Doc.: INS IVCOMB.CE/eng Page 1 of 11 Rev.

: 3 Date: 09/2011

HIV Ab&Ag
Fourth generation Enzyme Immunoassay
for the determination of antibodies to
Human Immunodeficiency Virus or HIV
type 1&2&O and P24 HIV-1 Antigen
in human serum and plasma

- for “in vitro” diagnostic use only -

DIA.PRO
Diagnostic Bioprobes Srl
Via G. Carducci n° 27
20099 Sesto San Giovanni
(Milano) - Italy
Phone +39 02 27007161
Fax +39 02 26007726
e-mail: info@diapro.it

REF IVCOMB.CE
96/192/480/960 Tests

1
Doc.: INS IVCOMB.CE/eng Page 2 of 11 Rev.: 3 Date: 09/2011

1. Microplate MICROPLATE
HIV Ab&Ag n° 2 microplates. 12 strips of 8 breakable wells c oated with HIV
specific gp36, gp41 and gp120 peptides and with a Monoclonal
Antibody specific to the HIV-1 p24 Ag. Plates are sealed into a
A. INTENDED USE bag with desiccant.
The kit is a solid phase enzyme immunoassay for the in-vitro
diagnostic screening of antibodies to all subtypes of HIV-1 and 2. Negative Control CONTROL -
HIV-2 and HIV-1 antigen (p24) in human serum or plasma. 1x4.0ml/vial. Ready to use control. It contains animal serum
This kit is intended exclusively for In vitro diagnostic use in an negative for HIV antibodies and for p24 antigen, and 0.1%
authorized clinical laboratory and the test has to be carried out by Kathon GC as preservatives. The negative control is yellow-
specifically trained health-care professional personnel. brown color coded.

3. Positive Control HIV-1 Ab CONTROL 1+

B. INTRODUCTION 1x4.0ml/vial. Ready to use control. It contains inactivated
Epidemiological evidence indicates that an infectious agent HIV 1 antibody positive serum, filtered HIV Ab&Ag negative
transmitted through intimate contact, intravenous drug use or use animal serum and 0.1% Kathon GC as preservative. The
of infected blood or blood products leads to Acquired Positive Control is light green color coded.
Immunodeficiency Syndrome (AIDS). Important Note: The positive control has been inactivated using
This disease affects T-cell mediated immunity, resulting in severe B-propionolactone BPL/UV. This does not fully ensure the
lymphopenia and a reduced subpopulation of helper absence of viable pathogens, and therefore, the control should be
T-lymphocytes. Destruction of this T-lymphocyte population by the handled as potentially biohazardous, in accordance with good
virus causes an immune deficiency, resulting in a reduced or laboratory practices.
deficient response to subsequent infections.
Consequently, infections become more severe and may cause 4. Positive Control HIV-2 Ab CONTROL 2+
death. At present, there is no successful treatment for AIDS. 1x4.0ml/vial. Ready to use control. It contains inactivated HIV2
antibody positive serum, filtered HIV Ab&Ag negative animal
The etiological agent has been identified as a retrovirus, human serum and 0.1% Kathon GC as preservatives. The Positive
immunodeficiency virus type 1 (HIV-1). Control is dark green color coded.
A closely related, but distinct type of immunodeficiency virus, Important Note: The positive control has been inactivated using
designated HIV-2, has also been isolated. This virus causes a B-propionolactone BPL/UV. This does not fully ensure the
disease that is indistinguishable from AIDS. absence of viable pathogens, and therefore, the control should be
Serological cross-reactivity between HIV-1 and HIV-2 has been handled as potentially biohazardous, in accordance with good
shown to be highly variable from sample to sample. laboratory practices.
This variability requires the inclusion of antigens to both HIV-1 and
HIV-2 for the screening of antibodies to HIV-1 and HIV-2. 5. HIV-1 P24 Ag Calibrator CAL Ag
The presence of anti-HIV-1 and/or anti-HIV-2 and/or HIV p24 2 vials. Lyophilized. It contains not infectious recombinant p24
antigen in the blood indicates potential infection with HIV-1 and/or antigen in a 10 mM phosphate buffer pH 7.0+/-0.2 with 0.3
HIV-2 and consequently this blood should not be used for mg/ml Gentamicine Sulphate and 0.1% Kathon GC as
transfusion or for manufacture of injectable products. stabilizers. This component is calibrated against the NIBSC 1st
International HIV-1 p24 Ag reference sample 90/636 (diluted
1:256) as well as the EFS HIV Ag performance panel (3015-
C. PRINCIPLE OF THE TEST 3022).
Synthetic peptides representing immunodominant epitopes of Important Notes:
HIV-1 and HIV-2 together with a monoclonal antibody to p24 HIV-1 1) The Calibrator contains p24 recombinant Ag with a
antigen are coated onto wells of a microplate. concentration of about 100 pg/ml, corresponding to about 4 IU/ml.
The peptides and the antibody have been carefully selected to 2) The volume necessary to dissolve the content of the vial may
ensure the screening of antibody and p24 antigen to all HIV-1 vary from lot to lot. Please use the right volume reported on the
subtypes, including subtype O and HIV-2. Serum or plasma label .
samples are added to these wells and, if antibodies specific to HIV-
1 and/or HIV-2 (IgG, IgM or IgA) are present in the sample, they 6. Wash buffer concentrate WASHBUF 20X
will form stable complexes with the HIV peptide antigens in the 2x60ml/bottle. 20x concentrated solution. It contains 0.1%
well. In case HIV-1 p24 is present in the sample, the antigen will Kathon GC. Once diluted, the wash solution contains 10 mM
be captured by the specific monoclonal antibody. phosphate buffer saline pH 7.0+/-0.2 and 0.05% Tween 20.
Antigen-antibody complexes are then identified through the
successive addition of: (1) biotinylated peptides, a biotinylated 7. Conjugate # 1 CONJ 1
monoclonal antibody to HIV-1 p24, and; (2) horseradish peroxidase 8 vials. The vial contains lyophilized biotinylated HIV1&2&0
HRP Streptavidin conjugate. gp36, gp41 and gp120 peptides and a biotinylated monoclonal
The hydrolytic activity of horseradish peroxidase allows for the antibody specific for HIV 1 p24 antigen. Vials are to be
quantification of these antibody-antigen complexes. resuspended with 6 ml of the Conjugate # 1 diluent.
Peroxidase substrate solution is then added.
During incubation, a blue color will develop in proportion to the
amount of anti-HIV-1/2 antibodies or HIV-1 p24 antigen bound to 8. Conjugate 1 Diluent CONJ 1 DIL
the well, thus establishing their presence or absence in the sample. 1x60ml/bottle. Used to dissolve the lyophilized powder of
Wells containing samples negative for anti-HIV antibody and/or Conjugate # 1, it contains Tris saline Buffer supplemented with
p24 antigen remain colorless. 0.05% Kathon GC, Tween 20 and BSA.
A stop solution is added to each well and the resulting yellow color
is read on a microplate reader at 450 nm. 9. Conjugate # 2 CONJ 2
1x25ml/cassette. The solution contains HRP conjugated with
streptavidin in Tris saline Buffer supplemented with 0.05%
Kathon GC, Tween 20 and BSA. This component is color coded
D. COMPONENTS
in red.
The standard format of the product code IVCOMB.CE contains
reagents for 192 tests.

