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VIDAS®Anti-HAV Total (HAVT)

VIDAS Anti-HAV Total (HAVT) is an automated test for use on the VIDAS family instruments for the quantitative
measurement of total immunoglobulins directed against the hepatitis A virus (HAV) in human serum or plasma (lithium
heparin EDTA and Citrate), using the ELFA technique (Enzyme Linked Fluorescent Assay).

SUMMARY AND EXPLANATION PRINCIPLE

The diagnosis of a recent hepatitis A viral infection is The assay principle combines a 2-step enzyme
usually indicated by a positive anti-HAV IgM serology. Anti- immunoassay competition method with a final fluorescent
HAV IgM is normally detected when the patient becomes detection (ELFA).
®
symptomatic. An ELISA technique (e.g. VIDAS HAV IgM-Ref. The Solid Phase Receptacle (SPR ) serves as the solid
30 307 is routinely used for detection). phase as well as the pipetting device for the assay.
The appearance of IgG signals recovery and immunity (1). Reagents for the assay are ready-to-use and pre-
In developed countries, improved hygiene conditions and dispensed in the sealed reagent strips.
water quality have decreased the prevalence of infections All of the assay steps are performed automatically by the
by this virus, which is exclusively transmitted by fecal-oral instrument. The reaction medium is cycled in and out of
contact. Adults are increasingly affected and outbreaks of the SPR several times.
severe fulminant, cholestatic or relapsing hepatitis A are The anti-HAV immunoglobulin in the sample binds with
observed (2). The recent development of vaccines, which the inactivated antigen fixed on the SPR by an antibody.
are more efficient than short-lived passive immunization, Unbound components are eliminated by washing.
eliminates the risk of contracting viral hepatitis A in Antigenic sites which have not reacted with the
exposed populations (travelers to endemic regions, immunoglobulin of the sample are next saturated with
sewermen, drug addicts, male homosexuals) (2, 3, 4). monoclonal antibody conjugated with alkaline
phosphatase.
Pre-vaccinal screening for anti-HAV total antibody is
During the final detection step, the substrate (4-Methyl-
justified when its prevalence is high (> 35%) in the country umbelliferyl phosphate) is cycled in and out of the SPR.
of origin of the patient (5). Regardless of the pre-vaccinal
The conjugate enzyme catalyzes the hydrolysis of this
strategy followed, the anti-HAV total antibody level substrate into a fluorescent product (4-Methyl-
measured after the last injection of vaccination will verify
umbelliferone), the fluorescence of which is measured at
the patient's seroconversion (6), the level of which has
450 nm. The intensity of the fluorescence is inversely
been determined at 20 mIU/ml by the manufacturers of proportional to the concentration of anti-HAV
Hepatitis A vaccine (7).
immunoglobulins present in the sample.
The detection of anti-HAV total immunoglobulin allows to At the end of the assay, results are automatically
determine the patient's immune status (6, 8). calculated by the instrument in relation to the calibration
curve stored in memory, and then printed out.

CONTENT OF THE KIT (30 TESTS):

30 HAVT strips STR Ready-to-use.
30 HAVT SPRs SPR Ready-to-use.
1 x 30 Interior of SPRs coated with inactivated HAV antigens.
HAVT positive control C1 Ready-to-use.
1 x 1 ml (liquid) Delipidated human* serum with anti-HAV Ig + 1 g/l sodium azide. The confidence
interval in mIU/ml (milli-International Units per milliliter) is indicated on the MLE card
after the following mention: "Control C1 Dose Value Range".
HAVT negative control C2 Ready-to-use.
1 x 1 ml (liquid) Delipidated human* serum + 1 g/l sodium azide.

HAVT calibrator S1 Ready-to-use.

1 x 2 ml (liquid) Delipidated human* serum with anti-HAV Ig + 1 g/l sodium azide.

1 MLE card (Master Lot Entry) Specifications for the factory master data required to calibrate the test: to read the
MLE data, please refer to the User’s Manual.
1 Package insert provided in the kit or downloadable from www.biomerieux.com/techlib
* This product has been tested and shown to be negative for HBs antigen, antibodies to HIV1, HIV2 and HCV. However, since no
existing test method can totally guarantee their absence, this product must be treated as potentially infectious. Therefore, usual safety
procedures should be observed when handling.

