INNOTEST HIV Antigen mab Neutralization Reagents
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1 KEY-CODE: FRI INNOTEST HIV Antigen mab Neutralization Reagents v p 1/6 English INNOTEST HIV Antigen mab Neutralization Reagents Manufactured by: Fujirebio Europe N.V. Technologiepark Gent Belgium BTW BE RPR Gent Distributed by: Fujirebio Germany GmbH Hans-Böckler-Allee Hannover Germany Fujirebio Italia S.r.l. Via Vaccareccia 39/A Pomezia (Roma) Italy Fujirebio France SARL Les Conquérants, Bât. Le Kilimandjaro 8/10, avenue des Tropiques Les Ulis France Fujirebio Iberia S.L. Calle Tarragona 161, Planta Barcelona Spain Fujirebio Europe N.V Technologiepark Gent Belgium Other languages see / Autres langues voir / Andere Sprachen siehe / Altre lingue vedere / Ver otros idiomas / Outras línguas ver: EUROPE GR IS LT RO SK TR LI MT non-europe US CA AR, BR, CO, UY, AU, NZ, RU :00 17:00 GMT+1 M T W T F S S Fujirebio Europe N.V.
2 80565 INNOTEST HIV Antigen mab Neutralization Reagents / v0 / KEY-CODE: FRI10285 p 2/6 TABLE OF CONTENTS Symbols used... 2 Intended use... 3 Test principle... 3 Reagents... 3 Description, preparation for use, and recommended storage conditions... 3 Materials required but not provided... 3 Safety and environment... 3 Specimen... 4 Test procedure... 4 Results... 4 Abbreviations... 4 Validation... 5 Interpretation... 5 Limitations... 5 Test performance... 5 Sensitivity... 5 Specificity... 5 Potentially cross-reactive samples and false-positive samples... 5 Precision... 5 Trademarks... 6 INNOTEST is a worldwide registered trademark such as in Europe, USA, Japan, China... 6 Symbols used Manufacturer In Vitro Diagnostic Medical Device Batch code Catalogue number Use By Consult Instructions for Use Temperature limitation Contains sufficient for <n> tests Control Solution Neutralization Solution
3 80565 INNOTEST HIV Antigen mab Neutralization Reagents / v0 / KEY-CODE: FRI10285 p 3/6 Intended use Reagents for the confirmation of HIV p24 antigens in specimens found repeatedly reactive with the INNOTEST HIV Antigen mab test. Test principle Samples found reactive with the INNOTEST HIV Antigen mab test are pre-incubated with a specific neutralizing reagent (human antibodies against HIV), followed by the INNOTEST HIV Antigen mab procedure. HIV antigen, if present in the sample, will form a complex with the specific "neutralizing" antibodies. This neutralized antigen is prevented from binding to anti-hiv antibodies coated on the microwells and will be eliminated by washing so that the HRP-labeled conjugate is no longer able to bind. The result is a reduction in coloration of 50% or more in the neutralization well compared to the control well. Reagents Description, preparation for use, and recommended storage conditions - If kept at 2-8 C, all test reagents are stable until the expiry date given on the pack. Do not freeze reagents. - All reagents must be brought to room temperature (15-30 C) approximately 30 minutes before use and returned to the refrigerator immediately after use. - Keep reagents in their original vials, which must be closed for storage. - Alterations in physical appearance of kit reagents may indicate instability or deterioration. Each pack contains a securitainer with reagents for 20 tests: 1. 1 vial containing 1 ml of Control Solution (phosphate buffer containing specimen inactivating NP40, 0.05% Proclin as preservative, bovine casein as stabilizer and bovine aprotinin as protease inhibitor) vial containing 1 ml of Neutralizing Solution (human anti-hiv antibodies, containing specimen inactivating NP40, 0.05% Proclin as preservative, bovine casein as stabilizer and bovine aprotinin as protease inhibitor). Following components are supplied with the INNOTEST HIV Antigen mab kit: 1. Positive control: Dilute Positive Control with Matrix. Prepare 120 µl of Positive Control. 