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1 bioelisa HIV x 96 TESTS x 96 TESTS (480) ELISA test for the detection of antibodies to Human Immunodeficiency Viruses (HIV) type 1 (group M-O) or type 2 in human serum or plasma samples in clinical laboratories and as a first-line screening assay in blood centres. Summary Serological evidence of infection with HIV may be obtained by testing for presence of HIV antigens or antibodies in serum of individuals suspected for HIV infection. Antigen can generally be detected during both acute phase and the symptomatic phase of AIDS only. The antibodies to HIV-1 and/or HIV-2 can be detected throughout virtually the whole infection period, starting at or shortly after the acute phase and lasting till the end stage of AIDS. Therefore, the use of highly sensitive antibody assays is the primary approach in serodiagnosis of HIV infection. Apart from sexual transmission, the principal route of infection with HIV is blood transfusion. HIV can present both in cellular and cell-free fractions of human blood. Therefore, all donations of blood or plasma should be tested because due to the risk of HIV transmission through contaminated blood. This can be effectively achieved by testing for the antibodies to HIV-1 and HIV-2 by using a highly sensitive ELISA tests. Principle READ HIGHLIGHTED CHANGES bioelisa HIV is a two step incubation antigen sandwich enzyme immunoassay kit, in which the wells of a microplate are coated with recombinant HIV antigens expressed in E. coli (recombinant HIV-1 gp41, gp120, and recombinant HIV-2 gp-36). Serum or plasma samples are added to these wells. If specific HIV1/2 antibodies are present in the sample, they will form stable complexes with the HIV recombinant antigens. The microwells are then washed to remove unbound serum proteins. A second set of recombinant antigens conjugated with peroxidase and expressing the same epitopes are added to the wells. During the second incubation step, these antigens will bind to the specific captured antibodies. After a second wash to remove unbound conjugate a Chromogen solutions are added to the wells. In wells containing the antigen-antibody-antigen sandwich immunocomplex, the colorless Chromogens are hydrolyzed by the bound conjugate to a blue colored product. The blue color changes to yellow after blocking the reaction with sulfuric acid. The intensity of colour can be measured and is proportional to the anti-hiv 1/2 antibodies concentration in the sample. Wells containing negative samples remain colourless. Components 1. MCPL MICROPLATE: 12 x 8 wells coated with recombinant HIV 1/2 antigens (recombinant HIV-1 gp41, gp120, and recombinant HIV-2 gp36). The microwell strips are for SINGLE USE only. Do not use if the vacuum sealing has been damaged when first time taken of out the box. 2. CONTROL + 1 HIV-1 POSITIVE CONTROL: Antibodies to HIV-1 diluted in protein stabilized buffer. Contains 0.1% ProClin TM 300 as preservative and red dye. Ready to use. 3. CONTROL + 2 HIV-2 POSITIVE CONTROL: Antibodies to HIV-2 diluted in protein stabilized buffer. Contains 0.1% ProClin TM 300 as preservative and red dye. Ready to use. 4. CONTROL NEGATIVE CONTROL: Protein stabilized buffer tested negative for HBsAg and for antibodies to HIV-1, HIV-2, HCV and TP. Contains 0.1% ProClin TM 300 as preservative and yellow dye. Ready to use. 5. CONJ CONJUGATE: HIV-1 and HIV-2 recombinant antigens conjugated with peroxidase. Contains 0.1% ProClin TM preservative and red dye. Ready to use. 300 as 6. WASH SOLN 20x CONCENTRATE WASHING SOLUTION: Concentrate PBS buffer ph 7.4 (20x). Contains Tween-20 as detergent. To be diluted 1/20 in distilled or deionised water before use. 7. CHROM A CHROMOGEN SOLUTION A: Urea peroxide solution. Ready to use R eng

2 8. CHROM B CHROMOGEN SOLUTION B: TMB solution (Tetramethyl benzidine dissolved in citric acid). 9. H 2 SO 4 0.5M STOPPING SOLUTION: 0.5M sulphuric acid. Ready to use. 10. SEALS ADHESIVE SEALS: To cover the microplate during incubations. 11. BAG RESEALABLE BAG: For storage of unused strips. Precautions bioelisa HIV is intended for IN VITRO diagnostic use. For professional used only. IVD WARNING: Materials from human origin may have been used in the preparation of the negative control of the kit. These materials have been tested with tests kits with accepted performance and found negative for antibodies to HIV 1/2, HCV, TP and HBsAg. However, there is no analytical method that can assure that infectious agents in the specimens or reagents are completely absent. Therefore, handle reagents and specimens with extreme caution as if capable of transmitting infectious diseases. Bovine derived sera have been used for stabilizing of the positive and negative controls. Bovine serum albumin (BSA) and fetal calf sera (FCS) are derived from animals from BSE/TSE free-geographical areas. The Negative Control, HIV-1 Positive Control, HIV-2 Positive Control and Conjugate contains 0.1% ProClin TM as preservative. The following are the appropriate risk (R) and safety (S) phrases: R43 May cause sensitisation by skin contact. S26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. S28 After contact with skin, wash immediately with plenty of water. S36/37/39 Wear suitable protective clothing, gloves and eye/face protection. S45 In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible). S60 This material and its container must be disposed of as hazardous waste. S61 Avoid release to the environment. Refer to special instructions/ Safety data sheets. The ELISA assays are time and temperature sensitive. To avoid incorrect result, strictly follow the test procedure steps and do not modify them. 1. Do not exchange reagents from different lots or use reagents from other commercially available kits. The components of the kit are precisely matched for optimal performance of the tests. 2. Make sure that all reagents are within the validity indicated on the kit box and of the same lot. Never use reagents beyond their expiry date stated on labels or boxes. 3. CAUTION - CRITICAL STEP: Allow the reagents and specimens to reach room temperature (18-30 C) before use. Shake reagent gently before use. Return at 2-8 C immediately after use. 4. Use only sufficient volume of sample as indicated in the procedure steps. Failure to do so, may cause in low sensitivity of the assay. 5. Do not touch the bottom exterior of the wells; fingerprints or scratches may interfere with the reading. When reading the results, ensure that the plate bottom is dry and there are no air bubbles inside the wells. 6. Never allow the microplate wells to dry after the washing step. Immediately proceed to the next step. Avoid the formation of air bubbles when adding the reagents. 7. Avoid assay steps long time interruptions. Assure same working conditions for all wells R eng

