MicroELISA

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Fourth generation Enzyme ImmunoAssay (ELISA) for the determination of antibodies to Hepatitis C Virus in human plasma (EDTA, Heparin and Citrate) and sera.
The kit may be used for the screening of blood units of HCV-infected patients.
For “in vitro” diagnostic use only

The World Health Organization (WHO) define Hepatitis C infection as follows: “Hepatitis C is a viral infection of the liver which had been referred to as parenterally transmitted "non A, non B hepatitis" until identification of the causative agent in 1989. The discovery and characterization of the hepatitis C virus (HCV) led to the understanding of its primary role in post transfusion hepatitis and its tendency to induce persistent infection.

HCV is a major cause of acute hepatitis and chronic liver disease, including cirrhosis and liver cancer. Globally, an estimated 170 million persons are chronically infected with HCV and 3 to 4 million persons are newly infected each year. HCV is spread primarily by direct contact with human blood. The major causes of HCV infection worldwide are use of unscreened blood transfusions, and re-use of needles and syringes that have not been adequately sterilized. No vaccine is currently available to prevent hepatitis C and treatment for chronic hepatitis C is too costly for most persons in developing countries to afford. Thus, from a global perspective, the greatest impact on hepatitis C disease burden will likely be achieved by focusing efforts on reducing the risk of HCV transmission from nosocomial exposures (e.g. blood transfusions, unsafe injection practices) and high-risk behaviours (e.g. injection drug use).

Hepatitis C virus (HCV) is one of the viruses (A, B, C, D, and E), which together account for the vast majority of cases of viral hepatitis. It is an enveloped RNA virus in the flaviviridae family which appears to have a narrow host range. Humans and chimpanzees are the only known species susceptible to infection, with both species developing similar disease. An important feature of the virus is the relative mutability of its genome, which in turn is probably related to the high propensity (80%) of inducing chronic infection. HCV is clustered into several distinct genotypes which may be important in determining the severity of the disease and the response to treatment.
The incubation period of HCV infection before the onset of clinical symptoms ranges from 15 to 150 days. In acute infections, the most common symptoms are fatigue and jaundice; however, the majority of cases (between 60% and 70%), even those that develop chronic infection, are a symptomatic. About 80% of newly infected patients progress to develop chronic infection.

Cirrhosis develops in about 10% to 20% of persons with chronic infection, and liver cancer develops in 1% to 5% of persons with chronic infection over a period of 20 to 30 years.

Most patients suffering from liver cancer who do not have hepatitis B virus infection have evidence of HCV infection. The mechanisms by which HCV infection leads to liver cancer are still unclear. Hepatitis C also exacerbates the severity of underlying liver disease when it coexists with other hepatic conditions. In particular, liver disease progresses more rapidly among persons with alcoholic liver disease and HCV infection. HCV is spread primarily by direct contact with human blood.

Transmission through blood transfusions that are not screened for HCV infection, through the reuse of inadequately sterilized needles, syringes or other medical equipment, or through needle-sharing among drug-users, is well documented. Sexual and perinatal transmission may also occur, although less frequently.

Other modes of transmission such as social, cultural, and behavioural practices using percutaneous procedures (e.g. ear and body piercing, circumcision, tattooing) can occur if inadequately sterilized equipment is used. HCV is not spread by sneezing, hugging, coughing, food or water, sharing eating utensils, or casual contact.

In both developed and developing countries, high risk groups include injecting drug users, recipients of unscreened blood, haemophiliacs, dialysis patients and persons with multiple sex partners who engage in unprotected sex. In developed countries, it is estimated that 90% of persons with chronic HCV infection are current and former injecting drug users and those with a history of transfusion of unscreened blood or blood products. In many developing countries, where unscreened blood and blood products are still being used, the major means of transmission are unsterilized injection equipment and unscreened blood transfusions.

In addition, people who use traditional scarification and circumcision practices are at risk if they use or re-use unsterilized tools.

WHO estimates that about 170 million people, 3% of the world’s population, are infected with HCV and are at risk of developing liver cirrhosis and/or liver cancer. The prevalence of HCV infection in some countries in Africa, the Eastern Mediterranean, South-East Asia and the Western Pacific (when prevalence data are available) is high compared to some countries in North America and Europe.

Diagnostic tests for HCV are used to prevent infection through screening of donor blood and plasma, to establish the clinical diagnosis and to make better decisions regarding medical management of a patient.

Diagnostic tests commercially available today are based on Enzyme immunosorbent assays (EIA) for the detection of HCV specific antibodies. EIAs can detect more than 95% of chronically infected patients but can detect only 50% to 70% of acute infections. A recombinant immunoblot assay (RIBA) that identifies antibodies which react with individual HCV antigens is often used as a supplemental test for confirmation of a positive EIA result. Testing for HCV circulating by amplification tests RNA (e.g. polymerase chain reaction or PCR, branched DNA assay) is also being utilized for confirmation of serological results as well as for assessing the effectiveness of antiviral therapy. A positive result indicates the presence of active infection and a potential for spread of the infection and or/the development of chronic liver disease.

Antiviral drugs such as interferon taken alone or in combination with ribavirin, can be used for the treatment of persons with chronic hepatitis C, but the cost of treatment is very high. Treatment with interferon alone is effective in about 10% to 20% of patients. Interferon combined with ribavirin is effective in about 30% to 50% of patients. Ribavirin does not appear to be effective when used alone.
There is no vaccine against HCV. Research is in progress but the high mutability of the HCV genome complicates vaccine development. Lack of knowledge of any protective immune response following HCV infection also impedes vaccine research. It is not known whether the immune system is able to eliminate the virus.

