ࡱ> I Wbjbj %0W]  $ Z88. `t7Contents Principles of the ExaVirTM Load Kit..........................................................2 Components............................................................................................3 Storage of the Kit.....................................................................................3 Intended Use...........................................................................................4 Precautions..............................................................................................4 Sample Collection and Preparation..........................................................4 Separation Equipment..............................................................................5 Symbols...................................................................................................5 Temperature Specification....................................................................... 5 Flow Chart of the ExaVirTM Load Kit Step-by-Step Procedure...................6 Step-by-Step Instructions..........................................................................7 A: Separation.....................................................................................7 B: RT-assay......................................................................................11 References.............................................................................................19 1 Issued November 2002 Revised December 2002 Principles of the ExaVirTM Load Kit In the ExaVirTM Load Kit the activity of the viral enzyme Reverse Transcriptase (RT) is measured to estimate the HIV viral load in plasma samples. The procedure is divided into two main parts: the Separation and the RT-assay. In the Separation part the plasma is first treated to inactivate cellular enzymes. The virus particles are then separated from the plasma by the use of a gel that binds the virion lipid membrane. At this stage disturbing factors present in the plasma, such as antibodies or anti-retroviral drugs, are washed away. To obtain the RT, the virion is then lysed and the lysate collected for further analysis in the RT-assay part. During the RT-assay the lysates are analysed in an ELISA set-up. The principle of this assay is illustrated in figure 1. The figure shows a well in the 96-well Poly A Plate, used for the RT-assay, with the RNA template bound to the bottom. A reaction mixture, containing primer and a non-radioactive RT substrate, is added to the plate together with the lysates. If the lysates contain any RT, the enzyme will synthesise a DNA-RNA hybrid. This product is detected with an alkaline phosphatase (AP) conjugated -BrdU antibody. The product can then be quantified by addition of a colorimetric or fluorimetric AP substrate. The lysates are added to the Poly A Plate in two different amounts, 75 and 15 l, to increase the range of the assay. A serially diluted standard, with a known concentration of recombinant HIV-1 RT, is also added to the plate for quantification of viral load in the samples. The lysates obtained in the Separation part can also be frozen at -70C and used e.g. for later drug susceptibility analyses1. The evaluation is carried out using the Evaluation Protocol for the ExaVirTM Load Kit. Figure 1: Schematic illustration over the RT-assay part of the ExaVirTM Load Kit. BrdUTP = RT substrate, mAb-AP = AP conjugated antibody, pNPP = Colorimetric substrate for AP. RT DNA-SYNTHESIS DNA-QUANTIFICATION BrdUTP mAb-AP pNPP pNP Primer Template 1 Cavidi Tech is releasing a Drug Susceptibility Kit, ExaVirTM Drug, in the beginning of year 2003. Contact Cavidi Tech for further information (see Ordering Information on last page). 2 Components One ExaVirTM Load Kit contains reagents for analysis of 32 plasma samples, including one positive and one negative plasma control. Separation part: RT-assay part: 1 Plasma Treatment Additive (1) (lyophilised) 1 High Sensitivity Poly A Plate (A) (96 wells) 1 Separation Gel (2) (52 ml) 1 Base Buffer HS-Lenti (B) (12,5 ml) 1 Conc. Gel Wash Buffer (3) (120 ml) 1 Lysate & Std Dilution Buffer (B1) (15 ml) 1 Conc. Gel Reconditioning Buffer (4) (50 ml) 1 RT Reaction Components HS (C) (lyophilised) 1 Lysis Buffer (5) (24 ml) 1 HIV-1 rRT Standard (D) (lyophilised) 1 Lysis Buffer Additive (5.1) (lyophilised) 1 Plate Wash Buffer Complete (E) (80 ml) 1 96-well Lysate & Standard Preparation Plate 1 RT Product Tracer HS (O) (lyophilised) 32 Lysate Collection Tubes 1 Product Tracer Dissolvent (O1) (12,5 ml) 32 Plasma Processing Tubes with separate caps 1 AP Coloro Substrate Tablet (P1) 32 Columns 1 AP Coloro Substrate Buffer (P2) (30 ml) Or 1 AP Fluoro Substrate Buffer (P) (15 ml) 3 Pieces adhesive tape 1 Plastic lid