anti-HCV All materials contaminated with patient specimens or controls should be

inactivated by validated procedures (autoclaving or chemical treatment)

Antibodies in Human Serum and Plasma [STOP], [NC], [SA], [SB] irritates eyes, skin and mucous membranes. Upon
contact, rinse thoroughly with copious amounts of water and consult a
Package Size doctor.
[REF] 51250 96 Tests Complete Test Kit [NC], [PC], [DIL] contain sodium azide which may react with metals of
[IVD] laboratory plumbing forming explosive azides. Flush the conduit with
copious amounts of water as preventive measure, in case sodium azide
Intended Use containing solution is disposed in the sink.
The hepatitis C virus (HCV) is known to be the causative agent for most
blood-borne non-A, non-B hepatitis (NANBH). Studies throughout the Stability
world indicate that HCV is transmitted by sexual contact, by exposure to The reagents are stable up to the stated expiry dates on the individual
contaminated blood or blood products. labels when stored at 2...8°C.
The use of different HCV antigens (recombinant) has shown to be more After opening reagents have to be stored at 2...8°C and used within 30
effective in identifying acute and chronic non-A, non-B hepatitis than days (see also "Note").
single antigen assays. [WS] (20x) should be stored at room temperature to avoid crystallisation.
The HUMAN ANTI-HCV ELISA is intended to identify potentially infectious
samples and prevent them from use as donor materials. [MIC]
Sealed in an aluminium bag with a desiccant.
Principle Must be at room temperature before opening!
The HUMAN ANTI-HCV ELISA is a 3rd generation indirect ELISA detecting
anti-HCV IgG antibodies (anti-HCV-ab). The test utilises specific HCV unused: return to the zip-lock bag and store in this way at 2...8°C.
recombinant antigens (core, NS 3, NS 4, NS 5) coated on microtiter wells. Do not touch the upper rim or the bottom of the wells with fingers.
Specimen’s antibodies, if present, or controls bind to the HCV antigen (1.
step) at the solid phase. After the incubation unbound specimen compo- Reagent Preparation
nents are removed by washing. For the second incubation step anti- Bring all reagents to room temperature (15...25°C) before use.
human IgG HRP conjugate is added, which binds specifically to the Reagents not in use should always be stored at 2...8°C.
immobilised human anti-HCV IgG antibodies, and forms a sandwich
immunocomplex. After a second washing step to remove excess conju- Working Conjugate Solution [WCON]
gate, TMB/Substrate is added (Step 3). A blue colour develops changing to Dilute [CON] 1 + 20 with [C-DIL], e.g. 50 µl [CON] + 1000 l [C-DIL] for
yellow after stopping the reaction. The intensity of the colours is directly 8 wells. (100 µl required for each well.)
proportional to the HCV-IgG-Ab concentration in the specimen.
Prepare fresh for each run.
The absorbance of controls and specimen is determined by using ELISA
Stability: 6 hours at room temperature (avoid contamination).
microplate readers or automated ELISA systems (like HUMAN´s Huma-
Reader or ELISYS line). Results for patient samples are obtained by Working wash solution [WASH]
comparison with the cut-off value.
Crystals in [WS] have to be dissolved by heating at 35...37°C
Reagents and Contents Dilute [WS] 1 + 19 with fresh deionised water, e.g. 50 ml [WS] + 950 ml
[MIC] 12 Microtiter Strips = 1000 ml.
breakable 8-well strips coated with recombinant HCV about 2.5 ml [WASH] required for each well
antigens (E. coli)
Prepare only the quantity required for the current run.
[NC] 1.5 ml anti-HCV Negative Control (yellow cap)
Stability: 1 week at 2...8°C.
ready for use, human serum
[PC] 1.5 ml anti-HCV Positive Control (red cap) Working substrate solution [SUB] (see also A 2)
ready for use, human serum - Prepare required volume by mixing equal volumes of [SA] and [SB] in a
[DIL] 36 ml Sample Diluent (black cap) clean plastic container, rinsed with deionised water, prior to use.
Tris-buffer pH 7.1 0.1 - [SB] should be colourless to light blue, otherwise it should be discarded.
Tween-20 0.2%
- Handle [SUB] carefully and avoid contamination! Do not use, if it
[CON] 2.5 ml anti-HCV Conjugate (black cap) appears blue!
coloured blue
- Store protected from bright light.
anti-human IgG (goat), peroxidase-conjugated
Phosphate buffered saline pH 7.0 0.1 - Stability: 120 minutes at room temperature (15...25°C)
[C-DIL] 24 ml Conjugate Diluent (white cap) Specimen, Controls
Tris-buffer pH 7.1 0.1 Serum and plasma with the anticoagulants citrate, heparin or EDTA.
Tween-20 0.2%
Do not use highly lipemic, hemolysed or Icteric specimens.
[WS] 110 ml Wash Solution (20x) (black cap) pH 6.5 1.0
Concentrate for ca. 2000 ml Undiluted specimens may be stored for 12 hours at room temperature
Phosphate buffered saline (15...25°C), for 24 hours at 2...8°C, or for one year at -20°C. Freeze and
Tween-20 0.2 % thaw once only. Thawed specimen should be carefully homogenised.
Eliminate particulate matter by centrifugation or filtration.
[SA] 12 ml Substrate Reagent A (white cap)
Hydrogen peroxide 0.03 % Procedure
Citric acid buffer 0.06 mol/l Follow the procedure exactly as described.
[SB] 12 ml Substrate Reagent B (black cap)
3,3', 5,5'-tetramethylbenzidin (TMB) 0.6 mg/ml Procedural Notes
DMSO 0.7 mol/l P1: Do not mix or use components with different lot numbers. Do not
[STOP] 12 ml Stop Solution (white cap) mix caps of vials (risk of contamination). Do not use reagents after
Sulphuric acid, ready for use 1.0 mol/l their expiration date.
(R36/38), Xi irritant P2: Do not use reagents that could be contaminated or look or smell
1 Strip Holder different than usual.

