DESCRIPTION OF SYMBOLS USED KIT COMPONENTS SUBSTRATE 1 bottle 1. Avoid microbial contamination of reagents when opening 4. Avoid microbial contamination of the reagents, when Solution of 5-bromo-4-chloro- (100 ml) and removing aliquots from the original vials or bottles. opening and removing aliquots from the original vials or The following are graphical symbols used in or found on MP 3-indolyl phosphate (BCIP) bottles. As this will prematurely reduce the shelf life of the Component Description Quantity Diagnostics products and packaging. These symbols are and nitroblue tetrazolium 2. Do not pipette by mouth. kits and give erroneous results. Use aseptic techniques Provided the most common ones appearing on medical devices and (NBT). including pipettes or disposable pipette tips when drawing

their packaging. Some of the common symbols are explained 3. Wear laboratory coats and disposable gloves while aliquots from vials. NITROCELLULOSE STRIPS Available in more detail in the British and European Standard BS EN BLOTTING POWDER 10 packets performing the assay. Discard gloves in biohazard waste- Incorporated with HTLV-I in 18 or 36 980: 2008. Non-fat dry milk (1g each) bags. Wash hands thoroughly afterwards. 5. The kit controls should be assayed concurrently with viral lysate and recombinant strips samples for each test run. Use by Consult Instructions envelope antigens, and a Incubation Tray, 2 or 4 trays 4. It is highly recommended that this assay be performed in Synonyms for this: for Use serum addition control (anti- a biohazard cabinet. Expire Date human IgG) band. Keep dry 9 wells each 6. Use a new pipette tip for each specimen aliquot to prevent cross contamination. HTLV BLOT 2.4 Catalogue and away from light. 5. Keep materials away from food and drink. Batch Code Number Instructions For Use 1 copy WESTERN BLOT ASSAY 7. For best results dispense all reagents while cold and return Synonyms for this are: NON-REACTIVE 1 vial 6. In case of accident or contact with eyes, rinse immediately Instructions For Use Lot Number Forceps to 2°C to 8°C storage as soon as possible. CONTROL (80 µl) 1 pair with plenty of water and seek medical advice. Batch Number Inactivated normal human FOR RESEARCH USE ONLY Caution serum non-reactive for anti- 7. Consult a physician immediately in the event that 8. It is recommended that glassware to be used with the NOT FOR USE IN DIAGNOSTIC PROCEDURES Temperature Limitation Note : Volume of reagents provided are suffi cient for 4 runs. reagents should be washed with 2M hydrochloric acid and HCV, anti-HIV-1/2, anti-HTLV- contaminated materials are ingested or come in contact I/II and HBsAg. Contains with open lacerations or other breaks in the skin. rinsed thoroughly with distilled or deionised water prior to Do not reuse REVISION DATE: 09/10 Note Changes Highlighted sodium azide and thimerosal use. MAK0012-ENG-3 Manufacturer as preservatives. WARNINGS AND PRECAUTIONS 8. Wipe spills of potentially infectious materials immediately 9. Use only reagent grade quality, deionised or distilled water Harmful (Xn) / with absorbent paper and swab the contaminated area with (18 tests kit) : 11082-018 1. For Research Use Only. It is not intended for use in the to dilute reagents. Contains suffi cient Irritant (Xi) STRONG REACTIVE 1 vial 1% sodium hypochlorite solution before work is resumed. (36 tests kit) : 11082-036 diagnosis or prognosis of disease. for tests CONTROL I (80 µl) Sodium hypochlorite should not be used on acid containing Inactivated human serum 2. For Professional use only. spills unless the area is wiped dry with absorbent paper 10. All reagents must be mixed well before use. with high titered antibodies fi rst. Material used (including disposable gloves) should to HTLV-I and non-reactive HEALTH AND SAFETY INFORMATION be disposed as potentially biohazardous material. Do not 11. Working Conjugate solution, Diluted Wash Buffer and CHEMICAL & BIOLOGICAL PRINCIPLES OF THE for anti-HCV, anti-HIV-1/2 autoclave material containing sodium hypochlorite. Blotting Buffer should be prepared fresh prior to use. NAME AND INTENDED USE CAUTION: This kit contains materials of human origin. PROCEDURE and HBsAg. Contains sodium The MP Diagnostics HTLV BLOT 2.4 is a qualitative enzyme azide and thimerosal as No test method can offer complete assurance that 9. Autoclave all used and contaminated materials at 121°C 12. The Working Conjugate solution should be prepared using human blood products will not transmit infection. at 15 p.s.i. for 30 minutes before disposal. Alternatively, immunoassay for the