Murex HIV Antigen mab

Transcription

1 8E77-02 E C11VK86GB Murex HIV Antigen mab VK86 Enzyme immunoassay for the qualitative detection of human immunodeficiency virus (HIV) antigens Note Changes Highlighted 2007 Abbott / Printed in the UK / Murex HIV Antigen mab Key to symbols used: 8 C Lot: Store at 2 C to 8 C 2 C Use by For in vitro diagnostic use Caution, see instructions for use (potentially infectious) Consult instructions for use Legal Manufacturer Catalogue number See Reagents section for a full explanation of symbols used in component naming ABBOTT Diagnostics Division

2 MUREX HIV ANTIGEN MAB INTENDED USE The Murex HIV Antigen mab assay (8E77-02) is an enzyme immunoassay (EIA) for the qualitative detection of p24 core antigens of the human immunodeficiency virus (HIV) in human serum, plasma or cell culture supernatant. SUMMARY AND EXPLANATION OF THE TEST HIV is recognised as the aetiological agent of the acquired immune deficiency syndrome (AIDS). The virus is transmitted by sexual contact, by using contaminated injection needles, through transfusion of contaminated blood and by transplacental passage. The clinical spectrum of HIV infection shows considerable variation: the virus can be present in a latent form or can cause clinical symptoms characterised by AIDS-related complex and AIDS. In patients with AIDS, the immune system is so damaged that they become the target of many types of opportunistic infections. At present, at least two different human AIDS viruses, HIV-1 and HIV- 2 have been identified 1. In addition, highly divergent strains within the HIV-1 family have been isolated, and designated as HIV-1 group O 2,3,4. The core region of these different viruses is the most conserved: HIV-1 group M genes coding for the core proteins show a homology of 60 and 70 percent with HIV-2 and HIV-1 group O, respectively 5,6. Therefore, tests with carefully selected antibodies to HIV-1 core proteins (as in the Murex HIV Antigen mab) will also detect the cross-reactive core antigen of HIV-1 group O and HIV-2. In many countries, blood donors are systematically tested for the presence of antibodies to HIV. These antibodies can appear as early as 3 to 4 weeks after infection. In some cases, however, it can take up to 6 to 14 months. The major viral core antigen can be detected before antibody 7,8, and antigen testing may enable earlier detection of HIV infection in persons at risk. The HIV antigen test can also be used as an aid in the prognosis and therapy of HIV-positive individuals 9. Furthermore, it can be of value in the detection of HIV viral antigen upon virus isolation in tissue culture. PRINCIPLE OF THE PROCEDURE The microtitre wells have been coated with human polyclonal antibodies to HIV. The test sample containing HIV antigen is incubated in the well together with biotinylated anti-p24 monoclonal antibodies. In a subsequent step the biotin will bind with peroxidase-conjugated streptavidin. Incubation with chromogen produces a blue colour in the test well which turns yellow when the reaction is stopped with sulphuric acid. If the sample contains no HIV antigen, the labelled antibody will not be captured onto the well and no colour or only a low background colour develops. REAGENTS SUPPLIED Each pack contains: 1. Coated Wells 1 plate of 96 wells coated with human anti- HIV polyclonal antibodies with silica gel as drying agent. 2. Negative Control 1 bottle containing 2.5 ml of human serum negative for HIV-antigens. Contains 0.1% sodium azide as preservative. 3. NP40 1 bottle containing 10 ml of NP40. Contains 0.05% Proclin as preservative, bovine casein as stabiliser and bovine aprotinin as protease inhibitor. 4. Conjugate 1 (20 x Conc) 1 bottle containing 0.4 ml of concentrated biotinylated murine anti-p24 monoclonal antibodies, to be diluted 20x before use. Contains 0.05% Proclin as preservative, bovine albumin as stabiliser and heat inactivated mouse serum. 