Aidscan HIV-1／2 Trispot Test Kit 说明书

IVD

For Professional Use -

HIV 1/2 TRISPOT TEST

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A Rapid Trispot Test to detect of antibodies to HIV 1 & 2 in Human Serum or Plasma

AIDSCAN

CATALOGUE NO. : ATS

INTENDED USE :

AIDSCAN HIV-1/2 TRISPOT is an immuno concentration based assay for the detection of antibodies to HIV-1 & HIV-2 in Human Serum or Plasma.

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INTRODUCTION :

AIDSCAN HIV-1 / 2 TRISPOT T est is an immunoassay which employs r-proteins for the detection of antibodies to HIV in human serum or plasma. These proteins, which are corresponding to highly antigenic segments of both the structural and non-structural proteins of the HIV constitute the solid phase antigenic absorbent. The use of r-proteins offers the advantage of high degree of specificity and sensitivity due to multiple epitopes.The epidemiological evidence indicates that an infectious agent transmitted through

intimate contact, intravenous drug use or use of infected blood or blood products, leads to Acquried Immunodeficiency Syndrome (AIDS). This infection affects T-Cell mediated immunity, resulting in severe lymphoperia and a reduced sub-population of helper T-lymphocytes.

Destruction of this T-lymphocyte population by the virus cause an irreversible damage to immune system, resulting in a reduced or deficient response to subsequent infections and hence the patient becomes vulnerable (prone) to all kind of infections and shows bizzare symptoms hence this condition called as Syndrome. Consequently, infections become more severe and may cause dealth. At present there is no successful treatment for AIDS.

The etiological agent has been identified as a retrovirus, human immunodeficiency virus type 1 (HIV-1). A closely related, but distinct second type of immunodeficiency virus, designated as HIV-2, has been isolated and causes a disease that is indistinguishable from AIDS. The only difference is in the potentiality of infection. Seroiogical cross-reactitivity between HIV-1 and HIV-2 has been shown to be highly variable from sample to sample. This variability necessitates the inclusion of antigens to both HIV-1 and HIV-2 for the detection of HIV-1 and HIV-2.

The HIV genome has outer structural (env-gp120, gp41), inner structural (gag p17, p24, p7, p6), pol-viral enzymes (protease, reverse transcriptase, integrase) and regulatory proteins (T at, Rev, Vif, Vpu, Vpr, Nef) and long terminal repeats on either end (Fig. 1).

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Nef

1 Kb

L TR

L TR

Fig. 1 Structure of HIV genome

(encodes the viral Enzymes: PR - protease, RT = Reverse Transcriptase, IN = integrase)

Index:

II.I.

III.p17p24p7p6PR RT IN

gp 120

gp 41

(Inner structural proteins of the retro virion)

(outer envelop glycoproteins - associated with lipid bilayer)

Encodes also 6 small proteins unique to the virus. Tat & Rev - positive Regulato

ry protein Vif. Vpu Vpr. Nef - proteins with accessory function LTR - Long terminal repeat at each end. The left or 5’LTR containing the sign

als for transcriptioninitiation & the right or 3’ LTR contains the signals for transcription termination.Pol =gag =Env =IV .V .Fig. 2Level of different type of antibodies and antigens of HIV in blood

AIDSCAN HIV-1/2 TRISPOT Test utilizes a unique combination of HIV-1 & 2 antigens of the virus to selectively detect all subtypes of HIV-1 & 2 Virus in human serum/plasma with a high degree of sensitivity and specificity.

The level of different type of antibodies and antigens of HIV in blood is as shown in Fig. 2

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PRINCIPLE OF THE TEST

HIV-1 & HIV-2 antigens (HIV-1 & HIV-2) and a Control antigen ( C ) are

immobilized on a porous immunofiltration membrane. Sample and the reagent pass through the membrane and are absorbed into the underlying absorbent pad.

As the patient’s sample drains through the membrane, HIV antibodies if present in

serum / plasma, bind to the corresponding immobilized antigens. Unbound serum / plasma proteins are washed off in the subsequent washing step.Addition of the protein-A conjugate results in binding of HIV to give distinct Red

Spot near the test region (HIV-1 & HIV-2). At the control region (”C”) a “Built-in-Quality Control Spot” has been coated to confirm the proper functioning of the device, reagent and correct procedural application.

HIV-1

HIV-2

C

Fig. 3

WARNINGS

1.2.3.1.2.3.4.5.6.7.8.For in vitro diagnostic use only.

Wear disposable latex gloves while handling specimens and kit reagents.After the test, wash hands carefully.