in vitro detection of antibodies to HTLV-I The nitrocellulose strips are incorporated with HTLV-I viral preservatives. a polypropylene container or beaker. and HTLV-II in human serum or plasma samples. This test kit proteins derived from native inactivated disrupted viral particles decontaminate materials in 5% sodium hypochlorite is supplied for Research Use Only. It is not intended for use in STRONG REACTIVE HANDLE ASSAY SPECIMENS, STRONG REACTIVE solution for 30-60 minutes before disposal in biohazard 13. Do not expose reagents or perform test in an area and genetically engineered proteins. Individual nitrocellulose 1 vial AND NON-REACTIVE CONTROLS AS POTENTIALLY the diagnosis or prognosis of disease. CONTROL II waste-bags. containing a high level of chemical disinfectant fumes (e.g. strips are incubated with diluted serum or plasma specimens (80 µl) INFECTIOUS AGENTS. It is recommended that the components and controls. Specifi c antibodies to HTLV-I/II, if present in the Inactivated human serum hypochlorite fumes) during storage or during incubation and test specimens be handled using universal precautions. 10. Decontaminate all used chemicals and reagents by adding specimen will bind to the HTLV-I/II proteins on the strips. The with high titered antibodies They should be disposed of in accordance with established steps. Contact inhibits colour reaction. Also do not expose INTRODUCTION strips are washed to remove unbound materials while antibodies suffi cient volume of sodium hypochlorite to make a fi nal to HTLV-II and non-reactive safety procedures. reagents to strong light. that bind specifi cally to the HTLV proteins can be visualized for anti-HCV, anti-HIV-1/2 concentration of at least 1%. Leave for 30 minutes to The HTLV Blot 2.4 is intended as a supplemental antibody using a series of reactions with goat anti-human IgG conjugated and HBsAg. Contains sodium ensure effective decontamination. 14. The assay should preferably be performed at room assay for Research Use Only. The possible serological profi les The Strong Reactive Control I, Strong Reactive Control II with alkaline phosphatase and the substrate, BCIP/NBT. azide and thimerosal as temperature (25°C ± 3°C). defi ned by the HTLV Blot 2.4 include the following: HTLV and Non-Reactive Control contain Thimerosal and Sodium preservatives. 11. We do not recommend re-use of incubation trays. Seropositive, HTLV-I Seropositive, HTLV-II Seropositive, Azide, the Stock Buffer Concentrate and Wash Buffer 15. Make sure that the test strips are laid with the numbers Seronegative or Indeterminate. Concentrate contain Thimerosal and the Conjugate contains ANALYTICAL PRECAUTIONS LYOPHILIZED STOCK 1 or 2 bottles Sodium azide. Sodium azide can react with copper and lead on the strips facing upwards. BUFFER (each to be used in some plumbing systems to form explosive salts. 1. Optimal assay performance requires STRICT To be reconstituted in reagent reconstituted The quantities used in this kit are small, nevertheless when 16. For Western Blot Assay, it is important to use a rocking ADHERENCE to the assay procedure described in this grade water. Tris buffer with to 100 ml) disposing of azide-containing materials they should be fl ushed platform shaker and not a rotary shaker. Otherwise, Instruction Manual. Deviations from the procedure may heat inactivated animal and away with relatively large quantities of water to prevent metal performance of the kit will be compromised. The lead to aberrant results. non-animal proteins. Contains azide buildup in plumbing system. recommended speed and tilt angle of the shaker are 12 to thimerosal as preservative. 16 cycles per minute, and 5 to 10 degrees, respectively. 2. DO NOT MODIFY OR SUBSTITUTE REAGENTS FROM ONE KIT LOT TO ANOTHER. Controls, conjugate and WASH BUFFER 1 bottle 17. Ensure that automated equipment if used is validated Western Blot strips are matched for optimal performance. CONCENTRATE (20x) (70 ml) before use. Tris with Tween-20. Contains Use only the reagents supplied with the kit. thimerosal as preservative. 18. Ensure that the specimens are added away from the strip. 3. Do not use kit components beyond the expiry date printed Tray can be tilted and specimen added where the buffer is on the kit box. CONJUGATE 1 vial collected at lower end. This prevents dark spot formation Goat anti-human IgG (160 µl) due to specimen addition on the strip. conjugated with alkaline phosphatase. Contains sodium 19. Avoid the use of self-defrosting freezers for the storage of azide as a perservative. reagents and samples.