2
Doc.: INS IVCOMB.CE/eng Page 3 of 11 Rev.: 3 Date: 09/2011

10. Chromogen/Substrate SUBS TMB The use of any sharp (needles) or cutting (blades) devices
1x45ml/cassette. Ready-to-use component. It contains 50 mM should be avoided. All the personnel involved should be trained
citrate buffer pH 3.5-3.8, 4% dimethylsulphoxide, 0.03% tetra- in biosafety procedures, as recommended by the Center for
methyl-benzidine or TMB and 0.02% hydrogen peroxide or Disease Control, Atlanta, U.S. and reported in the National
H2O2. Institute of Health’s publication: “Biosafety in Microbiological and
Note: To be stored protected from light as sensitive to strong Biomedical Laboratories”, ed. 1984.
illumination. 4. All the personnel involved in sample handling should be
vaccinated for HBV and HAV, for which vaccines are available,
11. Sulphuric Acid H2SO4 O.3 M safe and effective.
1x25ml/cassette. It contains 0.3 M H2SO4 solution. 5. The laboratory environment should be controlled so as to
Attention: Irritant (Xi R36/38; S2/26/30). avoid contaminants such as dust or air-born microbial agents,
when opening kit vials and microplates and when performing the
12. Sample Diluent: DILSPE test. Protect the Chromogen/Substrate from strong light and
1x14ml/cassette. Contains Tris saline buffer supplemented with avoid vibration of the bench surface where the test is
0.05% Kathon GC, anti HAMA blocker, and Tween 20; used for undertaken.
specimen dilution. This component is color coded in light blue. 6. Upon receipt, store the kit at 2..8°C into a tem perature
controlled refrigerator or cold room.
13. Plate sealing foils n° 4 7. Do not interchange components between different lots of
the kits. It is recommended that components between two kits
14. Package insert n° 1 of the same lot should not be interchanged.
8. Check that the reagents are clear and do not contain
15. Conjugate #1 MIX container: CONJ 1 MIX visible heavy particles or aggregates. If not, advise the
laboratory supervisor to initiate the necessary procedures for kit
1 empty container. Container to be used to unify Conjugate # 1
lyophilise and Conjugate # 1 diluent before starting the replacement.
automated assay 9. Avoid cross-contamination between serum/plasma
samples by using disposable tips and changing them after each
16. Container caps n° 5 sample.
Plastic cap to be used to firmly seal the container after first use. 10. Avoid cross-contamination between kit reagents by using
disposable tips and changing them between the use of each
Important note: Upon request, Dia.Pro can supply reagents for one.
96, 480, 960 tests, as reported below : 11. Do not use the kit after the expiration date stated on the
external container and internal (vials) labels.
12. Treat all specimens as potentially infective. All human
1.Microplate n°1 n°5 n°10 serum specimens should be handled at Biosafety Level 2, as
2.Negative Control 1x2.0ml/vial 1x10ml/vial 1x20ml/vial recommended by the Center for Disease Control, Atlanta, U.S.
3.Positive Control 1 1x2.0ml/vial 1x10ml/vial 1x20ml/vial
4.Positive Control 2 1x2.0ml/vial 1x10ml/vial 1x20ml/vial in compliance with what reported in the Institutes of Health’s
5.HIV p24 Calibrator n°1 vial n° 5 vials n° 10 vials publication: “Biosafety in Microbiological and Biomedical
6.Wash buff conc 1x60ml/bottle 5x60ml/bottles 4x150ml/bottles Laboratories”, ed. 1984.
7.Conjugate # 1 n°4 vials n°20 vials n°40 vials
8.Conjugate 1 Diluent 1x30ml/vial 3x50ml/bottles 2x150ml/bottles 13. The use of disposable plastic-ware is recommended in the
9.Conjugate # 2 1x15ml/vial 2x38ml/bottle 2x75ml/bottle preparation of the liquid components or in transferring
10.Chrom/Substrate 1x25ml/vial 3x42ml/bottle 2x125ml/bottles components into automated workstations, in order to avoid
11.Sulphuric Acid 1x15ml/vial 2x40ml/bottles 2x80ml/bottles
12.SampleDiluent 1x7ml/vial 1x35ml/bottle 1x70ml/bottle cross contamination.
Plate seal foils n° 2 n° 10 n° 20 14. Waste produced during the use of the kit has to be
Pack. insert n° 1 n°1 n° 1 discarded in compliance with national directives and laws
Number of tests 96 480 960
Code IVCOMB.CE.96 IVCOMB.CE.480 IVCOMB.CE.960
concerning laboratory waste of chemical and biological
substances. In particular, liquid waste generated from the
washing procedure, from residuals of controls and from samples
has to be treated as potentially infective material and inactivated
E. MATERIALS REQUIRED BUT NOT PROVIDED before waste. Suggested procedures of inactivation are
1. Calibrated Micropipettes (200ul and 10ul) and disposable treatment with a 10% final concentration of household bleach for
plastic tips. 16-18 hrs or heat inactivation by autoclave at 121°C for 20 min..
2. EIA grade water (bidistilled or deionised, charcoal treated to 15. Accidental spills from samples and operations have to be
remove oxidizing chemicals used as disinfectants). adsorbed with paper tissues soaked with household bleach and
3. Timer with 60 minute range or higher. then with water. Tissues should then be discarded in proper
4. Absorbent paper tissues. containers designated for laboratory/hospital waste.
5. Calibrated ELISA microplate thermostatic incubator capable 16. The Sulphuric Acid is irritant. In case of spills, wash the
to provide a temperature of +37°C. surface with plenty of water
6. Calibrated ELISA microwell reader with 450nm (reading) 17. Other waste materials generated from the use of the kit
and possibly with 620-630nm (blanking) filters. (example: tips used for samples and controls, used microplates)
7. Calibrated ELISA microplate washer. should be handled as potentially infective and disposed
8. Vortex or similar mixing tools. according to national directives and laws concerning laboratory
wastes.

F. WARNINGS AND PRECAUTIONS

1. The kit has to be used by skilled and properly trained G. SPECIMEN: PREPARATION AND RECOMMENDATIONS
technical personnel only, under the supervision of a medical 1. Blood is drawn aseptically by venepuncture and plasma or
doctor responsible of the laboratory. serum is prepared using standard techniques of preparation of
2. When the kit is used for the screening of blood units and samples for clinical laboratory analysis. No influence has been
blood components, it has to be used in a laboratory certified and observed in the preparation of the sample with citrate, EDTA
qualified by the national authority in that field (Ministry of Health and heparin.
or similar entity) to carry out this type of analysis. 2. Avoid any addition of preservatives to samples; especially
3. All the personnel involved in performing the assay have to sodium azide as this chemical would affect the enzymatic
wear protective laboratory clothes, talc-free gloves and glasses. activity of the conjugate, generating false negative results.

