KEY-CODE: INX53531 80541 INNO-LIA HTLV I/II Score 25415 v10 2011-02-16 p 2/12 English

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 .....................................................................................................................................4 Safety and environment .......................................................................................................................................................5 Specimen (collection and handling).....................................................................................................................................5 Remarks and precautions ....................................................................................................................................................5 Manual 16-hours test procedure..........................................................................................................................................6 Directions for washing ....................................................................................................................................................7 Directions for incubation.................................................................................................................................................7 Automated 16-hours test procedure: Auto-LIA................................................................................................................8 Results ..................................................................................................................................................................................9 Reading...........................................................................................................................................................................9 Validation ........................................................................................................................................................................9 Interpretation of the results ..........................................................................................................................................10 Interpretation software: LIRAS for infectious diseases ...........................................................................................10 Limitations of the procedure...............................................................................................................................................10 Test performance ...............................................................................................................................................................11 Sensitivity......................................................................................................................................................................11 Specificity......................................................................................................................................................................11 Precision .......................................................................................................................................................................12 Trademarks.........................................................................................................................................................................12

Symbols used Manufacturer In Vitro Diagnostic Medical Device Batch code Catalogue number Use By Consult Instructions for Use Temperature limitation Biological risks Contains sufficient for <n> tests Conjugate 100x Control Control +

KEY-CODE: INX53531 80541 INNO-LIA HTLV I/II Score 25415 v10 2011-02-16 p 3/12 English

Sample Diluent Stop Solution Strips Substrate BCIP/NBT Wash Solution 5x Intended use The INNO-LIA HTLV I/II Score is a Line Immuno Assay, to confirm the presence of antibodies against human T-cell lymphotropic virus type I (HTLV- I) and type II (HTLV- II) in human serum or plasma. The INNO-LIA HTLV I/II Score also differentiates between HTLV- I and HTLV- II infections. It is intended as a supplementary assay on specimens found to be reactive using an anti-HTLV screening procedure. Test principle INNO-LIA HTLV I/II Score is a Line Immuno Assay that uses well-defined antigens derived from HTLV-I and HTLV-II immuno-dominant proteins. The antigens used are either recombinant proteins or synthetic peptides highly purified and fixed on a nylon membrane. The sequences are selected to allow the detection of antibodies with a wide specificity to all known isolates of the HTLV strains. The antigenicity exhibited by these proteins and peptides is either common to HTLV-I and HTLV-II antibodies or type-specific to one of the two viruses to allow confirmation and discrimination in a single assay. Two gag (p19 I/II, p24 I/II) and two env (gp46 I/II, gp21 I/II) bands are applied as non-typespecific antigens, which are used to confirm the presence of antibodies against HTLV I/II. The type-specific antigens for HTLV-I (gag p19-I, env gp46-I) and for HTLV-II (env gp46-II), are applied to differentiate between HTLV-I and HTLV-II infections. In addition, four control lines are coated: one negative control (streptavidin), and three positive control lines, 3+ positive control (anti-human Ig) which is also used as sample addition control line, 1+ positive control (human IgG), and the cut-off line (human IgG). The INNO-LIA HTLV I/II Score is based on the enzyme immunoassay principle (EIA). A test sample is incubated in a test trough together with the multiple antigen-coated strip. Specific HTLV antibodies, if present in the sample, will bind to the HTLV antigen lines on the strip. Subsequently, a goat anti-human IgG labelled with alkaline phosphatase is added and will bind to any HTLV antigen/antibody complex previously formed. Incubation with a chromogenic substrate produces a dark brown color in proportion to the amount of specific antibodies present in the sample. The color development is stopped with sulfuric acid. If the sample contains no HTLV-specific antibodies, only a low background color develops. Reagents Description, preparation for use and recommended storage conditions - If kept at 2 - 8C, opened or unopened, all reagents are stable until the expiration date. Do not freeze reagents. Do not use the kit beyond the expiry date. - All reagents and the plastic tube containing the test strips must be brought to room temperature (18 - 25C) approximately 30 minutes before use and returned to the refrigerator (2 - 8C) immediately after use. - Alterations in physical appearance of kit reagents may indicate instability or deterioration.

