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Detection Kit For HCV Core Antigen (EIA) 

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Hepatitis C Virus (HCV) is transmitted through contaminated blood and blood products.Routine screening of blood donors for the presence of anti-HCV antibody has reduced the risk of post-transfusion HCV infection.However,the anti-HCV antibody is deficit in pre-seroconversion "window phase".The early negative "window phase" of infection may be significant residual risk of post-transfusion HCV disease.HCV infection in "window phase" donation can be identified by detection of core antigen.The HCV Ag EIA Kit is an enzyme immunoassay for the detection of antigen to hepatitis C virus (HCV) in human serum or plasma based on Enzyme-linked immunosorbent assay (EIA).Monoclonal antibodies with specificity to different regions of the HCV core antigen are coated onto the microwells to capture HCV core antigen that is detected by HRP conjugated antibody.HCV antigen1b,2a,1b/2a gene types can be detected by this test system.The HCV Ag test detecting HCV infection is approximately 40-70 days earlier than existing HCV antibody assays and is suitable for large scale screening of individual blood donors.

The Kit Components
1. Anti HCV Ag monoclonal antibody coated Microwell Plate ( 96 well )
2. Positive Control (1 ml)
3. Negative Control (1 ml )
4. Specimen Diluent (10 ml)
5. Enzyme Conjugate (20 ml)
6. Colorant (20 ml)
7. 20XWash Buffer (50 ml)
8. Stop Solution (5 ml)
All unopened components are suitable for use through the labeled expiration date, and should be stored at 2 to 8 Centigrade. Under this condition,the kit will retain activity.

Materials Required But Not Provided
1. Single wavelength microwell reader capable of reading at 450nm.
2. Adjustable single or multi-channel micropipettes capable of delivering 50 to 250ul.
3. Disposable pipettes tips of all types of pipettes required.
4. A 37 Centigrade Water bath / incubator capable of shaking the microwell plate with an orbital
    motion or a validated equivalent.
5. Microwell plate washing system (automated or manual).
6. Distilled or deionized water of high quality.
7. 5% sodium hypochlorite for sterilization.

Specimen Collection, Transport And Storage
1. Specimen collection
    Blood should be collected by approved medical techniques. Serum or plasma collected in
    EDTA, heparin or citrate may be used for this test and should be used as soon as possible
    following collection. Specimens should be thoroughly separated from all cellular material. 
    Failure to do so may lead to a falsely elevated result.
2. Specimen Transport And Storage
    Store specimens at 2-8 Centigrade.Specimens not required for assay within seven days should
    be removed from the clot or cell pellet and stored frozen ( -20 Centigrade or colder ). Avoid
    multiple freeze-thaw cycles.

Procedure
1. Approximately 15-30 minutes prior to the beginning of the test procedure, bring kit components
    to room temperature ( 15 to 30 Centigrade). Invert liquid reagents gently several times, but
    avoid foaming. Check the incubator temperature, maintain at 37? Centigrade.
2. Determine the total number of wells needed for the assay. In addition to specimens, two reagent
    blanks, two Negative Controls and two Positive Controls should be included on each plate or
    partial plate.
3. Add 100ul of Specimen Diluent to all wells, 200ul to the blank wells.
4. Using a clean tip for each addition. Add 100ul of Negative Control, Positive Control or
    specimen to the corresponding wells.
5. Cover the microwell plate with a plate sealer. Incubate at 37 Centigrade with an orbital shaking
    motion for 90 minutes ( The incubation can still be processed in case of no shaking ).
6. Dilute the 20XWash Buffer to 1XWash Buffer for use. With an aspirator-washer device,
    aspirate the solutions from the microwells, then fill completely with Wash Buffer,do not allow
    the wells to overflow. Keep approximately 20 seconds between the addition of Wash Buffer
    and subsequent aspiration. Complete the aspirate / fill sequence four additional times.
    Completely aspirate all the wells to make sure no liquid remains in the wells. If necessary, invert
    the plate and tap it dry on absorbent tissue. When using a manual washing procedure, wash five
    times and tap the plate to be dry after the last wash. Ensure that no liquid is left in the well, or it
    may result in false performance. Strict obeying the specified wash procedure is crucial to ensure
    optimum assay performance.
7. Add 200ul of Enzyme Conjugate to all wells.
8. Cover the microplate with a new plate sealer. Incubate at 37 Centigrade for 30 minutes.
9. After the second incubation, wash the wells six times as described in step 6.
10. Add 200ul of Colorant to all wells. Colorant should be kept away from direct sunlight. The
    surplus colorant is stable in refrigerate ( 2 to 8 Centigrade), but must be discarded when a color
    change occurs.
11. Incubate at 37 Centigrade in the dark for 10 minutes. A color change from transparent to blue
    (or light blue) indicates that the corresponding specimen is positive.
12. Add 50ul of Stop Solution to all wells. This step could result in a color change from blue to
    yellow for the positive specimen.
13. Wiping off the moisture from the bottom of the microwells with a soft absorbent tissue before
    reading.
14. Read the microwell strip plate at a single wavelength of 450nm.