Reagents have to be stored between +2 C and +8 C.Prewarm all reagents to 25+5 C before use.

The expiration date is printed on each component and on the package.Do not expose the conjugate to excessive light and high temperature.Once opened, the components must be closed tightly.

PACK SIZE : Available in packs of 10's, 20's, 50's and 100's.

KIT CONTENTS

MATERIALS REQUIRED BUT NOT PROVIDED

- Sodium hypochlorite solution (free available chlorine 50-500 mg/l).- Disposable latex gloves.

SPECIMEN COLLECTION AND HANDLING

DO NOT HEAT OR REPEATEDLY FREEZE/THAW SPECIMEN.Collect blood in a clean dry sterilized vial and allow it to clot. Separate

the serum by centrifugation at room temperature. It is recommended that FRESH samples should be used. If serum is not to be assay immediately it should be stored at 2-8 C or frozen at-20 C. Serum may

01.A. Frozen samples : (I) Allow the sample to thaw in a vertical position in the rack. Mix the sample thoroughly. If particles are seen, allow them to settle at the bottom or if a centrifuge is available, the sample can be centrifuged at 5,000 r.p.m. for 15 minutes.

(ii) Insert the dropper just below the top surface of the sample and withdraw two drops of the sample.

B. Thick or viscous samples : Whenever possible, clear specimen should be used. However, viscous, thick or turbid samples which may sometimes take more than 40-60 seconds to flow through the membrane should be centrifuged at 5,000 r.p.m. for 15 minutes and retested on a fresh device to avoid inconsistent results. AIDSCAN HIV-1/2 TRISPOT Test works best when used with fresh samples, however the frozen or viscous samples can also perform well if the following instructions are strictly adhered to :

2. Specimen Processing

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PRECAUTIONS : For in vitro diagnostic use only.

Pack Size 1.2.3.4.Test Device

Buffer Solution (Ready to Use)Gold Conjugate (Ready to use)Droppers 10 Test 10 Units 1 x 6.0 ml 1 x 1.5 ml 10 Units 20 Test 20 Units 1 x 12.0 ml 2 x 1.5 ml 20 Units 50 Test 50 Units 2 x15.0 ml 2 x 3.0 ml 50 Units 100 Test 100 Units 3 x17.5 ml 2 x 6.0 ml 100 Units

0All human serum and plasma samples should be considered as potentially infectious. It is recommended that all specimens of human origin should be handled as recommended for any potentially infectious human serum or blood specimen in the Centers for Disease control / national Institute of health manual “Biosafety in Microbiological and Biomedical Laboratories”, 1984.

1023

Never pipette by mouth.

Do not smoke, eat or drink in areas in which specimens or kit reagents are handled. Wear disposable latex gloves while handling specimens and kit reagents. Afterwards wash hands carefully.Avoid splashing or forming aerosols.

Discard all materials and specimens as if capable of transmitting infection The preferred method of disposal is autoclaving for a minimum of one hour at 121 C. Liquid wastes not containing acid may be mixed with sodium hypochlorite in volumes such that the final mixture contains 50-500 mg/l available chlorine. Allow 30 minutes for decontamination to be completed.

NOTE.: Liquid waste containing acid must be neutralized with a proportional amount of base prior to the addition of sodium hypochlorite. Spills should be wiped up thoroughly using either an iodophor disinfectant or sodium hypochlorite solution. Materials used to wipe up spills should be added to biohazardous waste matter for proper disposal.

Store reagents between +2°C and +8°C. Avoid unnecessary exposure to light. The light sensitive reagents is the conjugate. Storage of samples in self-defrosting freezers is not recommended.

Do not use reagents after the expiration date printed on the label.

Do not mix or interchange reagents from different kits or kit lots. Cross contamination of reagents or samples can cause erroneous results.

Do not interchange vial caps. Use a new dropper for each sample.

Optimal results will be obtained by strict adherence to the test protocol. Accurate and precise pipetting, as well as following the exact time and temperature requirements, are essential.

Once the assay has been started. all steps should be performed without interruption.

Reusable glassware must be disinfected. washed out and rinsed free of detergents.

STORAGE AND STABILITY Storage

Store the test devices at 2-30°C temperature. Store the Buffer Solution and Gold Conjugate bottles at 2-8°C temperature. Do not use the kit beyond the expiry date mentioned on it.

Before running the test bring all the kit components to room temperature (25+5°C) for best results. Return the Buffer Solution & Gold Conjugate bottles to 2-8°C. when not in use. DO NOT FREEZE KIT COMPONENTS.1. The un-opened kits are stable for 1? year from the date of manufacturing as indicated on the package.

