........................................................................5 Remarks and precautions ......................................................................................................................................................................................................................................................................11 Specificity..................................................................................................................................................8 Results .........................................................................................................................................................................10 Test performance .........................................................................................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 + ....................................6 Directions for washing ............................................2 Intended use ........................3 Materials required but not provided ...............4 Safety and environment ...................................................................................................................................................................................................................................11 Sensitivity.........5 Specimen (collection and handling)..........................................................................................................................................................9 Interpretation of the results ..........................................................................................................................3 Reagents ........................KEY-CODE: INX53531 80541 INNO-LIA™ HTLV I/II Score 25415 v10 2011-02-16 p 2/12 English TABLE OF CONTENTS Symbols used ............................................................................................................................................................................... preparation for use and recommended storage conditions..................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................11 Precision ..........................................5 Manual 16-hours test procedure............7 Directions for incubation...9 Validation ...............................................................................................................................................................................................................................3 Test principle .............................3 Description..................................................................................................................................7 Automated 16-hours test procedure: Auto-LIA™.....................................................................................................................................................................................12 Trademarks....................................................................................................................................................................................10 Interpretation software: LIRAS™ for infectious diseases ............................................................................................................................................10 Limitations of the procedure............................................................................................................................................................................9 Reading............................................................................................................................................................................

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

25.3% chloracetamide (CAA) as preservative.1% CAA as preservative. Vortex mixer or equivalent. sheet Reading card 1 For identification of reactive antigen lines. Precision pipettes with disposable tips capable of delivering 10 µl. Sample Diluent 30 ml 57305 Containing color-coded (green) phosphate buffer containing sodium chloride.1% CAA as preservative.200 µl.12 ml 57309 Containing inactivated human serum containing antibodies to HTLV with 0.02% bromo-nitro-dioxane as preservative. detergent and 0. detergent and 0. Orbital mixer or rocker (see Directions for incubation).1% CAA as preservatives. Conjugate. Incubation tray 2 With 11 troughs each.01% MIT/0. Ready-to-use 45 ml 57302 Containing 5-bromo-4-chloro-3-indolyl phosphate/nitroblue Substrate tetrazolium in dimethyl formamide. 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. Optional: • Hot air fan (hair dryer) or dry incubator at 37°C. • A repetitive pipette together with disposable vials for the addition of Stop Solution. 50. Materials required but not provided - Distilled or deionized water. Graduated cylinders: 10. to be diluted 5x in distilled water. Timer. and 200 1000 µl. respectively. Negative 0. Adhesive 5 sealers Data reporting 1 For storage of developed strips. • Vacuum aspirator which contains 5% sodium hypochlorite solution in a waste bottle.01% Control methylisothiazolone (MIT)/0.12 ml 57307 Containing basematrix of human origin with 0. Wash Solution 45 ml 57299 Containing color-coded (blue) Tris buffer containing sodium chloride.1 mol/l sulfuric acid. Description Strips 20 57331 Containing 20 INNO-LIA™ HTLV antigen coated test strips. bovine protein stabilizers and 0. and 100 ml. with 0. Diluted wash solution is stable for 2 weeks if kept at 2 8°C.01% MIT/0. Positive Control 0. Plastic or stainless tweezers for strip handling.KEY-CODE: INX53531 80541 INNO-LIA™ HTLV I/II Score 25415 v10 2011-02-16 p 4/12 English Reagents supplied: Component Quantity Ref. 20 .01% MIT/0. . detergent. Stop Solution 45 ml 57303 Containing 0.1% CAA as preservative. Substrate and Wash Solution.

which contains sodium hypochlorite. SAMP DIL R43 S23 S24 S37 S60 - May cause sensitization by skin contact. CAUTION: Neutralize liquid waste that contains acid before adding sodium hypochlorite. Wear suitable gloves. SUBS BCIP/NBT. freeze at -20°C or lower. Ensure that the samples and controls are homogeneous before use. No test method can offer complete insurance that blood products will not transmit infectious agents. and local environmental regulations should also be observed. CONTROL-. anti-HCV and HbsAg. stored too close to cooling element.com. and positive for anti-HTLV I/II. serum or plasma should be separated from blood clot or blood cells by centrifugation. Positive Control and Negative Control should always be handled as potentially infectious. Use of personal protective equipment is necessary: gloves and safety spectacles when manipulating dangerous or infectious agents.Before storage. .Store the specimens at 2 . heparin or EDTA as anticoagulants. . - Specimens.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. - Remarks and precautions - Do not mix reagents with different lot numbers. S23-24-37-60 Contains 2-Chloroacetamide: CONJ. The most recent MSDS version is available on the website www. Waste should be handled according to the institution's waste disposal guidelines.8°C. Specimen (collection and handling) The INNO-LIA™ HTLV I/II Score may be performed on human serum or plasma collected in tubes containing citrate. Allow to stand overnight before disposal. Avoid contact with skin. All blood components and biological materials should be disposed of in accordance with established safety procedures. All federal. can cause erroneous results! Avoid microbial contamination of reagents. Therefore. Do not breathe vapour/spray. Do not aspirate the stop solution in a waste bottle. • Mix liquid waste with sodium hypochlorite so that the final concentration is ± 1% sodium hypochlorite. The Positive Control has been found to be negative for anti-HIV 1/2. Frozen reagents. .Repeatedly (more than 3 times) frozen and thawed samples may produce erroneous results. • Autoclave for at least 15 minutes at 121°C. The Negative Control has been found to be negative for antiHIV 1/2. Only adequately trained personnel should be permitted to perform the test procedure. . state. For storage longer than one week.innogenetics. anti-HCV and HBsAg. eg. Irritant! (Xi) R43. This material and its container must be disposed of as hazardous waste. • Incinerate disposable material. all blood components and biological materials should be considered as being potentially infectious and should be handled as such.