Some studies, however, have shown the presence of virus neutralizing antibodies in patients with HCV infection. In the absence of a vaccine, all precautions to prevent infection must be taken including (a) screening and testing of blood and organ donors; (b) Virus inactivation of plasma derived products; (c) implementation and maintenance of infection control practices in health care settings, including appropriate sterilization of medical and dental equipment; (d) promotion of behaviour change among the general public and health care workers to reduce overuse of injections and to use safe injection practices; and (e) Risk reduction counselling for persons with high-risk drug and sexual practices.“

The genome encodes for structural components, a nucleocapsid protein and two envelope glycoproteins, and functional constituents involved in the virus replication and protein processing. The nucleocapsid-encoding region seems to be the most conservative among the isolates obtained all over the world.

Microplates are coated with HCV-specific antigens derived from “core” and “ns” regions encoding for conservative and immunodominant antigenic determinants (Core peptide, recombinant NS3, NS4 and NS5 peptides).

The solid phase is first treated with the diluted sample and HCV Ab are captured, if present, by the antigens.

After washing out all the other components of the sample, in the 2nd incubation bound HCV antibodies, IgG and IgM as well, are detected by the addition of polyclonal specific anti hIgG&M antibodies, labelled with peroxidase (HRP).

The enzyme captured on the solid phase, acting on the substrate TMB mixture, generates an optical signal that is proportional to the amount of anti HCV antibodies present in the sample. A cut-off value let optical densities be interpreted into HCV antibody negative and positive results.

The kit contains reagents for 192 tests (code 071064).

1. Microplate 12 strips of 8 microwells coated with Core peptide, recombinant NS3, NS4 and NS5 peptides. Plates are sealed into a aluminium pouch with desiccant.

Bring the microplate to room temperature (18…24°C) before opening the bag. Unused strips have to be returned into the pouch and the pouch has to be sealed and stored back to 2...8°C, in presence of the desiccant.

2. Negative Control Ready to use control. It contains 10 mM Na-citrate buffer pH 6.0 ± 0.1, 2% casein as protein base and 0.1% Proclin 150 as preservative. The negative control is olive green colour coded.

3. Positive Control Ready to use control. It contains 1% goat serum proteins, human antibodies positive to HCV, 10 mM Na-citrate buffer pH 6.0 ± 0.1, 0.5% Tween 20, 0.09% Na-azide and 0.1% Proclin 150 as preservatives. The Positive Control is dark green colour coded.

Important Note: The absence of viable pathogens in the Positive Control can not be fully ensured, and therefore, the reagent should be handled as potentially biohazardous, in accordance with good laboratory practices.

4. Calibrator Lyophilized calibrator. To be dissolved with the volume of EIA grade water reported on the label. It contains foetal bovine serum proteins, human antibodies to HCV whose content is calibrated on the NIBSC Working Standard code 06/188-006, 10 mM Na-citrate buffer pH 6.0 ± 0.1, 0.3 mg/mL gentamicin sulfate and 0.1% Proclin 150 as preservatives.
Important Note: The absence of viable pathogens in the Calibrator can not be fully ensured, and therefore, the reagent should be handled as potentially biohazardous, in accordance with good laboratory practices.
Note: The volume necessary to dissolve the content of the vial may vary from lot to lot. Please use the right volume reported on the label.

5. Wash Buffer Concentrate 20x 20x concentrated solution. Once diluted, the wash solution (wash buffer diluted) contains 10 mM phosphate buffer pH 7.0 ± 0.2, 0.05% Tween 20 and 0.05% Proclin 150.
Once diluted, the wash solution is stable for 1 week at 2...8°C.

6. Conjugate Ready to use and red colour coded reagent. It contains Horseradish Peroxidase conjugated goat polyclonal antibodies to human IgG and IgM, 5% BSA, 10 mM Citrate buffer pH 6.4 ± 0.1, 0.1% Proclin and 0.05% Tween 20 as preservatives.

7. Substrate TMB Ready-to-use component. It contains 50 mM citrate-phosphate buffer pH 3.5-3.8, 4% dimethylsulphoxide, 0.03% tetra-methyl-benzidine or TMB and 0.02% hydrogen peroxide or H2O2. Mix gently before use.
Note: To be stored protected from light as sensitive to strong illumination.

8. Assay diluent Ready-to-use component. It contains goat serum, 10 mM tris buffered solution pH 8.0 ± 0.1 containing 0.1% Proclin 150 and 0.09% Na azide for the pre-treatment of samples and controls in the plate, blocking interference.

9. Stop Solution Ready-to-use component.
It contains 0.3 M H2SO4 solution. Mix gently before use.

10. Sample Diluent Ready-to-use component and dark green colour coded. It contains 1% casein, 10 mM Na-citrate buffer pH 6.0 ± 0.1 and 0.1% Proclin 150 as preservative.
To be used to dilute the sample.

Note: The diluent changes colour from olive green to dark bluish green in the presence of sample.

1. Calibrated Micropipettes (200 µL and 10 µL) and disposable plastic tips.

2. EIA grade water (bidistilled or deionised, charcoal treated to remove oxidizing chemicals used as disinfectants).

3. Timer with 60 minute range or higher.

4. Absorbent paper tissues.

5. Calibrated ELISA microplate thermostatic incubator capable to provide a temperature of +37°C.

6. Calibrated ELISA microwell reader with 450nm (reading) and possibly with 620-630nm (blanking) filters and 405 nm filters.

7. Calibrated ELISA microplate washer. 8. Vortex or similar mixing tools.