Method and kit for the detection of hepatitis-specific antibodies

Abstract

Provided is an improved method for detecting antibodies to a hepatitis virus in a tissue sample from individuals, which can reliably detect antibodies in recently infected individuals and which provides much lower false positive results in individuals that have cleared their hepatitis infections. Further provided is an improved method and kit which utilizes an activator of (i) hepatitis virus-primed lymphocytes, (ii) memory cells specific for said hepatitis virus, (iii) hepatitis virus-specific antibody production, or (iv) a combination thereof in a tissue sample to stimulate the production of antibodies from newly primed B cells, if present.

1 .- 141 . (canceled) 
     
     
         142 . A method of differentiating between a cleared and chronic hepatitis infection in a subject comprising the steps of:
 a. obtaining a tissue sample from said subject;   b. determining the anti-hepatitis virus antibody level in a first aliquot of said sample, wherein a detectable anti-hepatitis virus antibody level indicates that a subject is seropositive;   c. stimulating a second aliquot of said sample to produce anti-hepatitis virus antibodies in vitro by incubating said second aliquot in a culture in the presence of a medium comprising a) one or more activators of (i) hepatitis virus-primed lymphocytes, (ii) memory cells specific for said hepatitis virus, (iii) hepatitis virus-specific antibody production, or (iv) a combination thereof, and b) an antibody against a B-lymphocyte membrane domain;   d. determining the anti-hepatitis virus antibody level in said second aliquot of said sample;   e. dividing a value representing the stimulated anti-hepatitis virus antibody level obtained in step (d) by a value representing the anti-hepatitis virus antibody level obtained in step (b) for each subject; and   f. determining if the quotient obtained in step (d) is above a pre-determined threshold value for each subject,   wherein a value below said threshold value indicates that the subject has a cleared hepatitis virus infection and a value close to said threshold value indicates that the subject has a chronic hepatitis infection, thereby differentiating between a cleared and chronic hepatitis infection in said subject.   
     
     
         143 . The method according to  claim 142 , wherein said tissue sample is a whole blood sample. 
     
     
         144 . The method according to  claim 143 , further comprising the step of collecting a whole blood sample from said subject, prior to step a) into a container. 
     
     
         145 . The method according to  claim 142 , wherein said hepatitis virus is a hepatitis C virus (HCV). 
     
     
         146 . The method according to  claim 142 , wherein said hepatitis virus is a hepatitis B virus (HBV). 
     
     
         147 . The method according to  claim 142 , wherein said hepatitis virus is a hepatitis A virus (HAV), hepatitis D virus (HDV), or hepatitis E virus (HEV). 
     
     
         148 . The method according to  claim 142 , wherein said medium comprises a non-carbon dioxide-dependent buffering agent. 
     
     
         149 . The method according to  claim 148 , wherein said buffering agent is HEPES ((4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid) or a phosphate-based buffering agent. 
     
     
         150 . The method according to  claim 142 , wherein the culture of step a) results in a supernatant, and the supernatant is exposed to a viral antigen in step b), thereby allowing an antigen-antibody immune complex to form. 
     
     
         151 . The method according to  claim 142 , wherein the culture of step a) further comprises an antibody against a B-lymphocyte membrane domain. 
     
     
         152 . The method according to  claim 151 , wherein said antibody is against a non-secreted protein. 
     
     
         153 . The method according to  claim 151 , wherein said antibody is anti-CD19. 
     
     
         154 . The method according to  claim 152 , wherein said antibody is anti-CD19. 
     
     
         155 . The method according to  claim 142 , wherein said activator is a viral-derived peptide, lectin, bacterial endotoxin, a virus, lipid A, a cytokine, a chain of nucleotides, or a lymphokine. 
     
     
         156 . The method according to  claim 142 , wherein said activator is pokeweed mitogen. 
     
     
         157 . The method according to  claim 142 , wherein said hepatitis virus-primed lymphocytes or memory cells are B-lymphocytes. 
     
     
         158 . The method according to  claim 142 , wherein said hepatitis virus-primed lymphocytes or memory cells are T-lymphocytes. 
     
     
         159 . The method according to  claim 142 , wherein said hepatitis virus-primed lymphocytes or memory cells are both B-lymphocytes and T-lymphocytes. 
     
     
         160 . The method according to  claim 142 , wherein said antigen-antibody immune complex is detected on a solid phase support. 
     
     
         161 . The method according to  claim 160 , wherein said solid phase support is a nitrocellulose strip, polymer beads, synthetic beads, or a combination thereof.