 
               
               
                 3808 
                 14B7C3 
                   
                   
                 peptide# 11 
               
               
                 PTA- 
                 Core#12, 
                 FLC 
                 IgG1 
                 HCV core 
                 156-170 
               
               
                 3799 
                 4A6H3 
                   
                   
                 peptide# 16 
               
               
                   
               
             
          
         
       
     
     EXAMPLE 4 
     Use of Modified Core Peptides in an Anti-HCV ELISA 
     A peptide consisting of HCV core immunodominant region amino acids 1-43 was chemically synthesized. Another peptide with certain deletions in this region, namely, amino acids 1-8 and 31-33 were deleted, was also synthesized. These peptides were used to test 40 chronic HCV patients&#39; serum samples for anti-HCV antibody status. The results indicated that 39 of the 40 patients were not affected by deletion of these sequences. The deletion peptides can be used in combination with anti-HCV monoclonal antibodies, such as the ones shown in Table 4, in a combination assay. Peptides with deletions of amino acids 31-33 can be used with any of the monoclonal antibodies shown in Table 4. 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 5 
               
               
                   
               
               
                 Peptide/Specimen ID 
                 265-2 
                 266-2 
                 271-2 
                 272-2 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 11 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 15 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 18 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 20 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 21 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 27 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 30 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 35 
                 0.762 
                 0.911 
                 0.507 
                 0.611 
               
               
                 36 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 37 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 39 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 40 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 42 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 43 
                 1.621 
                 1.255 
                 1.382 
                 1.443 
               
               
                 44 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 45 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 46 
                 2.003 
                 1.863 
                 1.058 
                 1.829 
               
               
                 47 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 48 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 49 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 50 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 52 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 53 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 54 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 55 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 58 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 59 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 60 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 62 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 65 
                 0.056 
                 0.045 
                 2.237 
                 1.704 
               
               
                 66 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 67 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 68 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 70 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 73 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 75 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 78 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 80 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                 84 
                 2.500 
                 2.500 
                 2.170 
                 2.500 
               
               
                 90 
                 2.500 
                 2.500 
                 2.500 
                 2.500 
               
               
                   
               
               
                 Peptide 265 HCV Polyprotein Amino Acid Sequence 10-43 
               
               
                 Peptide 266 HCV Polyprotein Amino Acid Sequence 8-43 
               
               
                 Peptide 271 HCV polyprotein Amino Acid Sequence 1-43 with AA 31, 32 and 33 deleted 
               
               
                 Peptide 272 HCV Polyprotein Amino Acid Sequence 1-43 Consensus sequence