3
Doc.: INS IVCOMB.CE/eng Page 4 of 11 Rev.: 3 Date: 09/2011

3. Samples have to be clearly identified with codes or names in than 12 hours. After this time it has to be discarded and the
order to avoid misinterpretation of results. When the kit is empty, used container has to be washed with EIA grade water
used for the screening of blood units, bar code labeling and and kept dry for any following re-use.
electronic reading is strongly recommended.
4. Haemolysed (red) and visibly hyperlipemic (“milky”) samples Conjugate # 2:
have to be discarded as they could generate false results. Ready to use reagent. Mix well end-over-end before use.
Samples containing residues of fibrin or heavy particles or
microbial filaments and bodies should be discarded as they Chromogen/Substrate:
could give rise to false results. Ready to use. Mix well end-over-end before use.
5. Sera and plasma can be stored at +2°..8°C for u p to five days Be careful not to contaminate the liquid with oxidizing chemicals,
after collection. For longer storage periods, samples can be air-driven dust or microbes.
stored frozen at –20°C for several months. Any fr ozen samples Do not expose to strong illumination, oxidizing agents and
should not be frozen/thawed more than once as this may metallic surfaces.
generate particles that could affect the test result. If this component has to be transferred use only plastic, possible
6. If particles are present filter using 0.2-0.8u filters to clean up sterile disposable container.
the sample for testing.
7. Do not use heat inactivated samples as they could give Sulphuric Acid:
origin to false reactivity. Ready to use. Mix well end-over-end before use.
Attention: Irritant (Xi R36/38; S2/26/30)
Legend: R 36/38 = Irritating to eyes and skin.
H. PREPARATION OF COMPONENTS AND WARNINGS S 2/26/30 = In case of contact with eyes, rinse immediately with
A study conducted on an opened kit has not shown any relevant plenty of water and seek medical advice.
loss of activity up to 2 months.
Sample Diluent:
Microplates: Ready to use. Mix well end-over-end before use.
Allow the microplate to reach room temperature (about 1 hr)
before opening the container. Check that the pouch is not I. INSTRUMENTS AND TOOLS USED IN COMBINATION
broken or that some defect is present indicating a problem of WITH THE KIT
storage. In this case call Dia.Pro’s customer service. 1. Micropipettes have to be calibrated to deliver the correct
Unused strips have to be placed back into the aluminum pouch, volume required by the assay and must be submitted to
in presence of desiccant supplied, firmly zipped and stored at regular decontamination (household alcohol, 10% solution
+2°..8°C. When opened the first time, residual str ips are stable of bleach, hospital grade disinfectants) of those parts that
up to two months. could accidentally come in contact with the sample. They
should also be regularly maintained in order to show a
Negative Control: precision of 1% and a trueness of +/-2%. Decontamination
Ready to use. Mix well on vortex before use. of spills or residues of kit components should also be carried
out regularly.
Positive Controls Ab: 2. The ELISA incubator has to be set at +37°C (tolerance of
Positive controls are ready to use. Handle Positive Controls Ab +/-0.5°C) and regularly checked to ensure the corre ct
as potentially infective, even if HIV, if present in the control, has temperature is maintained. Both dry incubators and water
been chemically inactivated. baths are suitable for the incubations, provided that the
instrument is validated for the incubation of ELISA tests.
Calibrator Ag 3. The ELISA washer is extremely important to the overall
The Lyophilized Calibrator Ag contains a non-infectious performances of the assay. The washer must be carefully
recombinant p24 antigen. The volume of EIA grade water to be validated and correctly optimized using the kit controls and
used for its dissolution and to reach the appropriate p24 reference panels, before using the kit for routine laboratory
concentration (about 100 pg/ml) is written on the vial label. tests. Usually 4-5 washing cycles (aspiration + dispensation
To help dissolve the lyophilized pellet, vortex a few times, at of 350ul/well of washing solution = 1 cycle) are sufficient to
regular intervals. Complete dissolution should be achieved ensure that the assay performs as expected. A soaking time
within 2-5 minutes. of 20-30 seconds between cycles is suggested. In order to
Note: When dissolved the Calibrator is not stable. Store in set correctly their number, it is recommended to run an
aliquots at –20°C. assay with the kit controls and well characterized negative
and positive reference samples, and check to match the
Wash buffer concentrate: values reported below in the sections “Validation of Test”
The 20x concentrated solution has to be diluted with EIA grade and “Assay Performances”. Regular calibration of the
water up to 1200 ml and mixed gently end-over-end before use. volumes delivered by, and maintenance (decontamination
As some salt crystals may be present into the vial, take care to and cleaning of needles) of the washer has to be carried out
dissolve all the content when preparing the solution. according to the instructions of the manufacturer.
In the preparation avoid foaming as the presence of bubbles 4. Incubation times have a tolerance of +5%.
could give origin to a bad washing efficiency. 5. The ELISA microplate reader has to be equipped with a
Important Note: Once diluted, the wash solution is stable reading filter of 450nm and ideally with a second filter (620-
for 1 week at +2..8° C. 630nm) for blanking purposes. Its standard performances
should be (a) bandwidth < 10 nm; (b) absorbance range
Conjugate # 1: from 0 to > 2.0; (c) linearity to > 2.0; repeatability > 1%.
The Conjugate # 1 mix solution must be prepared immediately Blanking is carried out on the well identified in the section
before starting the test. Add 6 ml of Conjugate 1 diluent directly “Assay Procedure”. The optical system of the reader has to
to one Conjugate # 1 vial to dissolve the lyophilized powder. be calibrated regularly to ensure that the correct optical
This preparation (a total of 6 ml in one vial) is sufficient for 32 density is measured. It should be regularly maintained
tests, or 4 complete vertical strips of the microplate. To help according to the manufacturer ‘s instructions.
dissolve the lyophilized powder, vortex a few times, at regular 6. When using an ELISA automated work station, all critical
intervals. steps (dispensation, incubation, washing, reading, data
Important Note: Any unused portion of this reconstituted handling) have to be carefully set, calibrated, controlled and
Conjugate # 1 Solution may be stored at +2..8°C for no more regularly serviced in order to match the values reported in

