Kit and Method for the Detection of Anti-Hepatitis C Virus (Hcv) Antibodies

Abstract

The present invention provides a kit for the detection of anti-hepatitis C virus (HCV) antibodies using a double-antigen sandwich technique, said kit carries out the detection by the process ‘solid support-first antigen-HCV antibody to be detected-second antigen-enzyme linked substance-recognizable signal’, wherein one or more coupling reactions between the second antigen and the enzyme linked substance occur, and the diluent for the second antigen and that for the enzyme linked substance are respectively placed into two container, they are added in order in two steps to reaction system and incubated respectively. The present invention can solve the following problems encountered by prior art: 1) the influence of the enzyme linked substance on the activity of the second antigen; 2) the mismatch of disulfide bonds in second antigen, thereby enhance the activity of the second antigen and obviate loss of enzyme activity, therefore, the present kit highly improves the sensitivity and specificity of the detection.

1 .- 17 . (canceled) 
     
     
         18 . A kit for detection of anti-HCV antibodies, said kit complete the detection in the form of ‘solid support-the primary antigen-HCV antibodies to be detected-the secondary antigen-enzyme label-recognizable signal’, wherein the secondary antigen carry on the combination reaction with the enzyme label in one step or several steps, said kit comprise sample diluent, the secondary antigen diluent and enzyme label diluent, wherein said secondary antigen diluent and enzyme diluent are packed separately. 
     
     
         19 . The kit of  claim 18 , wherein diluent of the secondary antigen contains the reducing agent. 
     
     
         20 . The kit of  claim 19 , wherein the reducing agent is β-mercaptoethanol, 1,4-dithiothreitol, dithio-erythritol, reduced glutathione, cysteine, thio-amine methylate, dithio-sulfinic acid sodium, ascorbate, stannous chloride or sodium borohydride. 
     
     
         21 . The kit of  claim 20 , wherein the reducing agent is β-mercaptoethanol. 
     
     
         22 . The kit of  claim 21 , wherein when specimen to be detected and the secondary antigen are added into reaction system simultaneously and incubated together, specimen diluent and the secondary antigen diluent can be the same solution, i.e. the secondary antigen diluent in one container, wherein the concentration of β-mercaptoethanol in the secondary antigen diluent is 0.01-50‰, prefers 0.05-20‰, further prefers 1-10‰, most prefers 5‰. 
     
     
         23 . The kit of  claim 21 , wherein specimen to be detected and the secondary antigen are added into reaction system by two steps successively and incubated respectively, wherein the concentration of β-mercaptoethanol in said secondary antigen diluent is 0.01-50‰, prefers 0.05-30‰, prefers 0.2-20‰, further prefers 0.5-10‰, further prefers 0.5-5‰, most prefers 1‰. 
     
     
         24 . The kit of  claim 18 , wherein specimen diluent contain reducing agent. 
     
     
         25 . The kit of  claim 23 , wherein specimen diluent contain reducing agent. 
     
     
         26 . The kit of  claim 24 , wherein the reducing agent is β-mercaptoethanol, 1,4-Dithiothreitol, dithio-erythritol, reduced glutathione, Cysteine, thio-amine methylate, dithio-sulfinic acid sodium, ascorbate, stannous chloride or sodium borohydride. 
     
     
         27 . The kit of  claim 26 , wherein the reducing agent is β-mercaptoethanol. 
     
     
         28 . The kit of  claim 27 , wherein the concentration of β-mercaptoethanol in specimen diluent is 0.01-50‰, prefers 0.1-20‰, further prefers 0.5-10‰, most prefers 5‰. 
     
     
         29 . The kit of  claim 18 , said kit include pre-coating ELIS A plate, the secondary antigen, washing solution, color solution, quench solution, negative control, positive control, plate sealer, plastic bag, introduction and package box. 
     
     
         30 . The kit of  claim 23 , said kit include pre-coating ELISA plate, the secondary antigen, washing solution, color solution, quench solution, negative control, positive control, plate sealer, plastic bag, introduction and package box. 
     
     
         31 . The kit of  claim 27 , said kit include pre-coating ELISA plate, the secondary antigen, washing solution, color solution, quench solution, negative control, positive control, plate sealer, plastic bag, introduction and package box. 
     
     
         32 . The kit of  claim 18 , said secondary antigen is conjugate of HCV protein and tag, said tag is peptide tag or non-peptide tag. 
     
     
         33 . The kit of  claim 32 , wherein peptide tag is the peptide or protein containing His Tag, T7 Tag, S Tag, Flag Tag, HA Tag or HCV peptide fragment. 
     
     
         34 . The kit of  claim 32 , wherein the non-peptide tag is compound, hapten, vitamin, steroid, dyestuff, hormone, antibiotic, nucleic acid or conjugate of all above component with peptide or protein. 
     
     
         35 . The kit of  claim 32 , wherein the compound is dinitrophenol, bromodeoxyuridine; vitamin is biotin or derivatives thereof; steroid is digoxin; dyestuff is acridinium ester, rhodamine, dansyl chloride or dihydroxyfluorane. 
     
     
         36 . The kit of  claim 32 , wherein the tag is biotin. 
     
     
         37 . A method of detection of a kit of claim  1 , wherein said method complete the detection in the form ‘solid support-the primary antigen-HCV antibodies to be detected-the secondary antigen-enzyme label-recognizable signal’, and comprise the following procedure:
 A. the primary antigen catch antibody: add the specimen to be detected and incubate, then the primary antigen on pre-coating ELSIA plate catch the HCV antibody of specimen;   B. the antibody catch the secondary antigen: add the secondary antigen solution and incubate, HCV antibody catch the secondary antigen;   C. detection: introduce enzyme label by tag in one or more incubating steps and then detect;   wherein the step B is carried out in the reduction condition, step A and B can merge, i.e. specimen to be detected and the secondary antigen can be added into the reaction system simultaneously and incubated together, wherein said secondary antigen and enzyme label are added into reaction system in two steps successively and incubated respectively.