HIV Ag & Ab Card (4th Generation)

Rapid visual test for the qualitative detection of HIV-1 p24 Antigen and Antibodies (IgM, IgG & IgA)
to HIV-1& HIV-2 in Human Serum/ Plasma/ Whole Blood

INTRODUCTION Single use only Keep away from sunlight

First confirmed case of AIDS was identified in 1983 and by 1984 the etiologic agent, the Human
Keep Dry Country of Manufacture
Immunodeficiency Virus (HIV), subsequently named HIV-1 was isolated. Shortly afterwards in 1985 another
retrovirus subsequently named HIV-2 was isolated in Africa. These two viruses belong to the retrovirus group and
WARNING & PRECAUTIONS FOR USERS
are slow viruses. The structure, gene organization and serological behavior of HIV-1 & HIV-2 and their complete
nucleotide sequence has been determined. The serological events following HIV infection are represented CAUTION: ALL THE SAMPLES TO BE TESTED SHOULD BE HANDLED AS THOUGH CAPABLE OF
graphically in fig.1. During initial infection, the HIV-1 p24 antigen appears (after 2 weeks) in detectable levels, TRANSMITTING INFECTION. NO TEST METHOD CAN OFFER COMPLETE ASSURANCE THAT HUMAN
before anti-HIV (after 4 weeks) but becomes undetectable as the antibodies are formed and their level increases BLOOD PRODUCTS WILL NOT TRANSMIT INFECTION.
(seroconversion). 4th generation HIV immunoassays incorporate HIV p24 antigen along with HIV antibody 1. The use of disposable gloves and proper biohazardous clothing is STRONGLY RECOMMENDED while
detection which allows the identification of acute HIV infections by reducing the window period. running the test.
2. In case there is a cut or wound in hand, DO NOT PERFORM THE TEST.
3. Mark the test card with patient's name or identification number. Improper identification may lead to
wrong result reporting.
4. All materials used in the assay and samples should be decontaminated in 5% sodium hypochlorite
solution for 30-60 minutes before disposal or any other suitable disinfectant or by autoclaving at
121ºC at 15psi for 60 min. Do not autoclave materials or solution containing sodium hypochlorite.
They should be disposed off in accordance with established safety procedures and state guidelines.
5. Spills should be decontaminated promptly with Sodium Hypochlorite or any other suitable
disinfectant.
6. Assay Buffer contains Sodium Azide as a preservative. If these materials are to be disposed off
through a sink or other common plumbing systems, flush with generous amounts of water to prevent
accumulation of potentially explosive compounds.
7. Do not open the foil pouch until it attains room temperature.
8. Wash hands thoroughly with soap or any suitable detergent, after the use of the kit.
9. Do not perform the test in area with strong air flow i.e. under fan or strong air conditioning.
INTENDED USE (fig.1) 10. Bring kit to room temperature (20-30ºC) before use.
HIV Ag & Ab Card (4th Generation) is a visual, rapid qualitative and sensitive solid phase 11. Do not use kit components beyond the expiration date, which is printed on the kit.
immunochromatographic assay for the differential detection of HIV-1 p24 Antigen and Antibodies (IgM, IgG & 12. Use a separate Sample Dropper for each specimen.
IgA) to HIV-1& HIV-2 in Human Serum/ Plasma/ Whole blood (collected by venipuncture or Fingerprick). The 13. Do not re-use the test kit.
test is a screening test for p24 antigen (HIV-1) and HIV antibodies (anti-HIV-1 & anti-HIV-2) and is for in vitro 14. Do not use the test kit if the pouch is damaged or the seal is broken.
diagnostic use only. It is intended for screening of blood donors or other individuals at risk for HIV-1 & HIV-2 15. Do not mix or use the assay buffer of different kits/lot.
infection and for clinical diagnostic testing. It is a screening test for p24 antigen (HIV-1) and HIV antibodies 16. Do not smoke, drink or eat while handling specimen.
(anti-HIV-1 & anti-HIV-2). The test is for in vitro diagnostic use only and is intended for screening of blood SAMPLE / SPECIMEN COLLECTION AND STORAGE
donors or others individuals at risk for HIV-1 and/or HIV-2 infection and for clinical diagnostic testing. a) Collect the whole blood in a clean container without anticoagulants for serum or containing
PRINCIPLE (ANTIGEN-ANTIBODY REACTION) anticoagulants (EDTA, citrate or heparin) for whole blood/plasma samples by venipuncture. Fresh
HIV Ag & Ab (4th Generation) Test kit consists two devices; one device for detection of HIV-1 p24 antigen and samples are preferred for testing as they perform best when tested immediately after collection. If