KEY-CODE: INX53531 80541 INNO-LIA HTLV I/II Score 25415 v10 2011-02-16 p 4/12 English

Reagents supplied: Component Quantity Ref. Description Strips 20 57331 Containing 20 INNO-LIA HTLV antigen coated test strips. Sample Diluent 30 ml 57305 Containing color-coded (green) phosphate buffer containing sodium chloride, detergent, bovine protein stabilizers and 0.3% chloracetamide (CAA) as preservative. Negative 0.12 ml 57307 Containing basematrix of human origin with 0.01% Control methylisothiazolone (MIT)/0.1% CAA as preservative. Positive Control 0.12 ml 57309 Containing inactivated human serum containing antibodies to HTLV with 0.01% MIT/0.1% CAA as preservatives. Ready-to-use 45 ml 57301 Containing color-coded (red) goat anti-human IgG labeled Conjugate with alkaline phosphatase in Tris buffer containing bovine stabilizers, detergent and 0.01% MIT/0.1% CAA as preservative. Ready-to-use 45 ml 57302 Containing 5-bromo-4-chloro-3-indolyl phosphate/nitroblue Substrate tetrazolium in dimethyl formamide, with 0.01% MIT/0.1% CAA as preservative. Stop Solution 45 ml 57303 Containing 0.1 mol/l sulfuric acid. Wash Solution 45 ml 57299 Containing color-coded (blue) Tris buffer containing sodium chloride, detergent and 0.02% bromo-nitro-dioxane as preservative, to be diluted 5x in distilled water. Diluted wash solution is stable for 2 weeks if kept at 2 8C. Incubation tray 2 With 11 troughs each. Adhesive 5 sealers Data reporting 1 For storage of developed strips. sheet Reading card 1 For identification of reactive antigen lines. Materials required but not provided
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Distilled or deionized water. Precision pipettes with disposable tips capable of delivering 10 l, 20 - 200 l, and 200 1000 l, respectively. Orbital mixer or rocker (see Directions for incubation). Vortex mixer or equivalent. Graduated cylinders: 10, 25, 50, and 100 ml. Timer. Plastic or stainless tweezers for strip handling. Optional: Hot air fan (hair dryer) or dry incubator at 37C. A repetitive pipette together with disposable vials for the addition of Stop Solution, Conjugate, Substrate and Wash Solution. Vacuum aspirator which contains 5% sodium hypochlorite solution in a waste bottle.

KEY-CODE: INX53531 80541 INNO-LIA HTLV I/II Score 25415 v10 2011-02-16 p 5/12 English

Safety and environment Please refer to the Material Safety Data Sheet (MSDS) and product labelling for information on potentially hazardous components. The most recent MSDS version is available on the website www.innogenetics.com.

Irritant! (Xi) R43, S23-24-37-60 Contains 2-Chloroacetamide: CONJ, SUBS BCIP/NBT, CONTROL-, SAMP DIL R43 S23 S24 S37 S60
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May cause sensitization by skin contact. Do not breathe vapour/spray. Avoid contact with skin. Wear suitable gloves. This material and its container must be disposed of as hazardous waste.

Specimens, Positive Control and Negative Control should always be handled as potentially infectious. The Positive Control has been found to be negative for anti-HIV 1/2, anti-HCV and HbsAg, and positive for anti-HTLV I/II. The Negative Control has been found to be negative for antiHIV 1/2, anti-HCV and HBsAg. No test method can offer complete insurance that 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. All blood components and biological materials should be disposed of in accordance with established safety procedures. Autoclave for at least 15 minutes at 121C. Incinerate disposable material. Mix liquid waste with sodium hypochlorite so that the final concentration is 1% sodium hypochlorite. Allow to stand overnight before disposal. CAUTION: Neutralize 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 institution's waste disposal guidelines. All federal, state, and local environmental regulations should also be observed. Do not aspirate the stop solution in a waste bottle, which contains sodium hypochlorite.