5. Conjugate Diluent 1 2 bottles each containing 4 ml of aggregated human IgG in phosphate buffer. Contains 0.05% Proclin as preservative and heat inactivated mouse serum. Used to dilute the concentrated Conjugate Conjugate 2 1 bottle containing 0.3 ml of concentrated peroxidase conjugated streptavidin, to be diluted 100x before use. 7. Conjugate Diluent 2 1 bottle containing 30 ml of phosphate buffer, used to dilute the concentrated Conjugate 2. Contains 0.05% Proclin as preservative, bovine casein as stabiliser and bovine aprotinin as protease inhibitor. 8. Chromogen Solution 1 bottle containing 0.3 ml of concentrated tetramethylbenzidine (TMB) dissolved in dimethyl sulphoxide (DMSO), to be diluted 100x before use. Contains 0.02% Thiomersal as preservative. 9. Substrate Buffer 1 bottle containing 30 ml of phosphate citrate buffer containing 0.006% hydrogen peroxide, used to dilute the concentrated chromogen solution 10. Wash Fluid 1 bottle containing 45 ml of concentrated phosphate buffer, to be diluted 25x before use. Contains 0.15% Proclin as preservative. 11. Standard Solution 1 bottle containing 2.5 ml of recombinant p24 diluted in matrix. The indicative concentration of the standard is indicated on the vial label for your information. Contains 0.05% Proclin as preservative, bovine casein as stabliliser and bovine aprotinin as protease inhibitor. 2

3 12. Matrix 1 bottle containing 15 ml of phosphate buffer containing bovine proteins. Contains 0.05% Proclin as preservative, bovine casein as stabiliser and bovine aprotinin as protease inhibitor 13. Plate Sealers 8 adhesive Plate Sealers. 14. Minigrip Bag 1 plastic bag for storage of unused strips. STORAGE AND STABILITY If kept at 2 to 8 C, all test reagents, including the Coated Wells, are stable until the expiry date given on the pack. Do not freeze reagents. All reagents and the bag containing the test wells, must be brought to room temperature (15 to 30 C) approximately 30 minutes before use and returned to the refrigerator immediately after use. Unused test wells are stable for 8 weeks if stored at 2 to 8 C in the plastic minigrip bag with silica gel. Conjugate Working Solution 1 is stable for 1 week at 2 to 8 C. Substrate Solution and Conjugate Working Solution 2 are stable at room temperature (15 to 30 C) for 24 hours if kept in the dark. Preferably use freshly prepared Wash Fluid. Unused prepared Wash Fluid can be stored at 2 to 8 C for 4 weeks. The Positive Control is stable for the life of the kit. Keep undiluted reagents in their original vials. WARNINGS AND PRECAUTIONS The materials are for in vitro diagnostic use only HEALTH AND SAFETY INFORMATION 1. The human polyclonal antibodies used on the Coated Wells have been prepared from material that was determined to be HBsAg and anti-hcv negative and subsequently inactivated. The Negative Control and the aggregated human IgG in Conjugate Diluent 1 have been prepared from material of human origin that was determined to be non-reactive for HBsAg and antibodies to HCV and HIV types 1 and 2. However, no test method or inactivation procedure can offer complete assurance that products derived from human sources will not transmit infection. Therefore all material should be considered as being potentially infectious and should be handled as such. They should be disposed of in accordance with established safety procedures. 2. The Chromogen Solution contains DMSO (99.1%) which is classified per applicable European Economic Community (EEC) Directives as irritant (Xi). The following are the appropriate Risk (R) and Safety (S) phrases. Xi R36/37/38 Irritating to eyes, respiratory system and skin. S23 Do not breath aerosol. S24 Avoid contact with skin. S26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. 3. The Negative Control contains 0.1% sodium azide which is classified per applicable European Economic Community (EEC) Directives as harmful (Xn). The following are the appropriate Risk (R) and Safety (S) phrases. Xn R22 Harmful if swallowed R32 Contact with acids liberates very toxic gas S35 This material and its container must be disposed of in a safe way S36 Wear suitable protective clothing S46 If swallowed, seek medical advice immediately and show this container or label Azides can react with copper and lead used in some plumbing systems to form explosive salts. The quantities used in this kit are small; nevertheless when disposing of azide-containing materials they should be flushed away with large volumes of water. 