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4. William AE., Fang CT., Slamon DJ. et al. Seroprevalence STORAGE PREPARATION OF REAGENTS Procedure: SUMMARY OF ASSAY PROTOCOLS LIMITATIONS OF THE PROCEDURE and epidemiological correlates of HTLV-I infection in U.S. 1. Using forceps, carefully remove required 1. Store HTLV BLOT 2.4 kit and its components at 2°C to 8°C 1. DILUTED WASH BUFFER Reagents Qty Duration blood donors. Science 1988; 240: 643-646. number of STRIPS from the tube and when not in use. (a) DILUTED WASH BUFFER should be prepared fresh Optimal assay performance requires the strict adherence to the place numbered side up into each well. Nitrocellulose strip 1 - assay procedure described. Deviation from the recommended prior to use. 5. Lee H., Swanson P., Shorty VS., Zack JA., Roseblatt JD. Include strips for Strong Reactive, Weak procedure may lead to aberrant results. 2. All test reagents and strips when stored at 2°C to 8°C, are (b) Dilute 1 volume of WASH BUFFER CONCENTRATE Wash Buffer 2 ml 5 mins and Chen ISY. High rate of HTLV-II infection in seropositive Reactive and Non-Reactive controls. stable until the expiry date given on the kit. Do not freeze (20x) with 19 volumes of reagent grade water. Mix IV drug abusers in New Orleans. Science 1989; 244: 471- reagents. well. Blotting Buffer 2 ml - 475. 2. Add 2ml of DILUTED WASH BUFFER to 2 ml Specimen 20µl 60 mins LIMITED EXPRESSED WARRANTY DISCLAIMER A. Antigen strips 2. BLOTTING BUFFER each well. 6. Lipka JJ., Bui K., Reyes GR, Moeckli R., Wiktor SZ., • Avoid unnecessary exposure of antigen strips to light. (a) Reconstitute each bottle of LYOPHILIZED STOCK Wash Buffer 3 x 2 ml 3 x 5 mins The manufacturer makes no express warranty other than Blattner WA., Murphy EL., Hanson CV., Shaw GM., BUFFER with 100ml reagent grade water. Mix well to 3. Incubate the strips for at least 5 minutes 5 minutes that the test kit will function as a Research Use Only assay Shinsky JJ. and Foung SKH. Determination of a unique B. Reagents at room temperature (25°C ± 3°C) on Conjugate 2 ml 60 mins dissolve. This RECONSTITUTED STOCK BUFFER is within the specifications and limitations described in the immunodominant epitope of HTLV-I. Infect Dis 1990; 162: • Store reagents in their original vials or bottles, and they stable for 6 weeks if stored at 2°C-8°C. a rocking platform (speed of 12 to 16 Wash Buffer 3 x 2 ml 3 x 5 mins product Instructions For Use when used in accordance with 353-357. should be closed for storage. (b) BLOTTING BUFFER should be prepared fresh prior oscillations per minute). Remove buffer the instruction contained therein. The manufacturer disclaims • Dispense all reagents while cold and return to 2°C to 8°C to use. Add 1 g of BLOTTING POWDER to every 20 ml by aspiration. Substrate (Ready to use) 2 ml 15 mins any warranty express or implied, including such express or 7. Wiktor SZ., Alexandra SS., Shaw GM. et al. Distinguishing storage as soon as possible. of the RECONSTITUTED STOCK BUFFER prepared implied warranty with respect to merchantability, fi tness for between HTLV-I and HTLV-II by Western Blot. Lancet • Precipitates may form when the Substrate is stored at 2°C in step 2(a) above. Mix well. Stir to ensure powder 4. Add 2ml of BLOTTING BUFFER to each 2 ml Distilled Water 3 x 2 ml - use or implied utility for any other purpose. The manufacturer 1990; 335: 1533. to 8°C. This will not affect the performance of the kit. dissolves completely. well. is limited to either replacement of the product or refund of the (c) Stir again before dispensing. purchase price of the product. The manufacturer shall not be 8. Samuel KP., Lautenberger JA., Jorcyk CL., Josephs CAUTION: Avoid unnecessary exposure of substrate 5. Add 20µl each of sera or controls to 20 Øl AMOUNT OF REAGENTS REQUIRED liable to the purchaser or third parties for any damage, injury 3. WORKING CONJUGATE SOLUTION S., Wong Staal F. and Papas TS. Diagnostic potential to light. appropriate wells. FOR VARIOUS NUMBER OF STRIPS or economic loss howsoever caused by the product in the for human malignancies of bacterially produced HTLV-I Note : Prepare solution in polypropylene container / use or in the application thereof. The manufacturer makes no enevelope protein. Science 1984; 226: 1094-1097. beaker. 