for 96-well plate Rubber bands 1 Standard Curve Sheet Start-up Material supplied with first order Sample Box & Lid Vacuum Tubing Column Holder 4 x 3-litres Wash Buckets Waste Collector 2 x 10 ml Bottle-top dispensers Sample Collector 2 Small tubing for dispenser Tube Rack 1- &2-litres Glass bottles Vacuum Pump Evaluation Protocol for the ExaVirTM Load Kit Waste Container Instructions for Evaluation Protocol Equipment required but not provided 1 In-house Positive Control (see page 4) End-over-end mixing table 1 In-house Negative Control (see page 4) Pipettes (single, 8 or 12 multi-channel) Distilled water Reservoirs for multi-channel pipettes Vircon or other relevant disinfectant Pipette filter tips (1000 l) ELISA-plate reader Pipette tips (200 l & 5 ml) Incubator set at 33C 5-litre Container Freezer set at -15 to -25C 25 ml Bottle/Tube Vortex Computer with Microsoft Excel, version 97 or later (Plastic Pasteur pipettes) Storage of the Kit The kit may be delivered frozen or unfrozen. Store the kit below -15C until use. If delivered unfrozen, the kit should be refrozen or used within a week. Keep unfrozen components at 4 to 8C. All kit components are for one-time-use only. Discard any leftovers after the ExaVirTM Load Kit procedure has been performed. 3 Intended use The ExaVirTM Load Kit is an in vitro test system for quantitative measurement of HIV RT activity in plasma samples for estimation of HIV viral load. The ExaVirTM Load Kit is intended for research purposes only. Precautions h The kit is intended for laboratory, research purposes only, NOT for human or household use. h Use of the kit is at the users own risk. h Do not combine components from kits with different Lot numbers. h Do not expose the kit components to direct sunlight or a temperature above 37C during laboratory work. h The Cavidi Separation equipment should be used with the supplied Vacuum Pump to standardise the system. h The ExaVirTM Load Kit is functional and evaluated in a temperature between 18 and 33C. h Avoid contact with skin and mucous membrane since some components contain hazardous and toxic substances, e.g. sodium azide. h The kit contains no material of human origin. h To avoid transmission of infection, handle samples according to laboratory rules and safety regulations. h Do not pipette by mouth. h Do not drink, eat or smoke in areas where specimens or reagents are handled. Sample Collection and Preparation PLASMA The plasma samples must be frozen once before analysed in the ExaVirTM Load Kit in order to optimise inactivation of disturbing cell enzymes with the Plasma Treatment Additive. We recommend the use of EDTA or sodium citrate anti-coagulated whole blood. For optimal results, plasma should be separated from cells within four hours of the collection of the blood. Significant delays (more than six hours) can lead to inaccurate results. It is important to avoid cell contamination, since cells may give non-HIV RT signals in assay. Long-term storage of plasma samples should be at or below -60C. PREPARATION OF IN-HOUSE HIV POSITIVE CONTROL As human material cannot be included in the kit, prepare about 100 ml of a pool of EDTA or sodium citrate plasma by mixing samples with high and low HIV RT activity levels. The pool should correspond to approximately 50 fg/ml of RT. If no plasma with determined RT amount is available we recommend preparing a pool that corresponds to 10500 copies/ml (N.B. value determined from B-isolates only). Aliquot the material into 1 ml portions and use one portion as positive control with every ExaVirTM Load Kit. PREPARATION OF IN-HOUSE NEGATIVE CONTROL Prepare about 100 ml of a pool of EDTA or sodium citrate plasma from healthy blood donors. Aliquot the material into 1 ml portions and use one portion as negative control with every ExaVirTM Load Kit. 4 Separation Equipment Symbols B, O1 etc: A letter and/or number marked in bold refer to the kit component marked with the same letter and/or number. !: A clock-symbol in the margin of the text indicates that the step includes an incubation. The time reference underneath the clock-symbol informs about the length of the incubation, e.g. "90 min." means that the incubation lasts for 90 minutes. : The figure is an illustration of a 96-well plate with well A1 in the upper left corner. Specific wells are marked with a colour and a circle. Bent arrows illustrate a transfer from one 96-well plate to another or from one well to another. : The figure is an illustration of a bottle or tube. Bent arrows illustrate a transfer from one bottle to another. Temperature Specification Room temperature = 18 to 33Celsius (C) = 64 to 91Fahrenheit (F) 56C = 133F 18C = 64F -20C = -4F 37C = 99F 8C = 46F -60C = -76F 33C = 91F 4C = 39F -70C = -94F 28C = 82F -15C = 5F Figure 2: A complete set of the Separation equipment. 