4. NP40, Conjugate 1, Conjugate Diluent 1, Conjugate Diluent 2, Standard Solution and Matrix contain 0.05% ProClin, and Wash Fluid contains 0.15% ProClin. All listed concentrations are classified per applicable European Economic Community (EEC) Directives as irritant (Xi). The following are the appropriate Risk (R) and Safety (S) phrases. Xi R43 S24 S35 S37 S46 May cause sensitisation by skin contact. Avoid contact with skin. This material and its container must be disposed of in a safe way. Wear suitable gloves. If swallowed, seek medical advice immediately and show this container or label. Information for European Customers; For product not classified as dangerous per European Directive 1999/45/EC - Safety Data Sheet available for professional user on request. 5. Sulphuric acid required for the Stop Solution is corrosive and should be handled with appropriate care. If it comes into contact with the skin or eyes, wash thoroughly with water. ANALYTICAL PRECAUTIONS Do not use the kit beyond the expiry date. Do not mix components from kits with different lot numbers. All vessels used to prepare conjugate and substrate solutions must be clean. Do not touch the top of the plates. Avoid microbial contamination of reagents. Ensure that samples, Negative Control and diluted Solutions are homogeneous before use. Use a new pipette-tip for each specimen pipetted. To avoid contamination, do not touch the top edge of the wells with the pipette tips when adding sample or conjugate. Do not expose Substrate Solution to strong light during incubation or storage. The prepared Substrate Solution must be colourless when used; if the solution turns blue, it must be replaced Solutions containing TMB, sulphuric acid or peroxide should not come into contact with metals or metal-ions. Remove any air bubbles present by tapping the microplate gently. Place the wells upside down on a wet absorbent tissue for no longer than 15 minutes if they cannot be filled with conjugate or substrate immediately after washing. Before reading the plate, check the bottom of the plate to ensure it is clean and dry. Do not use blood collection tubes for the preparation of the reagent working solutions. 3

4 SPECIMEN COLLECTION AND STORAGE The Murex HIV Antigen mab assay can be used with serum, citrate plasma, EDTA plasma, heparin plasma or cell culture supernatant. Do not use heat-inactivated specimens. Specimens should be free of microbial contamination when tested. Insoluble material should be removed from all samples by centrifugation before testing. Repeatedly frozen and thawed, lipaemic or haemolyzed samples may produce erroneous results. Cell culture medium should first be centrifuged to remove cells and debris. Supernatant is used for testing and should be tested with an HIV-negative cell culture supernatant as control. Store samples at 2 to 8 C. For storage of longer than one week, separate and freeze aliquots at 20 C or lower. Before storage, serum or plasma should be separated from the blood clot or blood cells by centrifugation. REAGENT PREPARATION Allow all test materials to reach room temperature (15 to 30 C) before use. Wash Fluid Dilute concentrated Wash Fluid 25x with distilled or deionized water, e.g. by adding 45 ml Wash Fluid to 1080 ml distilled water. Prepare at least 50 ml of wash solution for each 8-well strip. NOTE: salt crystals may have formed in the concentrated Wash Fluid after storage at 2 to 8 C. These crystals should be completely redissolved before dilution e.g. by placing the bottle in a water bath at 37 C. Wash Fluid must be at room temperature (15 to 30 C) when used. Diluted Conjugate 1 Dilute concentrated Conjugate 1 20x with Conjugate Diluent 1 e.g. by adding 25 µl Conjugate 1 to 475 µl Diluent 1 per 8-well strip or 300 µl Conjugate 1 to 5.7 ml Diluent 1 