6. Cover the tray with the cover provided and 60 minutes Reagents NUMBER OF STRIPS TO BE USED representation express or implied, that this product will not (a) WORKING CONJUGATE SOLUTION should be infringe the intellectual property rights of the third parties. SPECIMEN COLLECTION, TRANSPORT AND STORAGE incubate for 1 hour at room temperature 3 6 9 15 20 27 36 9. Hadlock KG., Goh CJ., Bradshaw PA., Perkins S., Lo J., prepared fresh prior to use. (25°C ± 3°C) on the rocking platform. (b) Prepare WORKING CONJUGATE SOLUTION by 1X Wash Buffer (ml) 60 100 140 240 300 400 520 Habbaz RK., Kaplan J. and Foung SKH. Delineation of an Serum or plasma samples collected in EDTA, heparin or sodium immunodominant and highly HTLV specifi c epitope within citrate may be used. Before storage, ensure that blood clot or diluting CONJUGATE 1:1000 into BLOTTING BUFFER, 7. Carefully uncover the tray to avoid 1X Blotting Buffer (ml) 20 40 60 80 100 120 160 the HTLV-I transmembrane glycoprotein. Blood 1995; blood cells have been separated by centrifugation. for example 10µl CONJUGATE to 10ml BLOTTING splashing or mixing of samples. Tilt the TECHNICAL PROBLEMS / COMPLAINTS 68(4): 1392-1399. BUFFER. tray to aspirate the mixture from the wells. Conjugate (Øl) 11 17 23 35 45 59 77 Samples should be stored at 2°C to 8°C if the test is to be run Change aspirator tips between samples Should there be a technical problem / complaint, please do Substrate (ml) 11 17 23 35 45 59 77 10. Varma M., Rudolph D., Knuchel M., Switzer W., Hadlock within 7 days of collection or frozen at -20°C or colder if the test 4. SUBSTRATE SOLUTION (ready to use) to avoid cross-contamination. the following: (a) Dispense directly the required volume from the bottle. KG., Velligan M., Chan L., Foung SKH., LaI RB. Enhanced is to be delayed for more than 7 days. Clear, non-hemolyzed Blotting Powder (g) 1 2 3 4 5 6 8 1. Note the kit lot number and the expiry date. specificity of truncated transmembrane protein for samples are preferred. Lipemic, icteric or contaminated Use a clean pipette. Cap tightly after use. 8. Wash each strip 3 times with 2ml of 2. Retain the kits and the results that were obtained. 3 x 2 ml serologic confi rmation of HTLV-I and HTLV-II infection (particulate) samples should be fi ltered (0.45µm) or centrifuged DILUTED WASH BUFFER allowing 5 3. Contact the nearest MP Biomedicals offi ce or your local by Western Blot assay containing recombinant envelope before testing. minutes soak on the rocking platform distributor. glycoproteins. J. Clin. Micro. 1995; 33(12): 3239-3244. between each wash. Sera can be inactivated but this is not a requirement for optimal ASSAY PROCEDURE - Manual Use Only QUALITY CONTROL test performance. Inactivated as follows: 9. Add 2 ml of WORKING CONJUGATE 2 ml NOTE: - The procedures identifi ed in this Instructions for Use SOLUTION to each well. We recommend that the Non-Reactive Control and both Strong REFERENCES 1. Loosen caps of serum containers. document are for manual testing only. Reactive Controls be run with assay regardless of the number of 2. Heat serum at 56°C for 30 minutes in a water bath. samples tested. In order for the results obtained from any assay 10. Cover tray and incubate for 1 hour at room 1. Towbin H., Staehlin T. and Gordan J. Electrophoretic 3. Allow serum to cool before retightening caps. Note: a) Aspirate all used chemicals and reagents into trap 60 minutes to be considered valid, the following conditions must be met: temperature (25°C ± 3°C) on the rocking transfer of proteins from polyacrylamide gels to 4. Serum can be stored frozen until analysis. containing sodium hypochlorite. platform. nitrocellulose sheets: procedure and some applications. 