3 8. Calibrate the pipette frequently to assure the accuracy of samples/reagents dispensing. Use different disposal pipette tips for each specimen and reagents in order to avoid cross-contaminations. 9. Assure that the incubation temperature is 37 C inside the incubator. 10. When adding specimens, do not touch the well s bottom with the pipette tip. 11. When measuring with a plate reader, determine the absorbance at 450 nm or at 450/ nm. 12. The enzymatic activity of the conjugate might be affected from dust and reactive chemical and substances like sodium hypochlorite, acids, alkalis etc. Do not perform the assay in the presence of these substances. 13. If using fully automated equipment, during incubation, do not cover the plates with the plate cover. The tapping out of the remainders inside the plate after washing, can also be omitted. 14. All specimens from human origin should be considered as potentially infectious. Strict adherence to GLP (Good Laboratory Practice) regulations can ensure the personal safety. 15. Never eat, drink, smoke, or apply cosmetics in the assay laboratory. Never pipette solutions by mouth. 16. Chemical should be handled and disposed of only in accordance with the current GLP (Good Laboratory Practices) and the local or national regulations. 17. The pipette tips, vials, strips and specimen containers should be collected and autoclaved for not less than 2 hours at 121 C or treated with 10% sodium hypochlorite for 30 minutes to decontaminate before any further steps of disposal. Solutions containing sodium hypochlorite should NEVER be autoclaved. Materials Safety Data Sheet (MSDS) available upon request. 18. Some reagents may cause toxicity, irritation, burns or have carcinogenic effect as raw materials. Contact with the skin and the mucosa should be avoided but not limited to the following reagents: Stopping solution, the Chromogens, and the washing solution. INDICATIONS OF INSTABILITY DETERIORATION OF THE REAGENT: Values of the positive or negative controls, which are out of the indicated quality control range, are indicators of possible deterioration of the reagents and/or operator or equipment errors. In such case, the results should be considered as invalid and the samples must be retested. In case of constant erroneous results and proven deterioration or instability of the reagents, immediately substitute the reagents with new one or contact your local distributor or Biokit technical support for further assistance. Storage and stability The components of the kit will remain stable through the expiration date indicated on the label and package when stored between 2-8 C, do not freeze. To assure maximum performance of bioelisa HIV kit, during storage protect the reagents from contamination with microorganism or chemicals. The microwell strips can be broken to be used separately. Place unused wells or strips in the plastic sealable storage bag together with the desiccant and return to 2-8 C. Once open the microplate is stable one month at 2-8 C. The negative control, HIV-1 positive control, HIV-2 positive control, conjugate, chromogen solution A, chromogen solution B and stopping solution, once open are stable one month at 2-8 C. The washing solution, once diluted, is stable for one week at room temperature or for two weeks at 2-8 C. Available packaging - 1 plate kit (96 tests), REF Contains: 1 plate, 1 x 1 ml negative control, 1 x 1 ml HIV-1 positive control, 1 x 1 ml HIV-2 positive control, 1 x 12 ml conjugate, 1 x 50 ml washing solution, 1 x 8 ml chromogen solution A, 1 x 8 ml chromogen solution B, 1 x 8 ml stopping solution, 1 plastic bag and adhesive seals (6 units). - 5 plates kit (5 x 96 tests), REF Contains: 5 plate, 3 x 1 ml negative control, 3 x 1 ml HIV-1 positive control, 3 x 1 ml HIV-2 positive control, 5 x 12 ml conjugate, 2 x 125 ml washing solution, 1 x 60 ml chromogen solution A, 1 x 60 ml chromogen solution B, 1 x 60 ml stopping solution, 1 plastic bag and adhesive seals (20 units) R eng

4 Material required not provided - Distilled or deionized water. - Disposable gloves and timer. - Appropriate waste containers for potentially contaminated materials. - Disposable V-shaped troughs. - Dispensing system and/or pipette (single or multichannel) and disposable pipette tips. - Absorbent tissue or clean towel. - Dry incubator or water bath, 37 ± 1 C. - Microshaker for dissolving and mixing conjugate with samples. - Microplate reader with a 450 nm filter. Reference filter of 620 nm or 630 is advisable. - Manual or automated wash system. Sample collection transporting and storage 1. No special patient s preparation required. Collect the specimen in accordance with the normal laboratory practice. Either fresh serum or plasma specimens can be used with this assay. Blood collected by venipuncture should be allowed to clot naturally and completely the serum/plasma must be separated from the clot as early as possible as to avoid haemolysis. Care should be taken to ensure that the serum specimens are clear and not contaminated by microorganisms. Any visible particulate matters in the specimen should be removed by centrifugation at 3000 RPM (round per minutes) for 20 minutes at room temperature or by filtration. 2. Plasma specimens collected into EDTA, sodium citrate or heparin may be tested, but highly lipaemic, icteric, or hemolytic specimens should not be used as they can give false results in the assay. Do not heat inactivate specimens. This can cause deterioration of the target analyte. Samples with visible microbial contamination should never be used. 3. bioelisa HIV is intended ONLY for testing of individual serum or plasma samples. Do not use the assay for testing of cadaver samples, saliva, urine or other body fluids, or pooled (mixed) blood. 