PEPTI-LAV determinations 72253

Transcription

1 PEPTI-LAV determinations DISCRIMINATION TEST FOR HIV-1 AND HIV-2 ANTIBODIES BY ENZYME IMMUNOASSAY Remark: this kit can only be used as complementary test for the HIV-1 and HIV-2 antibodies differentiation that have been primary detected with a screening test. In any case, it can be used as a confirmatory test. IVD /10

2 TABLE OF CONTENTS 1. INTENDED USE CLINICAL VALUE CONTENTS OF THE KIT EQUIPMENT REQUIRED BUT NOT SUPPLIED PRECAUTIONS HEALTH AND SAFETY INSTRUCTIONS COLLECTING AND HANDLING THE SAMPLES RECONSTITUTION OF THE REAGENTS AND STORAGE TEST PROCEDURE READING THE RESULTS INTERPRETATION OF RESULTS PERFORMANCES LIMITS OF THE TEST LITERATURE [EN]

3 1. INTENDED USE The identification in 1985 of a second virus associated with AIDS, called HIV type 2 (1), made the epidemiologic studies as well as the procedures of routine controls in blood banks and specific controls in analytical laboratories more complex. The investigations carried out in 1986, 1987 and 1988 evidenced the presence of the HIV-2 virus mainly in the West-African countries, but also in the European countries (France, Portugal, Italy, Greece, Crete, Germany, England, Sweden...) and in the United States. Serological studies conducted by different groups emphasized on the limits and inadequacies of the immunological reagents obtained only from HIV-1 antigen for the detection of HIV-2 infected patients (2). However, cross reactions can be explained by the high degree of homology between the coding genes for the internal proteins and for the envelope proteins of the HIV-1 and HIV-2 viruses. On the basis of such tests, numerous teams reported the existence of mixed contaminations, with a frequency which was rapidly considered too high, casting a doubt on the solidity of the conclusions (3). Owing to these two types of findings, the diagnosis of the HIV infection requires the simultaneous use of screening tests based on the use of antigens representative of both viruses and of highly specific discrimination tests to identify the virus which caused the infection. One neat way of solving the problem of the differential identification of the anti-hiv-1/hiv-2 antibodies consists in implementing a mono-epitope serological assay based on the use of synthetic peptides (4). The PEPTI-LAV 1-2 test, developed by Bio-Rad, is based on an immunoenzymatic strip method using two synthetic peptides allowing a mono-epitope assay. Peptide1, specific of the HIV-1 virus, imitates an immunodominant epitope of the transmembranar envelope glycoprotein: GP 41. Peptide2, specific of the HIV-2 virus, imitates an epitope of the transmembranar glycoprotein: GP 36. These two peptides fixed separately on the membrane allow the displaying in one test of the specificity of the response of the serum or plasma under test. 2. CLINICAL VALUE PEPTI-LAV 1-2 is a unit test using a membrane fixed on a plastic strip as a solid support. Two antigens are fixed on this membrane, located as follows: The HIV-1 specific peptide is fixed on the membrane extremity opposite the plastic support The HIV-2 specific peptide is fixed between the HIV-1 specific peptide and the control band The control band is located on the membrane extremity close to the plastic support 1 2 T Membrane Plastic support 1 = HIV-1 specific peptide 2 = HIV-2 specific peptide T = Control band The test is based on an immunoenzymatic technique comprising the following reaction phases: 1 - The samples under test are incubated with the strip. If HIV-1 and/or HIV-2 antibodies are present, they bind to the corresponding peptide fixed on the strip. 2 - The peroxidase-labeled anti-human IgG antibody is added after washing. It binds in turn to the specific IgG captured by the peptides on the solid phase and to the control band consisting of IgG. 3 - The presence of the enzyme fixed on the complex compounds is shown by the addition of substrate after the elimination by washing of the fraction of conjugate left free. 4 - Stopping of the development, reading of the developed bands on the solid support and interpretation of the results. [EN] 3