1. NON-REACTIVE CONTROL b) All incubations are to be carried out on a rocking Proc. Natl. Acad. Sci. U.S.A. 1976; 76 4350-4354. We recommend that the sera should not undergo repeated No HTLV-I/II viral specifi c bands, rpg46-I, rpg46-II or GD21 platform. 11. Aspirate WORKING CONJUGATE 3 x 2 ml freeze-thaw cycles prior to testing. should be observed on the Non-Reactive control strip. The SOLUTION from the wells. Wash as in 2. Poiesz BJ., Ruscetti FW., Gazdar AF., Bonn PA., Minna band for the serum control (anti-human IgG) should be Caution: step 8. JD. And Gallo RC. Detection and Isolation of type C visible. Some samples cause dark patches on the spot of the strip retrovirus particles from fresh and cultured lymphocytes where they are added. To avoid this problem, one should 12. Add 2 ml of SUBSTRATE SOLUTION to of a patient with cutaneous T-cell lymphoma. Proc. Natl. ADDITIONAL MATERIALS REQUIRED BUT NOT 2 ml 2. STRONG REACTIVE CONTROL I ensure the following:- each well. Acad. Sci. U.S.A. 1980; 77(12): 7415-7419. PROVIDED The serum control band and all relevant HTLV-I/II molecular weight bands must be evident. The relevant HTLV-I bands i. Sample should be added only after BLOTTING BUFFER is 13. Cover tray and incubate for 15 minutes 3. Kalyanaraman VS., Sarngadharan MG., Robert-Guroff M., Optimal assay performance requires STRICT ADHERENCE to 15 minutes that must be present are p19, p24, gp46, rgp46-I and GD21. added. on the rocking platform. Miyoshi I., Blayney D., Golde and Gallo RC. A new subtype the assay procedure described below. Deviations in procedure Note that the gp46 band is diffused. The serum control (anti- or equipment may produce aberrant results. of human T-cell leukemia virus (HTLV-II) associated with human IgG) band should be visible. ii. Tilt the tray slightly by elevating either the top or bottom end 14. Aspirate the SUBSTRATE and rinse the 3 x 2 ml a T-cell variant of hairy cell leukemia. Science 1982; 218: 1. Deionized or distilled water, reagent grade of the tray. The Blotting Buffer will fl ow to the lower end of the strips several times with reagent grade 571-573. 3. STRONG REACTIVE CONTROL II 2. Disposable gloves tray. Add the sample where the Blotting Buffer is collected. water to stop the reaction. The serum control band and all relevant HTLV-I/II molecular 3. Rocking platform (designed with a rocking speed of 12 to When all the samples are added, return the tray back to its Manufacturer: weight bands must be evident. The relevant HTLV bands 16 oscillations per minute and which moves through a 5° to original fl at position. Always ensure that the strips are kept 15. Using forceps, gently remove strips onto that must be present are p24, GD21 and rpg46-II. The serum 10° tilt to wash membranes evenly) wet during the process. paper towels. Cover with paper towels and MP Biomedicals Asia Pacifi c Pte Ltd control (anti-human IgG) band should be visible. 4. Pipettors and tips of appropriate volume dry. Alternatively, allow strips to dry in the 2 Pioneer Place 5. Aspirator with sodium hypochlorite trap iii. Alternatively, if tilting the tray is not desired, the samples wells of the tray. Singapore 627885 6. 56°C water bath (optional) may be added to the top or bottom end of the well. This way Tel. No. : +65 775 0008 7. Sodium hypochlorite for decontamination if dark patches showed, the reading of the strip results will 16. Mount strips on worksheet (non-absorbent Fax No. : +65 774 6146 not be affected. white paper). Do not apply adhesive tape Email: [email protected] over the developed bands. Observe the bands and record the result. For storage, keep the strips in the dark.