4
Doc.: INS IVCOMB.CE/eng Page 5 of 11 Rev.: 3 Date: 09/2011

the sections “Internal Quality Control”. The assay protocol Before the next sample is aspirated, needles have to be duly
has to be installed in the operating system of the unit and washed to avoid any cross-contamination among samples or
validated as for the washer and the reader. In addition, the tips have to be changed.
liquid handling part of the station (dispensation and For the next operations follow the operative instructions reported
washing) has to be validated and correctly set. Particular below for the Manual Assay.
attention must be paid to avoid carry over by the needles It is strongly recommended to check that the time lap between
used for dispensing and for washing. This must be studied the dispensation of the first and the last sample will be
and controlled to minimize the possibility of contamination of calculated by the instrument and taken into consideration by
adjacent wells. The use of ELISA automated work stations delaying the first washing operation accordingly.
is recommended for blood screening when the number of In case the instrument Dia.Pro workstation is used, follow the
samples to be tested exceed 20-30 units per run. indications that the computer provides automatically to the user
7. When using the Dia.Pro workstation, follow carefully the of DiaPro’s kits when the proper assay protocol is launched.
instructions provided by the instruments itself, guiding the Anyway, the correct number of lyophilized Conjugate # 1 must
operator in all the operative steps. be dissolved each with 6 ml Conjugate # 1 Diluent. Once the
8. When using automatic devices, in case the vial holder of the lyophilized powders are dissolved and mixed well, they are to be
instrument does not fit with the vials supplied in the kit, mixed together into the gray plastic container labeled
transfer the solution into appropriate containers and label “Conjugate # 1 MIX”. Once the Conjugate # 1 MIX has been
them with the same label peeled out from the original vial. transferred to the container the assay may begin.
This operation is important in order to avoid mismatching
contents of vials, when transferring them. When the test is 2. Manual assay:
over, return the secondary labeled containers to 2..8°C, 1. Dissolve the right number of lyophilized Conjugate # 1 with
firmly capped. Conjugate # 1 Diluent before starting to dispense the
9. Dia.Pro’s customer service offers support to the user in the samples and controls of the test.
setting and checking of instruments used in combination 2. Place the required number of strips in the microwell holder.
with the kit, in order to assure compliance with the Leave the 1st well empty for the operation of blanking.
requirements described. Support is also provided for the 3. Dispense 50 ul Sample Diluent in all the wells, except A1
installation of new instruments to be used with the kit. used for blanking.
4. Dispense 150 ul of Negative Control in triplicate, 150 ul
HIV1 Positive Control, 150 ul HIV2 Positive Control and
L. PRE ASSAY CONTROLS AND OPERATIONS 150 ul of Calibrator Ag in duplicate in proper wells.
1. Check the expiration date of the kit printed on the external 5. Dispense 150 ul of Sample in each properly identified well.
label of the kit box. Do not use if expired. Mix gently the plate on the work surface, avoiding
2. Check that the liquid components are not contaminated by overflowing and contaminating adjacent wells, in order to
naked-eye visible particles or aggregates. Check that the fully disperse the sample into the diluent.
Chromogen/Substrate is colorless or pale blue by aspirating 6. Incubate the microplate for 60 min at +37°C .
a small volume of it with a sterile transparent plastic pipette.
Check that no breakage occurred in transportation and no Important note: Strips have to be sealed with the adhesive
spillage of liquid is present inside the box. Check that the sealing foil, supplied, only when the test is carried out manually.
aluminum pouch, containing the microplate, is not punctured Do not cover strips when using ELISA automatic instruments.
or damaged.
3. Dilute all the content of the 20x concentrated Wash Solution 7. Wash the microplate with an automatic washer by
as described above. delivering and aspirating 350ul/well of diluted washing
4. Dissolve the Calibrator Ag. solution as reported previously (section I.3).
5. Dissolve the Conjugate # 1 vial containing lyophilzed 8. Pipette 150 ul Conjugate # 1 mix, prepared as described
powder with the Conjugate 1 Diluent (1 lyophilized before, into each well, except the 1st blanking well, and
Conjugate # 1 + 6ml Conjugate # 1 Diluent) to obtain the cover with the sealer.
Conjugate # 1 Mix as described in the proper section.
6. Allow all the other components to reach room temperature Important note: Be careful not to touch the plastic inner
(about 1 hr) and then mix as described. surface of the well with the tip filled with the Conjugate.
7. Set the ELISA incubator at +37°C and prepare the ELISA Contamination might occur.
washer by priming with the diluted washing solution,
according to the manufacturers instructions. Set the right 9. Incubate the microplate for 30 min at +37°C .
number of washing cycles as found in the validation of the 10. Pipette 100 ul of Conjugate # 2 in all the wells, except A1,
instrument for its use with the kit. and gently agitate the microplate to mix the two
8. Check that the ELISA reader has been turned on at least 20 conjugates.
minutes before reading.
9. If using an automated workstation, turn it on, check settings Important Note: This solution must be added to the bottom of
and be sure to use the right assay protocol. each well to ensure proper performance. Inadequate mixing of
10. Check that the micropipettes are set to the required volume. the two solutions (Conjugate 1 and Conjugate 2) may reduce
11. Check that all the other equipment is available and ready the binding of streptavidin HRP (Conjugate 2) to the biotinylated
to use. reagents and consequently affect the performance of the assay.
12. In case of problems, do not proceed further with the test and Be sure to provide an adequate mixing when the Conjugate # 2
advise the supervisor. is added, both in the manual and in the automated procedures.

M. ASSAY PROCEDURE 11. Incubate the microplate sealed for 30 min at +37°C .
The assay has to be carried out according to what reported 12. Wash as in section 7.
below, taking care to maintain the same incubation time for all 13. Dispense 200 ul of Chromogen/Substrate mixture into each
the samples in testing. well, the blank well included. Then incubate the microplate
at room temperature (18-25°C) for 30 minutes . Start
1. Automated assay: the timing immediately after addition of this component to
In case the test is carried out automatically with an ELISA the first well.
system, we suggest to make the instrument dispense 50 ul
Sample Diluent first and then 150 ul controls and samples.