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The SPR The strip

The interior of the SPR is coated during production with The strip consists of 10 wells covered with a labeled, foil
inactivated HAV antigens. Each SPR is identified by the seal. The label comprises a bar code which mainly
HAVT code. Only remove the required number of SPRs indicates the assay code, kit lot number and expiration
from the pouch and carefully reseal the pouch after date. The foil of the first well is perforated to facilitate the
opening. introduction of the sample. The last well of each strip is a
cuvette in which the fluorometric reading is performed.
The wells in the center section of the strip contain the
various reagents required for the assay.
Description of the HAVT strip
Wells Reagents
1 Sample well.
2 Sample diluent: TRIS buffer (0.2 mol/l, pH 6.2) + protein and chemical stabilizers +
0.9 g/l sodium azide (300 µl).
3-4-6-7-8-9 Wash buffer: TRIS HCl buffer (0.05 mol/l, pH 8) + Triton X 100 pH 8 + 8.78 g/l
sodium chloride + 0.9 g/l sodium azide (600 µl).
5 Anti-HAV mouse monoclonal antibody conjugated with alkaline phosphatase.
Diluent: TRIS buffer (0.05 mol/l, pH 6.3) + Tween 20 pH 6.2 + protein and chemical
stabilizers + 0.9 g/l sodium azide (400 µl).
10 Cuvette with substrate: 4-Methyl-umbelliferyl phosphate (0.6 mmol/l) +
diethanolamine (DEA*) (0.62 mol/l or 6.6%, pH 9.2) + 1 g/l sodium azide (300 µl).

* Signal Word: DANGER

Hazard statement
H318 : Causes serious eye damage.
Precautionary statement
P280 :Wear protective gloves/protective clothing/eye protection/face protection.
P305 + P351 + P338: IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present
and easy to do. Continue rinsing.
For further information, refer to the Material Safety Data Sheet.

MATERIALS AND DISPOSABLES REQUIRED BUT • Do not use visibly deteriorated STRs (damaged foil or
NOT PROVIDED plastic).
- Pipette with disposable tip to dispense 150 µl. • Do not use reagents after the expiration date indicated
- Powderless, disposable gloves. on the label.
- For other specific materials and disposables, please • Do not mix reagents (or disposables) from different lots.
refer to the Instrument User’s Manual. • Use powderless gloves, as powder has been reported
- VIDAS family instrument. to cause false results for certain enzyme immunoassay
tests.
WARNINGS AND PRECAUTIONS • Kit reagents contain sodium azide which can react with
• For in vitro diagnostic use only. lead or copper plumbing to form explosive metal azides.
• For professional use only. If any liquid containing sodium azide is disposed of in
• This kit contains products of human origin. No the plumbing system, drains should be flushed with
known analysis method can totally guarantee the water to avoid build-up.
absence of transmissible pathogenic agents. It is • The substrate in well 10 contains an irritant agent (6.6%
therefore recommended that these products be diethanolamine). Refer to the hazard statements "H"
treated as potentially infectious and handled and the precautionary statements "P" above.
observing the usual safety precautions (see • Spills should be wiped up thoroughly after treatment
Laboratory biosafety manual - WHO - Geneva - latest with liquid detergent or a solution of household bleach
edition). containing at least 0.5% sodium hypochlorite. See the
• This kit contains products of animal origin. Certified User’s Manual for cleaning spills on or in the instrument.
knowledge of the origin and/or sanitary state of the Do not autoclave solutions containing bleach.
animals does not totally guarantee the absence of • The instrument should be regularly cleaned and
transmissible pathogenic agents. It is therefore decontaminated (see the User’s Manual).
recommended that these products be treated as
potentially infectious and handled observing the usual
safety precautions (do not ingest or inhale).
• Do not use the SPRs if the pouch is pierced.