1. Vacuum Pump 3. Waste Container 5. Waste Collector 7. Tube Rack 2. Vacuum Tubing 4. Column Holder 6. Sample Collector 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H 1 2 4 5 3 2 6 7 X 5 Flow Chart of the ExaVirTM Load Kit Step-by-Step Procedure TAKE OUT components and thaw frozen ones. TAKE OUT components and thaw frozen ones. PREPARE & DISTRIBUTE the two Reaction mixtures in the Poly A Plate. ADD 100 l Plasma Treatment Additive and 1 ml sample or control to labelled Plasma Processing Tubes. SUCK dry & WASH each gel with 2 x 8 ml Gel reconditioning buffer. PREPARE Separation equipment. LABEL columns and Lysate Collection Tubes. TAKE OUT components and thaw frozen ones. PREPARE the Wash buffer, the RT Product Tracer and the AP Substrate. WASH the Poly A Plate using four buckets or ELISA-plate washer. ADD 100 l RT Product Tracer to all wells in the Poly A Plate. INCUBATE for 60 minutes in the dark at room temperature. INCUBATE for 90 min. at 33C. ADD 1.5 ml Separation Gel to each Plasma Processing Tube. PREPARE the Gel Wash, Gel reconditioning and Lysis buffer. VORTEX & ADD each sample to the corresponding column. SUCK dry & WASH each gel with 4 x 8 ml Gel wash buffer. SHAKE for 90 min. at room temperature. WASH the Poly A Plate using four buckets or ELISA-plate washer. RT-assay ADD 120 l AP Coloro OR Fluoro Substrate to all wells in the Poly A Plate. READ the Poly A Plate straight away (zero reading), after two to three hours and the following day. Separation Step 1, Page 7 Step 7, Page 9 SUCK dry & MOVE Column Holder to Sample Collector. ADD 600 l Lysis buffer to each column. APPLY vacuum gently, SUCK dry & TRANSFER lysates to the Preparation Plate. Step 8, Page 9 PROCEED to the RT-assay or seal and store the Preparation Plate, containing the lysates, at -70C for later analysis1. ADD backgrounds, lysates & standard to the Poly A Plate. MAKE a serial dilution of the HIV-1 rRT Standard. INCUBATE overnight at 33C. Step 6, Page 9 Step 5, Page 8 Step 4, Page 8 Step 3, Page 8 Step 2, Page 7 Step 9&10, Page 12 Step 11, Page 13 Step 12&13, Page 14-15 Step 14-17, Page 16 Step 18, Page 16-17 Step 19, Page 18 Step 20, Page 18 Step 21, Page 18 6 EVALUATE using the Evaluation Protocol for the ExaVirTM Load Kit. Step-by-Step Instructions A: Separation During the first part of determination of HIV viral load using the ExaVirTM Load Kit, the RT is isolated from the plasma sample. This is performed using a gel that binds the lipid membrane of the HIV virions. When the virus particles have been immobilised on the gel, all disturbing factors are washed away. After this, the RT is obtained by lysis of the virions. The lysates are collected for analysis in the RT-assay. Preparation 1. Take out and thaw components and samples Take out the following components and thaw the frozen ones at or below 37C: 1 Plasma Treatment Additive (1) 1 Separation Gel (2) 1 Conc. Gel Wash Buffer (3) 1 Conc. Gel Reconditioning Buffer (4) 1 Lysis Buffer (5) 1 Lysis Buffer Additive (5.1) 32 Plasma Processing Tubes and caps 1 Sample Box 32 Columns 32 Lysate Collection Tubes 1 Separation equipment set-up (page 5) 1 Piece Adhesive tape 1 96-well Lysate & Standard Preparation Plate (1-ml wells) 1 Positive control (page 4) 1 Negative control (page 4) 30 Plasma samples 2. Prepare the plasma samples Dissolve the contents of the Plasma Treatment Additive (1) in 4 ml distilled water and vortex to mix. Label Plasma Processing Tubes from 1 to 32 and put them in the Sample Box. Add 100 l of the dissolved Plasma Treatment Additive and 1 ml plasma sample to each tube (Add the positive and negative control to tubes 31 and 32, respectively.). Add a cap to each tube and vortex to mix before incubating the samples in the dark for one hour at room temperature. Perform step 3 during this time. Day One NOTE Print out the three sheets Plasma Sample List, Sample Tracking & ExaVir Load from the Evaluation Protocol. Use them as lab journal during the ExaVir Load procedure. Carry out step 9, page 12 now if performing the RT-assay directly after the Separation. ! 60 min. IMPORTANT Use filter tips when adding the samples. If less than 1 ml plasma is available, dilute the sample up to 1 ml with plasma from the negative control pool. Note the actual samplevolume in the lab journal. a) b) c) h 7 3. Prepare Separation equipment Make sure that all parts of the Separation equipment (figure 2, page 5) are clean and disinfected (see Hint on page 10). Connect the Vacuum pump, Waste Container and Waste Collector with the Vacuum Tubing, according to figure 3. Put the Column Holder on top of the Waste Collector. Label the columns and the Lysate collection tubes from 1 to 32. Put them in their appropriate position in the Column Holder and the Tube Rack, respectively. 