4 3 - CONTENTS OF THE KIT All the reagents are intended to in vitro diagnostic use only. Each kit contains reagents sufficient for 10 determinations. The determinations may be performed in multiple independent runs. a) Reagents Label Reagent Composition Presentation R1 HIV-1/HIV-2 peptides coated strips and internal anti-igg control band HIV-1 peptide: GP 41, HIV-2 peptide: GP 36 1 cartridge of 10 strips R2 R3 R4 Concentrated washing solution (20X) Tris NaCl buffer ph 7.4 Preservative: ProClin 300 (0.04%) Specimen diluent contains CHCl % Conjugate Peroxidase-labeled anti-human IgG goat antibodies Preservative: Thimerosal 0.01% b) Material supplied in the kit 1 empty vial for preparing the chromogenic solution. 10 graduated tubes with blue stopper for sample incubation. 10 graduated tubes with orange stopper for development. 10 colorless stoppers for conjugate incubation. 10 individual results sheets. 1 vial 70 ml 2 vials 2 x 40 ml 1 vial 2 ml R5 Diaminobenzidine (DAB) Chromogen 4 tablets Substrate 1 vial R6 0.6% H ml 4 - EQUIPMENT REQUIRED BUT NOT SUPPLIED Distilled or demineralized water. 100 ml graduated cylinders. 5 ml or 10 ml graduated pipettes. Automatic or semi-automatic pipettes, adjustable or fixed, allowing the measuring or dispensing of 30 μl and 150 μl. Disposable gloves. Liquid jet vacuum pump with safety bottle. Sodium hypochlorite (Bleach). Absorbent paper. Tweezers. 1, 2 or 3 dimensional agitators. Container for biohazardous waste. Protective glasses. 5 - PRECAUTIONS The reliability of results depends on correct observance of the following Good Laboratory Practices: Do not use expired reagents. Do not mix reagents from different lots within a given test run. Before use, it is required to wait 30 minutes to allow the reagents stabilizing at room temperature (18-30 C). Carefully reconstitute reagents avoiding any contamination. Use glassware thoroughly washed and rinsed with distilled water or preferably, disposable material. 4 [EN]

5 Use a new dispensing tip for each sample. Never use the same container to dispense conjugate and color development solution. Check pipettes for accuracy and precision and if the instruments being used are correctly working. Do not change the assay procedure. Do not allow strips to dry more than 10 minutes during the test. 6 - HEALTH AND SAFETY INSTRUCTIONS Never handle the strips with bare hands : Use plastic tweezers. Wear disposable gloves when handling reagents. Do not pipette by mouth. Because no known test method can offer complete assurance that the HIV, Hepatitis B or C virus or other infectious agents are absent, consider these reagents, as well as patient samples, as potentially infectious and handle them carefully. Any equipment directly in contact with samples and human source reagents as well as buffer solutions should be considered as contaminated products and treated accordingly. Avoid spilling samples or solutions containing samples. Contaminated surfaces should be cleaned 10% diluted bleach. If the contaminating fluid is an acid, the contaminated surfaces should be first neutralized with sodium bicarbonate, then cleaned with bleach, and dried with absorbent paper. The material used for cleaning should be discarded into a biohazardous waste container. Samples, human source reagents, as well as contaminated material and products should be discarded after decontamination: - either by soaking into bleach at a final concentration of 5% sodium hypochlorite (1 volume of bleach per 10 volumes of contaminated fluid or water) for 30 minutes - or by autoclaving at 121 C for 2 hours minimum. CAUTION: DO NOT PLACE SOLUTIONS CONTAINING SODIUM HYPOCHLORITE IN THE AUTOCLAVE. Do not forget to neutralize and/or autoclave the wash waste solutions or any fluid containing biological samples before discarding them into the sink. Chemicals should be handled and discarded in accordance with Good Laboratory Practices. Some reagents contain ProClin 300 (0.04%, 0.1% and/or 0.5%). 7 - COLLECTING AND HANDLING THE SAMPLES Collect a blood sample according to current practice. The tests should be performed with undiluted serum or plasma samples (collected with anticoagulants such as EDTA, heparin, citrate). Extract the serum or plasma from the clot or red cells as soon as possible in order to avoid hemolysis. Extensive hemolysis may affect test performance. Samples with aggregates should be clarified by centrifugation prior testing. Suspended fibrin particles or aggregates may yield falsely positive results. The samples can be stored at +2-8 C if the test is performed within 7 days or they may be deepfrozen at -20 C. Plasma samples should be quickly thawed by heating for a few minutes at 40 C (to limit fibrin precipitation). Samples that have been frozen and thawed more than 3 times should not be used. If the samples have to be shipped, they should be packaged in accordance with the regulations effective for the transport of etiological agents. DO NOT USE CONTAMINATED, HYPERLIPEMIC OR HYPERHEMOLYZED SERUM OR PLASMA SAMPLES. Note: Samples containing up to 90g/l albumin, 100 mg/l bilirubin, lipemic samples containing up to the equivalent of 36 g/l trioleine, and hemolyzed samples containing up to 10 g/l hemoglobin do not affect the test results. [EN] 5