5
Doc.: INS IVCOMB.CE/eng Page 6 of 11 Rev.: 3 Date: 09/2011

Important note: Do not expose to strong direct illumination. Check Requirements

High background might be generated. Blank well < 0.100 OD450nm value
Negative Control < 0.200 mean OD450nm value after blanking
14. Pipette 100 ul Sulphuric Acid into all the wells using the (NC) Absorbance of individual negative control
same pipetting sequence as in step 13 to stop the values must be less than or equal to 0.200. If
one value is outside this range, discard this
enzymatic reaction. Addition of acid will turn the positive value and recalculate mean. If two values are
controls and positive samples from blue to yellow. outside this range the run should be repeated.
15. Measure the color intensity of the solution in each well, as HIV-1 Ab Mean OD450nm > 0.700.
described in section I.5, at 450nm filter (reading) and Positive Control
possibly at 620-630nm (background subtraction), blanking HIV-2 Ab Mean OD450nm > 0.700.
the instrument on A1. Positive Control
HIV Ag Calibrator S/Co > 1
Important notes:
1. If the second filter is not available ensure that no finger
prints are present on the bottom of the microwell before If the results of the test match the requirements stated above,
reading at 450nm. Finger prints could generate false proceed to the next section.
positive results on reading. If they do not, do not proceed any further and operate as
2. Reading has to be carried out just after the addition of the follows:
Stop Solution and anyway not any longer than 30 minutes
after its addition. Some self oxidation of the chromogen can Problem Check
occur leading to high background. Blank well 1. that the Chromogen/Sustrate solution has not
> 0.100 OD450nm got contaminated during the assay
Negative Control 1. that the washing procedure and the washer
(NC) settings are as validated in the pre qualification
> 0.200 OD450nm study;
N. ASSAY SCHEME after blanking 2. that the proper washing solution has been
used and the washer has been primed with it
Method Operations before use;
3. that no mistake has been done in the assay
Sample Diluent 50 ul procedure (dispensation of positive control
Controls and calibrator 150 ul instead of negative control;
Samples 150 ul 4. that no contamination of the negative control
1st incubation 60 min or of their wells has occurred due to positive
Temperature +37°C samples, to spills or to the enzyme conjugate;
5. that micropipettes haven’t got contaminated
Wash step 4-5 cycles
with positive samples or with the enzyme
Conjugate # 1 150 ul conjugate
2nd incubation 30 min 6. that the washer needles are not blocked or
Temperature +37°C partially obstructed.
Conjugate # 2 100 ul Positive Controls 1. that the procedure has been correctly
3rd incubation 30 min < 0.700 OD450nm executed;
2. that no mistake has been done in the
Temperature +37°C distribution of controls (dispensation of negative
Wash step 4-5 cycles control instead of positive control. In this case,
TMB/H2O2 200 ul the negative control will have an OD450nm
4th incubation 30 min value > 0.200, too.
3. that the washing procedure and the washer
Temperature r.t.
settings are as validated in the pre qualification
Sulphuric Acid 100 ul study;
Reading OD 450nm 4. that no external contamination of the positive
control has occurred.
HIV Ag Calibrator 1. that the procedure has been correctly
An example of dispensation scheme is reported below: S/Co < 1 executed;
2. that no mistake has been done in the
distribution of controls (dispensation of negative
Microplate control instead of Calibrator Ag. In this case,
1 2 3 4 5 6 7 8 9 10 11 12 the negative control will have an OD450nm
A BLK CAL Ag value > 0.200, too.
B NC CAL Ag 3. that the washing procedure and the washer
C NC S1 settings are as validated in the pre qualification
D NC S2 study;
E POS 1 Ab S3 4. that no external contamination of the positive
F POS 1 Ab S4 control has occurred.
G POS 2 Ab S5 5.that the lyophilize powder was dissolved
H POS 2 Ab S6 correctly with the correct volume of water
written on the vial label.
Legenda: BLK = Blank NC = Negative Control POS 1 Ab = HIV -1 Ab Positive
Control, POS 2 Ab = HIV -2 Ab Positive, CAL Ag = HIV p24 Ag Calibrator, S = Should these problems happen, after checking, report any
Sample residual problem to the supervisor for further actions.

O. INTERNAL QUALITY CONTROL

A check is carried out on the controls and the calibrator any time
the kit is used in order to verify whether their OD450nm values P. CALCULATION OF THE CUT-OFF
are as expected and reported in the table below. The tests results are calculated by means of a cut-off value
determined with the following formula on the mean OD450nm
value of the Negative Control (NC):

NC + 0.125 = Cut-Off (Co)

6
Doc.: INS IVCOMB.CE/eng Page 7 of 11 Rev.: 3 Date: 09/2011

The performance evaluation was carried out both in an external

The value found for the test is used for the interpretation of centre of excellence for HIV diagnosis, that examined the device
results as described in the next paragraph. on a population of antibody positive and negative samples
against a CE-marked kit used in the laboratory as reference,
Important note: When the calculation of results is done by the and in DiaPro’s laboratories as well to complete the study.
operative system of an ELISA automated work station be sure
that the proper formulation is used to calculate the cut-off value R.1 ANALYTICAL SENSITIVITY
and generate the right interpretations of results. The limit of detection (or analytical sensitivity) of the assay has
been calculated by means of preparations specific for HIV-1 and
HIV-2 antibody and HIV-1 p24 Ag detection, supplied by NIBSC
Q. INTERPRETATION OF RESULTS Blanche Lane South Mimms Potters Bar Hertfordshire EN6
Test results are interpreted as ratio of the sample OD450nm 3QG, UK.
and the Cut-Off value (or S/Co) according to the following table: Samples were diluted in HIV Ab&Ag negative plasma to
generate limiting dilution curves and examined in duplicate.
The tables below reports the mean OD450nm values and the
S/Co Interpretation S/Co index:
<1 Negative
NIBSC anti-HIV 2 monitor sample
>1 Positive code 99/674 – 005
Sample Lot # 0506 Lot # 0706 Lot # 0906
A negative result indicates that the patient has not been infected Dilution OD450nm S/Co OD450nm S/Co OD450nm S/Co
by HIV. 1x over >14.5 over >15.0 over >15.1
If the initial absorbance value is equal to or greater than the cut- 2x 3.838 14.1 3.765 14.5 3.774 14.4
off value, retest the sample in duplicate. If both retest values are 4x 2.371 8.7 2.268 8.7 2.319 8.9
less than the cut-off, the interpretation is not reactive for HIV 8x 1.253 4.6 1.097 4.2 1.140 4.4
antibody and/or antigen (negative). 16x 0.700 2.6 0.712 2.7 0.735 2.8
If one or both retest values are equal to or greater than the cut- 32x 0.462 1.7 0.439 1.7 0.483 1.8
off the interpretation of the test results is repeatedly reactive. 64x 0.281 1.0 0.260 1.0 0.294 1.1
The sample should be considered reactive or positive for HIV 128x 0.189 0.7 0.171 0.7 0.174 0.7
diluent 0.140 0.5 0.122 0.5 0.131 0.5
antibody and/or antigen according to the criteria of this HIV
ELISA test.
A positive result is indicative of HIV infection and therefore the The device shows a limiting dilution value at 64x.
patient should be treated accordingly.
NIBSC British working standard for anti HIV 1
Important notes: code 99/750 –007
1. Interpretation of results should be done under the Sample Lot # 0506 Lot # 0706 Lot # 0906
supervision of the responsible of the laboratory to reduce Dilution OD450nm S/Co OD450nm S/Co OD450nm S/Co
the risk of judgment errors and misinterpretations. 1x 2.206 8.1 2.086 8.0 2.142 8.2
2x 0.999 3.7 0.925 3.6 1.027 3.9
2. Rrepeatedly reactive specimens should be submitted to a
4x 0.475 1.7 0.486 1.9 0.486 1.9
Confirmation Assay before diagnosis of HIV infection is 8x 0.295 1.1 0.301 1.2 0.289 1.1
released. 16x 0.212 0.8 0.206 0.8 0.220 0.8
3. When test results are transmitted from the laboratory to an diluent 0.140 0.5 0.122 0.5 0.131 0.5
informatics centre, attention has to be done to avoid
erroneous data transfer. The devise shows a limiting dilution value at 8x.
4. Diagnosis of HIV infection has to be done and released to
the patient only by a qualified medical doctor. NIBSC British working standard for anti HIV 1
code 99/710 – 007
An example of calculation is reported below: Sample Lot # 0506 Lot # 0706 Lot # 0906
Dilution OD450nm S/Co OD450nm S/Co OD450nm S/Co
The following data must not be used instead or real figures obtained by 1x 0.588 2.2 0.611 2.4 0.607 2.3
the user. 2x 0.301 1.1 0.309 1.2 0.312 1.2
4x 0.198 0.7 0.210 0.8 0.192 0.7
Negative Control: 0.110 – 0.120 - 0.115
diluent 0.140 0.5 0.122 0.5 0.131 0.5
OD450nm Mean Value: 0.115 OD450nm
Lower than 0.200 – Accepted
The devise shows a limiting dilution value at about 2x.
HIV 1 Ab Positive Control: 2.000 OD450nm mean value
Higher than 0.700 – Accepted NIBSC HIV-1 p24 Antigen Monitor Sample
code 02/146-002
HIV 2 Ab Positive Control: 2.100 OD450nm mean value Sample Lot # 0506 Lot # 0706 Lot # 0906
Higher than 0.700 – Accepted Dilution OD450nm S/Co OD450nm S/Co OD450nm S/Co
1x 3.664 13.4 3.650 14.0 3.552 13.5
Calibrator Ag: 0.322 OD450nm mean value
2x 2.151 7.9 2.133 8.2 2.086 8.0
S/Co > 1 - Accepted
4x 1.209 4.4 1.178 4.5 1.214 4.6
Cut-Off = 0.115 +0.125 = 0.240 8x 0.734 2.7 0.729 2.8 0.780 3.0
Sample 1: 0.070 OD450nm 16x 0.388 1.4 0.342 1.3 0.351 1.3
Sample 2: 1.690 OD450nm 32x 0.259 0.9 0.236 0.9 0.229 0.9
Sample 1 S/Co < 1 = negative diluent 0.140 0.5 0.122 0.5 0.131 0.5
Sample 2 S/Co > 1 = positive
The devise shows a limiting dilution value at about 16x.