Stability

2.Opened kits must be used with in 3 months of opening. Test device once opened from the pouch must be used immediately.

3.Repeated 'freeze-thaw cycles' i.e., bringing the kits to room temperature and back to the refrigerator several times will reduce the stability of the kit.INDICATION OF INSTABILITY OR DETERIORATION OF REAGENTS

Changes in the physical appearance of the reagents supplied may indicate deterioration of these materials. Do not use gold conjugate which are turning black or precipitating.Reading of the Results

Read the result immediately. Do not read after 5 minutes.1, Bring all the reagents. devices and specimens to room temperature (25+5°C).2. Add 2 drops of buffer solution to the test device.3. Add 2 drops of either serum or plasma.4. Add 4 drops of buffer solution.5. Add 2 drops of gold conjugate.6. Add 4 drops of buffer solution.Test Procedure

TROUBLESHOOTING FALSE POSITIVE Addition of more than 2 drop of sample or gold conjugate Cause / Error

Remedy

Use only 2 drops of sample or gold conjugate

Cause / Error Remedy

WEAK INTENSITY OF CONTROL SPOT Bring the sample, test device, buffer and gold conjugate

to room temperature before testing (25 + 5 C)

Very cold reagent Cause / Error

Remedy

POOR SENSITIVITY

Frozen sample not mixed properly after thawing.Mix well sample before pipetting.

PERFORMANCE CHARACTERISTICS

Accuracy : AIDSCAN HIV-1/2 TRISPOT T est meets the requirements when tested against DCI approved kit.Specificity

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No. of

Negatives T ested

68No. of Negatives by

AIDSCAN HIV-1/2 TRISPOT T est

68Specificity (%)

100

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Sensitivity No. of HIV-1 Positive Samples T ested

42

No. of Positives by

AIDSCAN HIV-1/2 TRISPOT T est

42

Sensitivity (%)

100

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No. of HIV-2 Positive Samples T ested

14No. of Positives by

AIDSCAN HIV-1/2 TRISPOT T est

14Sensitivity (%)

100

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a)

b)

LlMITATIONS OF THE TEST

RESULTS

Interpretation of Results

1. NEGATIVE: If only one red spot (control spot) appears at the control region "C" indicates that the specimen does not contain antibodies either to HIV-1 or HIV-

2. (Fig. a)

2. POSITIVE: (a) If two red spots (Control spot and HIV-1 or HIV-2 Spot) appear at the control region "C” and test region HIV-1 and/or HIV-2 indicates that the specimen is reactive for antibodies to HIV-1 and/or HIV-2. (Fig. b)

(b) If three red spots (Control. HIV-1 & HIV-2 Spot) appear at the control region "C" and test region HIV-1 & HIV-2

indicates that the specimen is reactive for antibodies to HIV-1 & HIV-2. (Fig. c)3. INVALID RESULT

If no spot appears after the completion of test. either with Clear background or with complete reddish background the test indicates ERROR. (Fig. d) This may indicate a procedural error or deterioration of specimen / reagents or particulate matter in the specimen. The specimen should be retested on a fresh device.IMPORTANT

(i) Test spot on "HIV-1 & HIV-2" area either dark or light in colour (red) should be considered reactive for antibodies to HIV.

(ii) Sometimes. if the sample solution does not soak-in within 40-60 seconds. the

C

TRISPOT TEST H I V -1H I V -2

Fig. d

sample should be observed for any suspended particulate matter; if it is present. centrifuge the sample at 5000 r.p.m. for 15 minutes. Use a fresh device to re-run the test.

(iii) Sample found to be reactive by the above screening test must be confirmed by standard supplemental assay. like ELISA, Radio immuno assay or Western blot.

1.Samgadharan MG, Markham PD: The role of human T-Iymphotropic retroviruses in leukemia and AIDS, in Wormser GP (ed): AIDS and Other

Manifestations of HIV Infection. New Jersey, Noyes Publications, 1987, pp 218-220

2.Barre-Sinoussi F, Chermann JC, Rey F, et at: Science 220:868-871, 198

3.3.Gallo RC, Salahuddin SZ, Popovic M, et at: Science 224:500-503, 198

4.4.Coffin J, Haase A, Levy JA, et at: What to call the AIDS virus? Nature

321:10, 1986.

5.Clavel F, Guetard D, Brun-Vezinet F: Science 233:343-346, 198

6.

REFERENCES

7.The kit work best when used with fresh sample and when all the kit component are at room temperature (25+5 C). Sample which have been frozen and thawed several times contain particulates which can block the membrane,hence resulting in improper flow of reagents and high background colour which may -R -03, 2010-09-28

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