per plate. Conjugate Working Solution 1 Mix 1 part of diluted Conjugate 1 with 1 part of NP40 e.g. by adding 0.5 ml NP40 to 0.5 ml diluted Conjugate 1 per 8-well strip or 6 ml NP40 to 6 ml diluted Conjugate 1 per plate. Conjugate Working Solution 2 Dilute concentrated Conjugate 2 100x with Conjugate Diluent 2, e.g. by adding 20 µl Conjugate 2 to 1.98 ml Diluent 2 per 8-well strip or 240 µl Conjugate 2 to ml Diluent 2 per plate. Substrate Solution Dilute concentrated TMB chromogen 100x with Substrate Buffer, e.g. by adding 20 µl TMB to 1.98 ml Substrate Buffer per 8-well strip or 240 µl TMB to ml Substrate Buffer per plate. NOTE: concentrated Chromogen Solution should be melted completely (melting point 18 C) before pipetting. Positive Control Dilute Standard Solution with Matrix, e.g. by adding 60 µl Standard Solution to 60 µl Matrix for one 8-well strip. For more than one strip, include at least two Positive Controls. Positive Control can be prepared by adding 2.5 ml of Matrix to the bottle of Standard Solution. Label the bottle to indicate that it now contains Positive Control. MATERIALS REQUIRED BUT NOT PROVIDED distilled or deionised water. sulphuric acid of analytical grade in the range of 1 to 2M. precision pipettes with disposable tips to deliver volumes in the ranges of 20 to 200 µl and 200 to 1000 µl. vortex mixer or equivalent. incubator at 37 C. instrumentation: a) microwell plate washing system (automated or manual) b) microwell plate reading system or c) automated processors. absorbent tissues. microplate reader to read the absorbance at 450 nm and at nm for dual wavelength analysis. optional, a repetitive or multichannel pipette together with V-shaped troughs for addition of conjugate solutions, Substrate Solution, Wash Fluid and Sulphuric acid. a plate shaker to ensure adequate mixing after sample and reagent addition. clean bottles to contain diluted conjugates, substrate and Wash Fluid. ASSAY PROCEDURE Before starting the assay, ensure that all reagents are at room temperature (15 to 30 C). Step 1 Prepare Wash Fluid, Positive Control and Conjugate Working Solution 1 (See Reagent Preparation). Step 2 Use only the number of wells required for the test, taking into account that for one strip one Negative and one Positive Control should be included. For more strips at least two Negative and two Positive Controls should be included in each strip-holder. Step 3 Add 100 µl of Conjugate Working Solution 1 to each 100 µl test well. Add 100 µl of the appropriate specimen or + control to each test well. Ensure specimens and controls 100 µl are adequately mixed with the Conjugate Working Solution 1 by pipetting up and down 5 times or by using a plate shaker at 1000 rpm for one minute. Step 4 Cover the strips with an adhesive plate sealer and 60 mins incubate for 60 minutes at 37 C. NOTE: Prepare Conjugate Working Solution 2 at the end of the incubation period. (See Reagent Preparation). Step 5 Wash each well 5 times with Wash Fluid according to Wash Procedures. Step 6 Add 200 µl of Conjugate Working Solution 2 to each 200 µl well. Step 7 Cover the strips with an adhesive sealer and incubate30 mins for 30 minutes at 37 C. NOTE: Prepare Substrate Solution at the end of the incubation period (See Reagent Preparation). Step 8 Wash each well 5 times with Wash Fluid according to Wash Procedures. Step 9 Add 200 µl of Substrate Solution to each test well. 200 µl Step 10 Incubate for 30 minutes at room temperature 30 mins (15 to 30 C). Step 11 To stop the reaction, add 50 µl sulphuric acid (1-2M) 50 µl to each well, in the same sequence and at the same time intervals as the Substrate Solution. Tap the strip-holder carefully to ensure thorough mixing. Step 12 Read the absorbance of the solutions in the wells 5-15 mins (after 5 minutes and within 15 minutes after step 11) at 450 nm with a microplate reader. For dual wavelength analysis, 620 to 690 nm should be used as reference wavelength. Blank the instrument on air (no plate in the carriage). 4