2. Diluted conjugate 1: Dilute Conjugate 1 20x with Conjugate Diluent 1. Prepare 1.5 ml of diluted Conjugate 1 for 20 tests. NOTE: Diluted Conjugate 1, used in the neutralization procedure, is different from Conjugate Working Solution 1, which can only be used in the INNOTEST HIV Antigen mab test procedure. Materials required but not provided The neutralization test is performed in combination with the INNOTEST HIV Antigen mab kit, 96T (Ref ) and 480T (Ref ). Safety and environment Please refer to the Safety Data Sheet (SDS) and product labelling for information on potentially hazardous components. The most recent SDS version is available on the website Warning: Contains mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-2Hisothiazol-3-one: CONTROL SOLN, NEUTR SOLN H319 H317 P280 P305+P351+P338 P363 P333+P313 P337+P313 P302+P352 Hazard statements H317 May cause an allergic skin reaction. H319 Causes serious eye irritation. Precautionary statements P280 Wear protective gloves/protective clothing/eye protection/face protection. P302+P352 IF ON SKIN: Wash with plenty of soap and water. P305+P351+P338 IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. P333+P313 If skin irritation or rash occurs: Get medical advice/attention. P337+P313 If eye irritation persists: Get medical advice/attention. P363 Wash contaminated clothing before reuse.
4 80565 INNOTEST HIV Antigen mab Neutralization Reagents / v0 / KEY-CODE: FRI10285 p 4/6 - The neutralizing solution, containing human anti-hiv antibodies, has been found negative for HBsAg and HCV. - No test method can offer complete insurance that human blood products will not transmit infectious agents. Therefore, all blood components and biological materials should be considered as being potentially infectious and should be handled as such. - Only adequately trained personnel should be permitted to perform the test procedure. They should be disposed of in accordance with established safety procedures. Autoclave for at least 15 minutes at 121 C. Incinerate disposable material. Mix liquid waste with sodium hypochorite so that the final concentration is ± 1% sodium hypochlorite. Allow to stand overnight before disposal. CAUTION: Neuralize liquid waste that contains acid before adding sodium hypochlorite. - Use of personal protective equipment is necessary: gloves and safety spectacles when manipulating dangerous or infectious agents. - Waste should be handled according to the institutions' waste disposal guidelines. Also observe all federal, state and local environmental regulations. Specimen - Repeatedly reactive specimens (serum, plasma or cell culture supernatant) with the INNOTEST HIV Antigen mab. - 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, lipemic or hemolyzed 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 run with a HIV-negative cell culture supernatant as control. - Store samples at 2-8 C. For storage longer than one week, freeze aliquots at -20 C. - Before storage, serum or plasma should be separated from blood clot or blood cells by centrifugation. Test procedure Before starting the test, be sure that all reagents (including those of the INNOTEST HIV Antigen mab) are at room temperature (15-30 C). 1. Reserve 2 wells of the microtiter plate for each sample. These wells are called control well A and neutralization well B, respectively. In addition, treat the Positive Control as a sample to check the proper activity of Neutralization and Control Solution. 2. Reserve 1 well for the Positive Control and 1 well for the Negative Control (supplied with INNOTEST HIV Antigen mab). 3. Add 50 µl of the Control Solution to each control well A. 4. Add 50 µl of the Neutralizing Solution to each neutralization well B. 5. Add 50 µl of NP40 solution (supplied with the INNOTEST HIV Antigen mab) to the wells reserved for the Negative and Positive Controls. 6. Add 50 µl of the diluted Conjugate 1 to each well. NOTE: Diluted Conjugate 1, used in the neutralization procedure is different from Conjugate working solution 1, which can only be used in the INNOTEST HIV Antigen mab test procedure. 7. Add 100 µl of each sample into the control well A and the neutralization well B. Add 100 µl of Positive and Negative Control in their respective wells. Mix the contents of the wells adequately by pipetting up and down 5 times, or by using a plate shaker at 1000 rpm for 1 minute. 8. Cover the strips with an adhesive sealer. 9. Incubate for 60 minutes at 37 C. Proceed with steps 3 to 7 of the INNOTEST HIV Antigen mab test procedure. 10. Read the absorbance of the solutions in the wells at 450 nm with a microplate reader. Do not use dual wavelength analysis ( or 620 nm). Results Abbreviations N = the absorbance of the Negative Control P = the absorbance of the Positive Control
