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Enzyme immunoassay for the detection of antibodies against Human Hepatitis Virus Type C (HCV)

INTRODUCTION                                                                               

Hepatitis C virus (HCV), which was formerly described as the parenterally transmitted form of non-A, non-B hepatitis (NANBH)¹, becomes a chronic disease in 50% of the cases.²   HCV can also be transmitted through intravenous drug abuse, sexual , and household contact.³ Hepatitis C virus is a single stranded RNA virus with some structural relations to the flavivirus family.  Nucleic acid sequences of HCV cDNA clones provided the basis for the construction of recombinant peptides representing putative hepatitis C virus proteins.^4,5  Anti-hepatitis C virus antibody screening of blood using synthetic or recombinant proteins, helped to identify apparently healthy blood donors with anti-HCV antibodies who otherwise might have transmitted the virus.⁶

This is an enzyme linked immunosorbent assay using recombinant proteins derived from core regions of HCV virus to detect the presence of HCV antibodies in human sera.

PRINCIPLE OF THE ASSAY

 Multiple epitopes of HCV proteins(Core, NS3, NS4 and NS5) are bound to the microtiter wells.  When antibodies to HCV are present in the test sample, they react with recombinant proteins and attach to the solid-phase.  Non-reactive antibodies are removed with the wash buffer.  Human IgGs bound to the antigen are reacted with anti-human IgG peroxidase conjugate and visualized by subsequent reactions with a chromogenic substrate.  Positive sample generates a medium to dark blue color.  No color or very pale blue color indicates a negative reaction.  The intensity of the reaction is photometrically quantitated.

Materials provided with the kits:

Materials required but not provided:

1.        Precision pipettes: 0.02, 0.05, 0.10, 0.15, 0.20, and 1.0 ml.

2.        Disposable pipette tips.

3.        Distilled water.

4.        Humidified Box capable of maintaining 37°C

5.        Absorbent paper or paper towel.

6.        Microtiter plate or strip-well washer

7.        Microtiter plate reader.