5 WASH PROCEDURES OPERATING INSTRUCTIONS FOR WASHING EQUIPMENT SHOULD BE CAREFULLY FOLLOWED. CONTAMINATION OF WASH FLUID AND WASHER CAN CAUSE EXTENSIVE PROBLEMS. a) Protocol for automated stripwasher Ensure that the stripwasher is set up for a flat-bottomed well format, incorporating a bottom sweep (if possible). Perform 5 wash cycles using working strength Wash Fluid ensuring that: i) The fill volume is 300 µl/well. ii) The dispense height is set to completely fill the well without causing an overflow but giving a positive meniscus. iii) The time taken to complete one aspirate/wash/soak cycle is approximately 30 seconds. iv) Where possible a double aspirate step is used on the final cycle to ensure that no liquid is left in the well. When using the automated wash procedure it is recommended that the wells are inverted and tapped on paper towel or tissue after the last aspirate. b) Protocol for manual washer (i) Aspirate the first row of wells. (ii) Completely fill this row with working strength Wash Fluid. (iii) Repeat this procedure for each row of wells in turn. (iv) Ensure that each row of wells is left to soak for 30 seconds. (v) Repeat (i) to (iv) a further 4 times. (vi) Aspirate the contents of the wells. It is recommended that the wells are inverted and tapped dry on paper towel or tissue after each wash. NOTE: The correct washing procedure is CRITICAL for the successful performance of the test. If the liquid is not completely aspirated from each well or if the wells are not completely filled or if the fill volume is less than 300 µl erroneous results are likely to be obtained. Do not allow the wells to become dry during the assay procedure. Pre-rinse the washer with diluted wash solution before use. Washers must be rinsed with distilled or deionised water at the end of the test to avoid blockage or corrosion. Detailed protocols for programming recommended automated washers can be found in the Wash Protocol Instructions available from your Murex representative. QUALITY CONTROL Calculate N, eliminating controls above Calculate P, eliminating controls under If more than half the number of either set of controls have to be eliminated, the test run should be repeated after careful investigation into the source of errors. The absorbance at 450/ nm of each Negative Control should be less than The absorbance at 450/ nm of each Positive Control should be greater than RESULTS CALCULATION OF RESULTS Abbreviations: N = the mean of the absorbance of the Negative Controls P = the mean of the absorbance of the Positive Controls S = the absorbance of the sample Cut-off Value Calculate the Cut-off value as N INTERPRETATION OF RESULTS A sample is NON-REACTIVE if S < N A sample is REACTIVE if S > N All reactive samples should be retested in duplicate. Repeatedly reactive samples can be further evaluated by neutralisation of HIV antigens, using the HIV Antigen mab Neutralization Reagents (1F98-02). Neutralisable samples should be considered as HIV antigen positive: If using the INNOTEST TM HIV Antigen mab Neutralization Reagents follow step 1 to 8 of the Test Procedure and then follow the Test Procedure in the Murex HIV Antigen mab IFU from step 5 to 12. LIMITATIONS OF THE PROCEDURE The Assay Procedure and Interpretation of Results must be followed. The sole use of this test is for work with individual serum or plasma samples or cell culture supernatant. AIDS and AIDS-related conditions are clinical syndromes and their diagnosis can only be established clinically. EIA testing alone cannot be used to diagnose AIDS, even if the recommended investigation of reactive specimens suggests a high probability that HIV antigen or antibody is present. A negative result with an antigen detection test does not preclude the possibility of infection with HIV. After seroconversion, p24 antigen may be complexed with antibodies and become undetectable. Samples with elevated levels of Rheumatoid Factor may yield false positive results with the Murex HIV Antigen mab. In these cases the HIV Antigen mab Neutralization Reagents will show the samples to be falsely positive. SPECIFIC PERFORMANCE CHARACTERISTICS Sensitivity