4. Add Separation Gel Take the Sample Box from the dark after the 1-hour incubation. Shake the Separation Gel bottle (2) to homogenise the gel slurry and add 1.5 ml to each tube. Shake the bottle between each transfer. This is important to ensure that each tube receives the same amount of gel. Put each cap back on the corresponding tube. Put on the lid of the Sample Box and tighten it with e.g. adhesive tape. Place the Sample Box on its side on the (end-overend) mixing table (fig. 4) and incubate at room temperature for 90 minutes. Perform step 5 during this time. 5. Prepare buffers GEL WASH BUFFER Mix contents of Conc. Gel Wash Buffer (3) and 1200 ml distilled water in a 2-litre bottle. Connect one of the dispensers to the bottle. Finally connect the small dispenser tubing to the dispenser. GEL RECONDITIONING BUFFER Mix contents of Conc. Gel Reconditioning Buffer (4) and 550 ml distilled water in a 1-litre bottle. Connect the other dispenser to the bottle. Finally connect the small dispenser tubing to the dispenser. LYSIS BUFFER Dissolve contents of Lysis Buffer Additive (5.1) using approximately 5 ml of Lysis Buffer (5). When dissolved, transfer the contents back to the Lysis Buffer bottle (5) and mix. ! 90 min. Figure 3: Vacuum Pump, Waste Container and Waste Collector connected by Vacuum Tubing. Figure 4: Sample Box on endover- end mixing table. a) a) b) b) 8 Separation 6. Wash gels After 90 minutes of incubation, vortex each tube and pour the contents into the corresponding column in the Column Holder. Start the pump, apply the vacuum by closing the valve of the Waste Collector and suck the gels dry. Wash the gels four times by turning off the vacuum, adding 8 ml Gel wash buffer to each column and applying the vacuum. 7. Recondition gels Recondition the gels two times by turning off the vacuum, adding 8 ml Gel reconditioning buffer to each column, waiting one minute, applying the vacuum and sucking the gels dry. 8. Lyse virions Put the Tube Rack with the labelled Lysate collection tubes into the Sample Collector. Disconnect the Vacuum Tubing from the Waste Collector and connect it to the Sample Collector (fig. 6). Make sure that the valve is open. Transfer the Column Holder, with the columns, from the Waste Collector to the Sample Collector. IMPORTANT Make sure that no buffer is left on top of the gels before proceeding. IMPORTANT When adding the buffers, dispense on the inner side of the column, not on top of the gel (fig. 5 a). If the gel clogs and blocks the column, use a Pasteur pipette to gently resuspend the gel (fig. 5 b). Figure 5 a and b: Wash of gels. a) Dispensing of Gel wash buffer. b) Resuspension of clogged gel in column. Figure 6: Vacuum Pump, Waste Container and Sample Collector, with Tube Rack, connected by Vacuum Tubing. b a) b) c) h a) a 9 Add 600 l Lysis Buffer (5) to each column and leave it for a few minutes before gently applying the vacuum and sucking the gels dry. Transfer all lysates from the Sample Collection Tubes to the 96-well Lysate & Standard Preparation Plate (fig. 7). Make sure that each lysate is transferred to the right position in the Lysate & Standard Preparation Plate and that the pipette filter tip is changed between each transfer. Proceed to the RT-assay part. Seal the plate with adhesive tape and store it at 4 to 8C until use (not more than 3 hours). If the RT-assay is not performed the same day, seal the plate and store it at -20C. 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H 1 9 17 25 2 10 18 26 3 11 19 27 4 12 20 28 5 13 21 29 6 14 22 30 7 15 23 P 8 16 24 N Preparation Plate Figure 7 a, b & c: Transfer of lysates. a & b) Transfer of lysates from Lysate Collection Tubes to the Lysate & Standard Preparation Plate. c) Sample layout after transfer (1, 2...= Sample number, P = Positive Control, N = Negative Control HINT - Disinfection Before Next Use The Separation equipment (number 3 to 7 in figure 2 on page 5) needs to be disinfected before next use. Rinse the equipment with 70% ethanol or keep it at 56C overnight. Rinse the equipment with water and let it dry before the next use. Empty the Waste Container (number 3 in fig. 2) after disinfection and add a suitable amount of relevant disinfectant before next use. a d) c) b) 10 b c B: RT-assay After isolation of the RT from the plasma samples, the enzyme activity is quantified in the RT-assay part. During this procedure the enzyme synthesises a product that can be detected by an AP conjugated antibody. First the lysates and a serially diluted standard are added to the Poly A Plate together with a Reaction mixture, which contains