4. Transportation and Storage: Store specimens at 2-8 C. Specimens not required for assaying within 3 days should be stored frozen (-20 C or lower). Multiple freeze-thaw cycles should be avoided. For shipment, samples should be packaged and labeled in accordance with the existing local and international regulations for transportation of clinical samples and etiological agents. Automatic processing Automated or semi-automated assay may be used with different instruments. It is very important to validate any automated system to demonstrate that results obtained for samples are equivalent to the ones obtained using manual assay. It is recommended that the user validate periodically the instrument. If there is any difficulty in the programming and setting of Biokit automatic processors, please contact your distributor. PROCEDURE (see procedural flow chart) Previous operations Allow all the reagents to reach room temperature (20-25 C) before running the assay. Check the washing solution concentrate for the presence of salt crystals. If crystals have formed, resolubilize by warming at 37 C until crystals dissolve. Dilute 1:20 the washing solution as indicated in the washing step instructions. Use distilled or deionized water and only clean vessels to dilute the buffer. All other reagents are READY TO USE. Washing step instructions A good washing procedure is essential in order to obtain correct and precise analytical data. Is therefore recommended to use a good quality ELISA microplate washer, maintained at the best level of washing performances. In general no less than 5 automatic washing cycles of µl/well are sufficient to avoid false positive reactions and high background. To avoid cross-contaminations of the plate with specimen or conjugate, after incubation do not discard the content of the wells but allow the microplate washer to aspirate it automatically. Assure that the microplate washer liquid dispensing channels are not blocked or contaminated and sufficient volume of washing solution is dispensing each time into the wells. In case of manual washing, we suggest to carry out 5 washing cycles, dispensing µl/well and aspirating the liquid for 5 times. If poor results (higher background) are observed, increase the washing cycles or soaking time per well. In any case, the liquid aspirated out the strips should be treated with a sodium hypochlorite solution at a final concentration of 2.5% for 24 hours, before they are wasted in an appropriate way. The concentrated washing solution should be diluted 1:20 before use. If less than a whole plate is used, prepare the proportional volume of solution R eng

5 Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Preparation: Mark three wells as negative control (e.g. B1, C1, D1), two wells as positive control (e.g. E1 for HIV-1 and F1 for HIV-2) and one Blank (e.g. A1, neither samples or conjugate should be added into the Blank well). If the results will be determined by using dual wavelength plate reader, the requirement for use of Blank well could be omitted. Use only number of strips required for the test. Adding Sample: Add 100 μl of samples, and 100 μl of positive and negative controls into their respective wells. NOTE: Use a separate disposable pipette tip for each specimen, negative and positive control as to avoid cross-contamination. Incubating Sample: Cover the plate with the plate cover and incubate at 37 C for 30 ± 1 minutes. It is recommended to use thermostat-controlled water tank to assure the temperature stability and humidity during incubation. If dry incubator is used, do not open the door frequently. Washing(1): After the end of the incubation, remove and discard the plate cover. Wash each well 5 (five) times with diluted Washing buffer. Each time allow the microwells to soak for seconds. After the final washing cycle, turn down the plate onto blotting paper or clean towel and tap it to remove any remainders. Adding Conjugate: Add 100 μl of conjugate reagent into each well except the Blank. Incubating Conjugate: Cover the plate with the plate cover and incubate at 37 C for 30 ± 1 minutes. Step 7 Washing(2): As Step 4. Step 8 Step 9 Step 10 Coloring: Add 50 μl of chromogen A and 50 μl of chromogen B solutions into each well including the Blank. Incubate the plate at 37 C for 15 minutes avoiding light. The enzymatic reaction between the chromogen solutions and the conjugate produces blue color in positive control and HIV 1/2 positive sample wells. Stopping Reaction: Using a multichannel pipette or manually, add 50 µl of stopping solution into each well and mix gently. Intensive yellow color develops in positive control and HIV 1/2 positive sample wells. Measuring the Absorbance: Calibrate the plate reader with the Blank well and read the absorbance at 450 nm. If a dual filter instrument is used, set the reference wavelength at 620 nm or 630 nm. Calculate the cut-off value and evaluate the results. (NOTE: read the absorbance within 10 minutes after stopping the reaction). Quality control It is recommended that each laboratory must establish appropriate quality control system with quality control material similar to or identical with the patient sample being analyzed. - The Absorbance value of the Blank well, which contains only chromogen and stopping solution, must be less than at 450 nm. - The Absorbance value of the positive control must be at 450/ nm or at 450 nm after subtracting the blank. - Each individual absorbance value of the negative control must be less than at 450/ nm or at 450 nm after subtracting the blank. If one of the negative control values does not meet the Quality Control criteria, it should be discarded and the mean value calculated again using the remaining two values. If more than one negative control Absorbance values do not meet the Quality Control Range specifications, the test is invalid and must be repeated. Results Each microplate should be considered separately when calculating and interpreting the results of the assay, regardless of the number of plates concurrently processed. The results are calculated by relating each specimen absorbance (S) value to the cut-off value (CO) of the plate. If the cut-off reading is based on single filter plate reader, the results should be calculated by subtracting the Blank well A value from the print report values of specimens and controls. In case the reading is based on dual filter plate reader, do not subtract the Blank well A value from the print report values of specimens and controls R eng