Specimen (collection and handling) The INNO-LIA HTLV I/II Score may be performed on human serum or plasma collected in tubes containing citrate, heparin or EDTA as anticoagulants. - Before storage, serum or plasma should be separated from blood clot or blood cells by centrifugation. - Store the specimens at 2 - 8C. For storage longer than one week, freeze at -20C or lower. - Repeatedly (more than 3 times) frozen and thawed samples may produce erroneous results.
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Remarks and precautions

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Do not mix reagents with different lot numbers. Frozen reagents, eg. stored too close to cooling element, can cause erroneous results! Avoid microbial contamination of reagents. Ensure that the samples and controls are homogeneous before use.

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Do not touch the membrane of the strip. Manipulate the strips always with the plastic backing. Use a new pipette tip for each specimen. Make sure that the test strips are placed in the troughs with their membrane side facing upwards. All incubation steps should be performed using an orbital shaker or rocker (use rocker only for overnight incubation). The shaking of the solutions over the strips is important in achieving even line staining and maximum sensitivity. During shaking, the strip surface should be completely submerged. Cover the throughs with an adhesive sealer to avoid drying of the strips during the overnight sample incubation. Unused and developed strips should be kept away from strong light and heat. This kit should only be used by personnel trained in clinical laboratory practices. Re-use of strips or troughs will result in erroneous results. Cutting strips will result in erroneous interpretation of the results.

Manual 16-hours test procedure Please read Remarks and precautions before performing the test. 1. Use the required amount of test troughs, taking into account that for each test run, a Positive and Negative Control should be assayed. Identify test troughs as controls and specimens, and place them in the tray. 2. Add 1 ml of Sample Diluent to each test trough. 3. Add 10 l of the appropriate specimen or control to their appropriately labelled troughs. 4. Remove the required amount of test strips from their container, and add one strip to each of the test troughs. The test strip is placed membrane side upwards into the trough using tweezers. THE STRIPS MUST BE COMPLETELY SUBMERGED. 5. Cover the troughs with an adhesive sealer (see Remarks and precautions). Incubate the samples by placing the tray on a shaker or rocker (see Directions for incubation) and agitate OVERNIGHT (16 2 h) at room temperature (18 - 25C). NOTE: Carefully remove the adhesive sealers to avoid cross-contamination. 6. Wash each test strip 3 times (5 minutes) with 1 ml Wash Solution (see Directions for washing). 7. Add 1 ml of Conjugate Solution to each test trough. 8. Incubate with the conjugate by placing the test tray on the shaker or rocker and agitate for 30 minutes at room temperature (18 - 25C). 9. Wash each test strip 3 times (5 minutes) with 1 ml Wash Solution (see Directions for washing). 10. Add 1 ml of Substrate Solution to each test trough. 11. Incubate with the substrate by placing the test tray on the shaker or rocker, and agitate for 30 minutes at room temperature (18 - 25C). 12. Aspirate liquid. Add 1 ml of Stop Solution to each test trough. 13. Incubate with the Stop Solution by placing the test trough on the shaker or rocker, and agitate for 10 - 30 minutes at room temperature (18 - 25C). 14. Aspirate the Stop Solution.