primer and RT substrate. Then the synthesis takes place during an over-night-incubation. During day two the AP conjugated antibody is added to the Poly A Plate. By addition of a colorimetric or fluorimetric AP substrate, the RT activity can be quantified. Standard Set-up of the 96-well Poly A Plate 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H 1 1 9 9 17 17 25 25 S1 S9 S1 S9 2 2 10 10 18 18 26 26 S2 S10 S2 S10 3 3 11 11 19 19 27 27 S3 S11 S3 S11 4 4 12 12 20 20 28 28 S4 Bg S4 Bg 5 5 13 13 21 21 29 29 S5 Bg S5 Bg 6 6 14 14 22 22 30 30 S6 Bg S6 Bg 7 7 15 15 23 23 P P S7 Bg S7 Bg 8 8 16 16 24 24 N N S8 Bg S8 Bg = 75 l Lysate = 15 l Lysate = 15 l Standard = 75 l Standard = 75 l Background = 15 l Background 1, 2...= Lysate number P = Positive Control S1, S2...= Standard number Bg = Background N = Negative Control 11 Preparation 9. Take out and thaw components Take out the following kit components and thaw the frozen ones at or below 37C: 1 High Sensitivity Poly A Plate (A) 1 Base Buffer HS-Lenti (B) 1 Lysate & Std Dilution Buffer (B1) 1 RT Reaction Components HS (C) 1 HIV-1 rRT Standard (D) 1 Piece of adhesive tape 1 Plastic lid for 96-well plate 10. Prepare Reaction mixtures and add to the Poly A Plate Add the contents of the Base Buffer HS-Lenti (B) to the RT Reaction Components (C) and mix thoroughly. In a small bottle or tube, mix 6 ml of this first Reaction mixture (C) with 3.6 ml of the Lysate & Std Dilution Buffer (B1) (fig. 8). Mix thoroughly and label this bottle/tube 2RM (2nd Reaction mixture). Add 100 l of the contents of bottle C to all wells in rows 1, 3, 5, 7, 9 and 10 of the Poly A Plate (A) (fig. 9 a). Add 160 l of the reaction mixture in bottle/tube 2RM to all wells in rows 2, 4, 6, 8, 11 and 12 of the Poly A Plate (A) (fig. 9 b). Cover the plate with a plastic lid. 2RM B1 B C All 6 ml 3.6 ml Figure 9 a & b: Addition of Reaction mixtures. a) Dark grey = Add 100 l from bottle C to these wells. b) Light grey = Add 160 l from bottle/tube 2RM to these wells. 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H C 2RM Figure 8: Preparation of the two Reaction mixtures. Poly A Plate Poly A Plate a b a) b) c) h 12 11. Prepare HIV-1 rRT Standard Dissolve the contents of the HIV-1 rRT Standard (D) in 4 ml Lysate & Std Dilution Buffer (B1) and mix. Cover the wells containing lysates in the 96-well Lysate & Standard Preparation Plate. Add 300 l Lysate & Std Dilution Buffer (B1) to wells B10 to H10 and A12 to H12 of the 96-well Lysate & Standard Preparation Plate. Add 500 l of the dissolved HIV-1 rRT Standard (D) to well A10. Make a dilution series by transferring 200 l from well A10 to B10 and mix, B10 to C10 and mix etc, all the way to C12 (fig. 10). Wells D12 to H12 are background controls and do not belong to the dilution series. IMPORTANT Keep the dissolved HIV-1 rRT Standard in a cool dark place until use. Add the dilutions to the Poly A Plate within 30 minutes. Mix thoroughly and change pipette tips after each transfer. Figure 10: Serial dilution of Standard. Orange = Add 500 l dissolved HIV-1 rRT Standard. Arrow = Transfer 200 l from one well to the next. 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H Lysates 200 l Preparation Plate b) c) a) 13 Addition 12. Add backgrounds and lysates to Poly A Plate Transfer 75 l from wells D12 to H12 of the Lysate & Standard Preparation Plate to wells D10 to H10 of the Poly A Plate. Transfer 15 l from wells D12 to H12 of the Lysate & Standard Preparation Plate to wells D12 to H12 of the Poly A Plate (fig. 11). Mix the lysates in the Lysate & Standard Preparation Plate with a pipette before transfer to the Poly A Plate. The lysates should be added one row at a time, starting with the 75-l and then the 15-l portion, according to figure 12 a (also see the Standard Set-up on page 11). Change pipette tips between the different lysate rows. Figure 11: Addition of backgrounds. Transfer first 75 l (dark blue) and then 15 l (light blue) from the wells marked with dark blue colour in the Lysate & Standard Preparation Plate to the Poly A Plate. 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H Lysates Poly A Plate Preparation Plate NOTE Let lid cover already added rows. Make sure not to pass the pipette over any uncovered wells in the Poly A Plate. The Lysate & Standard Preparation Plate can be sealed and stored at -70C for additional analyses1. a) b) Figure 12 a, b & c: Addition of lysates. a) Transfer first 75 l (dark colours) and then 15 l (light colours), from the Lysate & Standard Preparation Plate to the Poly A Plate, one row at a time. b & c) Transfer of lysates from Lysate & Standard Preparation Plate to Poly A Plate. 