second device for the detection of HIV-1 & HIV-2 antibodies in human serum/ plasma/whole blood. samples are not immediately tested, they should be stored at 2-8°C for not more than 3 days,
otherwise false / erroneous results may be obtained.
HIV Antigen device contains two lines; “C” (Control Line) & “Ag” (HIV-1 p24 Antigen detection Test Line). Test
b) Hemolyzed or clotted blood sample or sample with microbial contamination should not be used.
line is coated with anti-p24 antibodies. When a sample is added to the HIV Antigen device followed by the
c) Serum/ plasma specimens, if not tested immediately, should be refrigerated at 2-8°C. For storage for
addition of assay buffer, HIV-1 p24 antigen if present in the sample will bind to the anti-p24 gold colloid
more than 3 days, freeze the specimen at -20°C or below.
conjugate making antigen antibodies complex. This complex migrates along the membrane to the test region
d) Repeated freezing and thawing of the specimen should be avoided.
and forms the visible purple line at “Ag” region as antibody-antigen-antibody gold conjugate complex.
e) Specimens containing precipitate or particulate matter may yield inconsistent test results. Such
HIV Antibody test device contains two lines; “C” (Control line), & “Ab” (HIV-1 & HIV-2 Antibody detection Test specimens must be centrifuged at 10,000 RPM for 15 minutes and the clear supernatant should only
Line). The test line is coated with HIV-1 & HIV-2 Antigens for the detection of HIV Antibodies. When a sample is be used for testing.
added to the HIV Antibody device followed by the addition of assay buffer, anti-HIV-1 and/or anti-HIV-2 f) The use of hemolytic, lipaemic, icteric or bacterially contaminated specimens should be avoided as it
antibodies if present in the sample, bind to the respective HIV-1 and/or HIV-2 antigen gold colloid conjugate may lead to erroneous results.
making antigen-antibodies complexes. These complexes migrate along the membrane to the test region and
form the visible purple line at “Ab” region as antigen-antibody-antigen gold colloid conjugate complex. BEFORE YOU START
The intensity of the test bands in the respective device will vary depending upon the amount of 1. Bring the required number of HIV Ag & Ab card (4th Generation) test device foil pouches and specimen
antigen/antibody present in the sample. The appearance of any pink/purple color line in a specific test region to room temperature prior to testing.
should be considered as reactive for that particular antigen and/or antibody. A red procedural control line 2. The Assay Buffer Solution provided in the kit has closed nozzle and screw cap with pin (outside).
should always develop in the test device window to indicate that the test has been performed properly. Before using Assay Buffer, keep the vial vertically straight and tap down gently on the working
platform, so that Assay Buffer comes down at the bottom of the vial and orifice the closed nozzle, as
KIT PRESENTATION & MATERIALS PROVIDED illustrated below in Fig. iii & iv, before use:
HIV Ag & Ab Card (4th Generation) kit contains following components:
1. HIV Ag & Ab Card 50 nos.
2. Assay Buffer 2 vials (25 Tests each)
3. Sample Dropper 1 pack of 50 nos.
4. Instruction Manual 1 No.
Component available on request: Negative Control, HIV Antibody Positive Control and HIV Antigen
Positive Control.
Optional material required, but not provided: Swab and Sterile Lancet. Fig. iii Fig. iv Fig. v
Fig. i invert the cap & give a half screw the cap
KIT PRESENTATION
close vial provided Fig. ii press on the closed turn twist to tightly after
25 Test Pack 50 Test Pack 100 Test Pack open the cap puncture the nozzle
in the kit nozzle use
STORAGE AND STABILITY TEST PROCEDURE
HIV Ag & Ab Card (4th Generation) should be stored at 2-30ºC in the cool & dry area available. Expiry date on 1) Bring the complete kit and specimen to be tested to room temperature prior to testing.
the kit indicates the date beyond which the kit should not be used. The kit should not be frozen & must be 20-30ºC
protected from exposure to humidity.
2) Remove the test card from the foil pouch prior to use and place it on a flat and dry
DESCRIPTION OF SYMBOLS USED
surface. The test should be performed immediately after removing the
The following are graphical symbols used in or found on J. Mitra diagnostic products and packing. These IGE
N
test card from the foil pouch. Do not use the card if desicant is HIV
ANT
symbols are the most common ones appearing on medical devices and their packing. They are explained in Ag
pink in color. C