5 80565 INNOTEST HIV Antigen mab Neutralization Reagents / v0 / KEY-CODE: FRI10285 p 5/6 A = the absorbance of the control well A B = the absorbance of the neutralization well B BP = the absorbance of the neutralized Positive Control AP = the absorbance of the Positive Control in well A Validation - The test run is valid if N < 0.150, P > and BP 0.5 x AP. - If the absorbance of the control well A is higher than the limit of the microplate reader (overflow), the sample should be retested after dilution with an HIV negative human serum. Interpretation Calculate the cut-off value as N % reduction = (A - B) x 100 A A sample is confirmed as positive for HIV p24 antigen if the control well A has an absorbance above the cut-off value and there is a reduction in coloration of 50% or more in the neutralization well compared to the control well. A N and B 0.5 x A NOTE: The expected absorbance range at 450 nm of the Negative Control is ± 0.010, resulting in an expected cut-off value of: ± The expected absorbance range at 450 nm of the Positive Control is ± The expected absorbance range at 450 nm of the neutralized Positive Control is ± 0.016, corresponding with a reduction of about 90%. Neutralized samples should be considered as HIV antigen positive. A negative sample does not preclude the possibility of exposure to or infection with HIV. Limitations - Please read the limitations of the INNOTEST HIV Antigen mab assay carefully. - The provided protocol must be strictly followed to obtain optimum performance of the assay. - Repeatedly frozen and thawed, lipemic or hemolyzed samples may produce erroneous results. Test performance Sensitivity A total of 95 viral culture supernatants as well as 22 samples positive for HIV antibodies and reactive on the INNOTEST HIV Antigen mab, were tested. Upon neutralization, all but one sample gave a reduction in coloration of more than 50%. The sample that was not neutralized was negative on an alternative commercially available HIV Antigen assay. Two BBI HIV Antigen Mixed Titer panels (PRA201 and PRA202) were tested using INNOTEST HIV Antigen mab Neutralization Reagents. All positive samples gave a reduction in coloration of more than 50%. Specificity Healthy blood donors A total of 197 sera from healthy blood donors were tested, resulting in similar results for the absorbance of the control and neutralization well. Potentially cross-reactive samples and false-positive samples For 6 potentially cross-reactive samples showing a positive result with the INNOTEST HIV Antigen mab, less than 50% reduction in coloration was observed upon neutralization. Fourteen samples with no evidence for HIV infection and 4 from healthy blood donors found to be positive with the INNOTEST HIV Antigen mab were tested. None of these samples were neutralized by more than 50%, indicating that the initial reaction was false positive. Precision Intra-assay variability of the % neutralization was determined by testing 3 (high, medium and low) positive samples in 14 replicates in one run. The %CV varied between 0.3 and 3.7%
6 80565 INNOTEST HIV Antigen mab Neutralization Reagents / v0 / KEY-CODE: FRI10285 p 6/6 Inter-assay variability of the % neutralization was determined by testing 5 positive samples and the positive control on 6 different days. The %CV varied from 0.3 to 3.9%. Interlot variability of the % neutralization was determined by testing 14 positive samples and 1 negative sample on 3 different lots. The %CV varied from 0.1 to 3.2% Trademarks INNOTEST is a worldwide registered trademark such as in Europe, USA, Japan, China
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