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H Standard Preparation Plate Poly A Plate a 14 b c 13. Add HIV-1 rRT Standard to Poly A Plate Let lid cover lysates and add 75 l of the standard from wells A12 to C12 of the Lysate & Standard Preparation Plate to wells A10 to C10 of the Poly A Plate. Transfer 15 l of the standard from wells A12 to C12 to wells A12 to C12 of the Poly A Plate (fig. 13). This is easiest performed using an 8-channel pipette. Transfer 75 l of the standard from wells A10 to H10 to wells A9 to H9 of the Poly A Plate. Transfer 15 l of the standard from wells A10 to H10 to wells A11 to H11 of the Poly A Plate (fig. 13). Seal the Poly A Plate with adhesive tape. Press the tape down firmly over each well to ensure proper sealing. Incubate overnight (16 to 24 hours) at 33C. b) c) Figure 13: Addition of standard. First add 75 l (dark yellow) and then 15 l (light yellow) from wells A12 to C12 of the Lysate & Standard Preparation Plate to the Poly A Plate. Then add 75 l (dark orange) and then 15 l (light orange) from column 10 of the Lysate & Standard Preparation Plate to the Poly A Plate. 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H Lysates Poly A Plate Preparation Plate NOTE If a single-channel pipette is used, start adding the standard with the lowest concentration, i.e. C12 and then B12 etc. HINT - Poly A Plate Set-up The final set-up of the Poly A Plate is illustrated in the picture to the right. Green = Lysates, Yellow = HIV-1 rRT Standard, Blue = Background. Dark colours = 75-l portion, light colours = 15-l portion. (Also see page 11.) 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H 15 a) IMPORTANT To be able to use the Evaluation protocol the HIV-1 rRT Standard must be added to the wells described here. overnight ! Preparation 14. Collect and thaw components Take out the following components and thaw the frozen ones at or below 37C: 1 Plate Wash Buffer Complete (E) 1 RT Product Tracer HS (O) 1 Product Tracer Dissolvent (O1) 1 AP Coloro Substrate Tablet (P1) 1 AP Coloro Substrate Buffer (P2) Or 1 AP Fluoro Substrate Buffer (P) 1 Piece Adhesive Tape 1 Plastic Lid Rubber Bands 15. Prepare Plate Wash buffer Add two to three litres of distilled water to a 5-litre container. Add the contents of the Plate Wash Buffer Complete (E) to the container. Rinse the bottle with distilled water a few times and pour it into the container. Adjust the volume to five litres with distilled water and mix thoroughly. Pour approximately 0.5 litres into each of the four buckets. Save the rest of the Wash buffer for later use in step 20. 16. Prepare RT Product Tracer Transfer the contents of the Product Tracer Dissolvent (O1) to the RT Product Tracer (O) and vortex. Make sure that the lyophilised material is completely dissolved before use in step 19. This takes about ten minutes. Store it in the dark until use. 17. Prepare AP Substrate Add the AP Coloro Substrate Tablet (P1) to the thawed AP Coloro Substrate Buffer (P2). Store it in the dark until use. 18. Stop the RT reaction by washing Collect the Poly A Plate from the incubator. Remove the tape carefully, pulling it diagonally from one corner to the other. Secure the strips with a rubber band. Empty the plate into the waste sink. h Day Two h h NOTE The AP Fluoro Substrate Buffer (P), for fluorimetric detection, is ready to use after thawing. h NOTE The Plate wash buffer will give a final concentration of Triton X- 100 of 0.75%. a) 16 ! 5 x 5 min. Put the Poly A Plate in the first bucket and tap gently. Pick up the plate and empty it over the bucket. Repeat this action 10 to 15 times before moving the plate to the second bath. Repeat the procedure in the second bucket and move the plate to the third bucket. Repeat the procedure and leave the plate to soak for five minutes (only for buckets three and four). Keep the plate with the wells facing up and tap gently to make bubbles escape. Repeat the procedure for the last bucket. After washing in four baths, tap the plate upside-down on a dry absorbing paper. Leave it to dry upside-down on the paper for five minutes. Tap the plate again to make all bubbles in the wells disappear. Do not forget to take off the rubber band before proceeding to the next step. Throw away the used Wash buffer and rinse out the buckets, first with tap water and then with distilled water. Turn buckets upsidedown to dry. NOTE The Plate wash buffer should preferably hold a temperature between 18 and 28C. b) c) Figure 14: Wash of Poly A Plate in plastic buckets. HINT - Using a Plate Washer Machine A standard ELISA-plate washer machine can be used instead of the buckets, although the bucket wash gives better background values. If a machine is used, turn the plate 180 between the washes, if possible. d) 17 e) Quantification 19. Add RT Product Tracer Add 100 l of the dissolved RT Product Tracer (O) to each well of the Poly A Plate, without touching the bottom of the wells. Seal the plate with adhesive tape. Press the tape down firmly over each well to ensure proper sealing. Incubate for 90 minutes at 33C. 20. Remove RT Product Tracer Collect the Poly A Plate from the incubator and wash with the remaining Plate wash buffer according to step 18. 