6 Calculate the cut-off value by adding to the mean absorbance of the negative control. Cut-off = NCx Example: Blank well Absorbance value: A1= at 450 nm (NOTE: blanking is required only when reading with single filter at 450 nm). Well No.: B1 C1 D1 Neg Ctrl: Well No.: E1 F1 Pos Ctrl: All controls values are within the stated quality control range. Calculation of the NCx = ( )/3 = Calculation of the cut-off: (CO) = = Interpretation of results Divide the sample absorbance by the cut-off value. (S/CO) Negative Results (S/CO < 1): Specimens giving absorbance less than the cut-off value are negative for this assay, which indicates that no anti-hiv 1/2 antibodies have been detected with bioelisa HIV kit. A negative result does not exclude the possibility of exposure or infection with HIV. Positive Results (S/CO 1): Specimens giving an absorbance equal to or greater than the cut-off value are considered initially reactive, which indicates that anti-hiv 1/2 antibodies have probably been detected using bioelisa HIV All initially reactive specimens should be retested in duplicates using bioelisa HIV before the final assay results interpretation. Repeatedly reactive specimens can be considered positive for antibodies to HIV 1/2 with bioelisa HIV Borderline (S/CO = ): Specimens with absorbance to cut-off ratio between 0.9 and 1.1 are considered borderline and retesting of these specimens in duplicates is required to confirm the initial results. Follow-up, confirmation and supplementary testing of any positive specimen with other analytical system (e.g. WB, PCR) is required. Clinical diagnosis should not be established based on a single test result. It should integrate clinical and other laboratory data and findings. INITIAL RESULTS INTERPRETATION AND FOLLOW-UP ALL INITIALY REACTIVE OR BORDERLINE SAMPLES Rep. in duplicate,+,+,+,-,-,- Interpretation + S/CO 0.9 S/CO < 0.9 Follow-up Interpretation IND - IND = non interpretable - If, after retesting of the initially reactive samples, both wells are negative results (S/CO < 0.9), these samples should be considered as non-repeatable positive (or, false positive) and recorded as negative. As with many very sensitive ELISA assays, false positive results can occur due to the several reasons, most of which are connected with, but not limited to, inadequate washing step. For more information regarding bioelisa troubleshooting, please refer to bioelisa Troubleshooting Guide. - If after retesting in duplicates, one or both wells are positive results, the final result from this ELISA test should be recorded as repeatedly reactive. Repeatedly reactive specimens can be considered positive for antibodies to HIV 1/2 and therefore the patient is probably infected with HIV 1/2 and the blood unit must be discarded. - After retesting in duplicates, samples with values close to the cut-off value should be interpreted with caution and considered as " borderline" zone sample, or uninterpretable for the time of testing R eng

7 Limitations of the procedure 1. Positive results must be confirmed with another available method and interpreted in conjunction with the patient clinical information. 2. Antibodies may be undetectable during the early stage of the disease and in some immunosuppressed individuals. Therefore, negative results obtained with bioelisa HIV are only indication that the sample does not contain detectable level of anti-hiv 1 or 2 antibodies and any negative result should not be considered as conclusive evidence that the individual is not infected with HIV 1 or The most common assay mistakes are: using kits beyond the expiry date, bad washing procedures, contaminated reagents, incorrect assay procedure steps, insufficient aspiration during washing, failure to add specimens or reagents, improper operation with the laboratory equipment, timing errors, the use of highly heamolysed specimens or specimens containing fibrin, incompletely clotted serum specimens. 4. The prevalence of the marker will affect the assay s predictive values. 5. This assay cannot be utilize to test pooled (mixed) plasma. bioelisa HIV has been evaluated only with individual serum or plasma specimens. 6. bioelisa HIV is a qualitative assay and the results cannot be use to measure antibodies concentrations. This assay cannot distinguish between infections with HIV-1 and HIV-2. Performance characteristics Evaluation study carried in Alkmaar, the Netherlands, between April and November 2005, demonstrated the following performance characteristics of bioelisa HIV The diagnostic specificity of the kit was 99.85% as determined on all negative samples (5471) that were investigated. When examined on the unselected donors only (random and first time donors), the specificity was 99.92% (95% CI %). bioelisa HIV test results on unselected donors: Panel Number tested Positive (S/CO 1) Negative (S/CO < 1) Number % Number % Random serum donor 2, , Random plasma donor 1, , First time donor Total 5, , All panels of HIV-1, HIV-1 subtype O and HIV-2 confirmed antibody positive samples that were used in this study were also tested reactive with bioelisa HIV which resulted in diagnostic sensitivity of 100%. A total of 32 seroconversion panels, which represent 210 samples tested. 13 samples not classified from PRB918 and PRB917 because there are not data of Antigen or RNA determination required for the classification. 41 samples classified as negative. RNA and or Antigen negative. 61 samples classified as early-seroconversion. 95 samples classified as seroconversion. The testing results also show that bioelisa HIV is a state-of-the-art compare to most of the currently available on the market CE-marked tests. The analytical sensitivity was evaluated on PeliCheck anti-hiv panels. The analytical sensitivity of bioelisa HIV on the PeliCheck anti-hiv standard dilutions was comparable to other anti-hiv assays. Analytical specificity: bioelisa HIV test results on samples from hospitalised patients and samples containing potentially cross-reacting blood-specimens: R eng

