Blood virus screening
HCV-Ab
Diagnostic kit for Antibody to Hepatitis C Virus (ELISA)
Two-step incubation, indirect principle
INTENDED USE
This kit is an enzyme-linked immunosorbant assay (ELISA) for qualitative detection of
antibodies to hepatitis C virus in human serum or plasma. It is intended for screening of
blood donors and diagnosis of patients infected with hepatitis C virus.
PRINCIPLE OF THE ASSAY
By: Mr. Mohammed Abubasha
Polystyrene microwell strips
are pre-coated with
recombinant,
immunoreactive antigens
corresponding to the core
and the non-structural
(HCV-Ab) in the sample
serum or plasma
regions of HCV (HCV-Ag)
During the first incubation step, specific HCV-Ab, if present are captured on the solid phase
pre-coated with HCV antigens. The wells are washed to remove unbound serum proteins
Rabbit anti-human IgG
antibodies conjugated to the
enzyme horseradish
peroxidase
(HRP-Conjugate) are
added
During the second incubation step, these HRP-conjugated antibodies bind to any antigen-antibody
(IgG) complexes previously formed and unbound HRP-conjugate is then removed by washing
By: Mr. Mohammed Abubasha
Chromogen solutions
(substrate)
Chromogen solutions containing tetramethylbenzidine (TMB) and urea peroxide are added to the
wells. In presence of the antigen-antibody-anti-IgG (HRP) immunocomplex, the colorless chromogens
are hydrolyzed by the bound HRP conjugate to a blue-colored product.
The blue color turns yellow after the reaction is stopped with sulfuric acid.
By: Mr. Mohammed Abubasha
The intensity of color can be measured and it is proportional to the amount of antibody
captured in the wells, and to the amount of antibody in the sample respectively. Wells
containing samples negative for HCV-Ab remain colorless
SPECIMEN COLLECTION AND STORAGE
1. Sample collection:
Either fresh serum or plasma samples can be used for this assay. Blood
collected by venipuncture should be allowed to clot naturally and
completely- the serum/plasma must be separated from the clot as early
as possible to avoid hemolysis of RBC.
2. Storage:
Store samples at 2-8C. Samples not required for assaying within 3 days
should be stored frozen (-20C or below). Multiple freeze-thaw cycles
should be avoided.
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ASSAY PROCEDURE
Step 1: Reagent preparation:
Allow the reagents and samples to equilibrate at room temperature (1830C) for at least 15-30 minutes. Check the wash buffer concentrate for the
presence of salt crystals. If crystals have formed, resolubilize by warming
at 37C until crystals dissolve. Dilute the wash buffer 1 to 20 with distilled
or deionized water. Use only clean vessels to dilute the buffer. Shake the
reagents gently before, and return to 2-8C immediately after use.
Step 2: Numbering wells:
Set the strips needed in the strep-holder and number sufficient number of
wells including three of the negative controls (e.g. B1, C1, D1), two of the
positive controls (e.g. E1, F1) and one Blank (e.g. A1, neither samples nor
HRP-Conjugate should be added into the Blank well).
1
2
3
4
5
6
7
8
9 10 11 12
A
B
C
D
E
F
G
H
By: Mr. Mohammed Abubasha
Step 3: Adding diluent:
Add 100µl specimen diluent into each well except the blank.
Step 4: Adding sample:
Add 10µl of positive control, negative control, and specimens into their
respective wells. (Note: use a separate disposable pipette tip for each
specimen, negative and positive control as to avoid cross-contamination).
Mix by tapping the plat gently.
Step 5: Incubating (1)
Cover the plate with the plate cover and incubate for 30 minutes at 37C. It
is recommended to use a thermostat-controlled water tank to assure the
temperature stability and humidity during the incubation. If a dry
incubator is used, do not open the door frequently.
Step 6: Washing (1):
At the end of incubation, remove and discard the plate cover. Wash each
well 5 times with diluted wash buffer. Each time allow the microwells to
soak for 30-60 seconds. After the final washing cycle, turn the plate down
onto blotting paper or clean towel, and tap it as to remove any residues.
