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

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, 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.

Materials provided with the kits:

ASSAY PROCEDURE

It is strongly advised to analyze each specimen and controls in duplicate. All the reagents should equilibrate to room temperature before use.

1. Dispense 100ml(or 3 drops) of specimen diluent into individual test wells.
2. Dispense 100ml positive control and negative control duplicate into individual wells.
3. Add 10ml of each test sample into duplicate test wells; vortex to mix.
4. Incubate for 30 minutes at 37°C
5. Wash each well 5 times by filling each well with diluted wash buffer, then inverting the plate vigorously to get all water out and blocking the rim of wells on absorbent paper for a few seconds.
6. Add 100ml of Enzyme Conjugate to each well. Mix it gently by swirling the microtiter plate on flat bench for 1 minutes. Do not add Enzyme Conjugate to the blank well.
7. Incubate for 20 minutes at 37°C
8. Wash the plate 5 times as step 5.
9. Add one drop (50ml) of Substrate Solution A (HRP-substrate) to each well, then add one drop (50ml) of Substrate Solution B (TMB) to each well. Mix gently and incubate at 37°C for 10 minutes. .
10. Add one drop (50ml) of Stop Solution to each well to stop the color reaction. Read the OD value at 450 nm/630 nm with dual filter plate reader. It is option to read the OD value at 450 nm with single filter plate reader. (using the OD value of the blank well to correct all the OD reading from all wells)