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Manufacturer,provider and
supplier of
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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