The RetroSys™ RT Activity Kit
Product code: RT-001

Protocol for determination of IC₅₀ value of RT activity inhibiting substances.

23 substances and one reference sample may be analysed with one kit.

1. Collect kit components.
• Take out the following components from the refrigerator:

  2 PolyA Plates
  1 Sample Dilution Buffer (bottle B)
  1 RT Reaction Components (vial C1)
  1 Reconstitution Buffer (bottle C2)
  1 HIV-1 rRT-standard (vial D)
  1 Concentrated Washing Buffer (bottle E)
  4 pieces of adhesive tape
  

2. Prepare the diluted sample dilution buffer.
• Label a 100 ml glass beaker "1".
• Add 30 ml of Sample Dilution Buffer (B) to beaker 1.
• Add 30 ml of distilled water to beaker 1.

  
  

3. Dilute the substances.
   When diluting the substances, mix thoroughly and change pipette tips between each transfer.
• Add 135 µl of the diluted sample dilution buffer (beaker 1) to each well of two sample preparation plates (96-well mictrotiter plate, not supplied).
• Add 15 µl of the first substance to be analysed to well A1.
• Add 15 µl of the second substance to be analysed to well A2 and so on until the substances have been added to wells A1-12 of both plates.
• Transfer 15 µl from wells A1-12 down to their corresponding B-wells.
• Transfer 15 µl from wells B1-12 down to their corresponding C-wells of both plates and so on until the G-rows of both plates are reached.
  The H-row is used for references and background controls.

  
  

4. Prepare the reaction mixture.
• Add 2 ml of Reconstitution Buffer (C2) to the vial containing RT Reaction Components (C1). Make sure that the lyophilised material dissolves properly and mix thoroughly.
• Transfer 200 µl of the dissolved material back to the Reconstitution Buffer (C2) and mix thoroughly.

  
  

5. Prepare the PolyA Plates with samples.
   When transferring samples start with the H-row and work upwards through the plate as this prevents the need to change pipette tips. The pipette tips should however be changed between plates.
• Take out both PolyA Plates from their foil pouches.
• Add 100 µl of reaction mixture (C2) to each well of both plates.
• Transfer 50 µl from each well of the first sample preparation plate to its corresponding well of the first PolyA Plate.
• Transfer 50 µl from each well of the second sample preparation plate to its corresponding well of the second PolyA Plate.
• Seal the plates with adhesive tape. Press the tape down firmly over each well to ensure proper sealing.
• Incubate the PolyA plates for 30 minutes at 33°C.

  
  

6. Dilute the HIV-1 rRT-standard.
• Add 5 ml of diluted sample dilution buffer (beaker 1) to the vial containing lyophilised HIV-1 rRT-standard (D). Mix thoroughly and make sure that the lyophilised material dissolves properly
• Label a 50 ml glass beaker "2".
• Add 12 ml of diluted sample dilution buffer (beaker 1) to beaker 2.
• Add 1 ml of HIV-1 rRT-standard (D) to beaker 2 and mix thoroughly.

  
  

7. Start the RT reaction.
• Take out the PolyA Plates from the incubator.
• Add 50 µl of standard enzyme (beaker 2) to all wells except H8-12 of both PolyA plates.
• Add 50 µl of diluted sample dilution buffer (beaker 1) to wells H8-12 of both plates.
• Seal the plates with adhesive tape. Press the tape down firmly over each well to ensure proper sealing.
• Incubate the plates for three hours at 33°C on an orbital shaker set at gentle agitation.

  
  

8. Collect kit components.
• Take out the kit box from the refrigerator and take out the following components:

  1 Concentrated Washing Buffer (bottle E)
  2 RT Product Tracer (vials O)
  1 AP Substrate Tablets (tube P1)
  1 AP Substrate Buffer (bottle P2)
  2 pieces of adhesive tape
  2 pieces of plastic lids

The remaining components are not used in this application and may be discarded after performing the assay.


9. Prepare the wash fluid.
• Add 150 ml of Triton X-100 to 1 litre of distilled water and mix thoroughly with a magnetic spin bar.
  Make sure that the Triton X-100 dissolves completely which takes approximately 10 minutes.
• Add 20 ml of Concentrated Washing Buffer (E) to a 20 litre container.
• Add the dissolved Triton X-100 to the 20 litre container.
• Adjust the volume to 20 litres with distilled water and mix thoroughly.

  
  

10. Prepare the RT Product Tracer.
• Add 12 ml of distilled water to each of the two vials containing RT Product Tracer (O). Make sure that the lyophilised material dissolves properly and mix thoroughly.
• Transfer the contents of both O vials to a 50 ml glass beaker.

  
  

11. Stop the RT reaction of the plates.
    After three hours of incubation the RT reaction of the plates is stopped by washing. Use approximately 6 ml of wash fluid per well and wash.
• Wash both PolyA Plates twice. Turn the plates 180° between washes if possible.
• Remove residual fluid in the wells by tapping the plate upside down on absorbing cloth or paper.

  
  

12. Start the binding of RT Product Tracer.
    When dispensing the tracer, pipette tips should not touch the bottom of the wells.
• Add 100 µl to each well of both PolyA Plates.
• Seal the plates with adhesive tape. Press the tape down firmly over each well to ensure proper sealing.
• Incubate the plates for 90 minutes at 33°C on an orbital shaker set at gentle agitation.

  
  

13. Prepare the AP substrate solution.
• Add the AP Substrate Tablets (P1) to the AP Substrate Buffer (P2).
• Shake the bottle occasionally and the tablets will dissolve completely in approximately twenty minutes.

  
  

14. Remove excess tracer.
    After 90 minutes of incubation, excess tracer is removed by washing. Use approximately 6 ml of wash fluid per well and wash.
• Wash both PolyA Plates four times. Turn the plates 180° between washes if possible.
• Remove residual fluid in the wells by tapping the plate upside down on absorbing cloth or paper.

  
  

15. Start the alkaline phosphatase reaction.
    The AP substrate solution must be mixed thoroughly and equilibrated to room temperature before it is added to the PolyA Plates. When dispensing the substrate solution, pipette tips should not touch the bottom of the wells.
• Add 200 µl of AP substrate solution to each well of both PolyA Plates.
• Cover the plates with plastic lids and incubate them at room temperature under dark cover on an orbital shaker set at gentle agitation.

  
  

16. Read the plates.
    Set the filter of the plate reader to 405 nm.
• Read the absorbance of both plates after 30 minutes of incubation.
• Repeat the reading of the plates at intervals until wells H1-7 give values of approximately 75% of the maximum value within the linear reading range of the plate reader.

  
  

17. Process the data.
    Use an alkaline phosphatase reading time at which wells H1-7 give approximately 75% of the maximum value within the linear measuring range of the plate reader. Follow the instructions for each plate separately.
• Calculate the mean A₄₀₅ of the background controls (wells H8-12).
• Subtract the mean A₄₀₅ of the background controls from the obtained A₄₀₅ of the other wells.
• Calculate the mean A₄₀₅ of the HIV-1 rRT standard (wells H1-7).
• Express the RT activity for wells containing substance dilutions as a percentage of the mean A₄₀₅ HIV-1 rRT standard.
• Plot the percentage of residual RT activity against the concentrations of the substance dilutions for each of the tested substances.