8 Type of sample Number tested Positive (S/CO 1) Negative (S/CO < 1) Number % Number % Mononucleosis Pregnant woman RF Anti-TPO Anti-smooth muscle Elevated IgG Levels Total In a separate study, the following specificity results were obtained: - Possible high dose hook effect is eliminated due to the implementation of two-step procedure. - Frozen positive/negative specimens have been tested to check for interferences due to collection and storage. The performance characteristics of bioelisa HIV were not affected for at least 3 freeze/thaw cycles. - Samples from patients infected with hepatitis A,B,C and E viruses as well as samples from patients infected with Treponema pallidum were tested with no cross-reactive reactions observed positive fresh serum samples tested in INSTITUTE FOR TROPICAL MEDICINE, BELGIUM have been tested with bioelisa HIV All 25 positive fresh serum samples have been positive with bioelisa HIV Accuracy: The below tables represent the results of analytical sensitivity and reproducibility of bioelisa HIV as controlled with PeliSpyMulti-Marker run control and with Internal QC sample tested in every plate, the 1:2048 dilution of the anti-hiv standard in this PeliSpy sample was consistently detected in all plates. Internal QC sample was always detected in all plates. PeliSpyMulti-Marker results: Percentiles Measured Dilution n Average 5 th 95 th Min Max 1: Internal QC sample results: Percentiles Measured n Average 5 th 95 th Min Max EXAMPLE SCHEME OF CONTROL/SAMPLES DISPENSING A Blank SMP3 B Neg C Neg D Neg E Pos (HIV-1) F Pos (HIV-2) G SMP 1 H SMP R eng

9 bioelisa: Troubleshooting guide Problem Possible causes Solution 1. Controls out of validation. 1a. Incorrect temperature, timing or pipetting. Check procedure. Repeat assay. 1b. Improper preparation of reagents, error of dilution, reagents not well mixed. Check procedure. Repeat assay. 2. No colour or only a light colour developed at the end of the assay. 1c. Cross-contamination of controls. Pipette carefully. Do not interchange caps. Repeat assay. 1d. Incorrect reading filter. Check that the wavelenght of the filter used is 450 nm. If no reference nm is used, absorbance increases approximately e. Interference in the optical pathway. Check the reader. Clean or dry the bottom of microplate. Check for air bubbles. Repeat reading. 1f. Used components from Do not use components from different lots. different lots as they are adjusted for each batch released. 1g. Expired reagents. Check the kit expiry date. Use 2a. One or more reagents not added or added in wrong sequence. 2b. Inactive conjugate: improper conservation. 2c. Inactive microplate: Improper conservation. 2d. Inactive chromogen solutions A and/or B: improper conservation. The container used affects chromogen stability, crosscontamination with the stopping solution. a non- expired kit. Check procedure. Repeat assay. Check for contamination. Recheck procedure. Repeat assay. Always keep unused strips in the bag very well closed, with the desiccant inside. Repeat assay. Use disposable containers or wash with acid or ethanol and rinse with deionised water before re-use. Recheck procedure. Repeat assay R eng

10 bioelisa: Troubleshooting guide Problem Possible causes Solution 3. Too much colour in all microplate wells. 4. Poor reproducibility or high number of non-repeatable reactive samples. 3a. Contaminated, oxidised chromogen solutions A and/or B 3b. Contaminated or improperly prepared reagents. 3c. Contaminated washing solution (1x). 3d. Insufficient washing or washing not consistent: Filling volume and/or aspiration insufficient or not uniform. Insufficient number of washing cycles, contaminated device. Check that chromogen solutions A and B are colourless, discard if blue. Use acid or ethanol washed or disposable containers. Repeat assay. Check for contamination (turbid aspect). Check dilutions. Repeat assay. Check the quality of distilled or deionised water used for dilution. Repeat assay. Check the washing device. Fill wells with washing solution close to the top, aspirate completely. Increase the number of wash cycles. After washing, blot the inverted microplate on tissue paper. 3e. Using of a washing solution from other kit. Use only the washing solution supplied with the kit. 4a. Washing problems. See 3c, 3d, 3e. 4b. Uncalibrated pipettes Use only calibrated pipettes, or tips not well fitted. with well fitted tips and pipette Improper pipetting. carefully, without bubbles and 4c. Reagents and sera not at room temperature or not well mixed before using. 4d. Air currents over the microplate during incubations. 4e. Too long time for addition of samples and/or reagents. Inconsistency in time intervals. Air bubbles. 4f. Interference in the optical pathway. splashing. Repeat assay. Equilibrate reagents to room temperature and mix thoroughly before using. Keep the microplate protected from air currents. Develop consistent and uniform technique. See 1e R eng

11 LEER CAMBIOS SOMBREADOS bioelisa HIV x 96 TESTS x 96 TESTS (480) Test de ELISA para la detección de anticuerpos contra el virus de inmunodeficiencia humana (HIV) tipo 1 (grupos M-O) o tipo 2 en muestras de suero o plasma humano para ser utilizado en laboratorios clínicos y como análisis de cribado de primera línea en bancos de sangre. Sumario La demostración serológica de infección por HIV puede obtenerse comprobando la presencia de antígenos o anticuerpos HIV en el suero de personas con sospecha de infección por HIV. En general, el antígeno sólo se puede detectar en la fase aguda y en la fase sintomática del SIDA. Los anticuerpos frente a HIV-1 o HIV-2 pueden detectarse casi durante todo el periodo de infección, comenzando en la fase aguda o poco después de esta y manteniéndose hasta la fase terminal del SIDA. Por lo tanto, la utilización de análisis de anticuerpos altamente sensibles es el primer abordaje en el serodiagnóstico de la infección por HIV. Aparte de la transmisión sexual, la principal vía de infección por HIV es la transfusión de sangre. El HIV puede aparecer tanto en las fracciones globulares como en las no globulares de la sangre humana. Por ello deberían analizarse todas las donaciones de sangre o plasma debido al riesgo de transmisión de HIV a través de sangre contaminada. Esto se puede conseguir eficazmente mediante la detección de anticuerpos frente al HIV-1 y HIV-2 en pruebas ELISA de alta sensibilidad. Principio