Quality Control
1. Reagent Blank Acceptance Criteria
    A plate is considered valid if the value of the reagent blank is greater than 0.000 and less than or equal to 0.08.
2. Negative Control Acceptance Criteria
    Individual Negative Control absorbance values must be greater than 0.000 and less than 0.100.
    If the value of Negative Control is less than 0.06, presume it to be 0.06 for calculation of Cutoff
    value; if greater, use the true value.
3. Positive Control Acceptance Criteria
    Positive Control values must be greater than or equal to 0.6.
4. Cutoff Value
    Neal-x = Average Absorbance Value of Negative Control
    Cutoff value = Neal -X + 0.06
The assay is invalid and must be repeated if either absorbance value of Reagent Blank / Negative Control / Positive Control is outside of the appropriate range.

Interpretation Of Results
1. Specimens with absorbance value less than the cutoff value should be considered non-reactive.
2. Specimens giving an absorbance equal to or greater than the cutoff value are considered initially
    reactive in the assay. Such specimens should be retested in duplicate using the original source.
3. Specimens that are reactive in at least one of the duplicate retests are considered repeatedly
    reactive and are presumed to be positive for HCV core antigen; Specimens that are
    non-reactive in both wells on retest should be considered negative for HCV core antigen.

Precautions
1. Strictly follow the test procedure.
2. Microwell strips from different plates can be mixed to assemble full or partial plate as long as
    they are from the same lot, within the expiration date. If less than the whole plate is being used,
    reseal unused strips in the original storage bag or in the plastic seal bag provided along with the
    desiccant sachet and return to 2-8 Centigrade.
3. Do not touch the bottom exterior surface of the wells.
4. Do not allow microwells become dry, and all steps must be completed in order without
    interruption once the assay has begun.
5. Be careful not to fill liquid high up on the well rim or splash reagent outside the wells.
6. Care must be taken not to cross-contaminate reagents or specimens.
7. The target incubation temperature for this assay is 37 Centigrade. However, the assay can be
    successfully run at 37? Centigrade.

Bio-Hazard Precaution
The HCV Ag EIA kit contains components of human origin. No test method can offer complete
assurance that products derived from human sources will not transmit infection. So all materials of
human origin should be considered as potentially infectious. It is recommended that all those
reagents and test specimens be handled as if capable of transmitting infectious agents.
1. Non-disposable apparatus should be sterilized after use. The preferred method is to autoclave
    for 30 minutes at 121 Centigrade. Disposables should be autoclaved or incinerated.
2. Any glassware used with the reagents should be thoroughly washed with 2M hydrochloric acid
    and then rinsed with distilled water or high quality deionized water.
3. Wear disposable gloves and work clothes while handling kit reagents and specimens.
    Thoroughly wash hands afterward.
4. Spillage of potentially infectious materials should be removed immediately with absorbent paper
    tissue and the contaminated area swabbed with 5% sodium hypochlorite before work is
    continued.
5. Sulphuric acid required for the Stop Solution and hydrochloric acid used for washing glassware
    are corrosive and should be handled with appropriate care. If they come into contact with the
    skin or eyes, wash thoroughly with water.

Product Characteristics
1. This assay was designed to screen individual units of blood or plasma, but not for ascitic fluid,
    pleural fluid, saliva and non-human specimen.
2. The haemoglobins(<120g/L), cholesterols(<7.0mmol/L),and triglycerides (<1.8mmol/L) in
    specimens won't affect the test result.
3. The results of genotyping of 23 HCV Ag positive samples detected with the HCV Ag EIA kit
    are: 1b type: 17(73.91%), 2a type: 4(17.39%), 1b/2a type: 2(8.69%).
4. Sensitivity: The recombinant antigen was tested with three different lots of kit. The sensitivity is
    2.5pg (OD450 > Cutoff value).
    A total 219,634 donations from 17,358 donors was tested with the HCV Ag EIA kit and was
    also tested with PCR. Twenty nine donations are positive reactive with the HCV Ag EIA Kit.
    These 29 cases are also positive reactive with HCV RNA testing.
5. Specificity: A total sample population of 219,634 plasma at two sites was tested with the HCV
    Ag EIA kit. The following table gives the initially reactive, repeatedly reactive and confirm
    positive rate.

Test site Specimen Initially Nonreactive Initially Reactive Repeatedly Reactive Confirmed Positive
1            140587   139884                   703                    580                          19
2              79047     78575                   472                    380                            9
The specificity was calculated as the number of true negatives that tested non-reactive divided
by the total number of negative. Specificity is 99.7% (218459/219606).
A total of 90 potentially cross-reactive specimens was tested with the HCV Ag EIA kit. The
samples tested were from patients with the following diseases: cytomegalovirus (CMV) 3,
Epstein-Bar virus (EBV) 4, hepatitis A virus (HAV) 7, hepatitis B virus (HBV) 59, herpes simplex
virus (HSV) 5 and hepatocellular carcinoma 12. All samples were tested negative with the HCV
Ag EIA kit.

Key words:
Rapid Diagnostic Test Detection Kits Hepatitis C Virus HCV Core Antigen EIA blood safety

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