R. PERFORMANCES
Evaluation of Performances has been conducted in accordance
to what reported in the Common Technical Specifications or
CTS (art. 5, Chapter 3 of IVD Directive 98/79/EC).

7
Doc.: INS IVCOMB.CE/eng Page 8 of 11 Rev.: 3 Date: 09/2011

NIBSC 1st International reference Reagent for HIV 1 Ag BBI Anti-HIV 1 Low Titer
code 90/636 – (Version 4, 12 May 2009) Performance Panel - PRB 107
Sample Lot # Lot # Lot # (modified version)
IU/ml OD450nm S/Co OD450nm S/Co OD450nm S/Co Member IVCOMB.CE Ref. Kit
14.8 14.8 14.5 ID # S/Co S/Co
16 2.053 2.048 2.053
8.0 8.1 8.0 2 5.6 3.7
8 1.118 1.121 1.124
4 0.571 4.1 0.574 4.1 0.576 4.1 3 2.1 4.5
2 0.290 2.1 0.291 2.1 0.290 2.0 5 0.5 0.1
1 0.160 1.2 0.162 1.2 0.160 1.1 6 4.4 7.3
0.5 0.104 0.7 0.105 0.8 0.103 0.7 7 1.4 1.0
diluent 0.014 // 0.014 // 0.014 // 8 6.3 7.1
10 1.8 3.5
12 5.9 5.0
The devise shows a sensitivity < 2 IU/ml as required by 13 7.1 3.3
CTS:2009. 15 3.2 2.7

R.2 DIAGNOSTIC SPECIFICITY AND SENSITIVITY

R.2.1 Diagnostic Specificity:

BBI anti HIV-1 Low Titer
The diagnostic specificity of the device was assessed on a Performance Panel - PRB 106
population of more than 5000 unselected blood donors. Member IVCOMB.CE Ref. Kit
In addition, a population of more than 200 hospitalized patients, ID # S/Co S/Co
under examination for non HIV pathologies, was tested as well. 1 4.5 7.5
Moreover, the diagnostic specificity was assessed by testing 2 2.6 1.8
more than 100 potentially interfering specimens (other infectious 3 5.2 2.0
diseases, E.coli antibody positive, patients affected by non viral 4 6.1 8.9
hepatic diseases, dialysis patients, pregnant women, hemolized, 5 11.4 17.6
lipemic, etc.). 6 0.5 0.1
No false reactivity due to the method of specimen preparation 7 3.9 7.9
has been observed. Both plasma, derived with different 8 13.2 15.7
9 >14.5 16.6
standard techniques of preparation (citrate, EDTA and heparin),
10 6.1 12.7
and sera have been used to determine the value of specificity. 11 4.8 6.8
Frozen specimens have been tested, as well, to check for 12 3.0 2.4
interferences due to collection and storage. 13 5.9 10.5
No interference was observed. 14 7.7 6.0
15 >14.5 5.5
R.2.2 Diagnostic Sensitivity
The diagnostic sensitivity of the test was determined on a
population of HIV positive specimens.
Results are reported in the tables below.

Etablissement Francais du Sang

BBI Anti-HIV 1 Mixed Titer
Mixed titer Performance
Performance Panel - PRB 204
Panel HIV Ab (1-6) Lot # 11
Member IVCOMB.CE Ref. Kit
ID Composition Lot # 0506 Lot # 0706 Lot # 0906 S/Co
ID # S/Co S/Co
S/Co S/Co S/Co mean
1 1.2 1.0
1 HIV2(1/200) >14.5 >15.0 >15.1 >14.9
2 >14.5 65.2
2 HIV2(1/800) 10.0 10.2 10.2 10.1
3 0.3 0.2
3 negative 0.4 0.4 0.4 0.4
4 >14.5 65.1
4 HIV1(1/700) 8.9 9.0 9.1 9.0
5 >14.5 66.1
5 HIV1(1/160) >14.5 >15.0 >15.1 >14.9
6 >14.5 67.5
6 HIV1(1/200) 1.9 1.9 1.8 1.9
7 >14.5 66.0
8 >14.5 64.6
9 1.3 2.4
BBI Anti-HIV 1 Low Titer
Performance Panel - PRB 108 10 >14.5 65.1
Member IVCOMB.CE IVAB.CE 11 >14.5 65.4
ID # S/Co S/Co 12 >14.5 63.8
1 3.5 3.1 13 4.7 67.0
2 0.5 0.2 14 >14.5 63.8
3 2.3 2.5 15 >14.5 63.6
4 11.4 10.6 16 >14.5 66.6
5 3.8 2.9 17 >14.5 68.8
6 6.9 5.3 18 >14.5 71.2
7 4.0 3.7 19 >14.5 71.5
8 7.5 6.3 20 >14.5 68.5
9 6.3 3.2 21 >14.5 71.7
10 2.4 1.5 22 >14.5 68.5
11 8.8 7.2 23 0.3 0.2
12 4.1 2.9 24 2.3 2.3
13 3.7 2.3 25 0.8 0.9
14 7.2 6.1
15 10.9 6.3