6 8 - RECONSTITUTION OF THE REAGENTS - STORAGE Each kit contains reagents sufficient for 10 determinations. The determinations may be performed in multiple independent runs. Note: Before use, allow the reagents to reach room temperature (18-30 C). a) Reagents ready for use R1: HIV-1/HIV-2 peptides coated strips and one control band R3: Sample diluent R4: Conjugate R6: Substrate b) Reagents to be reconstituted R2: Concentrated washing solution (20X) Dilute 1:20 in distilled water to obtain the ready-for-use washing solution. (For one test, 5 ml of washing solution + 95 ml of distilled water). R5: Diaminobenzidine (DAB) Chromogen Dilute one DAB tablet with 30 ml of the reconstituted washing solution R2 ready for use in the smoked glass vial provided for that purpose. Date the reconstitution. Homogenize after the tablet is completely dissolved. STORAGE Store the kit at +2-8 C. Once opened, all the kit reagents may be stored at +2-8 C until the expiration date stated on the box (except for specific instructions) : R2: The diluted washing solution can be stored at C during 2 weeks. The concentrated washing solution (R2) can be stored at C. The diluted chromogen can be used 1 month at 4 C. When the validity period of the chromogen has expired or the reconstitution of a new volume of chromogen solution is wanted, carefully rinse the vial 3 times with distilled water before preparing a new portion. 9 - TEST PROCEDURE 1. Sample incubation In a graduated tube with blue stopper, transfer 3 ml of diluent R3. Distribute 30 μl of the sample to be examined. Homogenize. Introduce the strip and close the tube. Place the tube under shaking (1, 2 or 3-dimensional agitator). Incubate for one hour ± 5 minutes at room temperature (18-30 C). 2. Washing of the excess sample Remove the solution using a vacuum pump or by turning the tube upside down, while holding the strip with the hand (foresee gloves and tweezers during this process). Completely fill the tube containing the diluted washing solution R2. Stir the strip in the solution and leave to incubate for 1 to 2 minutes at room temperature (18-30 C). Empty the tube again. Repeat twice more the filling/emptying steps, i.e. a total of 3 washings. 3. Incubation of the conjugate In the same graduated tube, dispense 3 ml of sample diluent R3. Distribute 150 μl of conjugate (R4) in each tube. Close with a colorless stopper and homogenize. Incubate for 30 ± 5 minutes as in paragraph Washing of the excess conjugate Proceed as in paragraph 2. 6 [EN]

7 5. Development The development phase takes place in the graduated tube with orange stopper. It comprises two steps : the preparation of the development solution and the actual development of the strip. Preparation of the development solution (Foresee 1 tube by strip) Transfer 3 ml of the reconstituted chromogen solution in a tube. Add 150 μl of substrate R6. Stir the solution to homogenize. Actual development Dip the strip into the development solution thus prepared. Shake the tube. The development takes place within at least 3 minutes and maximum 15 minutes. 6. End of development To stop the development, dip the strip into distilled water, then dry at ambient temperature. Notes: the test procedure details the execution of a single test. In the case of multiple testing, a large volume of conjugate solution (R3 + R4) and of development solution (R2 + R5 + R6) can be prepared and 3 ml of these solutions dispensed in each tube at the time of the test. The test can take place in an incubation tray for Western-Blot READING OF THE RESULTS 1. Conditions of test validity Each strip has a control band used to validate the development phases. This band exhibits a high intensity and is located on the membrane extremity close to the plastic support. 2. Reference marks for the reading of band intensity The PEPTI-LAV 1-2 is a unit test which does not use a reading instrument. Therefore, the interpretation is made with the naked eye by the displaying or not of straight bands on the membrane. To limit as much as possible the subjectivity that goes along with this reading method, some criteria and reference marks are used: The control band corresponds to a response intensity arbitrarily marked + + Any clearly positive band, of an intensity equal or greater than that of the control band, shall be marked + + Any clearly positive band, of an intensity lower than that of the control band shall be marked + Any little distinct colouring (colouring in fine lines and not of the same width than the control band, hardly visible intensity...) shall be market ± and considered not interpretable. The absence of colour on the membrane shall be marked -- Positive response = + +, + Negative response = -- Uninterpretable response = ± 11 - INTERPRETATION OF THE RESULTS 1. General case Profile HIV-1 Response HIV-2 Response No peptide recognised One of the 2 peptides recognised Interpretation Negative result with PEPTI-LAV 1-2 Presence of anti-hiv-1 antibodies Presence of anti-hiv-2 antibodies [EN] 7