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STORAGE CONDITIONS Calibration

• Store the VIDAS Anti-HAV Total kit at 2-8°C. Calibration, using the calibrator provided in the kit, must
• Do not freeze reagents. be performed upon receipt of a new lot of reagents after
• Store all unused reagents at 2-8°C. the master lot data have been entered. Calibration should
• After opening the kit, check that the SPR pouch is then be performed every 14 days. This operation provides
correctly sealed and undamaged. If not, do not use the instrument-specific calibration curves and compensates
SPRs. for possible minor variations in assay signal throughout
• Carefully reseal the pouch with the desiccant inside the shelf-life of the kit.
after use to maintain stability of the SPRs and return The calibrator, identified by S1, must be tested in
the complete kit to 2-8°C. duplicate (see User’s Manual). The calibrator value must
• If stored according to the recommended conditions, all be within the set RFV "Relative Fluorescence Value"
components are stable until the expiration date indicated range. If this is not the case, recalibrate.
on the label.
Procedure
SPECIMENS 1. Only remove the required reagents from the
refrigerator and allow them to come to room
Specimen type and collection temperature for at least 30 minutes.
Serum or plasma (lithium heparin, EDTA and citrate). 2. Use one "HAVT" strip and one "HAVT" SPR for each
It is recommended that each laboratory checks the sample, control or calibrator to be tested. Make sure
compatibility of collection tubes used. the storage pouch has been carefully resealed
None of the following factors have been found to after the required SPRs have been removed.
significantly influence this assay: 3. The test is identified by the "HAVT" code on the
- hemolysis (after spiking samples with hemoglobin: 0 to instrument. The calibrator must be identified by "S1",
300 µmol/l (monomer)), and tested in duplicate. If the positive control is to be
- lipemia (after spiking samples with lipids: 0 to 2 mg/ml tested, it should be identified by "C1". If the negative
equivalent in triglycerides), control needs to be tested, it should be identified by
- bilirubinemia (after spiking samples with bilirubin: 0 to "C2".
400 µmol/l).
4. If necessary, clarify samples by centrifugation.
However, it is recommended not to use samples that are
clearly hemolyzed, or lipemic and, if possible, to collect a 5. Mix the calibrator, controls and samples using a
new sample. vortex-type mixer (for serum or plasma separated from
Do not inactivate samples. the pellet).
6. For this test, the calibrator, control, and sample
Specimen stability
test portion is 150 µl.
Samples can be stored at 2-8°C in stoppered tubes for up
7. Insert the "HAVT" SPRs and "HAVT" strips into the
to 7 days; if longer storage is required, freeze the sera or
instrument. Check to make sure the color labels with
plasma at -25 ± 6°C.
the assay code on the SPRs and the Reagent Strips
Avoid successive freezing and thawing.
match.
A study performed on frozen samples over a period of 2
months, showed that the quality of results is not affected. 8. Initiate the assay as directed in the User’s Manual. All
the assay steps are performed automatically by the
INSTRUCTIONS FOR USE instrument.
For complete instructions, see the User’s Manual. 9. Restopper the vials and return them to 2–8°C after
pipetting.
Master lot data entry
10. The assay will be completed within approximately 90
Before each new lot of reagents is used, specifications (or
minutes. After the assay is completed, remove the
factory master data) must be entered into the instrument
SPRs and strips from the instrument.
using the master lot entry (MLE) data. If this operation is
not performed before initiating the tests, the instrument 11. Dispose of the used SPRs and strips into an
will not be able to print results. The master lot data need appropriate recipient.
only be entered once for each lot.
It is possible to enter MLE data manually or automatically RESULTS AND INTERPRETATION
depending on the instrument (refer to the User’s Manual). Once the assay is completed, results are analyzed
automatically by the computer. Fluorescence is measured
twice in the Reagent Strip’s reading cuvette for each
sample tested. The first reading is a background reading
of the substrate cuvette before the SPR is introduced into
the substrate. The second reading is taken after
incubating the substrate with the enzyme remaining on
the interior of the SPR. The RFV (Relative Fluorescence
Value) is calculated by subtracting the background
reading from the final result. This calculation appears on
the result sheet.

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The results are automatically calculated by the instrument Precision

using calibration curves which are stored by the
Within-run reproducibility
instrument (4-parameter logistic model). The
concentrations are expressed in «mIU/mI». 3 samples were tested 30 times in a same run.
The patient RFV is interpreted by the VIDAS system. Both Sample 1 2 3
the results, expressed in mIU/mI (WHO reference
st
standard 1 Reference Preparation Hepatitis A Mean (mIU/ml) 24.2 44.4 231
immunoglobulin) (100 IU/ml), and their interpretation are CV % 8.5 4.2 2.3
printed on the result sheet. The results are interpreted as
follows: Between-run reproducibility
Concentration Interpretation 3 samples were tested singly in 29 different runs on the
< 15 mIU/ml Negative same VIDAS instrument.

