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ETI-DELTAK-2(P002097)EN:Pay attention to changes! IT:Attenzione alle modifiche! FR:Faire attention aux modifications! DE: Auf die Änderungen aufpassen! ES: ¡Atención a las modificaciones! PT: Cuidado com as modificações! RO: Atenie la modific ri! NO: Legg merke til endringene! SV:Uppmärksamma vidtagna ändringar! DA:Bemærk ændringerne! CS: Dávejte si pozor na zm ny!SK: Venujte pozornost™ zmenám! PL: Nale y zwróci uwag na zmiany! LT: Atkreipkite d mes pakeitimus! LV:Piev rsiet uzman bu izmai m!HU: Kérjük, figyeljen a változásokra! BG: !TR:De iikliklere dikkat edin !EL: !

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1HDAg ENZYME IMMUNOASSAY KIT Procedure for qualitative determination of hepatitis Delta antigen (HDAg) in human serum or plasma samples For in vitro use only 1. INTRODUCTION The hepatitis D virus (HDV) is a 36-nm diameter defective virus, which requires the help- er functions of hepatitis B virus (HBV) for successful replication. The HDV nucleocapsid is formed of a single-stranded genome (HDV-RNA), which is strictly associated to two specific proteins (p27 and p29) giving rise to the Delta antigen (HDAg) (1), and is sur- rounded by an HBsAg envelope (7). Consequently, detection of HDAg requires treatment of test samples with detergents to remove HBsAg and expose HDAg antigenic determi- nants (8). Hepatitis D presents three main clinical pictures, different in severity of symptoms; as a rule, hepatitis D symptoms are consistently severer than those observed in HBV infection or in other viral hepatitides. The finding of the three serological markers of hepatitis D (HDAg, anti-HD IgM and anti-HD total antibodies) after detection of HBsAg allows diag- nosis and characterization of various forms of hepatitis D to be established (4, 5, 9): ŒCoinfection , when the patient is infected by both HBV and HDV at the same time. The clinical course of the disease is acute, symptomatology is generally similar to that of HBV acute hepatitis. In most cases the disease progresses to full recovery; chronici- zation is rare. In acute HBV/HDV coinfection, IgM anti-HD is raised a few days to a few weeks after the onset of symptoms and seroconversion to IgG anti-HD occurs later. ŒSuperinfection , when the patient (HBsAg chronic carrier) had a previous HBV infection and is later infected by HDV. The disease is severer in this case and may evolve to a fulminant form of hepatitis (more frequent th an in case of coinfection) or may chroni- cize. As a rule, the antibody response to superinfection is quicker and brisker than in coinfection. IgM anti-HD is present at or raised soon after the onset of symptoms, fol- lowed shortly by the IgG antibody. ŒChronic infection , having a severer clinical course than HBV chronic hepatitis. The persistence of IgM anti-HD indicates that hepatitis D is becoming chronic, because it correlates directly with HDV replication and with the inflammatory activity of the under- lying liver disease. The finding of HDAg in the bloodstream allows the diagnosis of ongoing HDV acute in- fection to be established, though HDAg is found in some but not all acute D hepatitides. Its presence is short in duration and limited to the early stage of infection; the test can give negative results because viraemia is already over at the onset of clinical symptoms. Entity and duration of HD antigenaemia are usually proportional to the severity of hepa- titis (4). The finding of serum HDAg is in fact more frequent and conspicuous in fulminant and severe rather than in benign or mild forms of hepatitis to such an extent that serum HDAg represents a reliable prognostic marker of clinical evolution of the illness. As soon as the specific antibody response appears, HDAg becomes no longer detectable in serum, because its antigenic reactivity is masked by the endogenous antibodies. Con- sequently, HD antigenaemia is not detectable in patients with chronic D hepatitis as persistently high anti-HD titres are consistently present (4). Chronic HD antigenaemia can however be demonstrated in immunocompromized sub- jects, like those with HIV infection, as the antibody response is totally or partially sup- pressed (2, 6).