KEY-CODE: INX53531 80541 INNO-LIA HTLV I/II Score 25415 v10 2011-02-16 p 7/12 English

15. Remove the strips from the test troughs and place them, membrane side upwards, on absorbent paper using tweezers. As soon as the strips have dried completely, results can be interpreted. To accelerate the drying process, place strips in a dry incubator at 37C for 30 minutes or use a hair dryer for 1 minute. Developed strips will retain their color if stored in the dark. A summary of the test procedure is given in following table: 16-hours sample incubation procedure 1 ml Sample Diluent 10 l Specimen 10 l Controls 16-hours 2 hours LIA test strips 1 ml/3 x 5 min Washing 1 ml/30 min RTU* Conjugate 1 ml/3 x 5 min Washing 1 ml/30 min RTU* Substrate 1 ml/10 - 30 min Stop solution (*) RTU = Ready-to-use Directions for washing After 16-hours incubation, carefully remove the adhesive plate sealer. The liquid is aspirated from the trough with a pipette, preferentially attached to a vacuum aspirator, which contains 5% sodium hypochlorite solution in the waste bottle. The tray is held at an angle to allow all liquid to flow to one side of the trough (to the uncoated plastic backing part of each strip). - Add 1 ml of diluted wash solution to each trough and agitate on a shaker or a rocker. Shaking time is indicated in the assay procedure. - Repeat these steps as many times as indicated in the assay procedure. NOTE: Do not allow the strips to dry between the washing steps. Make sure not to damage the surface of the test strips when aspirating. Always use a clean aspiration device with disinfectant trap to avoid cross-contamination. Make sure the entire strip is thoroughly washed by complete submersion in the washing solution. Adapt the speed of the shaker or rocker when necessary. Avoid splashing of the Wash Solution over the edges of the troughs.
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Directions for incubation All the incubation steps (sample, conjugate, substrate, and stop solution incubation) and also the washing steps should be performed on a shaker or rocker (use rocker only for overnight sample incubation). - During incubation and washing steps, the strip surface should be completely submerged, with the membrane side facing upwards. - The shaker or rocker should allow a reciprocal (to- and- fro) motion of the strips in the trough, and a movement of the liquid over the strips without spilling over the trough. - The speeds generated by a shaker or rocker is critical in achieving even line staining and maximum sensitivity.
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KEY-CODE: INX53531 80541 INNO-LIA HTLV I/II Score 25415 v10 2011-02-16 p 8/12 English

Recommendations for an orbital shaker: Diameter of the circular motion should be equal or superior to 13 mm Recommended speed for a 13 mm circular motion is 160 rpm Recommended speed for a 24 mm circular motion is 90 rpm. Recommendations for a rocker: Recommended speed is 34 rpm at a shaking angle of around 9. Automated 16-hours test procedure: Auto-LIA The LIA test procedure can easily be automated using the Auto-LIA automate. This instrument is a walk-away system with automated aspiration, pipetting, and incubation. For more information on the Auto-LIA, please contact Innogenetics or your local distributor. Please read Remarks and precautions before performing the test. Detailed 16-hours sample incubation Auto-LIA procedures: 1. DISP CH1 Stpos: Begin Endpos: Till end 1000 l 2. INC 1 min, shake speed 4 3. PAUSE 4. INC 960 min, shake speed 4 5. WASH CH2 Stpos: Begin Endpos: Till end 1000 l 6. INC 6 min, shake speed 4 7. WASH CH2 Stpos: Begin Endpos: Till end 1000 l 8. INC 6 min, shake speed 4 9. WASH CH2 Stpos: Begin Endpos: Till end 1000 l 10. INC 6 min, shake speed 4 11. ASP 12. DISP CH4 Stpos: Begin Endpos: Till end 1000 l 13. INC 30 min, shake speed 4 14. WASH CH2 Stpos: Begin Endpos: Till end 1000 l 15. INC 3 min, shake speed 4 16. WASH CH2 Stpos: Begin Endpos: Till end 1000 l 17. INC 3 min, shake speed 4 18. WASH CH2 Stpos: Begin Endpos: Till end 1000 l 19. INC 3 min, shake speed 4 20. ASP 21. DISP CH6 Stpos: Begin Endpos: Till end 1000 l 22. INC 30 min; shake speed 4 23. ASP 24. DISP CH5 Stpos: Begin Endpos: Till end 1000 l 25. INC 20 min, shake speed 4 26. ASP 27. END CH1 = Sample Diluent CH2 = Wash Solution CH4 = Conjugate CH5 = Stop Solution CH6 = Substrate