bioelisa HIV es un método inmunoenzimático directo, tipo sándwich, de dos pasos de incubación, en donde los pocillos de un microplaca están recubiertos con antígenos recombinantes de HIV expresados en E. coli (recombinantes de HIV-1 gp41 y gp120 y recombinante de HIV-2 gp-36). A estos pocillos se les añade muestras de suero o plasma a analizar. Si en la muestra existen anticuerpos HIV-1/2 específicos, formarán complejos estables con los antígenos recombinantes del HIV. A continuación, se lavan los micropocillos para eliminar las proteínas séricas no ligadas. Se añade a los pocillos un segundo juego de antígenos recombinantes conjugados con la peroxidasa y que expresan los mismos epítopos. Durante la segunda fase de incubación, estos antígenos se unirán a los anticuerpos específicos capturados. Tras un segundo lavado para eliminar el conjugado no unido se añaden a los pocillos las soluciones de cromógeno. En los pocillos que contienen el inmunocomplejo "sándwich" antígeno-anticuerpo-antígeno, los cromógenos incoloros son hidrolizados por el conjugado ligado formando un producto de color azul. El color azul vira a amarillo tras bloquear la reacción con ácido sulfúrico. La intensidad del color es mensurable y proporcional a la concentración de anticuerpos anti-hiv 1/2 en la muestra. Los pocillos que contienen muestras negativas permanecen incoloros. Componentes 1. MCPL MICROPLACA: 12 x 8 pocillos recubiertos con antígenos HIV 1/2 recombinantes (HIV-1 recombinante gp41, gp120 y HIV-2 recombinante gp36). Las tiras de micropocillos son para UN SOLO USO. No utilizarla si el sellado al vacio ha sido dañado cuando se extrae por primera vez de la caja. 2. CONTROL + 1 CONTROL POSITIVO HIV-1: Anticuerpos frente a HIV-1 diluidos en tampón proteínico estabilizado. Contiene 0,1% de ProClin TM 300 como conservante y colorante rojo. Listo para usar. 3. CONTROL + 2 CONTROL POSITIVO HIV-2: Anticuerpos frente a HIV-2 diluidos en tampón proteínico estabilizado. Contiene 0,1% de ProClin TM 300 como conservante y colorante rojo. Listo para usar. 4. CONTROL CONTROL NEGATIVO: Tampón proteínico estabilizado negativo frente a HBsAg y anticuerpos HIV-1, HIV-2, HCV y TP. Contiene 0,1% de ProClin TM 300 como conservante y colorante amarillo. Listo para usar. 5. CONJ CONJUGADO: Antígenos recombinantes HIV-1 y HIV-2 conjugados con peroxidasa. Contiene 0,1% de ProClin TM 300 como conservante y colorante rojo. Listo para usar. 6. WASH SOLN 20x SOLUCIÓN DE LAVADO CONCENTRADA: Tampón PBS concentrado de ph 7,4 (20x). Contiene Tween-20 como detergente. Diluir 1/20 en agua destilada o desionizada antes del uso R spa

12 7. CHROM A SOLUCIÓN DE CROMÓGENO A: Solución de peróxido de urea. Listo para usar. 8. CHROM B SOLUCIÓN DE CROMÓGENO B: Solución TMB (tetrametilbencidina disuelta en ácido cítrico). 9. H 2 SO 4 0,5M SOLUCIÓN DE PARADA: Ácido sulfúrico 0,5M. Listo para usar. 10. SEALS LÁMINAS ADHESIVAS: Para cubrir la microplaca durante las incubaciones. 11. BAG BOLSA DE PLÁSTICO: Para guardar las tiras sin usar. Precauciones bioelisa HIV es para el diagnóstico IN VITRO. Para uso exclusivo por profesionales. IVD ADVERTENCIA: En la preparación del control negativo del kit se han podido utilizar materiales de origen humano. Estos materiales se han analizado con kits analíticos de eficacia comprobada y su resultado ha dado negativo a anticuerpos frente a HIV 1/2, HCV, TP y HBsAg. No obstante, no existe ningún método analítico que garantice la total ausencia de agentes infecciosos en muestras o reactivos. Por tanto, los reactivos y las muestras deben manejarse con extrema precaución, como si pudieran transmitir enfermedades. Para la estabilización de los controles positivos y negativos se han utilizado sueros de origen bovino. La albúmina de suero bovino (ASB) y el suero fetal bovino (SFB) provienen de animales de áreas geográficas libres de EEB/EET. El Control Negativo, el Control Positivo HIV-1, el Control Positivo HIV-2 y el Conjugado contienen 0,1% de ProClin TM como conservante. A continuación se presentan las frases de riesgo (R) y de seguridad (S) aplicables: R43 Posibilidad de sensibilización en contacto con la piel. S26 En caso de contacto con los ojos, lávense inmediata y abundantemente con agua y acúdase a un médico. S28 En caso de contacto con la piel, lávese inmediata y abundantemente con agua. S36/37/39 Úsense indumentaria y guantes adecuados y protección para los ojos/la cara. S45 En caso de accidente o malestar, acúdase inmediatamente al médico (si es posible, muéstresele la etiqueta). S60 Elimínense el producto y su recipiente como residuos peligrosos. S61 Evítese su liberación al medio ambiente. Recábense instrucciones específicas de la ficha de datos de seguridad. Los análisis ELISA son sensibles al tiempo y la temperatura. Para evitar un resultado incorrecto, seguir estrictamente los pasos del procedimiento de prueba y no modificarlos. 1. No intercambiar reactivos de diferentes lotes ni usar reactivos de otros kits comerciales. Los componentes del kit están ajustados con precisión para ofrecer un rendimiento óptimo de los tests. 2. Comprobar que todos los reactivos se encuentran dentro del periodo de validez indicado en la caja del kit y que pertenecen al mismo lote. No usar nunca reactivos después de la fecha de caducidad declarada en las etiquetas o las cajas. 3. PRECAUCIÓN - PASO CRÍTICO: Llevar los reactivos y las muestras a temperatura ambiente (18-30 C) antes de su uso. Agitar suavemente el reactivo antes de usarlo. Llevar de nuevo a 2-8 C inmediatamente después del uso. 4. Usar sólo el volumen suficiente de muestra según lo indicado en los pasos del procedimiento. En caso contrario, el análisis puede perder sensibilidad. 5. No tocar la base exterior de los pocillos; las huellas de dedos o los arañazos pueden interferir en la lectura. Al efectuar la lectura de los resultados, asegurarse de que la base de la placa está seca y de que no existen burbujas de aire dentro de los pocillos. 6. No dejar secar nunca los pocillos de la microplaca después del paso de lavado. Pasar inmediatamente al siguiente paso. Evitar la formación de burbujas de aire al añadir los reactivos R spa