21. Add AP Substrate and read the Poly A Plate Mix the AP Substrate thoroughly and add 120 l to each well of the Poly A Plate without touching the bottom of the wells. Remove bubbles, if any, in the wells with a clean pipette tip for each well. Read the plate straight after addition of the substrate (zero reading). Cover with a plastic lid and incubate in the dark at room temperature until next reading. Read the plate a second time after 2 to 3 hours and a third time the following day (16 to 24 hours after addition of AP substrate). Incubate in the dark between the readings. 22. Process the data Calculation of the viral load values of the plasma samples is performed using the Evaluation Protocol for the ExaVirTM Load Kit (see separate instruction manual). There is a separate Evaluation Protocol for using fluorimetry. 5x 5 min. ! 2 - 3 hours, overnight ! 90 min. ! HINT - Washing the Plate To avoid bubbles in the wells when adding the AP substrate in step 21, let the plate sit on the bottom of the last bucket. Remove any surface bubbles by scraping them away by hand. Lift the plate carefully out of the bucket ensuring that no bubbles are present in the wells. Empty the remaining wash buffer from the wells by turning the plate upside-down over the wash bucket. Do not forget to tap the plate upside-down and remove the rubber band before proceeding to step 21. VERY IMPORTANT If filter tips are not used, do not use the same pipette for the RT Product Tracer and the AP Substrate. HINT - Reader Information Colorimetry: Set the reader at A405 (reference at 620 nm) Fluorimetry: Exciting wavelength: 350-60 nm Fluorescence reading wavelength: 455-70 nm (Methylumbelliferone) a) a) b) b) h h 18 References TECHNICAL PAPERS (ExaVirTM Kits, Lenti-RT and C-type RT Activity Assay) Malmsten A., Shao X., Aperia K., Corrigan G.E., Sandstrm E., Kllander C.F.R., Leitner T., Gronowitz J.S. Features of HIV viral load determination based on Reverse Transcriptase activity recovered from human plasma. Under submission, December 2002. Shao X., Malmsten A., Lennerstrand J., Snnerborg A., Unge T., Gronowitz J.S., Kllander C.F.R. Use of HIV-1 reverse transcriptase recovered from human plasma for phenotypic drug susceptibility testing. Under submission to AIDS, December 2002. Lennerstrand J., Hertogs K., Stammers DK., Larder BA. Correlation between viral resistance to zidovudineand resistance at the reverse transcriptase level for a panel of human immunodeficiency virus type 1mutants. J Virol. 75(15): 7202-5, Aug 2001. Lennerstrand J., Stammers D.K., Larder B.A. Biochemical mechanism of human immunodeficiency virus type 1 reverse transcriptase resistance to stavudine. Antimicrob Agents Chemother. 45(7): 2144-6, July 2001. Corrigan G.E., Al-Khalili L., Malmsten A., Thorstensson R., Feny E-M., Kllander C.F.R. and Gronowitz J.S. Differences in reverse transcriptase activity versus p24 antigen detection in cell culture, when comparing a homogenous group of HIV-1 subtype B viruses with a heterogeneous group of divergent strains. AIDS Res. Hum. Retroviruses. 14(4), 347-352, 1998. Malmsten A, Ekstrand DHL, kerblom L, Gronowitz JS, Kllander CFR, Bendinelli M and Matteucci D. A colorimetric reverse transcriptase (RT) assay optimized for Moloney murine leukemia virus and its use for characterization of RTs of unknown identity. J.Virol. Meth.75, 9-20, 1998. Shao X, Rytting A-S, Ekstrand DHL, Vrang L, Kllander CFR and Gronowitz JS. Colorimetric assays for the evaluation of mode of action of human immunodefiency virus type 1 non-nucleoside reverse transcriptase inhibitors. Antivir. Chem. Chemother. 9(2):167-176, 1998. Awad RJ-K, Corrigan GE, Ekstrand D H L, Thorstensson R, Kllander CFR and Gronowitz JS. Measurement of levels of HIV-1 reverse transcriptase (RT) and RT activity blocking antibody in human serum by a new standardized colorimetric assay. J.Clin. Microb. 35, 1080-1089, 1997. Ekstrand DHL, Bttiger D, Anderson H, Gronowitz JS and Kllander CFR. Reverse transcriptase and corresponding activity blocking antibody in monitoring SIVsm infection in macaques. AIDS Res. Hum. Retroviruses. 13, 601-610, 1997. Shao X, Ekstrand DHL, Bhikhabhai R, Kllander CFR and Gronowitz JS. A non-radioactive microtitre plate reverse transcriptase (RT) assay, based on immobilised template, for screening of RT inhibitors and evaluation of their mode of action. Antivir. Chem. Chemother. 