8
Doc.: INS IVCOMB.CE/eng Page 9 of 11 Rev.: 3 Date: 09/2011

BBI anti HIV 1/2 Combo BBI HIV p24 Antigen Mixed Titer
Performance Panel - PRZ 206 Performance Panel - PRA 203
Member IVCOMB.CE Ref. Kit Member IVCOMB.CE Ref. Kit
ID # S/Co S/Co ID # S/Co S/Co
1 10.2 >15.7 1 2.3 4.3
2 0.4 0.2 2 4.1 12.2
3 >14.5 >15.7 3 1.4 6.0
4 >14.5 >15.7 4 1.9 3.0
5 6.6 9.2 5 Nd Nd
6 >14.5 9.1 6 1.0 1.3
7 0.4 0.2 7 2.9 5.7
8 >14.5 >15.7 8 3.0 1.5
9 11.4 15.5 9 0.4 0.5
10 >14.5 >15.7 10 3.6 12.2
11 >14.5 >15.7 11 2.4 3.1
12 >14.5 12.8 12 1.6 3.5
13 >14.5 15.7 13 1.2 2.1
14 0.4 0.9
15 3.3 2.5
16 1.1 1.8
BBI HIV-1 Incidence/Prevalence 17 2.5 6.3
Performance Panel - PRB 601 18 1.7 2.4
Member IVCOMB.CE Ref. Kit 19 0.4 0.5
ID # S/Co S/Co 20 4.6 17.0
1 >14.5 18.5
2 >14.5 17.4 Moreover, in the external Performance Evaluation a total of 651
3 >14.5 17.4
positive samples, including HIV type 1, HIV type 2, HIV type 1
4 >14.5 17.4
mixed subtypes (including 0), HIV 1 Antigen, more than 40 early
5 >14.5 15.8
6 >14.5 17.4
seroconversion HIV samples and cell culture supernatants were
7 >14.5 18.5 evaluated and a value of 100% was found.
8 >14.5 17.4
9 >14.5 17.4 Finally, more than 30 panels of seroconversion containing
10 >14.5 17.4 samples of HIV 1/2/0 Antibodies and/or HIV-1 p24 Antigen
11 >14.5 18.5 positive, obtained from BBI, USA, were evaluated using
12 >14.5 17.4 IVCOMB.CE lot # 0506. In the table below results are reported.
13 >14.5 17.4
14 >14.5 17.4 Seroconversion IVCOMB.CE IVAB.CE
15 >14.5 17.4 Panel 4th Generation 3rd Generation
HIV Ab&Ag HIV Ab
ID First specimen detected positive in the
panel
BBI Worldwide HIV PRB 910 (J) 2 3
Performance Panel - WWRB 303 PRB 916 (P) 4 5
Member IVCOMB.CE Ref. Kit PRB 917 (Q) 2 5
ID # S/Co S/Co PRB 919 (S) 1 2
1 >14.5 >15.9 PRB 922 (V) 1 1
2 >14.5 >15.9 PRB 924 (X) 5 6
3 >14.5 >15.9 PRB 926 (Z) 3 5
4 >14.5 >15.9 PRB 927 (AB) 2 3
5 4.1 >15.9 PRB 928 (AC) 2 3
6 >14.5 >15.9 PRB 929 (AD) 4 6
7 >14.5 >15.9 PRB 930 (AE) 2 3
8 >14.5 >15.9 PRB 931 (AF) 6 6
9 1.1 2.4 PRB 933 (AH) 2 2
10 >14.5 >15.9 PRB 935 (AJ) 6 7
11 11.3 0.3 PRB 937 (AL) 4 not detected
12 0.3 0.4 PRB 938 (AM) 1 3
13 >14.5 >15.9 PRB 939 (AN) 7 9
14 >14.5 >15.9 PRB 940 (AP) 2 4
15 >14.5 >15.9 PRB 941 (AQ) 4 5
PRB 942 (AR) 4 not detected
PRB 944 (AT) 3 5
PRB 946 (AV) 3 not detected
PRB 947 (AW) 2 4
Etablissement Francais du Sang PRB 948 (AX) 4 not detected
Performance Panel
PRB 949 (AY) 4 5
HIV Ag (3015-3022) lot 2004
PRB 950 (AZ) 3 4
Sample IVCOMB.CE Concentration HIV 1
PRB 952 (BB) 3 5
Lot # 0506 p24 Ag
S/Co [pg/ml] PRB 953 (BC) 3 4
8.7 PRB 955 (BE) 3 4
3015 500
4.2 PRB 956 (BF) 5 not detected
3016 250
3017 1.8 100
3018 1.2 50 The device shows a better sensitivity than the previous
3019 0.9 25 generation as it is able to detect the p24 antigen.
3020 0.6 10
3021 0.6 5
3022(diluent) 0.5 diluent