8 2. Special cases In some cases, a simultaneous reactivity to both peptides can be observed. To evaluate this type of results, it is necessary to estimate the relative intensity on each peptide: Case 1 - the two peptides are recognized with a differential intensity: + / + + ± / + + ± / + PEPTI-LAV 1-2 allows the diagnosis to be oriented towards a HIV-1 or HIV-2 preferred serology. Case 2 - the two peptides are recognized with the same intensity : + / / + + PEPTI-LAV 1-2 does not allow in these seldom cases a diagnosis to be made in favour of one virus or the other. Several hypotheses are possible: The patient has a dual infection The patient is infected by a particular virus Note: Inhibitions studies carried out on such sera showed the absence of a cross reaction between the peptides (5) PERFORMANCES Specificity 214 HIV antibody negative samples from whom 65 belong to potentially interfering (Toxoplasmose, Rubella, Rhumatoïde Factor, HTLV, MVC, HLA) were tested with PEPTI-LAV samples were found negative, 3 showed an indeterminate result. Only one sample was found dual reactive in HIV-1/2. It comes from a patient that showed Western-Blot profile with numerous bands of non classified bands. This patient was affected by several simultaneous infections (STD). The stimulation of the immune system could explain this non specific reaction. Sensitivity 306 positive samples with NEW LAV BLOT I and/or NEW LAV BLOT II were tested with PEPTI-LAV samples were found positive HIV-1 and/or HIV-2, 11 were indeterminate and 4 negative. Three of these 4 PEPTI-LAV 1-2 negative results showed particular profiles. One of these sample hardly recognizes other synthetic peptides. This sample is likely to have a unique epitope transmembrane glycoprotein. The second sample comes from an covert AIDS stage patient, none recognition of HIV-1 peptide can be explained by a decreased gp41 antibody level. One difficult sample, it is gp 160 and p25 western blot profile and does not very along with time. Finally the fourth samples is of African origin. Among the 306 positive samples, the 55 NEW LAV BLOT II exclusively positive were clearly identified HIV-2 reactive by PEPTI-LAV 1-2. HIV-1-HIV-2 discriminating power 78 doubly positive samples NEW LAV BLOT I and II were tested. 69 samples were clearly differentiated in 28 HIV-1 and 41 HIV-2 peptide positives. 9 samples came out peptide double reactive. 8 of them were of African origin, where the coexistence of two viruses HIV-1 and HIV-2. These results does not allow to conclude towards lack of discrimination power of the test and a double infection cannot be excluded. Indeed, the perfect specificity of the epitopes mimicked by the peptides used was demonstrated by indirect ELISA inhibition tests (reference 5). Reproducibility 4 samples, 1 negative, 1 positive HIV-1 and 2 positive HIV-2 (one weak and one medium) were tested 6 times in the same run and in 6 different days. The same intensity of the bands was observed. 8 [EN]

9 13 - LIMITS OF THE TEST It is required to follow the procedure in order to ensure optimal performance. Very low titer of antibodies gp41 (HIV-1) or gp36 (HIV-2) may not be detected. Consequently a negative result indicates that the tested sample does not contain anti-hiv antibodies detectable with PEPTI-LAV 1-2. Such a result does not exclude the possibility of exposure to an HIV-1/HIV- 2 infection. A new later sample collection is suggested. The variability of HIV-1 (group M and group O) and HIV-2 does not allow to exclude the possibility of false negative reactions. No known test method can offer complete assurance that the HIV virus is absent LITERATURE 1. CLAVEL F., GUETARD D., BRUN-VEZINET F. Isolation of a new human retrovirus from West African patients with AIDS Science, 1986, 223, BAYLISS G.J., PARRY J.V., MORTIMER P.P. HIV-2 in Britain: no evidence, yet Lancet, 1988, January 16, FOUCAULT C., LOPEZ O., JOURDAN G. Double HIV-1 and HIV-2 seropositivity among blood donors Lancet, 1987, July 18, GNANN J.W., MITCHELL J.B. Synthetic peptide immunoassay distinguishes HIV type 1 and HIV type 2 infections Science, 1987, 237, COT M.C., PEETERS M., POULAIN M., BRUN-VEZINET F., DELAGNEAU J.F., Dual HIV-1 and HIV-2 Infection in West Africa supported by synthetic peptide analysis. [EN] 9

10 (EN) This product contains human or animal components. Handle with care. H341-H350-H317 P201-P280-P308+P313-P302+P352-P333+P313-P501 (EN) Danger Suspected of causing genetic defects. May cause cancer <state route of exposure if it is conclusively proven that no other routes of exposure cause the hazard>. May cause an allergic skin reaction. Obtain special instructions before use. Wear protective gloves/protective clothing/eye protection/face protection. IF exposed or concerned: Get medical advice/attention. IF ON SKIN: Wash with plenty of soap and water. If skin irritation or rash occurs: Get medical advice/attention. Dispose of contents/container in accordance with local/regional/national/ international regulations. 10 [EN]

11 NOTES [EN] 11

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