> 15 and < 20 mIU/ml Borderline positive Sample 1 2 3

> 20 mIU/ml Positive Number 29 29 29

Samples with a concentration > 400 mIU/ml must be Mean (mIU/ml) 20.8 43.5 212.4
retested after dilution of 1/100 in negative human serum. CV % 10.7 3.5 3.3
If the dilution factor has not been entered when the Work
List was created (see User’s Manual), multiply the result Specificity
to obtain the sample concentration.
Interpretation of test results should be made taking into 1521 samples were tested in comparison with another
consideration the patient history, and the results of any commercially available EIA technique in 3 reference
other tests performed. laboratories. The discrepant samples were confirmed with
a modified RIA technique to obtain a detection threshold
Interpretation of borderline positive close to 10 mIU/ml. Equivocal samples were not used in
Samples with concentrations found between 15 and 20 the performance calculations.
mIU/ml contain anti-HAV antibodies. Such a concentration 1) Random population
does not enable patient immunity to be affirmed; it is
recommended to retest the patient after a few days. 1136 samples from blood donors were tested.
QUALITY CONTROL EIA 1
One positive and one negative control are included in positive negative
each VIDAS HAVT kit. VIDAS positive 625 0
These controls must be performed immediately after negative 1* 510
opening a new kit to ensure that reagent performance has * This sample was found negative with the RIA
not been altered. Each calibration must also be checked confirmation technique.
using these controls. The instrument will only be able to
Relative sensitivity after confirmation: 100%
check the control values if they are identified by C1 and
(95% Confidence interval: 99.4% -100%).
C2.
Relative specificity after confirmation: 100%
Results cannot be validated if the control values deviate
(95% Confidence interval: 99.2% -100%).
from the expected values.
2) Vaccination follow-up samples
Note
200 samples were tested:
It is the responsibility of the user to perform Quality
- 30 samples before the first injection.
Control in accordance with any local applicable
- 60 samples 1 month after the first injection.
regulations.
- 60 samples 1 month after the second injection.
LIMITATIONS OF THE METHOD - 50 samples 1 month after the third injection.
Interference may be encountered with certain sera EIA 1
containing antibodies directed against reagent positive negative
components. For this reason, assay results should be VIDAS positive 158 0
interpreted taking into consideration the patient history, negative 1* 35
and the results of any other tests performed. *This sample was found negative with the RIA
PERFORMANCE confirmation technique.
Studies performed using VIDAS HAVT gave the following
results:
Measurement range
The measurement range of the VIDAS Anti-HAV Total
reagent is 15 to 400 mIU/ml.

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6 samples tested using the EIA 1 method were excluded 3) Acute hepatitis A & natural immunity samples
from the evaluation: 51 anti-HAV IgM positive samples and 50 natural
- 5 were equivocal with this method. immunity to hepatitis A samples were tested:
- 1 could not be retested with the confirmation method
(insufficient quantity). EIA 2
positive negative
Relative sensitivity before confirmation: 99.4%.
VIDAS positive 91 10*
Relative sensitivity after confirmation: 100%
negative 0 0
(95% confidence interval: 97.5% -100%).
Relative specificity after confirmation: 100% * These samples were found positive with the RIA
(95% confidence interval: 89.7% -100%). confirmation technique.
Relative sensitivity after confirmation: 100%
(95% confidence interval: 96.2% -100%).

ACCURACY

Dilution test

3 samples were diluted in negative human serum and tested singly in 2 runs. The ratio of the mean concentration
measured over the expected concentration is expressed as a mean recovery percentage.