KEY-CODE: INX53531 80541 INNO-LIA HTLV I/II Score 25415 v10 2011-02-16 p 9/12 English

Results Reading The identity and location of the antigens and controls coated on the strip are as follows:

Figure 1: INNO-LIA HTLV I/II Score test strip The intensity of the reaction on the control lines on each strip is used to assign the reactivity ratings for each antigen on that strip: Intensity of antigen line reaction (R) Rating Lower than R< Equal to R= Higher than , but lower or equal to 1+ < R 1+ 1+ Higher than 1+ but lower than 3+ 1+ < R < 3+ 2+ Equal to 3+ R = 3+ 3+ Higher than 3+ R > 3+ 4+ A reactivity rating must be made separately for each strip. Use the reading card for correct interpretation. Identification of the lines is obtained by alignment of the 3+ control line on the developed strip with the corresponding 3+ control line on the reading card. Validation Validation of the test run: - The positive control strip must show a reaction of at least on p19 I/II, p24 I/II, gp46 I/II, gp21 I/II and gp46-I. The p19-I and gp46-II antigen lines may show a negative rating. - The negative control strip must show a negative reaction (no reaction at all or less than control level ) for all the HTLV antigen lines. Validation of a single strip: The control levels , 1+ and 3+ should be visible on all strips. The intensity of the control level 3+ should be greater than that of level 1+, and the intensity of the control level 1+ should be greater than that of level . - The streptavidin line should have a negative rating (the intensity is weaker than the control line). NOTE: The strip must be completely dried to avoid any misinterpretation due to faintly visible bands appearing after addition of stop solution. Do not place paper on top of the strips as long as they are wet. Weak control bands can be observed for samples containing high IgG levels (above the normal IgG range).
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KEY-CODE: INX53531 80541 INNO-LIA HTLV I/II Score 25415 v10 2011-02-16 p 10/12 English

Interpretation of the results Confirmation Only the 4 confirmation lines (gag p19 I/II, gag p24 I/II, env gp46 I/II, env gp21 I/II) should be taken into consideration. Result Recommendation Negative for HTLV antibodies No band: Single band ( ): - p19 I/II or p24 I/II or gp46 I/II Negative for HTLV antibodies Indeterminate for HTLV antibodies Further investigation * - gp21 Two bands ( ): Indeterminate for HTLV antibodies Further investigation * - gp21 NON reactive Positive for HTLV antibodies Discrimination (see below) - gp21 reactive Positive for HTLV antibodies Discrimination (see below) Three or more bands: (*) A supplementary assay is recommended for indeterminate samples or testing of a followup sample. Discrimination Discrimination is only possible with samples that scored "positive" on the confirmation bands. Only the ratings of the 3 discrimination bands (env gp46-I, gag p19-I, env gp46-II) are taken into consideration. NOTE: - Sum of intensities = sum of ratings, and a intensity counts as 0.5. When the intensity is lower than the cut-off line, it does not count (= 0). If sum (env gp46-I + gag p19-I) > env gp46-II If sum (env gp 46-I + gag p19-I) env gp46-II AND env gp46-II > env gp 46-I Other combinations Positive for HTLV-I antibodies Positive for HTLV-II antibodies Positive for HTLV antibodies (untypable)

