HCV- EIA

REPORT in Immunology-Serology

What is HCV?
y Hepatitis C virus y is a small (55-65 nm in size), enveloped, positive-sense

single-stranded RNA virus of the family Flaviviridae y Hepatitis C virus is the cause of hepatitis C in humans. y The hepatitis C virus particle consists of a core of genetic material (RNA), surrounded by an icosahedral protective shell of protein, and further encased in a lipid (fatty) envelope of cellular origin. Two viral envelope glycoproteins, E1 and E2, are embedded in the lipid envelope.

What is EIA
y Enzyme Immunoassay y A labeled reagent assay, based on the primary interaction

between Ag and Ab involved y Makes use of enzymes to detect the sought for analyte

HCV-EIA
y for the qualitative determination of antibodies to Hepatitis C Virus

(HCV) in human serum or plasma. y It is intended for screening of blood donors y for aid in the diagnosis of clinical conditions related to infection with HCV. y Sensitivity and Specificity At present there is no recognized standard for establishing the presence or absence of HCV antibodies in human blood; therefore, sensitivity and specificity have been estimated as described below. 1. The sensitivity of the HCV EIA can be defined as the ability to detect as reactive those specimens from individuals with diseases known to be associated with HCV infection. 2. The specificity of the HCV EIA can be defined as the ability to detect as non-reactive specimens from a low risk population.

How is it used?
y Hepatitis C tests are used to detect and diagnose an infection

and/or to monitor the treatment of hepatitis C virus (HCV). Tests are used to detect the condition if a person:
y Has been exposed to someone with HCV y Participates in high risk behaviors such as injecting street drugs y Has abnormal liver function tests y Has symptoms associated with liver disease, such as jaundice, dark urine,

nausea, or unexpected weight gain or loss

Steps
y Human serum or plasma is diluted in specimen diluent and incubated on a

microwell coated with recombinant HCV antigen. y Following a 1-hour incubation, the plate is washed to remove unbound material. A peroxidase-conjugated antibody directed against human IgG is added to each well on the microwell plate. y Following a 60-min incubation, the wells are washed again to remove unbound material. A peroxidase-specific chromogenic substrate solution is added to each well. The substrate solution consists of hydrogen peroxide and ophenylenediamine (OPD) in a citrate buffer. y Following a 30-min incubation at 20-25C, 1 N sulfuric acid is added to stop the enzyme-substrate reaction. Anti-HCV antibody will bind to the HCV antigen in the microwell. Subsequently, the conjugate binds to that antibody. The reaction of the conjugate with the substrate solution results in the generation of an orange color. Absence of color indicates the absence of antiHCV in the sample. The intensity of the color generated is measured spectrophotometrically at 492 nm.

Results
y Results are expressed as "positive" or "negative" for anti-

HCV.

The following tests may be used to screen for and/or detect HCV:
y Anti-HCV test detects the presence of antibodies to the virus, indicating exposure to HCV. This test cannot

distinguish between someone with an active or a previous HCV infection. Usually, the test is reported as "positive" or "negative." There is some evidence that if the test is "weakly positive," it may be a false positive.The Centers for Disease Control and Prevention (CDC) suggests that weakly positive tests be confirmed with the HCV RIBA test before being reported. y HCV recombinant immunoblot assay (RIBA) test is an additional test ordered to confirm the presence of HCV antibodies. In most cases, it can tell if the positive anti-HCV test was due to exposure to HCV (positive RIBA) or represents a false signal (negative RIBA). In a few cases, the results cannot answer this question (indeterminate RIBA). Like the anti-HCV test, the RIBA test cannot distinguish between a current or past infection.

The following tests may be used to diagnose a current infection and to guide and monitor treatment:
y HCV RNA test, Qualitative may be used to distinguish between a current or past infection. It is reported

as a "positive" or "detected" if any HCV viral RNA is found; otherwise, the report will be "negative" or not detected." It may also be ordered after HCV treatment is complete to see if the virus has been eliminated from the blood. These tests are seldom used any more. y HCV Viral Load (HCV RNA test, Quantitative) detects and measures the number of viral RNA particles in the blood. Viral load tests are often used before and during treatment to help determine response to treatment by comparing the amount of virus before and during treatment (usually at several time points in the first three months of treatment). Successful treatment causes a decrease of 99% or more (2 logs) in viral load soon after starting treatment (as early as 4-12 weeks) and usually leads to viral load being not detected even after treatment is completed. Some newer viral load tests can detect very low amounts of viral RNA. y Viral genotyping is used to determine the kind, or genotype, of the HCV virus present. There are 6 major types of HCV; the most common (genotype 1) is less likely to respond to treatment than genotypes 2 or 3 and usually requires longer therapy (48 weeks versus 24 weeks for genotype 2 or 3). Genotyping is often ordered before treatment is started to give an idea of the likelihood of success and how long treatment may be needed.

Notes:
y Anti-HCV ELISA kits employ solid phase, indirect ELISA

assay for detection of antibodies to HCV in two-step incubation procedure. y HCV antibodies usually do not appear until several months into an infection but will always be present in the later stages of the disease. y About 25% of those with HIV/AIDS also have an HCV coinfection, and their liver disease is likely to progress at an accelerated rate.