Step 7: Adding HRP-Conjugate:
Add 100µl HRP-Conjugate into each well except the blank.
Step 8: Incubating (2):
Cover the plate with plate cover and incubate for 30 minutes at 37C.
Step 9: Washing (2):
After the end of incubation, remove and discard the plate cover. Wash
each well 5 times with diluted wash buffer as in step 6.
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Step 10: coloring:
Dispense 50µl of Chromogen A and 50µl of chromogen B solutions into
each well including the blank (Note: Chromogen A must be added before
Chromogen B), and mix by tapping the plate gently. Incubate the plate at
37C for 15 minutes avoiding light. The enzymatic reaction between the
Chromogen solutions and the HRP-Conjugate produces blue color in
positive control and HCV-Ab positive sample wells.
Step 11: Stopping reaction:
Using a multichannel pipette or manually, add 50µl stop solution into
each well and mix gently (Stop enzyme-substrate reaction). Intensive
yellow color should appear in positive control and HCV-Ab positive
sample wells.
Step 12: Measuring the Absorbance:
Calibrate the plate reader with the blank well and read the absorbance at
450nm. If a dual filter instrument is used, set the reference wavelength at
600-650 nm. Calculate the cut-off value and evaluate the results. (Note:
read the absorbance within 10 minutes after stopping the reaction).
INTERPRETATION OF RESULTS AND QUALITY CONTROL
Each microplate should be considered separately when calculating and
interpreting results, regardless of the number of plates concurrently
processed. The results are calculated by relating each samples optical
density (OD) value to the Cut-off value (C.O.) of the plate.
1) Calculation of the Cut-off (C.O.) value:
Cut-off value (C.O.) = Nc + 0.12
Nc: is the mean absorbance value for three negative controls.
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Note: if the mean OD value of the three negative controls is lower than
0.02, take it as 0.02. If higher than 0.02, see the quality control range.
Example):
Well number
Negative controls OD value
B1
0.010
C1
0.012
D1
0.014
(1) Calculation of Nc:
Nc = 0.010 + 0.012 + 0.014 = 0.012 (the mean value is lower than 0.02,
3
so take it as 0.02)
(2) Calculation of Cut-off (C.O.) = 0.02 + 0.12 = 0.14
If one of the negative control values does not meet the quality control
range specifications, it should be discarded and the mean value is
calculated again using the remaining two values. If more than one negative
control OD value does not meet the quality control range specifications, the
test is invalid and must be repeated.
2) Quality control range:
The test results are valid if the quality control criteria are verified. It is
recommended that each laboratory must establish appropriate quality
control system with quality control material similar to or identical with the
patient sample being analyzed.
 The OD value of the blank well, which contains only chromogens and
stop solution, is less than 0.080 at 450nm.
 The OD value of the positive control must be equal to or greater than
1.000 at 450/600-650nm or at 450nm after blanking.
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 The OD value of the negative control must be equal to or less than 0.080
at 450/600-650nm or at 450nm after blanking.
3) Interpretation of the results:
NEGATIVE RESULTS:
Samples giving an absorbance less than the Cut-off value are negative for
this assay. This indicates that no antibodies to hepatitis C virus have been
detected with this kit; therefore the patient is probably not infected with
HCV.
POSITIVE RESULTS:
Samples giving an absorbance equal to or greater than the Cut-off are
considered initially reactive, which indicates that antibodies to hepatitis C
virus have probably been detected using this kit. Retesting in duplicates of
any initially reactive sample is recommended. And therefore the patient is
probably infected with the virus. Any blood unite containing antibodies to
HCV should be immediately discarded.
BORDERLINE:
Samples with optical density to Cut-off ratio equal to or less than 2 are
considered borderline and retesting of those samples in duplicates is
recommended to confirm the results. Repeating positive samples could be
considered positive for antibodies to HCV.
By: Mr. Mohammed Abubasha