Sample Dilution factor Expected Mean measured Mean recovery

concentration concentration percentage
(mIU/ml) (mIU/ml)

E1 1/10 - 234.8 -

1/20 117.4 119 101

1/40 58.7 59.9 102

1/80 29.4 32.4 110

1/160 14.7 17.9 122

E2 1/10 - 210.4 -

1/20 105.2 98.1 93.3

1/40 52.6 50 95.1

1/80 26.3 26.2 99.5

1/160 13.2 14.9 113

E3 1/10 - 248 -

1/20 124 130.6 105

1/40 62 68 110

1/80 31 35.4 114

1/160 15.5 19.5 126

CROSS REACTIVITY AND RELEVANT INTERFERENTS

VIDAS Anti-HAV
EIA 2 neg Total
positive negative
Anti-nuclear
3 0 3
antibody (ANA) +
CMV+/EBV+ 2 0 2
ANA+/HCV+ 35 0 35
HIV + 23 0 23
HCV + 33 0 33
HBV + 5 0 5
Rhumatoid factor + 1 0 1

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WASTE DISPOSAL INDEX OF SYMBOLS

Dispose of used or unused reagents as well as any other Symbol Meaning
contaminated disposable materials following procedures
for infectious or potentially infectious products. Catalog number
It is the responsibility of each laboratory to handle waste
and effluents produced according to their nature and In Vitro Diagnostic Medical Device
degree of hazardousness and to treat and dispose of
them (or have them treated and disposed of) in Manufacturer
accordance with any applicable regulations.
LITERATURE REFERENCES
Temperature limit
1. LUNEL F. Choix raisonné des outils diagnostics d'une
hépatite: apport de la biologie moléculaire. Les virus des
hépatites. Coll société française de microbiologie/virologie,
1991, 10: 41-53. Use by date
2. BUISSON Y. La vaccination contre l'hépatite A. La Lettre de
l'infectiologue, 1992, 7, 8: 282-285. Batch code
3. LEMON S.M., STAPLETON J.T.. Hepatitis A virus -
Prévention. Viral Hepatitis: Scientific Basis and Clinical Consult Instructions for Use
Management. Churchill Linvingstone, 1993: 61-79.
4. MARGOLIS H.S., SHAPIRO C. Who should receive hepatitis Contains sufficient for <n> tests
A vaccine, 1992, 10, suppl. 1: S85-S87.
5. VAN DOORSLAER E., THOMAS G., VAN DAMME P. Cost-
Effectiveness Analysis of Vaccination Against Hepatitis A in Date of manufacture
travellers. Journal of Medical Virology, 1994, 44: 463-469.
6. WIEDERMANN G., AMBROSCH F., ANDRE F.E., D'HONDT
E., DELEM A., SAFARY A. Persistence of vaccine - induced
antibody to hepatitis A virus. Vaccine, 1992, 10, suppl. 1: WARRANTY
S129-S131. bioMérieux disclaims all warranties, express or implied,
7. JUST M., BERGER R. Reactogenicity and immunogenicity of including any implied warranties of MERCHANTABILITY
inactivated hepatitis A vaccine. Vaccine, 1992, 10, suppl. 1: AND FITNESS FOR A PARTICULAR USE. bioMérieux
S110-S113. shall not be liable for any incidental or consequential
8. MILLER W.J., CLARK W., HURNI W., KUTER B., damages. IN NO EVENT SHALL BIOMERIEUX’S
SCHOFIELD T. and NALIN D. Sensitive Assays for Hepatitis LIABLITY TO CUSTOMER UNDER ANY CLAIM
A Antibodies. Journal of Medical Virology, 1993, 41: 201-204.
EXCEED A REFUND OF THE AMOUNT PAID TO
BIOMERIEUX FOR THE PRODUCT OR SERVICE
WHICH IS THE SUBJECT OF THE CLAIM.
REVISION HISTORY
Change type categories :
N/A Not applicable (First publication)
Correction Correction of documentation anomalies
Technical change Addition, revision and/or removal of information related to the product
Administrative Implementation of non-technical changes noticeable to the user
Note: Minor typographical, grammar, and formatting changes are not included in the
revision history.
Release
Part Number Change Type Change Summary
date
INDEX OF SYMBOLS
Administrative REVISION HISTORY
2015/01 08447K
CONTENT OF THE KIT (30 TESTS)
Technical
WARNINGS AND PRECAUTIONS

BIOMERIEUX, the blue logo, SPR and VIDAS are used, pending, and/or registered trademarks belonging to bioMérieux or one of its
subsidiaries or one of its companies.
Any other name or trademark is the property of its respective owner.

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