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22. PRINCIPLE OF THE ASSAY The method for qualitative HDAg determination is a direct, non-competitive sandwich as- say, illustrated in Fig. 1, based on the ELISA technique (Enzyme-linked Immunosorbent Assay). Fig. 1 Well coated with human IgG anti-HD .HDAg added with a non-ionic detergent (Igepal ® 300 CA-630) from sample or control .Enzyme tracer : human anti-HD Fab conjugated to horseradish peroxidase (HRP). The presence of HDAg allows the enzyme tracer to bind to the solid phase. The enzyme activity is therefore proportional to the HDAg concentration present in samples or controls. Enzyme activity is measured by adding a colourless chromogen/substrate solution. The enzyme action on chromogen/substrate produces a colour which is measured with a pho- tometer. 312++incubation (1 hour, 37°C) washing incubation (1 hour, 37°C) washing chromogen/substrate instrumental reading at 450/630 nm. incubation (30 min, R.T.) stop solution

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33. REAGENTS AND ACCESSORIES 3.1. Reagents provided in the kit STORAGE: Upon receipt, store all reagents at 2-8°C, away from intense light. Do not freeze. Once opened, the reagents of this ki t are stable for eight weeks when properly stored, unless otherwise stated. The kit is stable for one work week when used through- out the day for eight hours at room temperature and stored overnight at 2-8°C. Reagents should not be used past the expiry date. The expiry date of the kit is reported on the external label. The expiry date of each component is reported on the respective vial label. Specific reagents (see §4.a) from different batches must not be mixed. Common rea- gents (see §4.b) are interchangeable between batches .3.2. Materials provided with the kit Œ2 precut cardboard sealers suitable for 1 to 12 strips Œ2 cardboard sealers suitable for 12 strips (one plate) Œpouch sealer. 3.3. Equipment and materials required, but not supplied ŒVertical reading photometer (such as ETI-SYSTEM reader or equivalent) with the fol- lowing instrument specifications: wavelength: dual wavelength, 450 nm and 620-630 nm bandwidth: 10 nm absorbance range: 0 absorbance units to 2.5 absorbance units repeatability: better than or equal to 0.005 absorbance units, or 1%, whichever is greater linearity or trueness: better than or equal to 0.010 absorbance units, or 2%, whichever is greater drift: less than 0.005 absorbance units per hour. ŒThermostatically-controlled humid chamber with the following specifications: temperature: 37°C 1°C. ŒManual or automatic equipm ent for rinsing wells (suc h as ETI-SYSTEM washer or equivalent) with the following instrument specifications: volume dispensed: 300-370 Lnumber of wash cycles: 5 soak time: 30 seconds aspirate the last aliquot of dispensed liquid: yes. ŒMicropipettes with disposable tips (50, 100 L) (50 L: trueness 3%, precision 2%; 100 L: trueness 2%, precision 1%). ŒGlassware. ŒDistilled water. ETI-LAB or ETI-MAX 3000 equipment may be used for automated test processing. Coated strips12 8-removable well strips Enzyme tracer1 vial,0.4 mL Negative control1 vial,3.3 mL Positive control1 vial,2.5 mL Nonidet P-401 vial, 8 mL Tracer diluent1 vial,14.7 mL Wash buffer1 vial,40 mL Chromogen/substrate1 vial,16 mL Stop solution1 vial,30 mL Number of tests96

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44. COMPOSITION AND PREPARATION OF REAGENTS All serum and plasma units used to produce the components provided in this kit have been tested for the presence of HBsAg, anti-HCV and anti-HIV-1/2 and found to be non- reactive, except for the material positive for anti-HD, which is reactive for HBsAg and anti-HCV. The presence of HBsAg and HCV in the final reagents is however excluded, because HBsAg is removed by purification and HCV-RNA is found negative. As, however, no test method can offer absolute assurance that pathogens are absent, all specimens of human origin should be considered potentially infectious and handled with care. a) SPECIFIC KIT REAGENTS 4.1. Coated strips Removable wells are coated with human IgG anti-HD. The wells are ready to use and should be stored at 2-8°C. Bring the coated wells to room temperature before opening the pouch to avoid develop- ment of condensed water. Place unused wells in the pouch, securely reseal and store at 2-8°C. 4.2. Enzyme tracer (50x conjugate) The vial contains 0.4 mL anti-HD Fab (human), conjugated to horseradish peroxidase (HRP), TRIS buffer, BSA, stabilizers and preservatives. Before use, dilute the solution 1:50 with tracer diluent (4.6) (e.g. 100 L tracer + 4.9 mL tracer diluent). Prepare only the amount of working enzyme tracer needed for the run and keep the concentrated enzyme tracer at 2-8°C. The working enzyme tracer can be stored for one month at 2-8°C after preparation. 