8, 149-159, 1997. Ekstrand DHL, Awad RJ-K, Kllander CFR and Gronowitz JS. A sensitive assay for the detection and quantification of RT activity, based on the use of carrier bound template and non-radioactive product detection, with special reference to HIV isolation. BAB. 23, 95-105, 1996. APPLICATION PAPERS (ExaVirTM Kits, Lenti-RT Activity Assay, C-type RT Activity Assay and Cavidi HS-kit Mn RT) Braun J., Plantier J-C., Hellot M-F., Tuaillon E., Gueudin M., Damond F., Malmsten A., Corrigan G.E. Simon F. A new quantitative HIV load assay based on plasma virion reverse transcriptase activity for the different types, groups and subtypes. Accepted for publication in AIDS. Niebert M., Rogel-Gaillard C., Chardon P., Tonjes R.R. Characterization of chromosomally assigned replication-competent gamma porcine endogenous retroviruses derived from a large white pig and expression in human cells. J Virol 2002 Mar; 76(6): 2714-20. 19 Oldmixon B.A., Wood J.C., Ericsson T.A., Wilson C.A., White-Scharf M.E., Andersson G., Greenstein J.L., Schuurman H.J., Patience C. Porcine endogenous retrovirus transmission characteristics of an inbred herd of miniature Swine. J Virol 2002 Mar; 76(6): 3045-8. van der Laan L.J.W., Lockey C., Griffeth B.C., Frasier F.S., Wilson C.A., Onions D.E., Hering B.J., Long Z., Otto E., Torbett B.E. and Salomon D.R. Infection by porcine endogenous retrovirus after islet xenotransplantation in SCID mice. Nature. Vol 407, 90-4, 7 Sept, 2000. Tonjes R.R., Czauderna F., Fischer N., Krach U., Boller K., Chardon P., Rogel-Gaillard C., Niebert M., Scheef G., Werner A. and Kurth R. Molecularly cloned porcine endogenous retroviruses replicate on human cells. Transplant Proc. 32(5): 1158-61, 2000. Czauderna F., Fischer N., Boller K., Kurth R. and Tonjes R.R. Establishment of moleculer clones of porcine endogenous retrovirus replicating on human cells. J Virol. 74(9): 4028-38, 2000. Hill C.L., Bieniasz P.D., McClure M.O. Properties of human foamy virus relevant to its development as a vector for gene therapy. J. Gen. Virol. 80: 2003-2009, 1999. Dejucq N, Simmons G, Clapham PR. Expanded tropism of primary human immunodeficiency virus type 1 R5 strains to CD4(+) T-cell lines determined by the capacity To exploit low concentrations of CCR5. J Virol. 73(9):7842-7, 1999. Hibbitts S, Reeves JD, Simmons G, Gray PW, Epstein LG, Schols D, de Clercq E, Wells TN, Proudfoot AE, Clapham PR. Coreceptor ligand inhibition of fetal brain cell infection by HIV type 1. AIDS Res Hum Retroviruses. 15(11):989-1000, 1999. Lawoko AL, Johansson B, Hjalmarsson S, Christensson B, Ljungberg B, Al-Khalili L, Sjolund M, Pipkorn R, Fenyo EM, Blomberg J. Comparative studies on neutralisation of primary HIV-1 isolates by human sera and rabbit anti-V3 peptide sera. J Med Virol. 59(2):169-179, 1999. Reeves JD, Hibbitts S, Simmons G, McKnight A, Azevedo-Pereira JM, Moniz-Pereira J, Clapham PR. Primary human immunodeficiency virus type 2 (HIV-2) isolates infect CD4-negative cells via CCR5 and CXCR4: comparison with HIV-1 and simian immunodeficiency virus and relevance to cell tropism In vivo. J Virol. 73(9):7795-804, 1999. McKnight A, Dittmar MT, Moniz-Periera J, Ariyoshi K, Reeves JD, Hibbitts S, Whitby D, Aarons E, Proudfoot AE, Whittle H, Clapham PR. A broad range of chemokine receptors are used by primary isolates of human immunodeficiency virus type 2 as coreceptors with CD4. J Virol. 72(5):4065-71, 1998. Simmons G, Reeves JD, McKnight A, Dejucq N, Hibbitts S, Power CA, Aarons E, Schols D, De Clercq E, Proudfoot AE, Clapham PR. CXCR4 as a functional coreceptor for human immunodeficiency virus type 1 infection of primary data evaluation programphages. J Virol. 72(10):8453-7, 1998. Dittmar MT, Simmons G, Hibbitts S, OHare M, Louisirirotchanakul S, Beddows S, Weber J, Clapham PR and Weiss RA. Langerhans cell tropism of HIV-1 subtype A through F isolates derived from different transmission groups. J Virol. 71(10), 8008-8013, 1997. Simmons G, Clapham PR, Picard L, Offord RE, Rosenklide MM, Schwartz TW, Buser R, Wells TNC and Proudfoot AEI. Potent inhibition of HIV-1 infectivity in data evaluation programphages and lymphocytes by a novel CCRS antagonist. Science. 276, 276-9, 1997. Tacke SJ, Kurth R and Denner J. Porcine endogenous retroviruses inhibit human immune cell function: Risk for xenotransplantation? Virology. 268, 87-93, 2000. 20 Ordering Information Lot nr: VLIEng-021209 Orders and inquiries regarding this and other RT or DNA polymerase activity kits should be adressed to Cavidi Tech. Users of this kit are also advised to contact Cavidi Tech for technical support. 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