4 5 6 FIGURE 1 TROUBLE SHOOTING CHART

serum control Strips are Expected bands do Bands other than the HTLV-specifi c bands not develop or are of Serum Control band defective rgp46-l detected on negative weak intensity. develops on negative control and/or known rgp46-ll Dark spots develop control negative samples Watery marks on on strips Strong Background develops on developed strips Non-specifi c White patches develop strip in the absence or presence bands and/or on strips of positive bands. p53 dark background develop on strips gp46 Check positive control Absence of Serum Control Band 1. They are cracked. p36 1. Strips fl ipped over 2. They contain air 1.Strips left to dry after during assay. bubbles which cause pre-soaking step prior 2. Trays not properly to adding Blotting p32 the appearrance of washed before use. white spots in reactive Buffer. 3. Poor dissolution of Tray wells or Control zones big enough p28 Blotting Powder. to prevent 1. overdeveloped strips (stop may have been crossed 4. Electrotransblot any detection. p26 reaction sooner). contaminated. interference during 3. They show dark spots p24 2. Incomplete washing. manufacturing. due to fungal growth p19 upon initial opening of the strip tubes. 1. Bacterial or fungal However, if dark spots contamination of test develop sometime sample. later after initial 2. Precipitation of opening of the tube GD21 immune complexes 1. Serum not added. then the problem is in aged test sample. Positive control weak Positive control OK 2. Strips flipped over during due to improper strip 3. Bacterial or fungal assay. storage conditions at contamination on 3. Conjugate not added. the user’s site. strip due to improper 4. Substrate not added. storage. 4. Strips physically 1. Trays contaminated damaged, cracked or The problem is probably The problem is probably with positive test scratched. caused by the reagents. caused by test sample. sample or positive 5. Strips not properly a. b. c. d. control. washed between 1.Reagents not properly 1. Wrong test sample 2. Negative control/test assay steps. prepared. dilution. sample contaminated 2. Wrong conjugate 2. Test sample 1. Wrong test sample or with positive control/ dilution. contaminated with conjugate dilution. test sample. 3. Unstable reagents conjugate. 2. Test sample/reagent 3. Same pipette tip due to improper 3. Test samples severely Viral specific bands as visualized with: incubation too long. used for delivery temperature exposure. immune-complexed. a. A HTLV-l/ll dual infection serum. 3. Incomplete washing and/or removal of, test 4. Conjugate contaminated 4. Test sample IgG b. Strong Reactive Control l. (Reactive for HTLV-l only) during assay. samples/control. with human IgG. deteriorated or 4. Incubation 4. Test sample may c. Strong Reactive Control ll. (Reactive for HTLV-lI only) 5. Incorrect substrate denatured due to temperature greater be an ELISA “false” d. Non-reactive Control. pH due to exposure repeated freeze- thaw than 30˚C. negative. or improper storage. to strong UV light or 5.Test sample reactive 5. Rotary platform used reducing agent. with non-viral proteins. 6. Trays, reagent(s) instead of Rocking or water having platform. high phosphate 6. Test sample may concentration. be an ELISA “false” 7. Rotary platform used positive. instead of Rocking platform.

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