The detection limit of the Murex HIV Antigen mab was calculated based on three different batches, using a standard, HIV 1 Antigen (Sanofi Diagnostics Pasteur), diluted in human serum. An average detection limit of 10 pg/ml was reached. A total of 82 clinical samples found to be positive for p24 antigen with an alternative antigen test were tested on the Murex HIV Antigen mab. All 82 samples were found to be reactive and could be neutralized to more than 50% using the Innotest HIV Antigen mab Neutralization Reagents. A total of 79 HIV-1 and 2 HIV-2 cell culture supernatents were tested. All supernatents were found to be reactive and could be neutralized using the Innotest HIV Antigen mab Neutralization Reagents. A total of 35 seroconversion panels were tested on Murex HIV Antigen mab and on an alternative antigen test. The mean duration of antigenemia observed with the Murex HIV Antigen mab was 18.5 days while it was 14.9 days for the alternative test. One BBI HIV Antigen Mixed Titer panel (PRA203) was tested and all 18 positive samples were found to be reactive. Specificity A total of 2000 healthy blood donors were tested, After initial testing 7 were found to be reactive and 4 of these samples were found to be repeatedly reactive. None of these samples could be neutralized using the Innotest HIV Antigen mab Neutralization Reagents, resulting in a specificity of 100% after neutralization. A total of 300 samples from seronegative patients belonging to risk groups and 122 samples from seronegative patients with opportunistic infections were tested. After initial testing, 10 samples were found to be reactive. Eight of these 10 initially reactive samples were tested with the Innotest HIV Antigen mab Neutralization Reagents and none of these 8 was confirmed as positive. A total of 179 potentially interfering samples were tested. Thirteen of these samples were reactive after initial testing of which 4 were positive after repeat testing as well. These 4 samples were out of a total of 34 patients with rheumatoid factor. None of these samples could be neutralised. Precision Intra-assay variation was determined by testing one negative sample and 2 (high and medium) positive samples in 32 replicates in one run. The %CV was 10.4% for the negative sample, 2.3% for the highly positive sample, and 3.6% for the medium sample. Inter-assay variation was determined by testing 11 negative and 14 positive samples on 5 different days. The %CV varied between 6.5% and 14.6% for the negative samples and 4.6% to 10.2% for the positive samples. Interlot variation was determined by testing the BBI p24 Antigen Mixed Titer Performance panel on 3 different lots. The %CV varied between 1.5% and 20.3%. 5

6 BIBLIOGRAPHY 1 Clavel F., et al. Isolation of a new human retrovirus from West African patients with AIDS. Science, 1986;233: De Leys R., et al. Isolation and practical characterization of an unusual human immunodeficiency retrovirus from two persons of West Central African origin. J. Virology, 1990;65: Gürtler L.G., et al. A new subtype of immunodeficiency virus type 1 (MVP-5180) from Cameroon. J. Virology, 1994;68: Myers G. Tenth anniversary perspectives on AIDS. AIDS research and human retroviruses 1994;10. 5 Guyader M., et al. Genome organization and transactivation of the human immunodeficiency virus type 2. Nature, 1987;326: Vanden Haesevelde M., et al. Genomic cloning and complete sequence analysis of a highly divergent African human immunodeficiency virus isolate. J Virology, 1994;68: Coombs R.W., et al. Plasma viremia in human immunodeficiency virus infection. N Engl J Med., 1989;321: von Sydow M., et al. Antigen detection in primary HIV infection. BMJ., 1988;296: Eyster M.E., et al. Predictive markers for the acquired immunodeficiency syndrome (AIDS) in hemophiliacs: persistence of p24 antigen and low T4 cell count. Ann Intern Med., 1989;110: Proclin is not a trade mark of Abbott Manufacturer: Murex Biotech Limited Central Road, Temple Hill, Dartford DA1 5LR UK Distributed by: ABBOTT Max-Planck-Ring Wiesbaden Germany C11VK86GB February