9
Doc.: INS IVCOMB.CE/eng Page 10 of 11 Rev.: 3 Date: 09/2011

The results of the Performance Evaluation, correlate perfectly BIBLIOGRAPHY

with what stated by EU CTS and are summarized in the table 1. Alizon, M., Sonigo, P., Barré-Sinoussi, F., Chermann, J.-C.,
here below: Tiollais, P., Montagnier, L. and Wain-Hobson, S., 1984.
Molecular Cloning of Lymphadenopathy-Associated Virus.
Nature 312: 757-760.
Diagnostic Sensitivity 100 % 2. Hahn, B.N., Shaw, G.M., Arya, S.K., Popovic, M., Gallo, R.C.
Diagnostic Specificity > 99.5 % and Wong-Staal, F., 1984. Molecular Cloning and
Characterization of the HTLV-III Virus Associated with AIDS.
Nature 312: 166-169.
3. Luciw, P.A., Potter, S.J., Steimer, K., Dina, D. and Levy, J.A.,
1984. Molecular Cloning of AIDS-Associated Retrovirus.
R.3 PRECISION Nature 312: 760-763.
The precision of the device was assessed by determining its 4. Popovic, M., Sarngadharan, M.G., Read, E. and Gallo, R.C.,
values in a within and between runs. In the table below results 1984. Detection, Isolation, and Continuous Production of
are reported for a negative sample and a low positive sample. Cytopathic Retroviruses (HTLV-III) from Patients with AIDS and
Pre-AIDS. Science 224: 497-500.
5. Sarngadharan, M.G., Popovic, M., Bruch, L., Schüpbach, J. and
Average values Negative Low Gallo, R.C., 1984. Antibodies Reactive with Human
N = 48 Sample Positive T-Lymphotropic Retroviruses (HTLV-III) in the Serum of
OD450nm 0.136 0.916 Patients with AIDS. Science 224: 506-508.
Std.Deviation 0.011 0.022 6. Vézinet-Brun, F., Barré-Sinoussi, F., Saimot, A.G., Christol, D.,
Montagnier, L., Rouzioux, C., Klatzmann, D., Rozenbaum, W.,
CV % 7.6 4.0 Gluckmann, J.C. and Chermann, J.-C., 1984. Detection of IgG
Antibodies to Lymphadenopathy-Associated Virus in Patients
with AIDS or Lymphadenopathy Syndrome. Lancet:
1253-1256, June 9.
7. Spire, B., Montagnier, L., Barré-Sinoussi, F. and Chermann,
S. LIMITATIONS
J.-C., 1984. Inactivation of Lymphadenopathy Associated Virus
The user of this kit is advised to carefully read and understand this by Chemical Disinfectants. Lancet: 899-901, Oct. 20.
package insert. Strict adherence to the protocol is necessary in 8. Sehulster, L.M., Hollinger, F.B., Dreesman, G.R. and Melnick,
order to obtain reliable test results. In particular, accurate sample J.L., 1981. Immunological and Biophysical Alteration of
and reagent pipetting, along with careful washing and timing of Hepatitis B Virus Antigens by Sodium Hypochlorite Disinfection.
incubation steps is essential for accurate and reproducible App. and Envir. Microbiol. 42: 762-767.
detection of HIV-1 and HIV-2 antibodies and HIV-1 p24 antigen. 10. Barre-Sinoussi, F., Chermann, J.C., Rey, F., Nugeyre, M.T.,
Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Vezinet-
After the EIA test is performed, repeatedly reactive specimens
Brun, F., Rouzioux, C., Rozenbrum, W. et Montagnier, L. 1983.
should be submitted for additional testing using Western Blot (WB), Isolation of a T-lymphotropic retrovirus from a patient at risk for
Immunofluorescence Assay (IFA), Radioimmunoprecipitation acquired immune deficiency syndrome (AIDS). Science 220:
Assay (RIPA) tests and PCR for HIV nucleic acid. 868-871.
The determination that a person's serum contains antibodies or 11. Gallo, R.C., Salahuddin, S.Z., Popovic, M., Shearer, G.M.,
p24 antigen to HIV has extensive medical, social, psychological Kaplan, M., Haynes, B.F., Palker, T.J., Redfield, R., Oleske, J.,
and economic implications. Safai, B., White, G., Foster, P. and Markham, P.D. 1984.
Frequent detection and isolation of cytopathic retroviruses
It is recommended that confidentiality, appropriate counselling and
(HTLV-III) from patients with AIDS and at risk for AIDS.
medical evaluation be considered an essential aspect of the testing Science 224:500-503.
sequence. AIDS and AIDS-related conditions are clinical diseases 12. Gold, J., Dwyer, J. 1994. A short history of AIDS. Med. J. Aust.
and their diagnosis can only be established clinically. 160:251-252.
EIA testing alone cannot be used to diagnose AIDS. 13. Saville R. D., Constantine N. T., Cleghorn F. R. and Al. Fourth-
A non-reactive test result at any point in the testing sequence does Generation Enzyme-Linked Immunosorbent Assay for the
not preclude the possibility of exposure to or infection with HIV. simoultaneus Detection of Human Immunodeficiency Virus
Antigen and Antibody.J.of Clin Microbiology, July 2001, p.2518-
The risk of an asymptomatic person, who is repeatably reactive,
2524.
developing AIDS and/or AIDS-related conditions is not known. 14. Bernard Weber, El Hadij Mbargane Fall, Annemarie Berger,
Falsely reactive test results can be observed with a test kit of this Hans Wilhelm Doerr. Reduction of diagnostic Window by New
nature. The proportion of reactive samples will depend on the Fourth-Generation Human Immunodeficiency Virus Screening
sensitivity and specificity of the test kit and on the prevalence of Assays.J. of Clin. Microb. Aug. 1998, p. 2235-2239.
HIV-1 and HIV-2 antibodies in the population to be screened. 15. Clark J., Coates T. J., Lescano A. G., et Al. Different Positive
Antibodies to HIV may occur due to voluntary participation in an Predictive Values of commercially available Human
Immunodeficiency Virus Enzyme-Linked Immunosorbent
HIV vaccine study.
Assays. Clin. And Vacc. Immunology. Feb 2006 p.302-303.
Interpretation of this diagnostic test will depend on the type of 16. Novack L., Galai N., Yaari A., Orgel M., Shinar E., Sarov B. Use
vaccine given. Correlation with the medical history and additional of Seroconversion Panels to estimate Delay in the detection of
testing may be necessary to accurately diagnose HIV in vaccine Anti-Human Immunodeficiency Virus Antibodies by Enzyme-
volunteers. Linked Immunosorbent Assay of pooled Compared to Singleton
Serum Samples.J. of Clin Microbiol. Aug. 2006 p.2909-2913.
17. Apetrei C, Buzdugan I, Mitroi I, Duca M.The clinical and
immunological correlations between the p24 antigenemia
levels and those of anti-p24 antibodies in HIV-seropositive
childrenBacteriol Virusol Parazitol Epidemiol. 1995 Apr-
Jun;40(2):141-4. Romanian.
18. Shumai EP, Vorob'ev SM, Makarova NE, Tugizov ShM,
Zverev VV, Kushch AA.The immunoenzyme detection of the
HIV-1 antigen by using monoclonal antibodies to protein
p24Vopr Virusol. 1992 Sep-Dec;37(5-6):229-32. Russian.
19. d'Arminio Monforte A, Novati R, Marchisio P, Zanchetta N,
Uberti-Foppa C, Tornaghi R, Massironi E, Lazzarin A, Principi
N.Early diagnosis of HIV infection in infants.AIDS. 1989
Jun;3(6):391-5.
20. Borghi V, De Rienzo B, Pietrosemoli P, Pecorari M,
Mongiardo N, Pellegrino F, Zanchetta GP, Lami G, Squadrini
F.Detection of serum HIV-Ag related to the major core protein
(p24) in persons at risk for AIDS.
Microbiologica. 1989 Jan;12(1):81-3.

10
Doc.: INS IVCOMB.CE/eng Page 11 of 11 Rev.: 3 Date: 09/2011

21. Goudsmit J, Lange JM, Krone WJ, Teunissen MB, Epstein

LG, Danner SA, van den Berg H, Breederveld C, Smit L,
Bakker M, et al.Pathogenesis of HIV and its implications for
serodiagnosis and monitoring of antiviral therapy.J Virol
Methods. 1987 Aug;17(1-2):19-34.
22. Lyamuya E, Bredberg-Raden U, Massawe A, Urassa E, Kawo
G, Msemo G, Kazimoto T, Ostborn A, Karlsson K, Mhalu F,
Biberfeld G.Performance of a modified HIV-1 p24 antigen
assay for early diagnosis of HIV-1 infection in infants and
prediction of mother-to-infant transmission of HIV-1 in Dar es
Salaam, Tanzania.J Acquir Immune Defic Syndr Hum
Retrovirol. 1996 Aug 1;12(4):421-6.
23. Clavel, F., Mansinho, K., Chamaret, S. et al. 1987. Human
immunodeficiency virus type 2 infection associated with AIDS in
West Africa. N. Engl. J. Med. 316:1180-1185.
24. Sinicco, A., Fora, R., Sciandra, M., Lucchini, A., Caramello, P.
and Gioannini, P. 1993. Risk of developing AIDS after primary
acute HIV-1 infection. J. of Acquired Immune Deficiency
Syndromes 6:575-581.
25. Ju Lin, Hsiang. 1995. Laboratory tests for human
immunodeficiency viruses. J. Intl. Fed. Clin. Chem. 7(2):61-66.
26. IVD Directive 98/79/CE, Common Technical Specifications
(CTS) – Annex II, List A.

Produced by
Dia.Pro. Diagnostic Bioprobes Srl.
Via G. Carducci n° 27 – Sesto San Giovanni (MI) - Italy

|
0318

11