Wash each test strip 3 times (5 minutes) with 1 ml Wash Solution (see Directions for washing). a Positive and Negative Control should be assayed. Cover the throughs with an adhesive sealer to avoid drying of the strips during the overnight sample incubation. 8. Add 10 µl of the appropriate specimen or control to their appropriately labelled troughs. 6. Add 1 ml of Conjugate Solution to each test trough.25°C). Cover the troughs with an adhesive sealer (see Remarks and precautions). Aspirate the Stop Solution. and add one strip to each of the test troughs.30 minutes at room temperature (18 . 10. Incubate with the substrate by placing the test tray on the shaker or rocker. Unused and developed strips should be kept away from strong light and heat. 14. the strip surface should be completely submerged. This kit should only be used by personnel trained in clinical laboratory practices. Re-use of strips or troughs will result in erroneous results. 5. 7. 3. Manipulate the strips always with the plastic backing. 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 . and agitate for 10 . 13.KEY-CODE: INX53531 80541 INNO-LIA™ HTLV I/II Score 25415 v10 2011-02-16 p 6/12 English - - Do not touch the membrane of the strip. and agitate for 30 minutes at room temperature (18 . 11. All incubation steps should be performed using an orbital shaker or rocker (use rocker only for overnight incubation). Incubate with the conjugate by placing the test tray on the shaker or rocker and agitate for 30 minutes at room temperature (18 . Incubate with the Stop Solution by placing the test trough on the shaker or rocker. and place them in the tray.25°C). 4. Wash each test strip 3 times (5 minutes) with 1 ml Wash Solution (see Directions for washing). Add 1 ml of Stop Solution to each test trough. . 1. NOTE: Carefully remove the adhesive sealers to avoid cross-contamination. Cutting strips will result in erroneous interpretation of the results. THE STRIPS MUST BE COMPLETELY SUBMERGED. During shaking.25°C). Make sure that the test strips are placed in the troughs with their membrane side facing upwards.25°C). taking into account that for each test run. Use the required amount of test troughs. Identify test troughs as controls and specimens. Add 1 ml of Substrate Solution to each test trough. The test strip is placed membrane side upwards into the trough using tweezers. Manual 16-hours test procedure Please read Remarks and precautions before performing the test. 2. 12. Use a new pipette tip for each specimen. 9. The shaking of the solutions over the strips is important in achieving even line staining and maximum sensitivity. Remove the required amount of test strips from their container. Add 1 ml of Sample Diluent to each test trough. Aspirate liquid.

As soon as the strips have dried completely. . Remove the strips from the test troughs and place them.KEY-CODE: INX53531 80541 INNO-LIA™ HTLV I/II Score 25415 v10 2011-02-16 p 7/12 English 15. . membrane side upwards. substrate. 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). • Make sure not to damage the surface of the test strips when aspirating. A summary of the test procedure is given in following table: 16-hours sample incubation procedure 1 ml Sample Diluent 10 µl Specimen 10 µl 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 .Repeat these steps as many times as indicated in the assay procedure. Developed strips will retain their color if stored in the dark. . • Adapt the speed of the shaker or rocker when necessary. • Make sure the entire strip is thoroughly washed by complete submersion in the washing solution. the strip surface should be completely submerged.30 min Stop solution (*) RTU = Ready-to-use Directions for washing After 16-hours incubation.and. carefully remove the adhesive plate sealer. and stop solution incubation) and also the washing steps should be performed on a shaker or rocker (use rocker only for overnight sample incubation). .The speeds generated by a shaker or rocker is critical in achieving even line staining and maximum sensitivity.During incubation and washing steps. • Avoid splashing of the Wash Solution over the edges of the troughs. place strips in a dry incubator at 37°C for 30 minutes or use a hair dryer for 1 minute. - . The liquid is aspirated from the trough with a pipette.fro) motion of the strips in the trough. with the membrane side facing upwards. • Always use a clean aspiration device with disinfectant trap to avoid cross-contamination. conjugate. . NOTE: • Do not allow the strips to dry between the washing steps.The shaker or rocker should allow a reciprocal (to. and a movement of the liquid over the strips without spilling over the trough. results can be interpreted. on absorbent paper using tweezers.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. - Directions for incubation All the incubation steps (sample. which contains 5% sodium hypochlorite solution in the waste bottle. To accelerate the drying process. preferentially attached to a vacuum aspirator.