13 7. Evitar interrupciones muy prolongadas durante los pasos del análisis. Asegurar las mismas condiciones de trabajo en todos los pocillos. 8. Calibrar la pipeta con frecuencia para garantizar la exactitud de la dispensación de muestras/reactivos. Usar diferentes puntas de pipetas desechables con cada muestra y reactivo para evitar contaminaciones cruzadas. 9. Asegurar una temperatura de incubación de 37 C dentro del incubador. 10. Al añadir las muestras, no tocar la base del pocillo con la punta de la pipeta. 11. En la medición con un lector de placas, determinar la absorbancia a 450 nm o a 450/ nm. 12. La actividad enzimática del conjugado podría resultar afectada por el polvo y reactivos y sustancias químicas como hipoclorito sódico, ácidos, álcalis, etc. No realizar el análisis en presencia de estas sustancias. 13. Si se utiliza un equipo totalmente automático durante la incubación, no cubrir las placas con la lámina adhesiva. También puede omitirse la acción de golpear la placa después del lavado para eliminar los restos que quedan dentro de ella. 14. Todas las muestras de origen humano deben considerarse potencialmente infecciosas. El cumplimiento estricto de las normas BPL (Buena Práctica de Laboratorio) puede garantizar la seguridad del personal. 15. No comer, beber, fumar o maquillarse en el laboratorio de análisis. No pipetear las soluciones con la boca. 16. Los productos químicos deben manipularse y eliminarse sólo conforme a las normas BPL (Buena Práctica de Laboratorio) vigentes y a las regulaciones locales o nacionales. 17. Las puntas de pipetas, viales, tiras y envases de muestras deben recogerse y esterilizarse en autoclave durante no menos de 2 horas a 121 C o tratarse con hipoclorito sódico al 10% durante 30 minutos para descontaminar antes de desecharlas. Las soluciones que contienen hipoclorito sódico NUNCA deben esterilizarse en autoclave. Se dispondrá de una ficha de datos de seguridad (FDS). 18. Las materias primas de algunos reactivos pueden causar toxicidad, irritación, quemaduras o ser carcinógenos. Debe evitarse el contacto con la piel y las mucosas, entre otros, con los siguientes reactivos: solución de parada, cromógenos y solución de lavado. SIGNOS DE INESTABILIDAD O DETERIORO DEL REACTIVO: Los valores de los controles positivo o negativo que se encuentren fuera de los límites de control de calidad indicados son indicadores de un posible deterioro de los reactivos o errores del operario o del instrumento. En tal caso los resultados deben considerarse no válidos y las muestras deben ser reanalizadas. En el caso de resultados erróneos constantes y deterioro o inestabilidad demostrados de los reactivos, sustituir inmediatamente estos por unos nuevos o ponerse en contacto con su distribuidor local o servicio técnico de Biokit para más información. Conservación y estabilidad Los componentes del kit son estables hasta la fecha de caducidad indicada en la etiqueta y en el envase, si se almacenan entre 2-8 C y no se congelan. Para garantizar el máximo rendimiento del kit bioelisa HIV , durante el almacenamiento proteger los reactivos de la contaminación con microorganismos o productos químicos. Se pueden romper las tiras de micropocillos para usarlas por separado. Para el almacenamiento colocar los pocillos o tiras no utilizados en la bolsa de plástico sellable junto con el desecante y llevarlos de nuevo a 2-8 C. Una vez abierta, la microplaca es estable 1 mes a 2-8 C. Una vez abiertos, el control negativo, el control positivo HIV-1, el control positivo HIV-2, el conjugado, la solución de cromógeno A, la solución de cromógeno B y la solución de parada son estables 1 mes a 2-8 C. La solución de lavado, una vez diluida, es estable durante 1 semana a temperatura ambiente o durante 2 semanas a 2-8 C. Presentaciones disponibles - Kit de 1 placa (96 tests), REF Contiene: 1 placa, 1 x 1 ml control negativo, 1 x 1 ml control positivo HIV-1, 1 x 1 ml control positivo HIV-2, 1 x 12 ml conjugado, 1 x 50 ml solución de lavado, 1 x 8 ml solución cromógeno A, 1 x 8 ml solución cromógeno B, 1 x 8 ml solución de parada, 1 bolsa de plástico y láminas adhesivas (6 unidades). - Kit de 5 placas (5 x 96 tests), REF Contiene: 5 placas, 3 x 1 ml control negativo, 3 x 1 ml control positivo HIV-1, 3 x 1 ml control positivo HIV-2, 5 x 12 ml conjugado, 2 x 125 ml solución de lavado, 1 x 60 ml solución cromógeno A, 1 x 60 ml solución cromógeno B, 1 x 60 ml solución de parada, 1 bolsa de plástico y láminas adhesivas (20 unidades) R spa

14 Material necesario no incluido - Agua destilada o desionizada. - Guantes desechables y cronómetro. - Recipientes de desecho adecuados para materiales potencialmente contaminados. - Palanganas desechables en forma de V. - Sistema de dispensación o pipeta (simple o multicanal) y puntas de pipeta desechables. - Paños absorbentes o toallitas secas. - Incubador seco o baño maría, 37 C ± 1 C. - Microagitador para disolver y mezclar el conjugado con las muestras. - Lector de microplacas con filtro de 450 nm. Recomendable filtro de referencia de 620 ó 630 nm. - Sistema de lavado manual o automático. Recogida, transporte y almacenamiento de muestras 1. No se necesita una preparación especial del paciente. Recoger la muestra conforme a la práctica normal de laboratorio. En este análisis se pueden utilizar muestras de suero o plasma fresco. Se puede recoger la sangre por venopunción para su coagulación natural y completa: el suero/plasma debe separarse del coágulo lo antes posible para evitar la hemólisis. Asegurarse de que las muestras séricas son transparentes y no están contaminadas por microorganismos. Eliminar cualquier partícula visible en la muestra por centrifugación a 3000 rpm (revoluciones por minuto) durante 20 minutos a temperatura ambiente o por filtración. 2. Pueden utilizarse muestras de plasma recogidas en EDTA, citrato sódico o heparina, aunque no deben utilizarse muestras muy lipémicas, ictéricas o hemolíticas, ya que pueden dar resultados falsos en el análisis. No usar muestras inactivadas, ya que se puede deteriorar el analito diana. No usar nunca muestras con contaminación microbiana visible. 3. bioelisa HIV está diseñado para analizar ÚNICAMENTE muestras de suero o plasma. No usar para examinar muestras de cadáveres, saliva, orina u otros líquidos corporales, o sangre agrupada (mixta). 