more detail in the European Standard EN ISO15223-1:2021. ANT

IBO
DY
HIV
3) Label the test card with patient's name or identification number. Ab
Manufactured By In vitro diagnostic medical device C
(fig. a)
No. of tests Consult Instructions for use 4) The test card contains 2 devices - one HIV antigen detection device
Lot Number Temperature and one HIV antibody detection device placed in a single tray.
Batch Number Limit
N
IGE
ANT
Manufacturing Date Caution, see instruction for use HIV
C
Ag

5) Add 1 drop (20 μl) of the sample (Serum / Plasma / Whole Blood) IBO
DY
Expiry Date Catalogue Number HIV
ANT
using sample dropper / micropipette into the sample wells of both C
Ab

Do not use if package Contains biological Material the HIV Antigen and HIV Antibody device respectively. (fig. b)
is damaged of Animal Origin
 negative test result does not eliminate the possibility of HIV infection, and should be confirmed by
ELISA and/or RT-PCR.
OR
5. Positive test results do not rule out co-infections with other pathogens.
Whole blood sample collection from finger prick
PERFORMANCE CHARACTERISTICS OF HIV Ag & Ab CARD (4th GENERATION)
5a) Wipe the complete fingertip with the alcohol swab. Wait (fig. 5a) Analytical Sensitivity: The sensitivity of the HIV Ag & Ab CARD (4th Generation) has been determined for
until the finger has completely dried (minimum 30
HIV-1 p24 Antigen using WHO international standard: HIV-1 p24 antigen NIBSC Code No. 90/636 and it is
seconds).
equal to 100 IU/ml.
5b) Take the lancet and prick the side of the pulp (ball of the (fig. 5b) Sensitivity and Specificity studies were carried out on fresh as well as frozen samples from low risk as well as
finger) with the lancet, perpendicular to the lines of the fingerprint. high risk groups. Performance of the test with reference to sensitivity and specificity has been determined by
5c) Make sure a well formed drop of blood is present on the tip of the using clinical known negative and positive antibodies serum/plasma and whole blood samples and HIV p24
finger. Antigen control.
5d) Take the sample dropper/micropipette and collect 20 μl blood by The performance of the test was evaluated and compared with a licensed commercially available Elisa test.
dipping the tip of the sample dropper/ micropipette into the blood The results obtained are as follows:
drop. Add 1 drop of blood immediately in the sample well of the (fig. 5c)
HIV Antigen device. Sample Type No. of Samples Licensed Test HIV Ag & Ab Card
Tested (4th Generation)
5e) Repeat the above step (5d) for HIV Antibody device.
Caution: Care should be taken that the blood sample does not clot & the transfer to Negative 1200 1200 1199
the sample pad is immediate. Sample dropper should be discarded
immediately considering it to be biohazardous. HIV Positive 125 125 125
N
IGE
ANT
6) In case of blood samples, wait for 30 seconds (till the blood is HIV
Ag
Sensitivity: 100% Specificity: 99.91%
C
completely absorbed). IBO
DY
ANT
HIV Cross Reactivity: The cross reactivity of the kit has been tested using 60 samples from other diseases and
7) Add 1 drop Assay Buffer into the sample well of both device. C
Ab

(fig. c) results observed are as follows:

Addition of incorrect no. of drops of sample/Assay Buffer may
give invalid or false result. S. No. Sample Type No. of Samples HIV Ag and Ab Card (4th Generation)
8) Allow reaction to occur for 20 minutes. HIV Antigen card HIV Antibody Card
9) Read results at 20 minutes. Positive results may appear as early as 2-10 minutes. However negative
1 HCV 6 NEGATIVE NEGATIVE
results must be confirmed at 20 minutes. Do not read results after 30 minutes.
10) Discard the HIV Ag & Ab Card (4th Generation) immediately after reading results at 20 minutes 2 HBV 8 NEGATIVE NEGATIVE
considering it to be potentially infectious. Follow the local regulations for disposal of potential 3 Typhoid 6 NEGATIVE NEGATIVE
biohazardous material.
4 Syphilis 3 NEGATIVE NEGATIVE
INTERPRETATION OF THE RESULTS 5 Dengue NS1 Ag 4 NEGATIVE NEGATIVE
REACTIVE
HIV ANTIGEN REACTIVE: As shown in Fig. (d), appearance of purple colored HIV ANTIGEN 6 Dengue Antibody 4 NEGATIVE NEGATIVE
line in test region “Ag” and red line in control region “C” of the HIV Antigen C Ag
7 Scrub Typhus 2 NEGATIVE NEGATIVE
device and only one distinct red line in the control region “C” of the HIV HIV ANTIBODY

8 CRP 3 NEGATIVE NEGATIVE

Antibody device indicates that the sample is “REACTIVE” for HIV-1 p24 C Ab

Antigen. (fig. d) 9 Chikungunya 4 NEGATIVE NEGATIVE

HIV ANTIBODY REACTIVE: As shown in Fig. (e), appearance of only one HIV ANTIGEN
10 TB 3 NEGATIVE NEGATIVE
distinct red line in the control region “C” of the HIV Antigen device and purple C Ag
11 Leptospira 4 NEGATIVE NEGATIVE
colored line in test region “Ab” and red line in control region “C” of the HIV HIV ANTIBODY

12 Malaria P. falciparum 2 NEGATIVE NEGATIVE

Antibody device indicates that the sample is “REACTIVE” for HIV-1 and/or C Ab

HIV-2 Antibodies. (fig. e) 13 Malaria P. vivax 2 NEGATIVE NEGATIVE

HIV ANTIGEN & ANTIBODY REACTIVE As shown in Fig. (f), appearance of HIV ANTIGEN
14 HAMA 4 NEGATIVE NEGATIVE
purple colored line in test region “Ag” and red line in control region “C” of the C Ag
15 RA 2 NEGATIVE NEGATIVE
HIV Antigen device and purple colored line in test region “Ab” and red line in HIV ANTIBODY

control region “C” of the HIV Antibody device indicates that the sample is C Ab 16 ASO 3 NEGATIVE NEGATIVE
“REACTIVE” for both HIV-1 p24 Antigen and HIV-1 and/or HIV-2 Antibodies. (fig. f) External Evaluation: The performance of HIV Ag & Ab Card has been evaluated by National Institute of
NON-REACTIVE HIV ANTIGEN Biologicals, India. The result obtained of 3 different lots are as follows:
As shown in Fig. (g), appearance of only one distinct red line in the control C Ag
Sensitivity: 100% Specificity: 100%
region “C” of both the HIV Antigen and HIV Antibody device indicates that the HIV ANTIBODY