Interpretation software: LIRAS for infectious diseases The LiRAS for infectious diseases software is designed to assist with the interpretation of the LIA results. Please contact your local distributor to obtain the latest updated version. WARNING: - Do not use the automated interpretation without taking into account the limitations of the procedure as mentioned below. Limitations of the procedure The protocol provided must be strictly followed to obtain optimal performance of the assay. A negative result does not preclude the possibility of exposure to HTLV or infection with the virus. - A sample giving a positive reaction on the streptavidin control line may give cross-reactions with HTLV antigen lines which contain synthetic peptides (gag p19 I/II, env gp46 I/II, gag p19-I, env gp46-I, and env gp46-II) and can therefor not be determined as positive for HTLV antibodies. - The use of diluted samples may give erroneous results.
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KEY-CODE: INX53531 80541 INNO-LIA HTLV I/II Score 25415 v10 2011-02-16 p 11/12 English -

An indeterminate result on the INNO-LIA HTLV I/II Score needs follow-up. Additional testing is recommended (e.g. evaluation of a follow-up or PCR).

Test performance The results of the INNO-LIA HTLV I/II Score using the manual 16-hours sample incubation procedure were obtained by testing a collection of characterized samples including Panels from SFTS & BBI. Sensitivity A collection of 217 HTLV-I positive and 111 HTLV-II positive samples, originating from HTLVinfected patients that screened positive and further confirmed positive on INNO-LIA HTLV I/II, have been included in this comparative evaluation. A total of 328 samples were tested internally on the INNO-LIA HTLV I/II Score using the manual 16-hours sample incubation protocol. Of these 328 samples, 214 samples were correctly identified as positive for HTLV-I antibodies, 110 samples were correctly identified as positive for HTLV-II antibodies and 4 were positive for HTLV antibodies. Taken together, a sensitivity of 100% (328/328) was observed after initial testing. As in total 4 samples could not be differentiated as either HTLV-I (3 samples) or HTLV-II (1 sample), the differentiation capacity is 98.8% (324/328). Specificity Blood donors A total of 305 blood donor samples were analyzed internally on the INNO-LIA HTLV I/II Score using the manual 16-hours sample incubation procedure. After testing, 304 samples scored negative and one sample scored indeterminate. All together the calculated specificity is 99.7% (304/305) for normal blood donors. Clinical samples A collection of 206 clinical samples from various conditions were tested internally on INNOLIA HTLV I/II Score using the manual 16-hours sample incubation procedure. One sample (Geriatric clinic) gave an indeterminate pattern and one sample (Pregnant women) gave a positive pattern. This latter sample was found to be positive upon repeated testing in duplicate and upon testing on INNO-LIA HTLV I/II Score assay while a negative result was obtained by Murex screening ELISA and by HTLV I/II Western Blot. All together, these data indicate 99.0% (204/206) specificity for this sample set. Potentially interfering samples A total of 75 potentially interfering samples including infectious diseases markers (HBV, HIV, HCV, and Syphilis) and other immunological disorders (Rheumatoid factors, Autoimmune) were tested internally using the manual 16-hours sample incubation procedure. Seventy-two samples gave negative results on INNO-LIA HTLV I/II Score, two were indeterminate (Syphilis subset) and one was found HTLV-I positive (HIV subset). Repeated testing showed negative results for the initially indeterminate samples and positive result for the initially positive sample. The latter sample was positive on the Murex screening ELISA as well and scored indeterminate on HTLV-I/II Western Blot. For this sample set, the specificity after initial testing is calculated at 97.3% (72/74) upon excluding the positive sample and 100% (74/74) upon repeated testing.

KEY-CODE: INX53531 80541 INNO-LIA HTLV I/II Score 25415 v10 2011-02-16 p 12/12 English

Precision A panel of 7 HTLV-positive samples, together with positive and negative control, was tested by different experimenters on 3 different lots using the 16-hours sample incubation procedure. The HTLV-positive samples were diluted with a factor ranging from 1/7 to 1/25 before testing. The use of different kit lots and performance by different experimenters resulted in the same test outcome for each of the samples evaluated. Trademarks INNOGENETICS is a Registered Trademark of Innogenetics N.V. INNO-LIA is a trademark of Innogenetics N.V.