2 Adhesive Strips P3: Record specimens and controls carefully on the spread sheet
supplied with the kit.
Preservatives: Total concentration < 0,1%
P4: [MIC] - select the required number of Microtiter Strips.
Safety Notes P5: Run duplicates or triplicates for positive resp. negative control.
Do not swallow the reagents. Avoid contact with eyes, skin and mucous Pipette controls and specimen on the bottom in the microwells.
membranes. All patient specimens and controls should be handled as
potentially infectious. The positive control has been inactivated by
heating. The negative control has been checked on donor level for HCV
and HIV-1/2 antibodies and HBsAg and found negative. Wear protective
clothing and disposable gloves according to Good Laboratory Practices.
P6: Always add reagents in the same order and timing to minimise Calculation of Control Values, Cut-off and Cut-off index
reaction time differences between wells. This is important for Mean absorbance values of [NC] in wells B1 and C1 (MNC) and [PC] in
reproducible results. Pipetting of specimens should not exceed 5 wells D1, E1 and F1 (MPC) are calculated according to:
minutes. Otherwise pipette the controls in the indicated positions at
half way time of the series. If more than 1 plate is used, repeat the A450 (B1) + A450 (C1) A450 (D1) + A450 (E1) + A450 (F1)
controls for each plate. MNC = ──────────────── MPC = ───────────────----------------
P7: Avoid/remove air bubbles prior to incubations and reading of 2 3
absorbance.
P8: [SUB]- incubate in the dark. [SUB] initiates a kinetic reaction, which is Cut-off value COV = (0.25 x MPC) + MNC
terminated by [STOP]. Cut-off index S/Co = Aspecimen / COV
The test run may be considered valid provided that the following criteria
Wash Procedure
are met:
The wash procedure is critical. Insufficient washing will result in poor
precision or falsely high absorbances. 1. Colour in A1: colourless or light yellow, otherwise the test is invalid
and should be repeated.
W1: Remove Adhesive Strips, aspirate off the contents into 5% sodium
hypochlorite solution and add [WASH] to each well, aspirate off after 2. MNC 0.20
30 sec. soak time and repeat washing 7 times. 3. MPC 0.60
W2: In case of automatic washers fill and prime with [WASH]. Sub- 4. MPC – MNC 0.40.
sequently wash strips 8 times. Ensure the washer fills all wells
completely and aspirates off efficiently after 30 sec. (remaining Interpretation of Results
liquid: < 15 µl). Result Interpretation
W3: After washing, remove remaining liquid by tapping the plate upside anti-HCV ab-
down on tissue paper. A450 (specimen) COV
nonreactive
anti-HCV ab-
Pipetting Scheme A450 (specimen) COV
initially reactive: retest
Follow the procedure exactly as described. Pay particular attention to anti-HCV ab-
Retest in duplicate:
the washing procedure!