sample is “NON REACTIVE” for both HIV-1 p24 Antigen and HIV-1 and/or C Ab
Precision: Within run (Intra assay) & between run (Interassay) precision have been determined by testing 10
HIV-2 Antibodies. replicates of ten samples - four negative, one HIV antigen (p24) positive, one HIV-2 positive and four HIV-1
(fig. g)
Positive. The C.V. (%) of all the ten samples were within 10%.
INVALID
The test is invalid, if no control line appears HIV ANTIGEN HIV ANTIGEN LIMITED EXPRESSED WARRANTY DISCLAIMER
(with or without the appearance of test lines) C Ag C Ag
The manufacturer limits the warranty to the test kit, as much as that the test kit will function as an in vitro
diagnostic assay within the limitations and specifications as described in the product instruction-manual,
after the completion of test, either with clear HIV ANTIBODY HIV ANTIBODY
when used strictly in accordance with the instructions contained therein. The manufacturer disclaims any
background or with complete pinkish/ C Ab C Ab
warranty expressed or implied including such expressed or implied warranty with respect to merchantability,
purplish background (fig. h to k). (fig. h) (fig. i) fitness for use or implied utility for any purpose. The manufacturer’s liability is limited to either replacement of
Repeat the test with a new card. Invalid test HIV ANTIGEN HIV ANTIGEN
the product or refund of the purchase price of the product and in no case liable to claim of any kind for an
may be because of following reasons: C Ag C Ag amount greater than the purchase price of the goods in respect of which damages are likely to be claimed. The
(a) Improper storage at temperature other HIV ANTIBODY HIV ANTIBODY manufacturer shall not be liable to the purchaser or third parties for any injury, damage or economic loss,
than the recommended temperature. C Ab C Ab
howsoever caused by the product in the use or in the application there of.
(b) Wrong Procedure (fig. j) (fig. k) BIBLIOGRAPHY OF SUGGESTED READING
(c) Long atmospheric exposure of the test 1. Ajaka Z.L. et.al. (1994). HIV-1/HIV-2 seronegativity in HIV-1 subtype O infected patients. The Lancet,
device after opening the pouch. 343, 1393-1394.
(d) Use of turbid/ lipemic/ haemolyzed sample. 2. Granade, T. C., Nguyen, S., Kuehl, D. S., & Parekh, B. S. (2013). Development of a novel rapid HIV test
In case of invalid result, serum/ plasma test samples should be centrifuged at 10,000 rpm for for simultaneous detection of recent or long-term HIV type 1 infection using a single testing device.
15 minutes and retest using new Card. AIDS research and human retroviruses, 29(1), 61-67.
3. Nakatsuma, A., Kaneda, M., Kodama, H., Morikawa, M., Watabe, S., Nakaishi, K., ... & Ito, E. (2015).
LIMITATIONS AND INTERFERENCES
Detection of HIV-1 p24 at attomole level by ultrasensitive ELISA with thio-NAD cycling. PLoS One,
1. Any deviation from test procedure may lead to invalid/ erratic results.
10(6), e0131319.
2. Some samples show cross reactivity for HIV antibodies and/or p24 antigen. Following factors are 4. Parpia, Z. A., Elghanian, R., Nabatiyan, A., Hardie, D. R., & Kelso, D. M. (2010). p24 antigen rapid test
found to cause false positive HIV antigen and/or antibody test results: Naturally occurring antibodies, for diagnosis of acute pediatric HIV infection. JAIDS Journal of Acquired Immune Deficiency
Passive immunization, Leprosy, Renal Disorders, Mycobacterium avium, Herpes simplex, Syndromes, 55(4), 413-419.
Hypergammaglobulinemia, Malignant neoplasms, Tetanus vaccination, Autoimmune diseases,
VER-01 R-00

Blood Transfusion, Multiple myeloma, Haemophelia, Heat treated specimens, Lipemic serum, Anti-
nuclear antibodies, T-cell leukocyte antigen antibodies, Epstein Barr virus, HLA antibodies and other
retroviruses. in vitro diagnostic reagent, not for medicinal use
3. This is only a screening test. All samples detected reactive must be confirmed by using HIV Western
J. MITRA & CO. PVT. LTD.
Rev. Date: Jan.-24

Blot and/or PCR. Therefore for a definitive diagnosis, the patient's clinical history, symptomatology as
well as serological data, should be considered. The results should be reported only after complying A 180-181, Okhla Indl. Area, Phase-1, New Delhi-110 020, INDIA
MN/AAI/117

with above procedure. Ph: +91-11-47130300, 47130500

4. A negative result may occur if the concentration of antigen/antibody in a specimen is below the
e-mail: jmitra@jmitra.co.in Internet: www.jmitra.co.in
detection limit of the test or if the specimen was collected or transported improperly. Therefore a