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

NOTE: • The strip must be completely dried to avoid any misinterpretation due to faintly visible bands appearing after addition of stop solution. 1+ and 3+ should be visible on all strips. Validation of a single strip: The control levels ±. - . The p19-I and gp46-II antigen lines may show a negative rating. and the intensity of the control level 1+ should be greater than that of level ±. . • Do not place paper on top of the strips as long as they are wet. Validation Validation of the test run: . .The negative control strip must show a negative reaction (no reaction at all or less than control level ±) for all the HTLV antigen lines. 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. The intensity of the control level 3+ should be greater than that of level 1+. • Weak control bands can be observed for samples containing high IgG levels (above the normal IgG range). gp21 I/II and gp46-I. 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.The streptavidin line should have a negative rating (the intensity is weaker than the ± control line).The positive control strip must show a reaction of at least ± on p19 I/II.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 ±. p24 I/II. gp46 I/II.

gag p24 I/II. 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. and env gp46-II) and can therefor not be determined as positive for HTLV antibodies. - .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. . and a ± intensity counts as 0.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. Only the ratings of the 3 discrimination bands (env gp46-I. Result Recommendation Negative for HTLV antibodies No band: Single band (≥ ±): . gag p19-I.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. .gp21 Two bands (≥ ±): Indeterminate for HTLV antibodies Further investigation * . env gp46-II) are taken into consideration. NOTE: .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.p19 I/II or p24 I/II or gp46 I/II Negative for HTLV antibodies Indeterminate for HTLV antibodies Further investigation * . A negative result does not preclude the possibility of exposure to HTLV or infection with the virus. env gp46 I/II.gp21 NON reactive Positive for HTLV antibodies Discrimination (see below) .Sum of intensities = sum of ratings. Discrimination Discrimination is only possible with samples that scored "positive" on the confirmation bands. env gp46-I. WARNING: . it does not count (= 0). env gp46 I/II.5.The use of diluted samples may give erroneous results. gag p19-I. When the intensity is lower than the cut-off line. Please contact your local distributor to obtain the latest updated version. env gp21 I/II) should be taken into consideration.

g. have been included in this comparative evaluation. originating from HTLVinfected patients that screened positive and further confirmed positive on INNO-LIA™ HTLV I/II. and Syphilis) and other immunological disorders (Rheumatoid factors. 214 samples were correctly identified as positive for HTLV-I antibodies. Sensitivity A collection of 217 HTLV-I positive and 111 HTLV-II positive samples. 110 samples were correctly identified as positive for HTLV-II antibodies and 4 were positive for HTLV antibodies. Of these 328 samples. Repeated testing showed negative results for the initially indeterminate samples and positive result for the initially positive sample. Taken together. One sample (Geriatric clinic) gave an indeterminate pattern and one sample (Pregnant women) gave a positive pattern. 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. the differentiation capacity is 98.7% (304/305) for normal blood donors. The latter sample was positive on the Murex screening ELISA as well and scored indeterminate on HTLV-I/II Western Blot. two were indeterminate (Syphilis subset) and one was found HTLV-I positive (HIV subset). 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. A total of 328 samples were tested internally on the INNO-LIA™ HTLV I/II Score using the manual 16-hours sample incubation protocol.0% (204/206) specificity for this sample set.3% (72/74) upon excluding the positive sample and 100% (74/74) upon repeated testing. Additional testing is recommended (e. HIV. Potentially interfering samples A total of 75 potentially interfering samples including infectious diseases markers (HBV.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. After testing. the specificity after initial testing is calculated at 97. Seventy-two samples gave negative results on INNO-LIA™ HTLV I/II Score. 304 samples scored negative and one sample scored indeterminate. For this sample set. 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. 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. HCV. As in total 4 samples could not be differentiated as either HTLV-I (3 samples) or HTLV-II (1 sample).8% (324/328). . All together. Autoimmune) were tested internally using the manual 16-hours sample incubation procedure. evaluation of a follow-up or PCR). these data indicate 99. a sensitivity of 100% (328/328) was observed after initial testing. All together the calculated specificity is 99.

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