Reagents and specimens should be at room temperature before use. Both results: S/Co > 1.5 reactive: perform confirmatory
test
Sample Preparation: [For in plate dilution see A 1]
Both results: S/Co < 1.0 Nonreactive
Dilute specimen, [PC] and [NC] 1 + 20 with [DIL], e.g. 10 l sample, [PC] Both results: 1.0 < S/Co < 1.5 equivocal: monitoring of
or [NC] + 200 l [DIL]. Shake for 15 seconds. subsequent specimen; perform
100 l are needed for each well. confirmatory test
Step 1 Well [µl] Result 1: S/Co < 1.0 invalid, (procedural error): retest
Result 2: S/Co > 1.5
A1 B1/C1 D1/F1 G1...
Blank [NC] [PC] Sample Performance Characteristics
Diluted [NC] in duplicate -- 100 -- -- Sensitivity and Specificity
Diluted [PC] in triplicate -- -- 100 -- The HUMAN ANTI-HCV ELISA has been tested with 744 positive and 5061
Diluted samples -- -- -- 100 unselected specimens against commercially available ELISA tests.
Mix carefully (15 sec) Sensitivity: 100%; Specificity: 99.6%.
[MIC] cover with Adhesive Strips Seroconversion sensitivity of anti-HCV ELISA was evaluated using 31
Incubate 60 min. at 37°C seroconversion panels. Results were compared with a commercially
available, CE-marked assay resulting in an equivalence seroconversion
Wash 8 times as described (see W1 - W3)
sensitivity.
[WASH] 350 350 350 350 Typical performance data can be found in the Verification Report,
Step 2 accessible via:
[WCON] -- 100 100 100 www.human.de/data/gb/vr/el-hcv.pdf
Mix carefully (15 sec) www.human-de.com/data/gb/vr/el-hcv.pdf
[MIC] cover with Adhesive Strips
Incubate 30 min. at 37°C Note
Wash 8 times as described (see W1 - W3) The handling should always be in compliance with common GLP
[WASH] 350 350 350 350 requirements (*)! The validation criteria must be met!
(*This includes: Proper caps being replaced on the vials and firmly tightened / Remove only
Step 3
reagents required for a run from stock solutions if they could come into contact with other
[SUB] 100 100 100 100 contaminating solutions like patient specimens etc. / Stock solutions always returned to
2...8°C when not in use.)
Incubate 30 min. at 17...25°C (see P8)
[STOP] 100 100 100 100 References
1. Uyttendaele S. et al., Vox Sang 66, 122-129 (1994)
Mix carefully
2. McFarlane I. G. et al., Lancet 335, 754-757 (1990)
Measure the absorbance at 450 nm as soon as possible or within 15
min. after terminating the reaction, using a reference wavelength of 3. Tsutsumt M. et al., Hepatology 19, 265-272 (1994)
620 - 690 nm (if available). 4. Claeys H. et al., J. Med. Virol. 36, 259-264 (1992)
5. Kuog G. et al., Science 244, 362-364 (1989)
Alternative procedures intended for automated ELISA analyzers:
A 1:
(Step1)
- dispense 200 µl [DIL] into each required well
- add 10 µl [NC], [PC] or specimen,
EL-HCV INF 5125001 GB 10-2010-15 |
0483
- mix and proceed as described

A 2: dispense 50 µl [SA] first, then 50 µl [SB] into each

Human Gesellschaft für Biochemica und Diagnostica mbH