4. Transporte y almacenamiento: Almacenar las muestras a 2-8 C. Las muestras no necesarias para los análisis de los próximos 3 días deberán congelarse (-20 C o menos). Evitar repetir ciclos de congelación-descongelación. Para el envío embalar y etiquetar las muestras conforme a las normativas locales e internacionales existentes sobre transporte de muestras clínicas y sustancias etiológicas. Procesamiento automático Puede utilizarse un análisis automático o semiautomático con diferentes instrumentos. Es muy importante validar cualquier sistema automático para demostrar que los resultados obtenidos para las muestras son equivalentes a las obtenidas con el análisis manual. Se recomienda al usuario la validación periódica del instrumento. Si tiene dificultades para programar y ajustar los procesadores automáticos Biokit, póngase en contacto con su distribuidor. PROCEDIMIENTO (Ver esquema del procedimiento) Operaciones previas Antes de iniciar el análisis, llevar todos los reactivos a temperatura ambiente (20-25 C). Examinar el tampón de lavado concentrado en busca de sales cristalinas. Si se han formado cristales, disolver calentando a 37 C hasta que se disuelvan los cristales. Diluir 1:20 la solución de lavado según se indica en las instrucciones del paso de lavado. Usar agua destilada o desionizada y limpiar sólo los recipientes para diluir el tampón. Todos los demás reactivos están LISTOS PARA USAR. Instrucciones del paso de lavado Es esencial un buen lavado para obtener datos analíticos correctos y precisos. Por tanto, se recomienda usar un lavador de microplacas ELISA de buena calidad, mantenido en condiciones óptimas para ofrecer el mejor rendimiento de lavado. En general son suficientes no menos de 5 ciclos de lavado automático de µl/pocillo para evitar reacciones falsas positivas y un ruido de fondo elevado. Para evitar contaminaciones cruzadas de la placa con las muestras o el conjugado, después de la incubación no desechar el contenido de los pocillos pero dejar que el lavador de microplacas lo aspire automáticamente. Asegurar que los canales de dispensación del líquido de lavado de las microplacas no estén obturados o contaminados y que continuamente se dispensa un volumen suficiente de solución de lavado en los pocillos. En el caso de lavado manual sugerimos realizar 5 ciclos de lavado que dispensen µl/pocillo y aspiren el líquido 5 veces. Si se observan resultados insuficientes (ruido de fondo), aumentar los ciclos de lavado o el tiempo de inmersión por pocillo. En cualquier caso, el líquido aspirado de las tiras debe tratarse con una solución de hipoclorito sódico a una concentración final del 2,5% durante 24 horas, antes de eliminarlo adecuadamente. El tampón de lavado concentrado debe diluirse 1:20 antes del uso. Si no se utiliza la placa completa, preparar el volumen proporcional de la solución R spa

15 Paso 1 Paso 2 Paso 3 Paso 4 Paso 5 Paso 6 Preparación: Marcar 3 pocillos como control negativo (p. ej., B1, C1, D1), 2 pocillos como control positivo (p. ej., E1 para HIV-1 y F1 para HIV-2) y un blanco (p. ej., A1; no deben añadirse muestras o conjugado en el pocillo del blanco). Si los resultados se determinarán utilizando el lector de placas de longitud de onda doble, puede suprimirse el uso del pocillo del blanco. Utilizar sólo el número de tiras necesarias para el test. Adición de la muestra: Añadir 100 μl de las muestras y 100 μl de los controles positivo y negativo en sus respectivos pocillos. NOTA: utilizar una punta de pipeta desechable nueva para cada muestra, control negativo y positivo para evitar la contaminación cruzada. Incubación de la muestra: Cubrir la placa con la lámina adhesiva e incubar a 37 C durante 30 ± 1 minutos. Se recomienda usar un recipiente con agua controlado con termostato para garantizar la estabilidad de la temperatura y la humedad durante la incubación. Si se utiliza un incubador seco, intentar abrir la puerta lo menos posible. Lavado (1): Después del final de la incubación, retirar y eliminar la lámina adhesiva de la placa. Lavar cada pocillo 5 (cinco) veces con solución de lavado diluida. Remojar cada vez los micropocillos durante segundos. Después del ciclo de lavado final, volcar la placa sobre papel secante o un paño limpio y golpear para desprender los restos. Adición del conjugado: Añadir 100 μl del reactivo conjugado en cada pocillo excepto el del blanco. Incubación del conjugado: Cubrir la placa con la lámina adhesiva e incubar a 37 C durante 30 ± 1 minutos. Paso 7 Lavado (2): Como el Paso 4. Paso 8 Paso 9 Paso 10 Tinción: Añadir 50 μl de solución de cromógeno A y 50 μl de solución de cromógeno B en cada pocillo, incluido el blanco. Incubar la placa a 37 C durante 15 minutos en oscuridad. La reacción enzimática entre las soluciones de cromógeno y el conjugado produce un color azul en los pocillos del control positivo y de la muestra positiva a HIV-1/2. Parada de la reacción: Con una pipeta multicanal o manualmente, añadir 50 µl de solución de parada en cada pocillo y mezclar suavemente. Se desarrolla un color amarillo intenso en los pocillos del control positivo y de la muestra positiva a HIV-1/2. Medición de la absorbancia: Calibrar el lector de placas con el pocillo del blanco y efectuar la lectura de la absorbancia a 450 nm. Si se usa un instrumento de filtro doble, ajustar la longitud de onda de referencia a 620 nm o 630 nm. Calcular el valor umbral y evaluar los resultados. (NOTA: Efectuar la lectura de la absorbancia en el plazo máximo de 10 minutos después de parar la reacción). Control de calidad Se recomienda a los laboratorios establecer un sistema de control de calidad adecuado con material de control de calidad similar o idéntico a la muestra del paciente a analizar. - El valor de la absorbancia del pocillo del blanco, que contiene sólo solución de cromógeno y solución de parada, debe ser inferior a 0,080 a 450 nm. - El valor de la absorbancia del control positivo debe ser 0,800 a 450/ nm o a 450 nm después de restar el blanco. - Cada valor de la absorbancia individual del control negativo debe ser menor que 0,100 a 450/ nm o a 450 nm después de restar el blanco. Si uno de los valores del control negativo no cumple los criterios del control de calidad, debe desecharse y calcular de nuevo el valor medio de los dos valores restantes. Si más de un valor de absorbancia del control negativo no cumple las especificaciones de los límites del control de calidad, el test no es válido y debe repetirse R spa