4.3. Negative control The vial contains 3.3 mL human serum/plasma non-reactive for anti-HD and preserva- tives. The reagent is ready to use and should be stored at 2-8°C. 4.4. Positive control The vial contains 2.5 mL recombinant HDAg (3), human serum/plasma, BSA, phos- phate-citrate buffer and preservatives. The reagent is ready to use and should be stored at 2-8°C. 4.5. Nonidet P-40 The vial contains 8 mL of a 2% solution of non-ionic detergent Igepal ® 300 CA-630, PBS buffer at pH 7.4, preservatives and an inert blue dye. The reagent is ready to use and should be stored at 2-8°C. 4.6. Tracer diluent The vial contains 14.7 mL human serum/plasma, newborn calf serum, PBS buffer and preservatives. The reagent is ready to use and should be stored at 2-8°C. The solution is used to dilute the enzyme tracer (4.2). b) REAGENTS COMMON TO OTHER ETI KITS 4.7. Wash buffer (25x)The vial contains 40 mL PBS buffer, Tween ® 20 and preservatives. Before use, dilute the vial contents to one litre with distilled water. The working wash buffer is stable for one week at 2-8°C, and is used to rinse wells. If crystallization occurs at 2-8°C, warm the wash buffer to 37°C and mix well before diluting.

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54.8. Chromogen/substrate The vial contains 16 mL of a tetramethylbenzidine/hydrogen peroxide system. The rea- gent is ready to use and should be stored at 2-8°C, away from the light. The solution should be colourless or have a slightly blue tinge. If the chromogen/sub- strate turns a darker blue, it may have become contaminated and should be discarded. 4.9. Stop solution The vial contains 30 mL 0.4N sulphuric acid solution. The reagent is ready to use and should be stored at 2-8°C. The stop solution is defined as irritant in the U.S. law (29 CFR 1910.1200, App. A). It is not a harmful or toxic substance as per Council Directive 99/45/EC. 5. SPECIMEN COLLECTION AND PREPARATION Either human serum or plasma may be used. The anticoagulants citrate, EDTA and heparin have been tested and may be used with this assay. Blood should be collected aseptically by venipuncture, allowed to clot, and the serum separated from the clot as soon as possible. Samples having particulate matter, turbidity, lipaemia, or erythrocyte debris may require clarification by filtration or centrifugation before testing. Grossly haemolyzed or lipaemic samples as well as samples containing particulate matter or ex- hibiting obvious microbial contamination should not be tested. If the assay is performed within 48 hours of sample collection, the samples should be kept at 2-8°C; otherwise they should be aliquoted and stored deep-frozen (Œ20°C or below). If samples are stored frozen, mix thawed samples well before testing. Performance is not affected by samples that have undergone up to four freeze-thaw cycles. Results obtained in heparinized plasma positive specimens may score absorbance val- ues above those of serum samples. 6. ASSAY PROCEDURE Bring all reagents to room temperature (20-25°C) before assaying. Perform all assay steps in the order given and without any appreciable delays between the steps. Number sufficient wells to run 3 replicates of the negative control and 2 replicates of the positive control with each run of samples in singlicate . Negative and positive con- trols must be run with each plate of patient specimens. Controls and samples should be subjected to the same process and incubation time. Prepare one substrate blank well containing chromogen/substrate only. A clean, disposable tip should be used for dispensing each control and sample. Identify wells with data sheet for testing controls and samples. Dispense controls and samples as illustrated in the scheme overleaf:

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7tion, because of the possible formation of precipitates that lower the initial absorb- ance values. Such phenomenon, however, does not result in sample misclassifi- cation. When dedicated equipment is used, absorbance values are provided automatically after selecting the suitable protocol. When another vertical reading photometer is used, blank the instrument with the blank, record the absorbance at 450/630 nm for each specimen and subtract the 630 nm absorbance value from the 450 nm absorb- ance value. ETI-LAB or ETI-MAX 3000 equipment may be used for automated test processing. 7. CALCULATION OF RESULTS 7.1. Calculation of cut-off value The cut-off value is determined by adding 0.100 to the mean absorbance for the negative control values (NC). Cut-off value = NC + 0.100. 7.2. Run validation criteria The following criteria should be used to validate quality control when evaluating results. Always calculate and evaluate mean control values for each plate, even if plates are combined to form a single batch. The absorbance value for the blank well must range between 0.000 and 0.150. 0.000 BLK 0.150. The mean absorbance for the negative control must be less than 0.100. NC < 0.100. Each negative control absorbance value must be less than 0.110. NC < 0.110. If any one of the negative control absorbance values does not meet these criteria, it should be discarded and the mean value recalculated using the remaining two values. If more than one negative control absorbance value does not meet these criteria, the run is invalid and must be repeated. The difference between the mean positive control absorbance value and the mean neg- ative control absorbance value must be greater than or equal to 0.500. PC NC 0.500. If not, the run is invalid and must be repeated. 7.3. Interpretation of results The presence or absence of HDAg is determined by comparing the absorbance value of unknown samples to that of the cut-off value. The unknown samples with absorbance values greater than or equal to the cut-off value should be considered reactive for HDAg. The unknown samples with absorbance values less than the cut-off value should be considered non-reactive. Samples with absorbance values within 10% of the cut-off value must be retested in or- der to confirm the initial result. Samples which are repeatedly reactive should be considered positive. Samples which are non-reactive at the second test should be con- sidered negative. xxxxx PAGE - 9 ============ 87.4. Calculation example The following data must only be considered an example and should not be employed in- stead of the data obtained by the user. Absorbance for the negative control Mean absorbance, NC = 0.015. Absorbance for the positive control Mean absorbance, PC = 1.782. Cut-off value (NC + 0.100) = 0.015 + 0.100 = 0.115. Positive Negative control difference (P N) = 1.782 0.015 = 1.767. In the given example, the P N difference meets the run validation criteria because it is greater than 0.500; thus the technique is acceptable and data should be considered valid. Screening of unknown samples Sample no. 1 = absorbance 0.910 Sample no. 2 = absorbance 0.053. Sample no. 1 should be considered reactive for HDAg and sample no. 2 non-reactive, as the cut-off value is 0.115. 8. SPECIFIC PERFORMANCE CHARACTERISTICS 8.1. Analytical specificity Analytical specificity may be defined as the ability of the assay to accurately detect spe- cific analyte in the presence of potentially interfering factors in the sample matrix (e.g., anticoagulants, haemolysis, effects of sample treatment), or cross-reactive antibodies. Controlled studies of potentially interfering substances or conditions showed that the as- say performance was not affected by anticoagulants (EDTA, heparin, citrate), haemolysis (up to 100 mg/dL haemoglobin), lipaemia (up to 3000 mg/dL triglycerides), bilirubinaemia (up to 20 mg/dL bilirubin), freezing of samples, or by antibodies to other infectious agents. Negative control sample No.Net absorbance at 450/630 nm 10.017 20.013 30.015 Positive control sa mple No.Net absorbance at 450/630 nm 11.852 21.712 xxx PAGE - 10 ============ 98.2. Precision Different sample pools (from A to D), containing different titres of specific analyte, were assayed to determine repeatability and reproducibility of the assay (i.e., within- and be- tween-assay variability). The variability shown in the tables below did not result in sam- ple misclassification. 8.3. Diagnostic specificity Diagnostic specificity is defined as the probability of the assay of scoring negative in the absence of specific analyte. Diagnostic specificity was assessed by testing 715 specimens selected from an expected HDAg-negative population (blood donors; dialysis patients; intravenous drug users; pa- tients affected by infectious diseases with similar symptomatology, such as acute or chronic HBV infection, and past HDV infection; patients affected by HIV and HCV infec- tions; patients affected by other hepatic dise ases; subjects positive for rheumatoid factor or heterophile antibodies). Seven positive results were observed at first test in the popu- lation studied - diagnostic specificity: 99.02% (95% confidence interval: 98.00-99.61%). At retest, those samples scored correctly negative. 8.4. Diagnostic sensitivity Diagnostic sensitivity is defined as the probability of the assay of scoring positive in the presence of specific analyte. Diagnostic sensitivity was assessed by testing 27 specimens selected from an expected HDAg-positive population (patients affected by acute HDV infection). No negative results were observed in the population studied - diagnostic sensitivity: 100% (95% confidence interval: 87.23-100%). 8.5. High-dose saturation effect Whenever samples containing extremely high antigen concentrations are tested, the sat- uration effect can mimic concentrations lower than real. However, a well-optimized two- step sandwich method excludes grossly underestimated results, because the analytical signals remain consistently high (saturation curve). Analysis of possible prozone effect was evaluated by testing four high-titred HDAg-posi- tive samples. All samples resulted in very high absorbance values that would be ex- pected with high-titred sera, indicating no prozone effect and, therefore, no sample mis- classification. RepeatabilityPool AP ool BPool CPool D Number of determinations26262626 Mean absorbance0.2780.6200.9561.079 Standard deviation0.0220.0500.0750.066 Coefficient of variation, % ReproducibilityPool APool BPool CPool D Number of determinations26262626 Mean absorbance0.3010.6281.0491.036 Standard deviation0.0350.0770.1470.184 Coefficient of variation, %11.512.314.017.7 PAGE - 11 ============ 109. LIMITATIONS OF THE PROCEDURE Bacterial contamination or heat inactivation of the specimens may affect the absorbance values of the samples with consequent alteration of HDAg levels. Diagnosis of an infectious disease should not be established on the basis of a single test result. A precise diagnosis, in fact, should take into consideration the patient's clinical history, symptomatology, as well as serological data. Serological data, however, should be interpreted with care in immunocompromized patients. 10. WARNINGS AND PRECAUTIONS A skillful technique and strict adherence to the instructions are necessary to obtain reli- able results. In particular, precise pipetting, aspiration and washing are essential. Non-repeatedly reactive samples might derive from various methodological factors, such as: Œcross-exchange of vial caps Œuse of the same tip when withdrawing from different vials or dispensing different samples Œexposure of reagents or samples to intense heat or heavy sources of bacterial conta- mination Œinadequate rinsing of wells Œcontamination of well rims by tracer or samples Œuse of reagents from different master lots. In addition the following precautions are required :Œto avoid contamination, use a clean, dedicated dispenser for the enzyme tracer solution Œwash buffer should be stored in clean containers to prevent contamination with en- zyme-inactivating substances. 11. SAFETY PRECAUTIONS ŒHandle with care chromogen/substrate and stop solution. Avoid chromogen/substrate and stop solution coming into contact with oxidizing agents or metallic surfaces. ŒDo not eat, drink, smoke or apply cosmetics in the assay laboratory. ŒDo not pipette solutions by mouth. ŒAvoid direct contact with all potentially infectious materials by using protective clothing such as lab coats, protective glasses and disposable gloves. Wash hands thoroughly at the end of each assay. ŒAvoid splashing or forming an aerosol. Any reagent spills should be washed with a 5% sodium hypochlorite solution and disposed of as though potentially infectious. ŒAll samples, biological reagents and materials used in the assay must be considered potentially able to transmit infectious agents. They should therefore be disposed of in accordance with the prevailing regulations and guidelines of the agencies holding ju- risdiction over the laboratory, and the regulations of each Country. Disposable ma- terials must be incinerated; liquid waste must be decontaminated with sodium hypo- chlorite at a final concentration of 5% for at least half an hour. Liquid waste containing acid must be neutralized before treatment. Any materials to be reused must be auto- claved using an overkill approach (USP 24, 2000, p. 2143). A minimum of one hour at 121°C is usually considered adequate, though the users must check the effectiveness of their decontamination cycle by initially validating it and routinely using biological indicators. 28 KB – 13 Pages